Molecular and
Experimental Medicine
Within the “neurovascular unit” neuron activation controls microvascular vasoreactivity via the astrocytes, which are a necessary part of the microvessel. During
ischemic stroke, microvessel and neuron activation occur rapidly and simultaneously. Members of Gregory del Zoppo’s laboratory have demonstrated that loss of
vascular basal lamina matrix with the rapid generation of pro-MMP-2, its activation systems, and other proteases accompany neuron injury. Furthermore, when
the endothelial blood-brain barrier becomes permeable, fibrin is deposited in
microvessels where thrombin is generated when plasma contacts perivascular tissue factor (TF). Recently, the laboratory has shown that tissue factor pathway
inhibitor (TFPI) is also generated by the endothelium and by activated microglial
cells in the ischemic regions. These alterations are part of the evolution of injury
in the neurovascular unit. Illustration prepared by Janet Hightower.
Eric F. Johnson, Ph.D.
Professor
Acting Head, Division of Biochemistry
MOLECUL AR AND EXPERIMENTAL MEDICINE
DEPAR TMENT OF
MOLECULAR AND
E X P E R I M E N TA L
MEDICINE
S TA F F
Ernest Beutler, M.D.*
Chairman and Professor
Head, Division of Hematology
Masahiro Aoki, M.D., Ph.D.
Adjunct Assistant Professor
Hiroshi Asahara, M.D., Ph.D.
Assistant Professor
Bonno N. Bouma, Ph.D.
Adjunct Professor
Joel N. Buxbaum, M.D.
Professor
Head, Division of Research
Rheumatology
Dennis A. Carson, M.D.
Adjunct Professor
Sergio D. Catz, Ph.D.
Assistant Professor
Francis V. Chisari, M.D.
Professor
Head, Division of
Experimental Pathology
Clifford W. Colwell, Jr., M.D.
Adjunct Professor
Laura M. Crisa, M.D.
Assistant Professor
Arthur D. Dawson, M.D.
Adjunct Professor
Albert B. Deisseroth, M.D.,
Ph.D.
Adjunct Professor
Gregory J. del Zoppo, M.D.**
Associate Professor
Thomas F. Deuel, M.D.
Professor
Head, Division of Molecular
Oncology
Darryl D’Lima, M.D.
Assistant Professor
Darlene J. Elias, M.D.
Adjunct Associate Professor
Brunehilde FeldingHabermann, Ph.D.
Associate Professor
Mitchell H. Friedlaender,
M.D.
Adjunct Professor
Jeffrey S. Friedman, M.D.,
Ph.D.
Assistant Professor
Theodore Friedmann, M.D.
Adjunct Professor
Andrew J. Gale, Ph.D.
Assistant Professor
Roberta A. Gottlieb, M.D.
Associate Professor
John H. Griffin, Ph.D.**
Professor
Andras Gruber, M.D.
Adjunct Assistant Professor
Luca G. Guidotti, D.V.M.,
Ph.D.
Associate Professor
2006
THE SCRIPPS RESEARCH INSTITUTE
Eric F. Johnson, Ph.D.
Professor
Acting Head, Division of
Biochemistry
Thomas J. Kipps, M.D.,
Ph.D.
Adjunct Professor
Lawrence E. Kline, D.O.
Adjunct Associate Professor
James A. Koziol, Ph.D.
Professor
Head, Division of
Biomathematics
Daniel F. Kripke, M.D
Adjunct Professor
Thomas J. Kunicki, Ph.D.*
Associate Professor
Pauline L. Lee, Ph.D.
Associate Professor
Stuart A. Lipton, M.D., Ph.D.
Adjunct Professor
Martin Lotz, M.D.
Professor
Head, Division of Arthritis
Research
Christopher Lee Marsh, M.D.
Adjunct Associate Professor
239
Giuseppe Remuzzi, M.D.
Adjunct Professor
Michael W. Robertson, Ph.D.
Associate Professor
Zaverio M. Ruggeri, M.D.**
Professor
Head, Division of
Experimental Hemostasis
and Thrombosis
Enrique Saldivar, M.D., Ph.D.
Adjunct Assistant Professor
Daniel R. Salomon, M.D.
Associate Professor
Alessandro Sette, Ph.D.
Adjunct Professor
Farhad F. Shadan, M.D.,
Ph.D.
Adjunct Assistant Professor
Sanford J. Shattil, M.D.
Adjunct Professor
Alexander R. Shikhman,
M.D., Ph.D.
Adjunct Assistant Professor
Inmaculada Silos-Santiago,
M.D., Ph.D.
Adjunct Associate Professor
Robert McMillan, M.D.
Professor Emeritus
Gregg J. Silverman, M.D.
Adjunct Professor
William E. Miller, M.D.
Adjunct Assistant Professor
Ronald A. Simon, M.D.
Adjunct Professor
Kevin V. Morris, Ph.D.
Assistant Professor
Jorge J. Nieva, M.D.
Assistant Professor
Peter J. Sims, M.D.,
Ph.D.***
Professor
University of Rochester
Rochester, New York
Marta Perego, Ph.D.
Associate Professor
Jack C. Sipe, M.D.
Associate Professor
Frank M. Huennekens, Ph.D.
Professor Emeritus
Paul J. Pockros, M.D.
Adjunct Assistant Professor
Donald D. Stevenson, M.D.
Adjunct Professor
Shaun Phillip Jackson, Ph.D.
Adjunct Associate Professor
K. Michael Pollard, Ph.D.
Associate Professor
Eng M. Tan, M.D.
Professor Emeritus
Asa B. Gustafsson, Ph.D.
Assistant Professor
Anne M. Hanneken, M.D.
Associate Professor
Mary J. Heeb, Ph.D.**
Associate Professor
James A. Hoch, Ph.D.
Professor
Head, Division of Cellular
Biology
240 MOLECUL AR AND EXPERIMENTAL MEDICINE
Bruce E. Torbett, Ph.D.
Associate Professor
Susan L. Uprichard, Ph.D.
Assistant Professor
Kottayil I. Varughese,
Ph.D.**
Associate Professor
Peter K. Vogt, Ph.D.
Professor
Head, Division of Oncovirology
Matthias G. von Herrath,
M.D.
Adjunct Associate Professor
Therese Wiedmer, Ph.D.***
Associate Professor
University of Rochester
Rochester, New York
Xiaohua Wu, Ph.D.
Assistant Professor
2006
THE SCRIPPS RESEARCH INSTITUTE
Uzen Savas, Ph.D.
Giulio Cattarossi, Ph.D.
Sharookh B. Kapadia, Ph.D.
Stefan Wieland, Ph.D.
David M. Cauvi, Ph.D.
Jung Hwan Kim, Ph.D.
Akemi Yagi, Ph.D.
Yunchao Chang, Ph.D.
Joseph S. Krueger, Ph.D.
Ji Zhao, Ph.D.***
Department of Biomedical
Sciences
Scripps Florida
Emily I. Chen, Ph.D.
Pablo G. Landart, Ph.D.
Guofeng Cheng, Ph.D.
Alan Yueh-Luen Lee, Ph.D.
Stephanie Cherqui, Ph.D.
Shi-Sheng Li, Ph.D.
Ian D. Dang, Ph.D.***
United States Patent and
Trademark Office
Rockville, Maryland
Enbo Liu, Ph.D.***
The Burnham Institute
La Jolla, California
Quansheng Zhou, Ph.D.
SENIOR RESEARCH
A S S O C I AT E S
Miao-Chia Lo, Ph.D.
Hiroshi Deguchi, M.D., Ph.D.
Chinh T. Dao, Ph.D.
Yuichi Kamikubo, Ph.D.
Maria F. Del Papa, Ph.D.
Richard D. Milner, M.D.,
Ph.D.
Adam Denley, Ph.D.
Jiann-Kae Luo, Ph.D.
Holly N. Maier, Ph.D.
Mathieu Marella, Ph.D.
Alejandra R. Diaz, Ph.D.
Laurent O. Mosnier, Ph.D.
Florent M. Martin, Ph.D.
Jonathon M. Flanagan, Ph.D.
Takao Yagi, Ph.D.
Associate Professor
Deirdre M. O’Sullivan, Ph.D.
Tatsuya Fukushima, Ph.D.
Keith Stephenson, Ph.D.
Dong-Er Zhang, Ph.D.
Associate Professor
Yuri Martina, Ph.D.***
Adaltis
Rome, Italy
Michael J. Giffin, Ph.D.
Jill M. Waalen, M.D.
Yasunori Mishima, M.D.
Shawn Patrick Grogan, Ph.D.
S TA F F S C I E N T I S T S
Subramanian Yegneswaran,
Ph.D.
Andreas G. Bader, Ph.D.
R E S E A R C H A S S O C I AT E S
Joseph R. Biggs, Ph.D.
Eun-Young Ahn, Ph.D.
Marco Gymnopoulos, Ph.D.
Wolf-Achim Hassenpflug,
M.D.
Daniela Beatriz Munafo,
Ph.D.
Eiko Nakamaru-Ogiso, Ph.D.
Akiko Okumura, Ph.D.
Reha Celikel, Ph.D.
Shinichi Asabe, Ph.D.
Dominik R. Haudenschild,
Ph.D.
Mei-Hui Hsu, Ph.D.
Dong Bai, Ph.D.
Gonzalo Herradon, Ph.D.
Erin N. Olson, Ph.D.
Chengqun Huang, M.D.,
Ph.D.
Jennifer L. Barber-Singh,
Ph.D.
Matteo Iannacone, M.D.
Mee Young Park, Ph.D.
Masanori Isogawa, M.D.
Natalie M. Pecheniuk, Ph.D.
Jennifer L. Johnson, Ph.D.
Cristina Bongiorni, Ph.D.
Tatsuo Ito, M.D.
Luke F. Peterson, Ph.D.
Klaus Kuhn, Ph.D.
Kristen E. Bower, Ph.D.
Hao Jiang, Ph.D.
Pablo Perez Pinera, M.D.
Sunil M. Kurian, Ph.D.
Anita Y. Boyapati, Ph.D.
Sohye Kang, Ph.D.
Gian Marco Podda, M.D.
Patrizia Marchese, Ph.D.
Nathan R. Brady, Ph.D.
Katia Maria Cabral, Ph.D.
Brian Savage, Ph.D.
Anna E. Cartier, Ph.D.
Mou-Chieh Kao, Ph.D.***
National Tsing Hua
University
Hsinchu, Taiwan
Natalia Reixach, Ph.D.
Tsaiwei Olee, Ph.D.
Fumihiko Okumura, Ph.D.
Rosamund Leila Reynald,
Ph.D.
MOLECUL AR AND EXPERIMENTAL MEDICINE
Bruno Sainz, Jr., Ph.D.
Stefaan J. Sansen, Ph.D.
Francesca Scaramozzino,
Ph.D.
Jason K. Yano, Ph.D.***
Takeda San Diego, Inc.
San Diego, California
Zhengyi Ye, Ph.D.
Jinseong Yi, Ph.D.
Jin Shi, Ph.D.
Xiaoyan Yin, Ph.D.
Misako Shibakura, Ph.D.***
Okayama University Medical
School
Okayama, Japan
Antonella Zampolli, Ph.D.
Wei Zhang, Ph.D.
Christina H. Swan, Ph.D.
Li Zhao, Ph.D.
Hendrik Szurmant, Ph.D.
Jin Zhong, Ph.D.
Noboru Taniguchi, M.D.
Weiguo Zou, Ph.D.
Jesus Torres-Bacete, Ph.D.
Masahiko Zuka, M.D., Ph.D.
Jaroslav Truksa, Ph.D.
S C I E N T I F I C A S S O C I AT E S
Masanao Tsuda, Ph.D.
Fanny E. Almus, Ph.D.
Billyana C. Tsvetanova, Ph.D.
Jose A. Fernandez, Ph.D.
Ji Wang, Ph.D.
Yang Wang, Ph.D.***
University of Texas
MD Anderson Cancer Center
Houston, Texas
Gabriele E. Foos, Ph.D.
Terri P. Gelbart, B.S., M.T.
Byoung Boo Seo, Ph.D.
Zhuangzhi Wang, Ph.D.
* Joint appointment in The Skaggs
Martin R. Weber, Ph.D.***
Bristol Myers-Squibb
Pharmaceutical Research
Institute
Wallingford, Connecticut
Andrea K. White, Ph.D.***
Chico State University
Chico, California
Robert A. White, Ph.D.
Adam C. Wilson, Ph.D.
Tetsuo Yamashita, Ph.D.
Ming Yan, Ph.D.
Xia Yang, Ph.D.
Institute for Chemical Biology
** Joint appointment in Department
of Cell Biology
*** Appointment completed, new location shown
2006
THE SCRIPPS RESEARCH INSTITUTE
241
242 MOLECUL AR AND EXPERIMENTAL MEDICINE
Ernest Beutler, M.D.
Chairman’s Overview
he faculty of the Department of Molecular and
Experimental Medicine comprises a group of 44
eclectic investigators whose focus is often in the
area referred to as translational medicine. Despite the
“translational” nature of much of our research, only 18
of our faculty members hold an M.D. degree, and only 3
of these are involved in direct patient care. This reflects
a longstanding national trend in which M.D.s bifurcate
their careers into either bench or bedside, and only rarely
both. Most, but not all, of the research in the department
is preclinical or even pre-preclinical. Indeed, only about
5% of the research support in the department comes from
industry; virtually all the rest comes from the National
Institutes of Health, mostly to individual investigators in
the form of R01 grants.
The pages that follow this overview contain the summaries of the year’s work by the investigators themselves.
These summaries demonstrate the breadth of biomedical
problems currently under investigation in this department.
But significant biomedical research rarely advances in
1-year segments. Some members of our faculty have
played a significant role in developing whole areas of
knowledge. Here I try to encapsulate what some of our
scientists are doing and, to some extent, what they have
done leading up to this work.
T
2006
THE SCRIPPS RESEARCH INSTITUTE
John Griffin and his colleagues have been leaders in
the area of thrombotic diseases (thrombophilia) for many
years. Dr. Griffin’s discoveries 25 years ago led to the
understanding of the physiologic function of protein C. A
deficiency of this anticoagulant protein is associated with
increased risk of venous thrombosis. As a result of some
of these early studies, activated protein C is now used in
the treatment of endotoxin shock. Recently, Dr. Griffin and
his colleagues have shown that activated protein C can
reduce bleeding induced by tissue plasminogen activator,
one of the treatments for stroke. Members of the Griffin
laboratory are currently also studying the role of lipoproteins in the risk of venous thrombosis; Dr. Griffin and his
colleagues at the Green Hospital have found that low levels of high-density lipoprotein are an important risk factor.
Bruce Torbett’s group is continuing to make progress toward the development of an innovative treatment
for HIV infections. Their approach is based on the finding that CCR5 is 1 of the 2 main chemokine receptors
for HIV entry into cells. Delivering a single-chain antibody against this receptor protects cells against entry
of HIV. Torbett uses HIV-derived vectors for gene delivery, a technology that his laboratory helped to pioneer.
Most of the work in Daniel Salomon’s laboratory is
directed at improving surgical transplantation of organs
such as liver, heart, or kidney. One experimental approach
to overcoming the shortage of human organs is to transplant pig organs into human patients. Pigs are similar in
size to humans, and if such xenotransplantation could be
achieved, there would be a virtually unlimited supply of
organs. Although some of the formidable immunologic
barriers to the transplantation of pig organs into humans
have been overcome, pigs harbor endogenous retroviruses. This has raised concerns not only about the
safety of patients who are receiving these transplants but
conceivably about the safety of the human species as a
whole. Animal viruses can wreak havoc on humankind, as
exemplified by bird influenza. Scientists in Dr. Salomon’s
laboratory have engineered a murine model consisting of
mice transgenic for the human porcine retrovirus receptor,
which his laboratory helped to identify and characterize.
His recent finding that these animals are infected with
retrovirus has very important implications for the use of
porcine organs in human organ transplantation.
Peter Vogt, head of the Division of Oncovirology, is
a preeminent pioneer in the field of viruses and cancer,
having discovered that viruses can “steal” genes from
vertebrates and turn them into cancer-causing genes.
Scientists in his laboratory have continued to expand our
MOLECUL AR AND EXPERIMENTAL MEDICINE
understanding of how mutated genes cause cancer. In
the past year, he and his colleagues demonstrated that
the phosphatidylinosital-3′-kinase that is mutated in
human tumors actually gains in enzymatic function and
that it can cause malignant transformation both in cell
culture and in intact animals.
Dong-Er Zhang and her group have continued to
expand our understanding of how the fusion gene AML1ETO causes acute myeloid leukemia in humans. In the
past year, Dr. Zhang and her group have been able to
show that an alternately spliced form of this fusion gene
is a potent inducer of leukemia. Members of the Zhang
laboratory have also pioneered the study of UBP43, an
enzyme that removes an ubiquitin-like protein from its
targets. They have now found that it serves as a novel
inhibitor to regulate interferon signaling.
The cytochrome P450s are a large group of enzymes
that function to metabolize both endogenous and exogenous compounds that need to be degraded. Eric Johnson,
acting head of the Division of Biochemistry, has pioneered
our understanding of the structure of these compounds,
an important step in understanding of how they actually
function. In the past year, Dr. Johnson has succeeded
in determining the structure of the cytochrome P450
that metabolizes nicotine in humans. This discovery
could aid in the design of drugs that may help smokers
kick the habit.
Brunhilde Felding-Haberman has developed a murine
model of human breast cancer. Having isolated human
antibodies directed at the activated confirmer of integrin
αVβ3 on human cells, she has used this model to study
the effectiveness of the antibody both in preventing and
in treating metastases. She is also using it to determine
whether neural stem cells may be effective in inhibiting
brain metastases of breast cancer cells.
My own laboratory has a long history of studying
single-gene diseases. The discovery of glucose-6-phosphate dehydrogenase deficiency was the first of these
and led to my origination of the X-inactivation hypothesis.
Since that time my colleagues and I have investigated
many of the inherited red-cell enzyme deficiencies, the
glycolipid storage disorders, particularly Gaucher disease,
and inborn errors of iron metabolism. It is the latter area
that is currently receiving most attention from my group.
The pathways that regulate the amount of iron absorbed
from the intestine and therefore maintain total body iron
within normal limits have proved to be much more complex than anyone had imagined. The 25 amino acid peptide hepcidin seems to play a particularly important role,
2006
THE SCRIPPS RESEARCH INSTITUTE
243
and we are striving to untangle the role of a complex series
of factors including inflammatory cytokines IL-6 and IL-1,
bone morphogenic proteins, and iron itself that serve to
regulate the production of hepcidin. These studies may
lead to more efficient treatment of diseases in which body
iron content is increased (hemochromatosis) or deficient
and of the treatment of the anemia of chronic inflammation, in which hepcidin is also involved.
Roberta Gottlieb and her colleagues demonstrated
that the death of cardiomyocytes after a heart attack
was not necessarily due to necrosis but was largely a
result of apoptosis. This opened the door to a variety of
therapeutic strategies. Members of her laboratory are now
focused on the study of autophagy in this process.
James Hoch and Marta Perego have pioneered phosphorelay signal transduction pathways in sporulating
bacteria and the regulation of phosphate flow in these
phosphorelays by a large number of phosphatases. They
have now turned their attention to unraveling the cellular regulatory mechanisms that control transcription of
the anthrax toxin genes.
Pleotrophin is a cytokine identified and cloned by
Thomas Deuel, head of the Division of Molecular Oncology. Pleotrophin has many different functions because
it inactivates a phosphatase that has several different
substrates, which have been identified by Dr. Deuel and
his colleagues. Among its various functions are the disruption of normal cytoskeletal architecture, inhibition of
neurite outgrowth in PC12 cells, and angiogenesis, particularly in tumor growth. Further understanding of the
functioning of pleotrophin and developing methods for
enhancing or inhibiting its activities offer many possibilities for treatment of several different disorders.
Joel Buxbaum, head of the Division of Research
Rheumatology, has a longstanding interest in the misfolding of proteins giving rise to deposits generically known
as “amyloid.” A single amino acid substitution may greatly
increase the propensity of a protein to misfold. One such
substitution is very common among people of African
origin as a result of a single base-pair change in the gene
encoding transthyretin. In the case of this particular misfolding protein, deposits occur in the heart, and scientists in the Buxbaum laboratory have recently shown that
10% of African Americans over the age of 65 with congestive heart failure carry this mutation. This is quite a
remarkable finding, with obvious health implications for
this ethnic group.
Francis Chisari, head of the Division of Experimental
Pathology, is an internationally recognized expert in the
244 MOLECUL AR AND EXPERIMENTAL MEDICINE
field of hepatitis. More than 20 years ago, using then
very new transgenic mouse technology, he produced the
first small animal model of hepatitis B infection—actually
the first transgenic mouse model of any human pathogen. In more recent years, scientists in his laboratory
have also been studying hepatitis C. The agent for this
important infection was discovered in 1989, and the
development of a tissue culture model of this infection
has eluded investigators in this field until last year, when
Chisari’s group and two other laboratories simultaneously developed the first tissue culture model of infection. Dr. Chisari and his group are now using this model
to identify entry and egress mechanisms in the virus, to
define metabolic and signaling pathways that regulate
the infection, and ultimately to develop antiviral drugs
to treat chronic infection.
Osteoarthritis, essentially the wearing out of joints,
affects most people sooner or later. One of the major
underlying problems is that as cartilage cells are destroyed
or die, they are not replaced. Scientists in the Division
of Arthritis Research, headed by Martin Lotz, are studying animal models to develop means of preventing cartilage loss, and they have recently found that inhibitors
of caspase injected into the joint in animals in these
models of osteoarthritis prevent the destruction of cartilage. This opens up the possibility of a new treatment
for this common, debilitating disorder, which Dr. Lotz
and his group will study in animals with the hope that a
human therapeutic may emerge.
Zaverio Ruggeri, head of the Division of Experimental
Hemostasis and Thrombosis, is internationally known for
his insights into the mechanism of thrombosis. He has
shown that the process of platelets adherence to endothelium is very different in the static systems that were
once used than in the dynamic flow systems that he has
pioneered. Working together with Tom Kunicki in his division, Dr. Ruggeri is mapping new genes that are important in the regulation of clot formation at the sites of
blood vessel injury. In collaboration with Luca Giodotti,
in the Division of Experimental Pathology, members of
Dr. Ruggeri’s group are studying an unexpected role of
platelets in immune-mediated processes, and particularly
in viral clearance.
To some extent, one may dichotomize biomedical
research into big research projects in which many principal investigators, often at several different institutions,
are addressing a problem of some significance, and
smaller projects headed by a single principal investigator.
The prototype of big science—a large-scale scientific
2006
THE SCRIPPS RESEARCH INSTITUTE
effort—is the Manhattan project of World War II, the
project that engineered and built the first atomic bombs.
Those leading the National Institutes of Health seem to
have become enamored of this approach to bioscience,
with the idea that large networks, interdisciplinary
approaches, and extensive collaboration are needed to
better understand Nature. Indeed, some problems require
this sort of approach. They include the genome project,
multicenter clinical trials, and large epidemiologic studies. But what must not be forgotten is that truly great,
innovative ideas do not usually arise in committees, and
that such large efforts are generally based on fundamental ideas and techniques developed by single investigators heading small research grants. Most of the research
in the Department of Molecular and Experimental Medicine is of the latter ilk. Members of our faculty have
contributed some of the fundamental ideas on which
today’s science is based, and this is what we continue
to try to achieve.
MOLECUL AR AND EXPERIMENTAL MEDICINE
INVESTIGATORS’ R EPORTS
D IVISION
OF
ARTHRITIS RESEARCH
Martin Lotz, M.D., Division Head
2006
THE SCRIPPS RESEARCH INSTITUTE
245
that mechanical dynamic compression of chondrocytes
led to rearrangement of the actin cytoskeleton. Currently
we are investigating the role of a signaling pathway that
involves Rho kinase and the actin-regulating protein
cofilin. These studies may reveal novel pathways and
therapeutic targets for stimulating formation of the extracellular matrix of cartilage.
Joint Injury and Osteoarthritis
D. D’Lima, C.W. Colwell, Jr., M. Lotz
cute or chronic mechanical injury is a risk factor
for the development of osteoarthritis. We are
investigating mechanisms that mediate the effects
of mechanical injury on joint integrity and potential therapeutic approaches that target these mechanisms. We
have established in vitro models to apply mechanical
stress to cartilage explants and cells in 3-dimensional
cultures. Application of high-intensity mechanical stress
causes cell death that is mediated in part by apoptotic
mechanisms. Cell death after mechanical injury can be
prevented by pharmacologic inhibitors of caspases; this
treatment results in the maintenance of biosynthetically
active cartilage cells.
In an animal model of posttraumatic osteoarthritis
induced by transection of the anterior cruciate ligament,
intraarticular injection of caspase inhibitors reduced the
severity of cartilage lesions. We plan to examine new
chemical classes of caspase inhibitors in this model. Our
long-term goal is to use the inhibitors to treat patients
with posttraumatic arthritis.
A
Mechanotransduction in
Chondrocytes
D. D’Lima, D. Haudenschild, M. Lotz
hysiologic levels of mechanical load are important in stimulating cartilage cells to maintain
the composition of the extracellular matrix. We
are elucidating the signaling mechanisms that transduce mechanical stimuli into biochemical responses
in cartilage cells. Dynamic compression of cartilage
in vitro exerts anabolic effects and stimulates the production of proteins that make up the cartilage extracellular matrix. Mechanical deformation of cells can
directly or indirectly through receptor-mediated events
affect the organization of the cytoskeleton. We found
P
Physiology of Facilitated Glucose
Transporter GLUT1 in Cartilage
Homeostasis and Osteoarthritis
A.R. Shikhman, D.C. Brinson, J. Valbracht, M. Lotz
rticular cartilage is an avascular tissue that functions under nearly anaerobic conditions and therefore depends on glucose supply for the generation
of energy. Glucose is the main precursor for UDP-hexosamines and UDP-uronic acids, which are used by
chondrocytes in the synthesis of glycosaminoglycans.
Transmembranous glucose transport facilitated by a
group of glucose transporter proteins termed GLUTs is
the first rate-limiting step in glucose metabolism. Human
articular chondrocytes express several specific GLUTs,
including GLUT1, GLUT3, GLUT6, GLUT8, and GLUT10.
GLUT1 is the most abundant glucose transporter in
human articular chondrocytes. Expression of GLUT1 in
chondrocytes is regulated by proinflammatory cytokines
via signaling pathways that depend on protein kinase C
and p38 MAP kinase. Overexpression of GLUT1 occurs
in osteoarthritic cartilage. Small interfering RNA inhibits
expression of GLUT1 in unstimulated and IL-1β–stimulated human articular chondrocytes. Inhibition of this
expression does not significantly change basal facilitated glucose transport, but it abrogates surplus glucose transport induced by cytokines or growth factors,
indicating that GLUT1 is required to provide glucose
supply in activated cells but not in resting cells.
Inhibition of GLUT1 expression in chondrocytes is
associated with suppression of glycolysis and lactate
production. GLUT1-regulated lactate production is more
sensitive to the inhibition of GLUT1 expression than is
transmembranous glucose transport, indicating that one
of the potential functions of GLUT1 is regulation of intracellular glucose flow through the glucose-consuming
metabolic pathways (glycolysis, pentose-phosphate
shunt, and hexosamine pathway).
A
246 MOLECUL AR AND EXPERIMENTAL MEDICINE
Inhibition of GLUT1 expression suppresses basal
and growth factor–stimulated thymidine transport and
chondrocyte proliferation and depends on phosphorylation of AMP-activated protein kinase. Chondrocytes with
suppressed GLUT1 are also characterized by an increase
in basal and growth factor–stimulated production of
hyaluronan and expression of hyaluronan synthase 2
that does not depend on activation of the kinase. Furthermore, inhibition of GLUT1 expression by small interfering RNA also reduces IL-1β–induced production of
nitric oxide. These data indicate that GLUT1 is a novel
regulator of chondrocyte activation and that its functions extend beyond its glucose-transporting activity.
PUBLICATIONS
Cecil, D.L., Johnson, K., Rediske, J., Lotz, M., Schmidt, A.M., Terkeltaub, R.
Inflammation-induced chondrocyte hypertrophy is driven by receptor for advanced
glycation end products. J. Immunol. 175:8296, 2005.
D’Lima, D., Hermida, J., Hashimoto, S., Colwell, C., Lotz, M. Caspase inhibitors
reduce severity of cartilage lesions in experimental osteoarthritis. Arthritis Rheum.
54:1814, 2006.
Hiraoka, K., Grogan, S., Olee, T., Lotz, M. Mesenchymal progenitor cells in adult
human articular cartilage. Biorheology, in press.
Shikhman, A.R. Glucosamine and osteoarthritis. Future Rheumatol. 1:67, 2006.
Transcriptional Regulation of
Chondrogenesis via Chromatin
Modification
H. Asahara, T. Ito, K. Yoshida, N. Taniguchi, M. Tsuda
hondrogenesis is a multistep pathway in which
multipotential mesenchymal stem cells differentiate into chondrocytes. The transcription factor
Sox9 regulates chondrocyte differentiation and cartilagespecific expression of genes, such as COL2A1, which
encodes collagen type II α1. During the past year, we
used an in vitro chromatin assembly model to investigate
the function of p300 in Sox9-dependent transcription.
Using chromatin templates, we determined whether
Sox9 transcriptional activity requires p300. We found
that addition of p300 upregulated the transcriptional
activation by recombinant Sox9, showing that both
p300 and Sox9 are necessary for activation of chromatin-mediated transcription.
Sox9-dependent transcription is regulated by p300mediated histone acetylation on chromatin. Using naked
or chromatinized DNA, we explored the relationship
between p300-induced histone acetylation and Sox9dependent transcriptional activation. Because p300-
C
2006
THE SCRIPPS RESEARCH INSTITUTE
mediated acetylation requires acetyl coenzyme A as a
substrate, we performed in vitro transcription assays
in the presence and absence of this coenzyme. We
found that p300 did not stimulate Sox9-dependent
transcription in the absence of the coenzyme. In histone acetyltransferase assays, both p300 and Sox9
synergistically acetylated histones, which were assembled on a chromatin template. In reporter assays, the
addition of p300 increased the relative luciferase activity
in a Sox9-dependent manner but not in assays in which
we used a mutant p300 deficient in histone acetyltransferase. These results suggest that the altered
chromatin structure caused by the histone acetyltransferase activity of p300 may have an important role in
Sox9-dependent transcription.
In chondrocytes, histone hyperacetylation activates
the expression of genes that encode the extracellular
matrix of cartilage. To assess the relationship between
expression of COL2A1 and histone acetylation on the
COL2A1 enhancer region, we used trichostatin A, a
histone deacetylase inhibitor, to induce histone hyperacetylation in human chondrocytes. We found that
treatment with trichostatin A stimulated COL2A1 expression in chondrocytes.
Taken together, these findings indicate that p300
stimulates Sox9-dependent transcription by modifying
histone acetylation. These results suggest that chromatin
regulation, such as regulation via histone deacetylase-1
inhibitors in chondrocytes, could be a new therapeutic
strategy for treatment of arthritis.
PUBLICATIONS
Furumatsu, T., Tsuda, M., Yoshida, K., Taniguchi, N., Ito, T., Hashimoto, M., Ito, T.,
Asahara, H. Sox9 and p300 cooperatively regulate chromatin-mediated transcription. J. Biol. Chem. 280:35203, 2005.
DIVISION
OF
BIOCHEMISTRY
Eric F. Johnson, Ph.D., Acting Division Head
Cytochrome P450: Regulation,
Structure, and Function
E.F. Johnson, K.J. Griffin, M.-H. Hsu, R.L. Reynald, S.
Sansen, Ü. Savas, J.K. Yano
E
nzymes in the cytochrome P450 superfamily primarily serve 2 purposes in human physiology.
Some P450s catalyze specific biotransforma-
MOLECUL AR AND EXPERIMENTAL MEDICINE
tions in autocrine, paracrine, and endocrine signal
transduction pathways. A second, relatively large group
of P450 monooxygenases play defensive roles by converting toxic compounds to less toxic forms that are
more soluble and more easily excreted than are the parent compounds. Each xenobiotic-metabolizing P450
generally oxidizes structurally diverse substrates, leading to a wide-ranging protective capacity for elimination of toxic chemicals. Often the expression levels of
these enzymes are increased in response to exposure
to xenobiotics or altered physiologic states. We wish
to understand how the structural diversity and genetic
regulation of P450s that metabolize xenobiotics contribute to a person’s ability to avoid the adverse effects
of environmental chemicals and alter the clearance and
bioavailability of therapeutic drugs.
Although extensive information on the conditional
expression of P450 genes in experimental species is
available, in humans the transcriptional responses of
P450 genes to environmental stimuli and to physiologic
changes are poorly understood. To address this problem,
we use human cell lines, primary cultures of human
cells, and transgenic mice to study mechanisms that
regulate human family 4 P450 genes. These genes
encode enzymes that are involved in both signal transduction and the metabolism of endogenous lipids and
xenobiotics. Studies with cell lines are providing new
information about endocrine and autocrine signal transduction pathways that govern the conditional expression
of these genes in response to nutritional, hormonal,
and xenobiotic signals. Research is in progress to test
whether more complex physiologic conditions such as
pregnancy or energy (caloric) restriction alter the expression of the human enzymes in transgenic mice.
Recently, we discovered that the human long chain
fatty acid ω-hydroxylase, CYP4F2, is induced in primary
cultures of human hepatocytes and in cell lines by
several drugs, termed statins, that are used to lower
serum levels of cholesterol. The induction of CYP4F2
could contribute to the reported reduction by statins
of long chain fatty acids that accumulate in X-linked
adrenoleukodystrophy.
In collaboration with C.D. Stout, Department of
Molecular Biology, we are defining the atomic structures
of individual human P450s to understand the structural basis for the broad yet unique catalytic selectivity of each enzyme. This information can be used to
better understand the adverse affects of the oxidation
of drugs and toxins and the potential for metabolic
2006
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247
drug-drug interactions that can arise from inhibition of
P450s if multidrug therapies are used.
Mammalian P450s are tethered to the endoplasmic
reticulum by a transmembrane segment at the amino
terminus and by additional interactions of the catalytic
domain with the cytoplasmic side of the membrane.
Although membrane proteins are difficult to crystallize,
we developed methods to express, purify, and crystallize
genetically modified mammalian P450s that retain a
native catalytic domain. Using this approach, we have
determined the atomic structures of several of the most
important drug-metabolizing P450s: 1A2, 2A6, 2C8,
2C9, and 3A4. Through these studies, we discovered
how the flexibility of the P450s and the diversity of their
amino acid sequences can shape catalytic specificity.
The P450 2A6 is also the principal nicotine-oxidizing enzyme. Although 2A6 plays a prominent role in
detoxification of nicotine, it also can activate the tobacco
smoke–specific carcinogen nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone to its carcinogenic
form. Several reports indicate that because of the
increased side effects of nicotine, persons who are
genetically deficient in 2A6 activity are less likely to
smoke than are persons not genetically deficient in this
activity. In collaboration with J. Cashman, Human Biomolecular Research Institute, La Jolla, California, we
are developing inhibitors of 2A6 that could reduce smoking behavior and diminish the likelihood of tobaccorelated lung cancers.
PUBLICATIONS
Johnson, E.F., Stout, C.D. Structural diversity of human xenobiotic-metabolizing cytochrome P450 monooxygenases. Biochem. Biophys. Res. Commun. 338:331, 2005.
Yano, J.K., Hsu, M.-H., Griffin, K.J., Stout, C.D., Johnson, E.F. Structures of
human microsomal cytochrome P450 2A6 complexed with coumarin and
methoxsalen. Nat. Struct. Mol. Biol. 12:822, 2005.
Neutrophil Dysfunction and
Human Immunodeficiencies
S.D. Catz, B.A. Ellis, D.B. Munafo, M. Park, S. Pacquelet,
J.L. Johnson
RAB27A AND THE SECRETORY MACHINERY
IN GRANULOCYTES
eutrophils kill microorganisms via microbicidal
products released to the phagosome or to the
extracellular space. In resting neutrophils, these
microbicidal molecules are segregated in secretory
N
248 MOLECUL AR AND EXPERIMENTAL MEDICINE
organelles, thus protecting the host from uncontrolled
activation. The secretory machinery used by these organelles is poorly characterized.
We discovered that the small GTPase Rab27a, which
is absent in patients with the immunodeficiency Griscelli
syndrome, is a main component of the secretory machinery in granulocytes. We also found that Rab27a is present in a large proportion of granules that contain matrix
metalloproteinase 9 and in a minor subpopulation of
granules that contain myeloperoxidase. Interference
with the Rab27a secretory machinery impaired secretion of the metalloproteinase and myeloperoxidase in
permeabilized neutrophils. In HL-60 promyelocytic cells,
the expression of Rab27a was dramatically increased
when the cells differentiated to granulocytes but not
when they differentiated to monocytes, supporting a
role for this small GTPase in the secretory machinery
of granulocytes.
We developed a RNA interference approach to
downregulate Rab27a in HL-60 cells and showed that
Rab27a-deficient cells have impaired myeloperoxidase
secretion. Similarly, we discovered that Rab27a-deficient
mice have a marked decreased in myeloperoxidase secretion in response to lipopolysaccharide. We concluded
that Rab27a is a main component of the secretory
machinery of the secretory organelles in neutrophils.
C R O S S TA L K B E T W E E N I L - 1 R E C E P T O R – A S S O C I AT E D
KINASE-4 AND NADPH OXIDASE
Exposure of neutrophils to lipopolysaccharide amplifies their oxidative response to formylated peptides in
a process referred to as “priming.” The relationship
between the signaling downstream of Toll-like receptor
4 after lipopolysaccharide stimulation and the activation
of NADPH oxidase remains elusive. Phosphorylation of
the NADPH oxidase cytosolic factor p47 phox is essential for activation of the NADPH oxidase. We examined
the hypothesis that IL-1 receptor–associated kinase-4
(IRAK-4), the main regulatory kinase downstream of
Toll-like receptor 4 activation, regulates NADPH oxidase
through phosphorylation of p47phox.
We discovered that p47phox is a substrate for IRAK-4
and that IRAK-4–phosphorylated p47phox can be subsequently phosphorylated by protein kinase C. We identified, by mass spectrometry, a novel threonine-rich
regulatory domain in p47phox. Lipopolysaccharide-dependent phosphorylation of p47phox was enhanced by the
inhibition of p38 MAP kinase, confirming that the kinase
responsible for p47 phox phosphorylation operates
upstream of p38 MAP kinase. IRAK-4–phosphorylated
2006
THE SCRIPPS RESEARCH INSTITUTE
p47phox activated NADPH oxidase in a cell-free system
and IRAK-4 overexpression increased NADPH oxidase
activity in response to lipopolysaccharide.
We concluded that IRAK-4 is responsible for the
phosphorylation of p47phox and NADPH oxidase activation after lipopolysaccharide stimulation. This finding may
have physiologic connotations in the human immunodeficiency triggered by IRAK-4 deficiency that is characterized by susceptibility to pyogenic bacterial infections.
PUBLICATIONS
Johnson, J.L., Ellis, B.A., Noack, D., Seabra, M.C., Catz, S.D. The Rab27a-binding protein, JFC1, regulates androgen-dependent secretion of prostate-specific antigen and prostatic-specific acid phosphatase. Biochem. J. 391(Pt. 3):699, 2005.
Preserving Vision in Glaucoma
and Macular Degeneration
A. Hanneken, J. Johnson
etinal nerve cell damage is the primary cause
of visual loss in patients with glaucoma and macular degeneration. Recent evidence suggests
that under certain circumstances, retinal cells can be
protected from dying and nerve cells can be rescued
from death by specific dietary flavonoids found in plant
extracts. We have identified a group of flavonoids that
are particularly effective in protecting eye-derived cells
from the type of injury associated with macular degeneration and glaucoma. The ability of flavonoids to
restore the health of injured retinal cells and induce
the outgrowth of neurites gives these compounds a
unique set of advantages.
Macular degeneration leads to the death of the
retinal pigment epithelial cells, whereas glaucoma leads
to the death of retinal ganglion cells. We have screened
multiple different flavonoids for their ability to protect
retinal pigment epithelial cells and retinal ganglion
cells from cell death induced by oxidative stress (Fig. 1).
Figure 2 shows the protective effect of luteolin on cultures of retinal pigment epithelial cells exposed to oxidative stress, the type of injury associated with macular
degeneration. Luteolin also prevents cell death induced
by oxidative stress in retinal ganglion cells.
Recently, we found that the effective flavonoids have
specific mechanisms of action. Some enhance the production of glutathione, the cell’s primary defense against
oxidative injury. Others block the production of reactive oxygen species, which cause cellular injury and
R
MOLECUL AR AND EXPERIMENTAL MEDICINE
2006
THE SCRIPPS RESEARCH INSTITUTE
249
F i g . 2 . Luteolin protects retinal pigment epithelial cells from oxi-
dative stress–induced cell death. H2O 2 = hydrogen peroxide;
t-BOOH = tert-butyl hydroperoxide.
F i g . 1 . Chemical structures of the dietary flavonoids. EGCG =
(–)-epigallocatechin gallate.
death. Additionally, some flavonoids can activate the
antioxidant response element in cells and so induce the
expression of genes that increase resistance to oxidative
injury (Table 1). We are investigating these flavonoids
in more detail to determine whether these compounds
are also capable of protecting cells in long-term protection assays.
At this point, we have compiled a list of specific
dietary flavonoids that protect both retinal pigment
T a b l e 1 . Protective mechanisms of different flavonoids.
Flavonoid
Involved in
glutathione
metabolism
Scavenges
reactive
oxygen species
epithelial cells and retinal ganglion cells from oxidative stress–induced death (Table 2). We are validating
and expanding these results; we hope to identify additional compounds and combinations that have greater
potency and efficacy.
This research is the result of a partnership formed
between the Scripps Mericos Eye Institute and Scripps
T a b l e 2 . Dietary flavonoids that protect retinal cells from injury
and death in macular degeneration.
Flavonoid
Dietary source
Luteolin
Spinach, wild greens, hot peppers,
celery, thyme, parsley, mint
Quercetin
Onions (especially yellow), cranberries,
cocoa, wild greens, capers, fennel,
spinach, chives, celery, cherries,
blueberries, apples, kale, red wine
Eriodictyol
Peppermint, citrus juices (lemon, lime,
sour orange)
Fisetin
Strawberries, tomatoes, onion, oranges,
apples, peaches, grapes, kiwifruit,
persimmons
Activates
antioxidant
response
element
Luteolin
No
Yes
No
Fisetin
Yes
Yes
Yes
Quercetin
Yes
Yes
Yes
Eriodictyol
Yes
Yes
Yes
250 MOLECUL AR AND EXPERIMENTAL MEDICINE
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THE SCRIPPS RESEARCH INSTITUTE
Research aimed at bringing together the promise of
biomedical research and the practice of medicine.
PUBLICATIONS
Hanneken, A., Lin, F.-F., Johnson, J., Maher, P. Flavonoids protect human retinal
pigment epithelial cells from oxidative stress-induced cell death. Invest. Ophthalmol. Vis. Sci., in press.
Maher, P., Hanneken, A. Flavonoids protect retinal ganglion cells from oxidative
stress-induced cell death. Invest. Ophthalmol. Vis. Sci. 46:4796, 2005.
NADH Dehydrogenases
T. Yagi, A. Matsuno-Yagi, B.B. Seo, E. Nakamaru-Ogiso,
M.-C. Kao, T. Yamashita, M. Marella, J. Barber-Singh,
J. Torres-Bacete
STRUCTURE AND FUNCTION OF PROTONT R A N S L O C AT I N G N A D H - Q U I N O N E O X I D O R E D U C TA S E
he proton-translocating NADH dehydrogenase of
mitochondria (complex I) is responsible for energy
coupling in the respiratory chain. Complex I is
composed of 46 unlike subunits and contains 1 FMN
and 8 iron-sulfur clusters as cofactors. The protontranslocating NADH dehydrogenase of bacteria, NDH-1,
is similar to complex I in terms of electron carriers and
inhibitor specificity. However, in contrast to complex I,
NDH-1 is composed of 14 unlike subunits (NuoA–NuoN).
Both NDH-1 and complex I consist of 2 major
domains: the peripheral segment and the membrane
segment. The peripheral domain of NDH-1 contains
7 subunits (NuoB–NuoG and NuoI) and bears all the
cofactors, so this domain participates in electron transfer. The membrane domain of NDH-1 appears to be
composed of 7 subunits (NuoA, NuoH, and NuoJ–NuoN),
which are homologs of mitochondrial DNA-encoded
subunits (ND1–ND6 and ND4L) of complex I. This
domain catalyzes proton translocation.
In one of our current projects, we are identifying
amino acid residues in the membrane subunits essential for proton translocation. For these studies, we use
a chromosomal DNA mutagenesis approach. We found
that in the NuoK subunit, mutations in glutamic acid
at positions 36 and 72 and in arginine at positions 25
and 26 (Fig. 1) suppress the energy-transducing activity of NDH-1, suggesting that these residues may be
directly involved in proton translocation.
In another project, we are characterizing the cofactors of NDH-1. As described earlier, NDH-1 contains
8–9 iron-sulfur clusters. We attempted to identify the
amino acid residues that coordinate cluster N3 in the
NuoF subunit. The NuoF subunit contains 5 conserved
T
F i g . 1 . Essential amino acid residues of the membrane domain
subunit NuoK of the proton-translocating NADH-quinone oxidoreductase from Escherichia coli. E indicates glutamic acid; R, arginine.
cysteine residues: at positions 180, 351, 354, 357,
and 398. We mutated the individual cysteines to alanine.
The mutated NuoF subunits were isolated and subjected to various physicochemical analyses including
electron paramagnetic resonance spectroscopy. The
data indicate that the cysteines at positions 351, 354,
357, and 398, but not the cysteine at position 180,
are involved in the ligation of cluster N3.
MOLECULAR REMEDY OF COMPLEX I DEFECTS
Studies suggest that defects in mitochondrial complex I are involved in many human diseases, such as
Leigh syndrome and sporadic Parkinson’s disease. However, no effective remedies for complex I deficiencies
have been established. We have adopted a gene therapy approach in which we use the gene NDI1, which
encodes Ndi1, the single polypeptide NADH dehydrogenase of Saccharomyces cerevisiae.
Our earlier experiments indicated that Ndi1 can
replace or supplement the functionality of complex I in
various cultured cells. In addition, by using NDI1–recombinant adeno-associated virus particles, we found that
Ndi1 can be expressed in mitochondria in skeletal muscles and brains of rats and mice. The expressed Ndi1
was functionally active.
For this approach to be useful, the mature protein
must have protective effects against complex I defects
in vivo. Currently, well-established animal models of
complex I diseases are not available. However, the
parkinsonian signs in mice treated with 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) might be
due to inhibition of complex I by MPTP. We determined
whether the expressed Ndi1 enzyme has protective
effects against the parkinsonian signs in MPTP-treated
mice. As shown in Figure 2, Ndi1 expressed in mouse
substantia nigra suppressed dopaminergic neuronal
deficits induced by MPTP such as decreases in tyrosine hydroxylase in the substantia nigra and the stria-
MOLECUL AR AND EXPERIMENTAL MEDICINE
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251
Asymmetries in the Spatial
Distributions of Enhancing
Lesions and Hypointense
Lesions in Relapsing-Remitting
Multiple Sclerosis
J.A. Koziol, S. Wagner,* D.F. Sobel,** A.C. Feng, H.P. Adams
* Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany
** Scripps Clinic, La Jolla, California
e examined the spatial distributions of enhancing lesions and hypointense lesions (“black
holes”) in 24 patients with relapsing-remitting
multiple sclerosis enrolled in a clinical study at Scripps
Clinic. We tested the hypotheses that lesions occur randomly throughout the brain and that the spatial patterns
of lesions are homogeneous among patients.
We investigated within patients whether enhancing
lesions and black holes have bilateral symmetry about
the following planes: midtransaxial, midcoronal, and
midsagittal. Using patients’ monthly magnetic resonance
images, we counted lesions in 10 locations: brain stem,
cerebellum, periventricular region (frontal, temporal,
parietal, and occipital), and other white matter (frontal,
temporal, parietal, and occipital).
We found a pronounced lack of midtransaxial symmetry in the locations of the lesions; most of the lesions
were supratentorial. We also noted lack of symmetry
about the coronal and sagittal axes, although it was
more subtle than the lack of symmetry about the midtransaxial plane. In addition, lesions preferentially
appeared in the anterior rather than the posterior part
of the brain. We found no consistent pattern of left- or
right-sided predominance in the locations of lesions,
even within periventricular or other white matter, across
all patients. But, we found a distinct lack of symmetry
about the midcoronal plane (anterior vs posterior) and
the midsagittal plane.
We next investigated whether patients have similar
patterns of locations of lesions. We used cluster analyses to detect subsets of patients that might have similar
patterns of lesion locations. Figure 1 shows the location patterns for the 4 tightest nondegenerate clusters
of patients on the basis of enhancing lesions and for
the 3 tightest nondegenerate clusters on the basis of
black holes.
W
F i g . 2 . Protective effects of Ndi1 expressed in mouse striatum
against decreases in tyrosine hydroxylase induced by treatment with
MPTP. Upper panel, control; middle panel, MPTP treatment; lower
panel, MPTP treatment after injection of NDI1–recombinant adenoassociated virus particles into the left substantia nigra in mouse brain.
tum and decreases in striatal levels of dopamine. The
data indicate that NDI1 will be a promising therapeutic tool in the treatment of diseases caused by impairments in complex I.
PUBLICATIONS
Betarbet, R., Canet-Aviles, R.M., Sherer, T.B., Mastroberardino, P.G., McLendon,
C., Kim, J.-H., Lund, S., Na, H.-M., Taylor, G., Bence, N.F., Kopito, R., Seo,
B.B., Yagi, T., Matsuno-Yagi, A., Klinefelter, G., Cookson, M.R., Greenamyre, J.T.
Intersecting pathways to neurodegeneration in Parkinson’s disease: effects of the
pesticide rotenone on DJ-1, α-synuclein, and the ubiquitin-proteasome system.
Neurobiol. Dis. 22:404, 2006.
Kao, M.-C., Nakamaru-Ogiso, E., Matsuno-Yagi, A., Yagi, T. Characterization of the
membrane domain subunit NuoK (ND4L) of the NADH-quinone oxidoreductase
from Escherichia coli. Biochemistry 44:9545, 2005.
Seo, B.B., Nakamaru-Ogiso, E., Flotte, T.R., Matsuno-Yagi, A., Yagi, T. In vivo
complementation of complex I by the yeast Ndi1 enzyme: possible application for
treatment of Parkinson disease. J. Biol. Chem. 281:14250, 2006.
Velazquez, I., Nakamaru-Ogiso, E., Yano, T., Ohnishi, T., Yagi, T. Amino acid residues
associated with cluster N3 in the NuoF subunit of the proton-translocating NADHquinone oxidoreductase from Escherichia coli. FEBS Lett. 579:3164, 2005.
Yagi, T., Seo, B.B., Nakamaru-Ogiso, E., Marella, M., Barber-Singh, J., Yamashita,
T., Kao, M.-C., Matsuno-Yagi, A. Can a single subunit yeast NADH dehydrogenase
(Ndi1) remedy diseases caused by respiratory complex I defects? Rejuvenation Res.
9:191, 2006.
Yagi, T., Seo, B.B., Nakamaru-Ogiso, E., Marella, M., Barber-Singh, J., Yamashita,
T., Matsuno-Yagi, A. Possibility of transkingdom gene therapy for complex I diseases. Biochim. Biophys. Acta, in press.
252 MOLECUL AR AND EXPERIMENTAL MEDICINE
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metries. We found an extreme lack of bilateral symmetry about the midtransaxial, midcoronal, and midsagittal
planes in terms of frequencies of both enhancing lesions
and black holes in our cohort. These asymmetries suggest that in individual patients, particular lobes or
regions might be more vulnerable than other regions to
pathologic changes associated with the formation of
lesions, even if the processes leading to lesions are
assumed to be ubiquitous. Perhaps asymmetries in the
locations of lesions reflect various asymmetries within
the CNS white matter and consequent heterogeneities
in the histopathologic features of lesions. An immediate
implication is that potential therapeutic agents could
have differential effects on these processes, so that the
treatment of choice for an individual patient with multiple sclerosis may depend on identifying the underlying mechanisms of disease.
PUBLICATIONS
Koziol, J.A., Wagner, S., Sobel, D.F., Feng, A.C., Adams, H.P. Asymmetries in the
spatial distributions of enhancing lesions and black holes in relapsing-remitting MS.
J. Clin. Neurosci. 2:895, 2005.
DIVISION
OF
CELLULAR BIOLOGY
James A. Hoch, Ph.D., Division Head
F i g . 1 . Distinct clusters of patients with relapsing-remitting mul-
tiple sclerosis have similar patterns of locations of lesions on magnetic resonance images. A, Total numbers of enhancing lesions. B,
Total numbers of black holes. Abbreviations: BS, brain stem; CE,
cerebellum; FP, frontal periventricular region; TP, temporal periventricular region; PP, parietal periventricular region; OP, occipital periventricular region; FW, frontal white matter; TW, temporal white matter; PW, parietal white matter; OW, occipital white matter.
Sensor Kinases That Regulate
Sporulation and the Synthesis
of Toxins
J.A. Hoch, M. Perego, T. Fukushima, F. Scaramozzino, H.
Szurmant, B. Tsvetanova, A. Wilson
The clusters are quite distinctive. In all of the clusters of enhancing lesions, parietal lesions dominate; in
particular, both periventricular and other white matter
lesions in clusters 1 and 2, but solely other white matter
lesions in clusters 3 and 4. Occipital lesions are also
present in clusters 2 and 4 and frontal white matter
lesions in cluster 3. Cluster 1 of black holes constitutes
solely parietal white matter lesions. Cluster 3 is slightly
expanded relative to cluster 1, encompassing both
frontal and parietal white matter lesions, whereas cluster 2 is rather dispersed throughout the white matter.
We developed statistical techniques to characterize
the asymmetries of the locations, and we investigated
statistical measures of individual and subgroup asym-
ormation of endospores in Bacillus subtilis is a
model for understanding the mechanism of developmentally programmed gene expression. Several dozen genetically dispersed sporulation operons are
regulated coordinately as temporal classes during the
time required to complete the formation of spores. This
complex developmental program is under the control of
the spo0 genes, which control entry of the cell into
sporulation and the production of toxins and virulence
factors in pathogens such as Bacillus anthracis.
The transcription factor Spo0A is the key master
regulator of the initiation of developmental transcription. The activity of the protein is controlled by a rever-
F
MOLECUL AR AND EXPERIMENTAL MEDICINE
sible phosphorylation-dephosphorylation mechanism.
The pathway to Spo0A activation is a series of phosphorylation reactions involving sequentially the Spo0F
and Spo0B proteins, for which we coined the term
phosphorelay. The probability that sporulation will be
initiated depends on the competition between kinases
and phosphatases.
Previously, we identified 5 sensor kinases involved
in signaling the initiation of sporulation in B subtilis.
Our studies in B anthracis revealed 9 sensor kinases
for sporulation that differ from those of B subtilis in the
signal-sensing domains. Deletion studies indicated that
sporulation in B anthracis is a consequence of the concerted activities of several of these sensor kinases. The
sporulation pathway regulates the production of the
genes for anthrax toxin through at least one Spo0Acontrolled regulator. Studies on the transcription of the
gene that encodes the protective antigen component
of the toxin revealed the extent of the promoter region
responsible for gene activation and the relationship of
the region to the positive transcription activator AtxA.
The goal of these studies is to understand the specific
and global controls that regulate virulence in B anthracis.
We have initiated a program to study the regulation
of sporulation in clostridia, which are increasingly prevalent as causes of nosocomial infections and as producers
of extremely lethal toxins. Genomic data on these anaerobic bacteria indicated that the sporulation phosphorelay
pathway of aerobic Bacillus species is partially absent in
a species-specific manner in clostridia. Some species, for
example, Clostridium tetani, have the spo0B gene but no
spo0F gene, whereas others, including Clostridium difficile and Clostridium botulinum, lack both genes. In
studies of C botulinum, we identified a sensor kinase
for sporulation that phosphorylates Spo0A directly. The
Spo0B protein of C tetani is fully functional in B subtilis,
but the pathway to the phosphorylation of this protein in
C tetani is not understood. The potential for therapeutic
applications based on these studies is excellent, because
the regulation of sporulation also is involved in the
expression of genes that encode clostridial toxins and
perhaps genes for other virulence factors.
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253
Computational Analysis of
Molecular Specificity in
2-Component Signaling
J.A. Hoch, R.A. White, H. Szurmant, T. Hwa*
* University of California, San Diego, California
n both prokaryotes and eukaryotes, a large number
of pathways with proteins with identical structural
folds are used to interpret and propagate vastly different signals specific for unique targets. A central question in understanding signal transduction is how does
a signaling protein distinguish its true partner from the
much larger number of similar partners present in the
cell. Without this specificity, unintended cross talk
among the pathways will greatly reduce the fidelity of
signal transduction. On the other hand, designed cross
talk at specific stages between specific pathways provides a means of combinatorial signal integration that
may greatly increase the signal-processing capability
of the cell. In collaboration with T. Hwa, University of
California, San Diego, we are examining the molecular
interactions that underlie partner recognition; the focus
of these studies is the 2-component system, the prevalent signaling system used in bacteria.
We have developed a sequence-based method, independent of structural considerations, for identifying specificity-determining interactions between proteins for
which genomic data indicate a large number of functionally coupled pairs. This method was applied to the
phosphotransfer domains of 2-component signaling proteins, primarily in the OmpR/EnvZ family. Using the
method, we identified a network of residue-residue interactions and generated a 3-dimensional structure consistent with the exemplary cocrystal structure obtained
for the Spo0B-Spo0F complex and mutation studies of
this pair of proteins. We also identified an interaction
network that links long-distance interactions with pair
specificity of 2-component signaling proteins.
The method provides a simple scoring procedure
that can be used to identify potential cross-phosphorylation between functional pairs and to assign orphan
2-component signaling proteins with no known mate
to their signaling partners. Applied to the interconnected
triad of 2-component signaling protein pairs in Bacillus
subtilis, the procedure produced results consistent with
recent discoveries of phosphotransfer cross talk, indicating that higher order signaling networks can be elu-
I
254 MOLECUL AR AND EXPERIMENTAL MEDICINE
cidated. Although currently we are applying the method
to 2-component signal transduction systems for which
structural and mutational data allow proof of principle,
the method may generate interaction structures for less
characterized protein pairs if sufficient functional pairs
exist in genomic data.
Molecular Dynamics of
Response Regulators
J.A. Hoch, J. Cavanagh*
* North Carolina State University, Raleigh, North Carolina
n the basis of studies of the backbone dynamics
of the response regulator Spo0F, we proposed
a model in which communication of information through the core of the Spo0F protein, between
buried and surface-bound residues, is responsible for
the dissociation of sensor kinases from response regulators after phosphorylation. We defined a region on
Spo0F that moves in a dynamically concerted fashion,
driven by the motion of the imidazole ring of histidine
at position 101.
The imidazole ring moves in response to a conformational change in the aspartic acid binding pocket upon
phosphorylation. Movement of the ring disrupts packing
interactions, a condition that alters the topology of the
kinase recognition site, thereby causing the kinase to
dissociate. Low concentrations of copper ions are potent
inhibitors of sporulation. Using nuclear magnetic resonance and micro electrospray ionization–mass spectrometry, we found multiple metal-bound species of Spo0F
in the presence of copper. One of the copper-binding
sites was histidine at position 101, where bound copper
alters the dynamics and conformation of the active site
residues of Spo0F, making Spo0F nonfunctional.
O
Negative Regulation of
Development in Bacilli
2006
THE SCRIPPS RESEARCH INSTITUTE
kinases and aspartyl phosphate phosphatases. Phosphatases belong to 2 families: Rap and Spo0E.
Rap proteins are characterized by a structural organization in tetratricopeptide repeats. Tetratricopeptide
repeat domains are thought to be ancient modules that
promote protein-protein interactions. The Rap proteins
act as negative regulators of the initiation of sporulation
by dephosphorylating the Spo0F response regulator intermediate of the phosphorelay. The activity of Rap proteins is generally inhibited by specific Phr pentapeptides
encoded within precursor proteins that follow an exportimport processing pathway that generates the active
inhibitor. The Spo0E proteins act as negative regulators of the phosphorelay by dephosphorylating the
Spo0A response regulator and master transcription factor for the initiation of sporulation.
We have identified the Rap and Spo0E proteins that
control the phosphorelay in Bacillus subtilis and Bacillus
anthracis. Bacillus anthracis has 6 genes that encode
Rap proteins; each of these genes is followed by a gene
that encodes a Phr pentapeptide. Five Rap-Phr genes
are chromosomally located; the sixth gene is present on
the pXO1 plasmid, which also carries the genes that
encode toxin protein components of B anthracis.
Through genetic and biochemical analysis, we determined that 1 chromosomally encoded system and the
plasmid-encoded Rap-Phr system regulate initiation of
sporulation in B anthracis and thus may influence
toxin production and virulence. We also identified the
products of 4 chromosomally located genes as members of the Spo0E family that dephosphorylate the
Spo0A protein. We are now focusing on the molecular
mechanism of interaction between Rap proteins and
their specific Phr peptide inhibitors or their target
Spo0F. We are also examining the surfaces used by
Spo0E and Spo0A to interact with each other.
Signal Transduction in
Enterococcus faecalis
M. Perego, F. Del Papa
M. Perego, C. Bongiorni, A. Diaz
nitiation of sporulation in gram-positive bacilli is
regulated by a multicomponent signal transduction
system called a phosphorelay. Multiple positive and
negative signals are integrated by the phosphorelay
through the opposing activities of histidine protein
I
nterococci are commensal bacteria within the
intestinal tract in mammals but also can cause
disease in compromised hosts. The acquisition
of resistance to multiple antibiotics by enterococci
makes infections caused by these microorganisms
clinically challenging. The ability of the bacteria to
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adapt and respond to different environmental stimuli,
including the host environment, led us to investigate
the role of 2-component signal transduction in the
physiology and pathogenesis of Enterococcus faecalis.
Using a bioinformatic approach, we identified 17
2-component systems consisting of a sensory histidine
kinase and a cognate response regulator and an additional orphan response regulator. We inactivated each
response regulator with the exception of the ortholog
of the YycF essential protein of gram-positive organisms. We tested the effect of the deletions on a number of physiologic conditions and detected defects in
growth, antibiotic resistance, stress response, and formation of biofilms. We are using these mutant strains
to analyze the role of signal transduction in pathogenesis in vivo and to determine the extent of the regulon
controlled by each 2-component system.
Analysis of the 2-component system encoded by the
gene fsr revealed that this system is the only one that
affects growth of enterococci as a biofilm on solid surfaces. The role of the fsr system in biofilms is to activate transcription of the gene that encodes gelatinase,
a zinc-metallo protease. Gelatinase is required for the
formation of biofilms by E faecalis. We also found that
full activation of gelatinase activity depends on a selfcleavage process at the C-terminal end of the gelatinase protein. Because growth of bacteria as biofilm is
strongly associated with the development of human
infections, such as infective endocarditis and urinary
tract infections, our findings suggest that gelatinase
may present a unique target for therapeutic intervention against biofilm-based enterococcal infection.
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255
lution of molecular specificity in protein-protein interactions. In the past year, we switched our attention to
proteins that regulate essential and important signal
transduction systems.
The YycFG 2-component signal transduction system
is essential for the growth of gram-positive bacteria,
including pathogenic staphylococci and streptococci.
This system regulates and coordinates the synthesis of
the constituents of cell walls and membranes with growth
and division. The activity of this system is regulated by
at least 2 proteins located on the outer surface of the
cellular membrane, and one of these, YycH, was purified
and crystallized. The crystal structure of YycH was solved
by using 2-wavelength selenium anomalous dispersion
data and was refined by using 2.3-Å data to an R-factor
of 25.2%. The molecule consists of 3 domains with a
unique 3-dimensional structure (Fig. 1). Although a cal-
Regulatory Proteins: Structure,
Molecular Recognition, and
Phosphosignaling
K.I. Varughese, H. Szurmant, J.A. Hoch
n recent studies, we have focused on the crystal
structures of the phosphorelay proteins Spo0F and
Spo0B, Spo0A bound to its target DNA, and the complex consisting of Spo0F and Spo0B. The structure of
the Spo0F-Spo0B complex showed for the first time
how response regulator proteins bind to histidine phosphotransfer domains of sensor kinases and was the
key initial finding that opened up the study of the evo-
I
F i g . 1 . YycH is a membrane-bound periplasmic protein that reg-
ulates the activity of YycG kinase, perhaps by interacting with the
sensing domain of the kinase. The YycH molecule is made up of 3
domains and has a novel 3-dimensional structure.
cium-binding site was also discovered in this structure,
the orthologs in other bacterial species do not contain
this motif. This structure has been extremely useful in
studies to determine the function of YycH.
256 MOLECUL AR AND EXPERIMENTAL MEDICINE
The regulation of toxin production by Bacillus
anthracis is critical for the ability of this organism to
invade and grow in the body. The regulatory circuits
for the expression of the genes for anthrax toxin and
the secretion of the toxin are complex, but a key protein
is the product of the gene pagR. We were able to express
and crystallize the 99 amino acid PagR transcription
factor encoded by this gene. Crystals of the selenomethionine protein diffracted to 1.75 Å, and the structure was determined by using the multiple anomalous
diffraction technique.
We found that the PagR protein consists of 5
α-helices and a 2-stranded β-sheet. Two monomers
form an elongated dimer. This structure is highly similar to that of the metalloregulatory proteins of the
Smt/ArsR family. However, PagR lacks the critical residues required for metal binding, making it unlikely that
metals are involved in its regulatory properties. The
structure provides a platform for mutagenesis experiments to uncover the role of this protein in the complex regulation of anthrax toxin.
PUBLICATIONS
Bongiorni, C., Stoessel, R., Shoemaker, D., Perego, M. Rap phosphatase of virulence
plasmid pXO1 inhibits Bacillus anthracis sporulation. J. Bacteriol. 188:487, 2006.
Brunsing, R.L., La Clair, C., Tang, S., Chiang, C., Hancock, L.E., Perego, M.,
Hoch, J.A. Characterization of sporulation histidine kinases of Bacillus anthracis.
J. Bacteriol. 187:6972, 2005.
Kojetin, D.J., Thompson, R.J., Benson, L.M., Naylor, S., Waterman, J., Davies,
K.G., Opperman, C.H., Stephenson, K., Hoch, J.A., Cavanagh, J. Structural analysis of divalent metals binding to the Bacillus subtilis response regulator Spo0F: the
possibility for in vitro metalloregulation in the initiation of sporulation. Biometals
18:449, 2005.
Low, L.Y., Yang, C., Perego, M., Osterman, A., Liddington, R.C. Structure and
lytic activity of a Bacillus anthracis prophage endolysin. J. Biol. Chem.
280:35433, 2005.
Musumeci, L., Bongiorni, C., Tautz, L., Edwards, R.A., Osterman, A., Perego, M.,
Mustelin, T., Bottini, N. Low-molecular-weight protein tyrosine phosphatases of
Bacillus subtilis. J. Bacteriol. 187:4945, 2005.
Szurmant, H., Nelson, K., Kim, E.J., Perego, M., Hoch, J.A. YycH regulates the
activity of the essential YycFG two-component system in Bacillus subtilis. J. Bacteriol. 187:5419, 2005.
Szurmant, H., Zhao, H., Mohan, M.A., Hoch, J.A., Varughese, K.I. The crystal structure of YycH involved in the regulation of the essential YycFG two-component system
in Bacillus subtilis reveals a novel tertiary structure. Protein Sci. 15:929, 2006.
White, A.K., Hoch, J.A., Grynberg, M., Godzik, A., Perego, M. Sensor domains
encoded in Bacillus anthracis virulence plasmids prevent sporulation by hijacking a
sporulation sensor histidine kinase. J. Bacteriol., in press.
Worner, K., Szurmant, H., Chiang, C., Hoch, J.A. Phosphorylation and functional
analysis of the sporulation initiation factor Spo0A from Clostridium botulinum.
Mol. Microbiol. 59:1000, 2006.
2006
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DIVISION OF EXPERIMENTAL
HEMOSTASIS AND THROMBOSIS
Zaverio M. Ruggeri, M.D., Division Head
Regulation of Allogeneic
Immune Responses to Cell
Transplants
L. Crisa, R. Prinsen, V. Cirulli,* B.E. Torbett
* Whittier Institute, La Jolla, California
lass I and class II MHC antigens are the primary
barrier to acceptance of allografts. However, certain class I MHC antigens may also trigger regulatory immune responses. Thus, in humans, HLA-G, a
nonpolymorphic class Ib HLA molecule, may mediate
immunologic tolerance at sites of immune privilege, such
as the anterior chamber of the eye, the testis, the thymus, and the cytotrophoblast.
Several explanations for the immunoregulatory
functions of HLA-G have been considered. The limited polymorphism of HLA-G in humans may allow
the recognition of tissues expressing high levels of this
molecule as “self,” thereby preventing the activation
of autoreactive or alloreactive T cells and natural killer
cells. Alternatively, HLA-G may foster the development
of specific immunoregulatory lymphocytes capable of
downregulating alloreactivity. Our previous finding that
HLA-G is expressed in the thymic medullary epithelium
in humans strongly supports both possibilities. Thus,
the purpose of HLA-G expression in the thymic medulla
may be to both educate developing T cells to recognize
HLA-G as self and induce the selection of HLA-G–specific immunoregulatory T-cell populations.
We are investigating the immune responses elicited
by HLA-G in human thymocytes and peripheral T cells.
Our goals are to dissect the molecular mechanisms of
HLA-G immune functions and then use this information
to bioengineer HLA-G expression in tissues suitable for
transplantation. Particular emphasis is given to models
of pancreatic islet transplantation for the treatment of
diabetes. For this purpose, we have generated lines of
human pancreatic cells that express either low or high
levels of membrane-bound or soluble recombinant HLAG. These HLA-Glow and HLA-Ghigh cell lines are useful
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tools for studies of HLA-G functions both in vitro and
in vivo in models of cell transplantation.
Another promising line of research for the bioengineering of cells for transplantation was provided by our
work on the identification of endothelial cell progenitors in human cord blood. While studying human thymopoiesis in a chimeric mice model in which mice are
reconstituted with human cord blood, we discovered
that cord blood hematopoietic stem cells engrafted in
these mice not only reconstituted the bone marrow and
repopulated the human thymic grafts but also contributed to the formation of new blood vessels at sites of
wound healing.
We are characterizing this population of putative
endothelial progenitors to be used as another target
cell type for transplantation. Specifically, we have defined
some of the growth and differentiation signals required
for the expansion ex vivo of human bone marrow–derived
endothelial progenitors. Currently, using a mouse model
of bone marrow–derived vasculogenesis, we are characterizing the immunologic and angiogenic properties of
bone marrow–derived endothelium. Ultimately, cotransplanting HLA-G–transduced allogeneic tissue along with
HLA-G–bioengineered endothelial cell progenitors and/or
enhancing recruitment of bone marrow–derived endothelium with intrinsic immunomodulatory properties
may endow tissue grafts with an additional level of
immunoprotection. This approach may be useful in
developing novel strategies for the induction of immunologic tolerance and/or the avoidance of rejection
after transplantation.
PUBLICATIONS
Cirulli, V., Zalatan, J., McMaster, M., Prinsen, R., Salomon, D.R., Ricordi, C.,
Torbett, B.E., Meda, P., Crisa, L. The class I HLA repertoire of pancreatic islets
comprises the nonclassical class Ib antigen HLA-G. Diabetes 55:1214, 2006.
Cerebral Microvessel-Neuron
Responses to Ischemia
G.J. del Zoppo, R. Milner, J. Hallenbeck,* E. Lo**
* National Institutes of Health, Bethesda, Maryland
** Massachusetts General Hospital, Boston, Massachusetts
nderstanding the interactions between neurons
and their supply microvessels can provide insight
into communication and control of neuronal
activation and the coordinate responses of neurons and
microvessels to local injury, as during ischemic stroke.
U
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257
Stroke is a vascular disorder that causes neuronal injury.
The social impact of the neurologic and behavioral consequences of this injury is enormous. We hypothesized
that alterations in the intercellular matrix of cerebral
microvessels and their cellular adhesion receptors by
active proteases are reflected by injury to neighboring
neurons and the extracellular matrix of the neurons. The
results of our studies have supported the concept of
the neurovascular unit.
We have extended our studies to 3 related areas:
(1) the distribution of expression of adhesion receptors
in the matrix of the neurovascular unit, (2) the impact
of hypoxia with glucose deprivation on cellular components of the microvascular permeability barrier, and
(3) the responses of intrinsic CNS inflammatory cells
(microglia and oligodendroglia) to focal ischemia and
other types of injury and the impact of the responses
on the neurovascular unit.
Our results have provided cell models that clearly
indicate the relationship between the neurovascular
unit and ischemic stroke. The cellular models help us
understand how the microvasculature and the related
neurons within the neurovascular unit interact.
The integrity of microvessels depends on adhesion
of endothelial cells and astrocytes to the basal lamina.
We have shown that in addition to β1 integrins, αβ-dystroglycan is expressed predominantly on the luminal side
of astrocyte end-feet in cerebral microvessels. In vivo,
dystroglycan is found on the entire microvasculature.
Both integrin α 6 β 4 and dystroglycan appear throughout the microvasculature of the striatal gray matter,
but only α6β4 is found on large penetrating vessels of
the cortical gray matter. Dystroglycan is a functional
laminin receptor for astrocytes in vitro. The reasons
for the significantly different distributions of these 2
receptors are being studied.
The effects of focal ischemia on brain microvessels
can be studied in vitro by depriving endothelial cells
and astrocytes of oxygen and glucose. The expression
of β 1 integrins on endothelial cells is relatively resistant to oxygen and glucose deprivation and actually
increases in response to this treatment. But dystroglycan expression decreases significantly after oxygen and
glucose deprivation. These changes recapitulate the
loss of dystroglycan expression within the brain microvasculature that occurs in focal ischemia. The disappearance of dystroglycan from astrocytes during oxygen
and glucose deprivation can be blocked by some protease inhibitors.
258 MOLECUL AR AND EXPERIMENTAL MEDICINE
A common early event in many brain injuries is the
breakdown of the blood-brain barrier, a situation that
leads to deposition of the serum proteins fibronectin
and vitronectin. We have shown that fibronectin and
vitronectin stimulate microglial activation and expression of the proteolytic enzymes matrix metalloproteinase 9 and matrix metalloproteinase 12 and that
this activation is mediated via integrins α 5 β 1 and
αvβ5, respectively.
Other experiments have revealed that interference
with cellular adhesion in the neurovascular unit significantly disrupts tight junctions within the blood-brain
barrier. These studies suggest several novel therapeutic avenues to reduce the pathologic changes that
occur after ischemic injury in the brain.
PUBLICATIONS
Adams, H., Adams, R., del Zoppo, G., Goldstein, L.B., Stroke Council of the
American Heart Association, American Stroke Association. Guidelines for the early
management of patients with ischemic stroke: 2005 guidelines update a scientific
statement from the Stroke Council of the American Heart Association/American
Stroke Association [published corrections appear in Stroke 36:1352 and 1626,
2005]. Stroke 36:916, 2005.
del Zoppo, G.J. Stroke and neurovascular protection. N. Engl. J. Med. 354:553,
2006.
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THE SCRIPPS RESEARCH INSTITUTE
grin adhesion receptors in a constitutively activated,
high-affinity format. Activation-inducing mutant integrin subunits endowed human breast cancer cells with
a highly aggressive, metastatic phenotype when tested
in immunodeficient mice, establishing a cause-and-effect
relationship between integrin activation and metastatic
activity in breast cancer. A clinical relevance of this
finding was evident from a screening of malignant tumor
cells that we isolated from blood samples and malignant effusions of patients with advanced breast cancer.
Functional analyses of tumor cell migration and the
ability of the cells to interact with components of the
vessel wall under blood flow conditions such as those
that occur in the venous circulation revealed that integrins are expressed in a constitutively activated form
exclusively in metastatic and invasive cells. Analyses
of the development of metastasis in the animal model
revealed that activated integrin αvβ3 promotes tumor
cell survival in the absence of an adhesive matrix and
supports the initial steps of tumor cell colonization of
target organs distant from the bloodstream.
I N H I B I T I O N O F B R E A S T C A N C E R M E TA S TA S I S B Y
A N T I B O D I E S F R O M C A N C E R PAT I E N T S
Mabuchi, T., Lucero, J., Koziol, J.A., del Zoppo, G.J. Focal cerebral ischemia preferentially affects neurons distant from their neighboring microvessels. J. Cereb.
Blood Flow Metab. 25:257, 2005.
NINDS ICH Workshop Participants. Priorities for clinical research in intracerebral
hemorrhage: report from a National Institute of Neurological Disorders and Stroke
workshop. Stroke 36:e23, 2005.
Harnessing Host Defense and
Regeneration to Target Breast
Cancer Metastasis
B.F. Felding-Habermann, J.S. Krueger, D. O’Sullivan,
W. Hassenpflug, J.S. Forsyth, B.M. Maruszak, E.I. Chen,
J.R. Yates, K.D. Janda, R.A. Lerner, J.F. Kroener,*
E.Y. Snyder**
* Scripps Clinic, La Jolla, California
** Burnham Institute, La Jolla, California
After we discovered that the activated conformer
of integrin αvβ3 was a functional target on metastatic
breast cancer cells, we used our breast cancer cell
model to isolate human antibodies directed against
this target. We isolated 2 antibodies that specifically
recognize the activated conformer of integrin α v β 3 .
These antibodies mimic natural ligands of the receptor;
they express an arginine–glycine–aspartic acid integrin
recognition motif in the third complementarity-determining region of the heavy chain. The antibodies can
be used to detect metastatic human tumor cells and
inhibit critical tumor cell functions that depend on
activation of integrin αvβ3. Importantly, treatment of
experimental mice with the antibodies prevented formation of metastases and reduced existing metastatic
tumor burden (Fig. 1). Our current goal is to optimize
these antibodies and, by using chemical modification,
harness them for treatment of metastatic breast cancer.
F U N C T I O N A L TA R G E T S I N B R E A S T C A N C E R
HARNESSING NEURAL STEM CELLS FOR THERAPY
M E TA S TA S I S
O F B R E A S T C A N C E R B R A I N M E TA S TA S E S
ur goals are to understand mechanisms of tumor
metastasis and to develop novel approaches for
early detection and therapeutic inhibition of
metastatic disease. Our studies on adhesive, migratory,
and invasive properties of human tumor cells revealed
that a metastatic subset of cancer cells expresses inte-
Improved diagnosis and treatment prolong the lives
of patients with breast cancer, but brain metastases
eventually develop in nearly 30% of patients who have
advanced disease. No current therapy can prevent or
effectively eliminate brain metastases. Therefore, we
seek to develop a novel treatment approach that is
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259
to reach metastatic brain lesions and locally convert a
harmless prodrug to highly toxic 5-fluorouracil at the site
of tumor growth. With this interdisciplinary team effort,
we hope to provide a new opportunity for treatment of
brain metastases in patients who have breast cancer.
F i g . 1 . Treatment with human antibodies against activated integrin
αvβ3 reduces the growth rate of lung metastases in advanced breast
cancer. Human breast cancer cells isolated from a blood sample from
a patient with breast cancer were labeled with a bioluminescent
marker and then injected into mice. The growth rate of lung metastases (tumor burden) in the animals was monotired by using weekly
noninvasive bioluminescence imaging. On day 56, treatment groups
were selected so that each group contained mice with light, intermediate, or heavy tumor burdens. The groups were treated with daily
injections of wild-type (WT) phage, phage displaying antibody 1 (Ab1),
or phage displaying antibody 5 (Ab5). The growth rate of the tumor
burden was determined for each animal at the beginning and the end
of treatment. The graph shows data points for changes in metastatic
lung signal reflecting the growth rate of the tumor burden in each
animal; a horizontal line indicates the median measurement. Overall,
the 3 groups differed significantly (F = 5.50, P = .04). Pairwise
comparison via a multiple comparison procedure indicated that the
Ab1 group differed significantly from the WT group (.05 α level).
PUBLICATIONS
Chen, E.I., Hewel, J., Felding-Habermann, B., Yates, J.R. III. Large scale protein
profiling by combination of protein fractionation and multidimensional protein identification technology (MudPIT). Mol. Cell. Proteomics 5:53, 2006.
Lillo, A.M., Kim, Y., Liu, Y., Ballatore, C., Anichini, A., Mortarini, R., Zhou, B.,
Felding-Habermann, B., Janda, K.D. Targeting heat-shock proteins on cancer cells:
selection, characterization, and cell-penetrating properties of a peptidic GRP78
ligand. Biochemistry, in press.
The Antithrombotic,
Anti-inflammatory, and
Antiapoptotic Protein C Pathway
J.H. Griffin, B.N. Bouma, M. Chopp,* H. Deguchi, D.J. Elias,
S. Eichinger,** F. Espana,*** J.A. Fernández, P. Kyrle,**
S. Li, Y.M. Lee, L. Mosnier, S. Navarro, N. Pecheniuk, X. Xu,
X. Yang, S. Yegneswaran, B.V. Zlokovic****
* Henry Ford Hospital, Detroit Michigan
based on the body’s own mechanisms for healing and
regeneration. We have established unique, trackable
human breast cancer cell models in which noninvasive
bioluminescence imaging is used to detect cancer spreading, onset and development of brain metastases, and
response to treatment (Fig. 2).
** Universität Wien, Vienna, Austria
*** Universitat de València, València, Spain
**** University of Rochester, Rochester, New York
arious host defense systems act in concert in normal physiology. Coagulation pathways, fibrinolysis pathways, and anticoagulant mechanisms
prevent bleeding while avoiding harmful blood clots.
The protein C pathway provides antithrombotic, antiinflammatory, and antiapoptotic activities and is a
focus of our research.
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ANTIAPOPTOTIC AND CYTOPROTECTIVE EFFECTS
F i g . 2 . Cell and animal model for brain metastasis of human
breast cancer. Noninvasive bioluminescence imaging is used to
monitor widespread metastatic colonization of breast cancer target
organs in immunodeficient mice injected with human breast cancer
cells labeled with a bioluminescent marker. Breast cancer cells isolated from a brain lesion home to various regions of the brain (A)
and maintain expression of epithelial antigens (B).
In collaboration with E.Y. Snyder of the Burnham
Institute, La Jolla, California, a pioneer and expert in the
biology of neural stem cells, we are testing the hypothesis that brain metastases can be inhibited with neural
stem cells because the stem cells have a proven propensity to seek out diseased areas in the brain. We are
using neural stem cells armed with cytosine deaminase
O F A C T I VAT E D P R O T E I N C
The antiapoptotic activity of activated protein C
(APC), first described in 2001, may provide cytoprotective activity that reduces cell death after a variety
of cellular injuries. Recombinant APC, a well-defined
anticoagulant enzyme, reduced mortality in patients
with severe sepsis in a phase 3 clinical trial. However,
2 potent anticoagulants, antithrombin III and recombinant tissue factor pathway inhibitor, did not, suggesting
the physiologic relevance of APC’s less well-defined
anti-inflammatory and antiapoptotic activities. Therapy
with recombinant APC is associated with an increased
risk for serious bleeding complications because of the
anticoagulant activity of the enzyme.
260 MOLECUL AR AND EXPERIMENTAL MEDICINE
To generate recombinant APC variants that have
reduced anticoagulant activity and thus are less likely
to cause bleeding, we dissected APC’s anticoagulant
activity from its cytoprotective activity by using sitedirected mutagenesis. Using staurosporine-induced
endothelial cell apoptosis assays, we showed that mutations to alanine in 2 APC surface loops that severely
reduced anticoagulant activity resulted in two APC
variants that retained normal antiapoptotic activity.
Like wild-type APC, these 2 mutants require protease
activated receptor-1 and endothelial cell protein C
receptor for cytoprotective activity.
Thus, it is possible to reduce anticoagulant activity
while preserving antiapoptotic activity of recombinant
APC variants. We are using animal models of injury to
determine if therapy with such APC variants can reduce
serious risks for bleeding while providing the beneficial effects of APC acting directly on cells.
N E U R O P R O T E C T I V E A C T I V I T I E S O F A C T I VAT E D
PROTEIN C
Stroke is a major cause of morbidity and mortality.
In collaboration with B. Zlokovic and colleagues, University of Rochester, we used human brain endothelium in vitro and murine in vivo stroke models to study
the neuroprotective activities of the protein C pathway.
Previously we showed that intravenous infusions of
recombinant APC reduced the size of brain infarctions
and brain edema induced by ischemia. Although thrombolytic effects of tissue plasminogen activator (tPA)
are beneficial, its neurotoxic effects are a problem. We
found that APC reduces the neurotoxic effects of tPA
and blunts tPA-induced apoptosis of both endothelial
cells and neurons.
Remarkably, new studies in murine and rat models
of ischemic stroke indicate that recombinant murine
APC reduces bleeding induced by the thrombolytic agent
tPA. APC stabilizes the blood-brain barrier against bleeding because it blunts the tPA-induced increases in mRNA
and protein levels of matrix metalloprotease-9, which
causes breakdown of the blood-brain barrier.
Thus, we think that APC merits clinical trials as
a neuroprotective agent in patients with ischemic
stroke. Furthermore, we speculate that APC may add
substantially to the effectiveness of tPA therapy for
stroke in humans.
I N F L U E N C E O F L I P I D S O N B L O O D C O A G U L AT I O N
Lipid-containing surfaces, including cell membranes
and lipoproteins, provide sites where both procoagulant and anticoagulant enzymes, cofactors, and sub-
2006
THE SCRIPPS RESEARCH INSTITUTE
strates can be assembled to express their activities.
Although dyslipoproteinemia is associated with arterial
atherothrombosis, little is known about plasma lipoproteins in patients with venous thrombosis. We used
nuclear magnetic resonance spectroscopy and antigenic
levels of apolipoproteins AI and B to determine the
concentrations of subclasses of lipoproteins in blood
samples from 49 men less than 45 years old who had
venous thrombosis and from matched control subjects.
Patients with venous thrombosis had significantly lower
levels of high-density lipoprotein (HDL) particles, large
HDL particles, HDL-cholesterol, and apolipoprotein AI
and significantly higher levels of low-density lipoprotein (LDL) particles and small LDL particles. The quartile-based odds ratios for decreased levels of HDL
particles and apolipoprotein AI were 6.5 for patients
and 6.0 for control subjects.
In collaboration with S. Eichinger and P. Kyrle, Universität Wien, we did similar new studies to determine
if dyslipoproteinemia exists in patients who have recurrent venous thrombosis. The results provided strong evidence supporting the hypothesis that HDL, notably large
HDL particles, protects against recurrence of venous
thrombosis. These clinical findings plus the results of
other in vitro experiments support the emerging concept
that procoagulant and anticoagulant lipids and lipoproteins may contribute to a yin-yang balance that influences the upregulation and downregulation of thrombin
generation and that alterations of this lipoprotein balance
may alter the hemostatic balance in patients who have
life-threatening thrombotic events.
Antithrombotic Mechanisms
M.J. Heeb, B.N. Bouma, K.M.S. Cabral, M.O. Hall,* L. Tonnu
* University of California, Los Angeles, California
e study plasma proteins that regulate blood
coagulation and can prevent thrombosis, a
factor in half of all deaths in the United
States. Knowledge of the mechanisms of action of
these anticoagulant proteins may lead to improved
antithrombotic therapies and preventive measures.
W
PROTEIN S
Protein S, a vitamin K–dependent protein, is a cofactor for activated protein C during inactivation of procoagulant factors Va and VIIIa. We showed that protein
S also exerts direct anticoagulant activity by inhibiting
MOLECUL AR AND EXPERIMENTAL MEDICINE
factors Va, VIIIa, and Xa and by competing with procoagulant factors for limiting phospholipids.
Because the finding that protein S has direct anticoagulant effects was questioned, we determined the
molecular forms of protein S in plasma and the activity
of the forms. Multimers of protein S in plasma were
detected by using several methods, including an enzymelinked immunosorbent assay that could in principle be
used to detect multimers of any protein. We developed
a novel assay for detecting direct anticoagulant activity
of protein S and showed that plasma protein S had
strong activity and that monomers, multimers, and
complexes consisting of protein S and C4b-binding
protein all had similar direct anticoagulant activity that
matched the activity of affinity-purified protein S monomers and multimers.
Protein S purified by methods used by most other
laboratories had weak direct anticoagulant activity that
did not match the activity of plasma protein S, explaining a conflicting report in the literature. That report
maintained that all plasma protein S is monomeric but
that only artifactual multimers of protein S, not monomers, have good direct anticoagulant effects. We are
investigating why some purification methods lead to
low direct inhibition by protein S; the findings should
reveal structural features of protein S that are important in its direct anticoagulant effects.
Protein S inhibition of prothrombinase (factor
Xa–factor Va) in plasma or in a purified system did
not depend on tissue factor pathway inhibitor. We are
examining whether other modes of protein S activity
depend on this inhibitor, as recently reported. In collaborative studies, protein S served as a ligand for
receptor tyrosine kinase Mer, promoting necessary
phagocytosis of shed outer rod segments of rat retinal
pigment epithelium. We are continuing structure-function
studies of protein S and studies of protein S in animal
models of thrombosis.
PROTEIN Z–DEPENDENT PROTEASE INHIBITOR
The unusual serpin protein Z–dependent protease
inhibitor (ZPI) requires protein Z, negatively charged
phospholipids, and calcium ions to inhibit procoagulant factor Xa. We found that ZPI also inhibited factor
IXa, a close homolog of factor Xa, independently of
protein Z. We sought to understand the requirement
for phospholipids for inhibition of factor Xa and to learn
the requirements for inhibition of factor IXa by ZPI and
protein Z. ZPI and protein Z inhibited factor Xa with
equal efficiency in the presence of either small, solu-
2006
THE SCRIPPS RESEARCH INSTITUTE
261
ble phospholipids or phospholipid vesicles. Formation
of complexes of Xa and ZPI was promoted by either
phospholipid species. Thus, phospholipids most likely
are required to promote a necessary conformation of
ZPI or factor Xa, and not to colocalize ZPI with factor
Xa on a surface. Using surface plasmon resonance binding techniques and activity measurements, we found
that calcium ions, but not protein Z or phospholipids,
were required for ZPI inhibition of factor IXa and for
association of factor IXa with ZPI.
Although factor Va, the cofactor for factor Xa, protected factor Xa from inhibition by ZPI or protein Z, the
same was not true for factor VIIIa, the cofactor for factor
IXa. Rather, low concentrations of factor VIIIa promoted
ZPI binding to and inhibition of factor IXa. Surface plasmon resonance studies indicted that ZPI binds to factor VIIIa with nanomolar affinity. Possibly, factor VIIIa
replaces the function of protein Z during ZPI inhibition
of factor IXa.
We had reported that low levels of protein Z were
associated with the risk for ischemic stroke in men but
not in women. Surprisingly, further analysis of data for
women revealed that low levels of the protein were
associated with the risk for stroke in the younger half
of women subjects (24–57 years old) but not in the
older half. We will determine whether lipid profiles that
change with estrogen levels can explain this finding.
Structure and Function of
Coagulation Cofactors
A.J. Gale, T. Cramer, J. Riceberg, D. Rozenshteyn,
J.-L. Pellequer*
*CEA/DSV/DIEP, Bagnols ser Cèze, France
oagulation factors Va and VIIIa are highly homologous cofactors of the serine proteases factor Xa
and factor IXa, respectively. These cofactors are
the primary targets of activated protein C (APC) in its
downregulation of the procoagulant pathway. In collaboration with J.-L. Pellequer in France, we used homology modeling techniques to model the 3-dimensional
structures of these multidomain proteins. We are using
these models and other structures to develop models of
various coagulation complexes. We use the models as
guides to create mutants of these cofactors and activated protein C in order to investigate mechanisms of
cofactor function and downregulation. For example, we
C
262 MOLECUL AR AND EXPERIMENTAL MEDICINE
engineered disulfide bonds between domains in both
factor Va and factor VIIIa. In factor Va, the disulfide
bond facilitated investigation of the mechanisms of
inactivation of factor Va by APC cleavage.
Factor VIIIa however, is inactivated by 2 mechanisms. Thrombin activation of factor VIII results in a
heterotrimer that consists of the A1 subunit, the A2
subunit, and the light chain. Both spontaneous dissociation of the A2 subunit and proteolytic cleavage of
factor VIIIa by APC inactivate factor VIIIa. Hemophilia
A, a deficiency of factor VIII, is treated by infusions of
purified recombinant factor VIII. But the usefulness of
factor VIII is limited because it is unstable after activation by thrombin as a result of the spontaneous dissociation of the A2 subunit.
We generated 2 mutants of factor VIII in which 2
newly introduced cysteine residues form a de novo disulfide bridge that cross-links the A2 and A3 domains.
These interdomain disulfides prevent the spontaneous
dissociation of the A2 subunit. We are using the mutants
as tools to investigate mechanisms of factor VIIIa inactivation alone and in combination with mutants of APC
cleavage sites. Additionally, the mutants may provide a
new, improved therapy for hemophilia A. Therefore, we
are investigating the function of these stabilized variants
in mice to determine if the variants have improved functional properties in vivo.
We are also characterizing the interactions of APC
with factor VIIIa by using mutants of residues on the
surface of APC that may be involved in factor VIIIa
binding. Conversely we will make mutants of factor
VIIIa residues that may be involved in APC binding.
Analysis of these mutants will allow us to delineate the
binding interaction between APC and factor VIIIa. In
other studies, we are investigating how the neutrophil
proteases cathepsin G and elastase modulate the functions of factor VIII and factor VIIIa, and we are characterizing the APC cofactor activity of factor V.
2006
THE SCRIPPS RESEARCH INSTITUTE
Gruber, A., Fernández, J.A., Bush, L., Marzec, U., Griffin, J.H., Hanson, S.R.,
DeCera, E. Limited generation of activated protein C during infusion of the protein
C activator thrombin analog W215A/E217A in primates. J. Thromb. Haemost.
4:392, 2006.
Hall, M.O., Obin, M.S., Heeb, M.J., Burgess, B.L., Abrams, T.A. Both protein S
and Gas6 stimulate outer segment phagocytosis by cultured rat retinal pigment epithelial cells. Exp. Eye Res. 81:581, 2005.
Heeb, M.J., Schuck, P., Xu, X. Protein S multimers and monomers each have
direct anticoagulant activity. J. Thromb. Haemost. 4:385, 2006.
Ilmakunnas, M., Pesonen, E.J., Hockerstedt, K., Makisalo, H., Fernández, J.A.,
Griffin, J.G., Repo, H., Siitonen, S., Petaja, J. Graft protein C entrapment is associated with reduced phagocyte activation during reperfusion in human liver transplantation. Crit. Care Med. 34:426, 2006.
Lindstrom, O., Kylanpaa, L., Mentula, P., Puolakkainen, P., Kemppainen, E.,
Haapiainen, R., Fernández, J.A., Griffin, J.H., Repo, H., Petaja, J. Upregulated
but insufficient generation of activated protein C is associated with development of
multiorgan failure in severe acute pancreatitis. Crit. Care 10:R16, 2006.
Mosnier, L.O., Griffin, J.H. Protein C anticoagulant activity in relation to antiinflammatory and anti-apoptotic activities. Front. Biosci. 11:2381, 2006.
Seligsohn, U., Griffin, J.H. Hereditary thrombophilia. In: William’s Hematology,
7th ed. Beutler, E., et al. (Eds.). McGraw-Hill, New York, 2005, p. 1981.
Tanaka, K.A., Fernández, J.A., Marzec, U.M., Kelly, A.B., Mohri, M., Griffin, J.H.,
Hanson, S.R., Gruber, A. Soluble thrombomodulin is antithrombotic in the presence of neutralising antibodies to protein C and reduces circulating activated protein C levels in primates. Br. J. Haemotol. 132:107, 2006.
Turunen, A.J., Fernández, J.A., Lindgren, L., Salmela, K.T., Kyllonen, L.E., Makisalo, H., Griffin, J.H., Siitonen, S.M., Petaja, J., Pesonen, E.J. Activated protein
C reduces graft neutrophil activation in clinical renal transplantation. Am. J. Transplant. 5:2204, 2005.
von dem Borne, P.A.K., Cox, L.M., Bouma, B.N. Factor XI enhances fibrin generation and inhibits fibrinolysis in a coagulation model initiated by surface-coated tissue factor. Blood Coagul. Fibrinolysis 17:251, 2006.
Zlokovic, B.V., Zhang, C., Liu, D., Fernández, J., Griffin, J.H., Chopp, M. Functional
recovery after embolic stroke in rodents by activated protein C. Ann. Neurol.
58:474, 2005.
Molecular Insights Into
Platelet Adhesion
T.J. Kunicki, Y. Cheli, L. Baronciani, M.T. Canciani,
F. Gianniello, S.T. Head, T.S. Mondala, D.R. Salomon,
A.B. Federici, O. Inoue,* K. Suzuki-Inoue,* O.J. McCarty,
M. Moroi,* Z.M. Ruggeri, Y. Ozaki,* S.P. Watson, K. Furihata
* Yamanashi Medical University, Yamanashi, Japan
PUBLICATIONS
Bouma, B.N., van Mourik, J.A. Unraveling the mystery of von Willebrand factor. J.
Thromb. Haemost. 4:489, 2006.
Bouma, B.N., von dem Borne, P.A.K., Meijers, J.C.M. Discovery of thrombin activatable fibrinolysis inhibitor. J. Thromb. Haemost. 4:257, 2006.
Gale, A.F., Radtke, K.P., Cunningham, M.A., Chamberlain, D., Pellequer, J.-L.,
Griffin, J.H. Intrinsic stability and functional properties of disulfide bond-stabilized
coagulation factor VIIIa variants. J. Thromb. Haemost. 4:1315, 2006.
Griffin, J.H. Control of coagulation reactions. In: William’s Hematology, 7th ed.
Beutler, E., et al. (Eds.). McGraw-Hill, New York, 2005, p. 1695.
Griffin, J.H., Fernández, J.A., Mosnier, L.O., Liu, D., Cheng, T., Guo, H., Zlokovic,
B.V. The promise of protein C. Blood Cells Mol. Dis. 36:211, 2006.
R E G U L AT I O N B Y H E T E R O G E N E O U S N U C L E A R
RIBONUCLEOPROTEIN L OF DIFFERENCES IN THE
EXPRESSION OF MOUSE INTEGRIN α2β1
n mice, heritable variations οf the integrin subunit
gene ITGA2 control cellular expression of the integrin
α2β1 and in blood platelets, significantly influence
adhesion to collagens in vitro. We found that a 2-fold
difference in platelet levels of α2β1 between 11 inbred
mouse strains is controlled by the length of a cytosineadenine (CA) dinucleotide repeat at a single site within
I
MOLECUL AR AND EXPERIMENTAL MEDICINE
intron 1. A total of 7 strains with a 21 CA repeat
sequence expressed twice the level of platelet α 2 β 1
as did 4 strains with a 6 CA repeat sequence and had
an equivalent gain of platelet adhesive function. These
differences in expression and function coincided with
an increase in the affinity of heterogeneous nuclear
ribonucleoprotein L for the 21 CA repeat sequence
relative to the 6 CA repeat sequence.
Using cell-free in vitro mRNA splicing, we found
that increased binding of the ribonucleoprotein results
in increased splicing efficiency and fidelity. Treatment
with short interfering RNA specific for the ribonucleoprotein abolished the splicing enhancer activity of the
21 CA repeat sequence in vivo and attenuated the
expression of endogenous α2β1 by established murine
cell lines. Our findings indicate that the increase in
surface α2β1 on mouse platelets is a consequence of
increased efficiency of ITGA2 pre-mRNA splicing through
a mechanism that is regulated by heterogeneous nuclear
ribonucleoprotein L and that depends on the length of
the CA repeat sequences at this specific site in intron 1.
A S S O C I AT I O N O F I T G A 2 H A P L O T Y P E S A N D B L E E D ING SEVERITY IN VON WILLEBRAND DISEASE
Diagnosis of von Willebrand disease is difficult
because of low heritability and the influence of modifier
genes. Using a qualitative trait locus method, we analyzed the association of bleeding severity with 8 candidate gene haplotypes within pedigrees of 11 index
cases of von Willebrand disease type 2: 2 type 2A, 3
type 2B, and 6 type 2M. These pedigrees included 47
affected and 49 unaffected relatives. As expected, the
bleeding score was most strongly influenced by the
serum level of von Willebrand factor. After Bonferroni
correction for multiple testing, only a single candidate
gene haplotype was associated with a significant increase
in bleeding severity: the ITGA2 promoter haplotype –52T.
Our findings support the hypothesis that genetic
differences in the expression of the integrin subunit α2
influence the bleeding phenotype of von Willebrand
disease. Thus, attenuation of the expression of receptors for collagen on platelets can influence risk for
morbidity in patients, such as those with von Willebrand disease, who have compromised hemostasis.
L A M I N I N - S T I M U L AT E D S P R E A D I N G O F P L AT E L E T S
T H R O U G H I N T E G R I N α6β1- D E P E N D E N T A C T I VAT I O N
O F G LY C O P R O T E I N V I
Glycoprotein VI (GPVI) is an important platelet receptor for collagens. Laminin also supports platelet adhesion through the integrin α6β1. We observed that laminin
2006
THE SCRIPPS RESEARCH INSTITUTE
263
stimulates spreading of human and mouse platelets
through a pathway that depends not only on α6β1 but
also on GPVI. Studies with GP6–/– mouse platelets indicated that GPVI is not required for platelet adhesion to
laminin. However, formation of lamellipodia on laminin
is completely inhibited in the absence of GPVI, whereas
formation of filopodia remains normal. Direct binding
and surface plasmon resonance spectroscopy confirmed
that GPVI is a receptor for laminin. In our model, α6β1
brings laminin in proximity to GPVI, which then binds
to laminin and mediates formation of lamellipodia and
platelet spreading.
I N F L U E N C E O F N - L I N K E D G LY C O S Y L AT I O N O N T H E
F U N C T I O N O F P L AT E L E T G P V I
The binding sites on GPVI for collagens and the
snake venom C-type lectin convulxin have not been
determined precisely. We used recombinant human
GPVI to evaluate the effect of N-linked glycosylation of
asparagine 92–glycine–serine 94 on binding to type I
collagen, collagen-related peptide, and convulxin. Deglycosylation of GPVI with peptide-N-glycosidase F (specific for complex N-linked glycans) or tunicamycin but
not endoglycosidase H (specific for N-linked glycans
rich in mannose) decreased the molecular weight of the
glycoprotein and attenuated cell binding to both collagen-related peptide and convulxin. Replacing asparagine
or serine with alanine significantly decreased cell adhesion to collagen-related peptide and to a lesser degree
to type I collagen and convulxin, but neither amino acid
change altered surface expression of GPVI. Our results
clearly implicate N-linked glycosylation at asparagine
92 as an important factor contributing to maximal GPVI
adhesion to type I collagen, collagen-related peptide,
and convulxin, in that order.
PUBLICATIONS
Cheli, Y., Kunicki, T.J. hnRNP L regulates differences in expression of mouse integrin α2β1. Blood 107:4391, 2006.
Inoue, O., Suzuki-Inoue, K., McCarty, O.J., Moroi, M., Ruggeri, Z.M., Kunicki,
T.J., Ozaki, Y., Watson, S.P. Laminin stimulates spreading of platelets through integrin α6β1-dependent activation of GPVI. Blood 107:1405, 2006.
Kunicki, T.J. Platelet immunology. In: Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 5th ed. Colman, R.W., Marder, V.J., Clowes, A.W.,
George, J.N., Goldhaber, S.Z. (Eds.). Lippincott Williams & Wilkins, Philadelphia,
2006, p. 461.
Kunicki, T.J., Baronciani, L., Canciani, M.T., Gianniello, F., Head, S.R., Mondala,
T.S., Salomon, D.R., Federici, A.B. An association of candidate gene haplotypes
and bleeding severity in von Willebrand disease type 2A, 2B, and 2M pedigrees. J.
Thromb. Haemost. 4:137, 2006.
Kunicki, T.J., Cheli, Y., Moroi, M., Furihata, K. The influence of N-linked glycosylation on the function of platelet glycoprotein VI. Blood 106:2744, 2005.
Kunicki, T.J., Nugent, D.J. Human platelet antigens. In: Blood Banking and Transfusion Medicine: Basic Principles and Practice, 2nd ed. Hillyer, C.D., Silberstein, L.E.,
Ness, P.M., Anderson K.N., Robach, J.D. (Eds.). Saunders, Philadelphia, 2006 p. 63.
264 MOLECUL AR AND EXPERIMENTAL MEDICINE
Functional Genomics in Organ
Transplantation and Islet Cell
Xenotransplantation
D.R. Salomon, S.M. Kurian, S. Cherqui, Y. Martina,
K. Marcucci, D. Valente, H. Ospina, D. Campbell, C. Marsh,
S. Head,* C. Lanigan,* J.R. Yates,** J. Hewel,**
P.Y. Kwok,*** J. Warrington,**** S. Horvath,*****
J. Papp,***** C.A. Wilson,† R. Bartel,†† J. Gavin††
* DNA Microarray Core, Scripps Research
** Department of Cell Biology, Scripps Research
*** University of California, San Francisco, California
**** Affymetrix, Santa Clara, California
***** University of California, Los Angeles, California
†
Food and Drug Administration, Bethesda, Maryland
††
MicroIslets, La Jolla, California
uccessful transplantation requires the orchestration of complex mechanisms set in motion by
surgical implantation of cells or organs into a
patient. Regulation of the immune response with
immunosuppressive drugs has received the most attention. But equally important is the unique cell biology
of the transplanted tissue that evolves under stress
after transplantation and ultimately determines the
function of the transplant.
One challenge, called functional genomics, is to
understand the expression and function of genes and
proteins after transplantation. How do immunosuppressive drugs work at this fundamental level? What is the
difference between a successful and an unsuccessful
transplant? Another challenge is to develop an unlimited
supply of healthy tissue for transplantation, for example, pancreatic islet cells to cure diabetes. Animals could
be used as donors, called xenotransplantation, although
the potential risks for infectious disease inherent in using
animal donors need to be better understood so that
this method can be used safely. One strategy would be
to create technologies to protect cell transplants from
rejection and optimize the function of the cells. Delivering therapeutic molecules to the transplanted tissue
could enhance the success of engraftment and function.
Finally, progenitor cells could be used to enhance the
formation of new blood vessels, called angiogenesis.
Revascularization of cell transplants is a critical step
in successful engraftment and function.
S
2006
THE SCRIPPS RESEARCH INSTITUTE
ics based on single nucleotide polymorphisms to establish profiles to diagnose acute and chronic transplant
rejection. These studies include patients with both kidney
and liver transplants. A major objective of these efforts
is to identify new pathways that drive the immune
response and cell biology of organ transplants that
might be used as the next generation of targets for therapy. For example, with all current drug therapies, the
target is the patient’s immune response; none target the
transplant itself, even though the function of the transplant is the ultimate determinant of success or failure.
We would like to test the hypothesis that gene
expression profiles can be used to create a metric or
simple diagnostic test for adequate immunosuppression.
Physicians could then adjust a patient’s drugs on the
basis of an objective measure. Our long-term goal is to
identify genes, proteins, and genetic polymorphisms
that determine the outcome of a transplant to create a
systems biology–based understanding of clinical transplantation at the molecular level.
P I G I S L E T X E N O T R A N S P L A N TAT I O N A N D T H E R I S K
FOR INFECTIOUS DISEASE
We are using novel technology to (1) create a protective alginate capsule around pig islets to prevent
rejection and (2) modify the capsule with therapeutic
molecules to enhance islet survival and function after
transplantation. Pig insulin works well in humans with
diabetes, and pigs can be genetically engineered and
can be available in great numbers. We are also using
our genomics tools to study how endothelial progenitors,
the progenitor cells for blood vessels, can be included
to further advance the success of cell transplantation,
a proof of concept for composite tissue engineering.
Although xenotransplantation is a logical strategy
to address current shortages of human donor organs,
a critical concern is the potential of moving infections
from the animals to humans. We established a new
mouse model for pig islet xenotransplantation, showed
that multiple tissues become infected with pig endogenous retrovirus, identified the human receptors for this
retrovirus, identified functional defects in nonhuman
primate cells for viral entry and assembly, and continued to refine our understanding of the viral biology and
potential risks. We think these studies are a necessary
complement to our work in safely advancing clinical
islet xenotransplantation.
FUNCTIONAL GENOMICS IN ORGAN
T R A N S P L A N TAT I O N
We are using high-density gene chip arrays, tandem
mass spectrometry proteomics, and complex trait genet-
PUBLICATIONS
Cherqui, S., Kurian, S.M., Schussler, O., Hewel, J.A., Yates, J.R. III, Salomon,
D.R. Isolation and angiogenesis by endothelial progenitors in the fetal liver. Stem
Cells 24:44, 2006.
MOLECUL AR AND EXPERIMENTAL MEDICINE
Cirulli, V., Zalatan, J., McMaster, M., Prinsen, R., Salomon, D.R., Ricordi, C.,
Torbett, B.E., Meda, P., Crisa, L. The class I HLA repertoire of pancreatic islets
comprises the nonclassical class Ib antigen HLA-G. Diabetes 55:1214, 2006.
Gemeniano, M., Mpanju, O., Salomon, D.R., Eiden, M.V., Wilson, C.A. The infectivity and host range of the ecotropic porcine endogenous retrovirus, PERV-C, is
modulated by residues in the C-terminal region of its surface envelope protein.
Virology 346:108, 2006.
Kunicki, T.J., Baronciani, L., Canciani, M.T., Gianniello, F., Head, S.R., Mondala,
T.S., Salomon, D.R., Federici, A.B. An association of candidate gene haplotypes
and bleeding severity in von Willebrand disease type 2A, 2B, and 2M pedigrees. J.
Thromb. Haemost. 4:137, 2006.
Kurian, S.M., Flechner, S.M., Kaouk, J., Modlin, C., Goldfarb, D., Cook, D.J.,
Head, S., Salomon, D.R. Laparoscopic donor nephrectomy gene expression profiling reveals upregulation of stress and ischemia associated genes when compared
to control kidneys. Transplantation 80:1067, 2005.
Martina, Y., Marcucci, K.T., Cherqui, S., Szabo, A., Drysdale, T., Srinivisan, U.,
Wilson, C.A., Patience, C., Salomon, D.R. Mice transgenic for a human porcine
endogenous retroviral receptor are susceptible to productive viral infection [published correction appears in J. Virol. 80:5100, 2006]. J. Virol. 80:3135, 2006.
Control of HIV Type 1, Gene
Delivery, and Regulation of
Hematopoietic Development
B.E. Torbett, K.S. Barnett, L. Crisa, K.M. Fischer, G.E. Foos,
M.J. Giffin, V.D. Kutilek, P.A. McClintock, R.C. Prinsen,
J.H. Savage, C.H. Swan, M.P. Tschan, J.A. Witkowski,
S. De Rozieres,* J.H. Elder,* H. Heaslet,* Y.-C. Lin,*
C.D. Stout*
* Department of Molecular Biology, Scripps Research
ur research interests include the structural and
biochemical evolution of the resistance of HIV
type 1 (HIV-1) proteases, gene delivery strategies to disrupt cellular entry of HIV-1, and normal and
abnormal regulation of myeloid development by the
transcription factors PU.1 and cyclin D–interacting
Myb-like protein (DMP1).
O
H I V - 1 P R O T E A S E R E S I S TA N C E
In patients infected with HIV-1, treatment with inhibitors of HIV reverse transcriptase and protease suppresses replication of the virus. However, HIV-1 variants
evolve that escape the approved drug treatments by
developing a broad-based resistance to the protease
inhibitors. A molecular understanding of the resistance
to protease inhibitors is needed so that new inhibitors
can be developed that target drug-resistant viruses
and, importantly, are less likely to induce inhibitorresistant viruses.
In collaboration with J.H. Elder, C.D. Stout, and
H. Heaslet, Department of Molecular Biology, we showed
that evolution of HIV protease from a form susceptible
2006
THE SCRIPPS RESEARCH INSTITUTE
265
to inhibitors to a form that is broadly resistant resulted
in profound changes in the protease structure. Structural changes in the resistant proteases included alterations in the flap and basal regions and alteration from
a symmetric to an asymmetric protease. These structural changes provide insight into the biochemical basis
for the loss of activity of protease inhibitors. To better
understand how structure contributes to the biochemical basis of resistance, we are continuing investigations
on the relationship between structure and function in
wild-type proteases and in mutant proteases that are
broadly resistant to inhibitors.
HIV-1 VECTOR DELIVERY OF CCR5-INTRABODY
G E N E S T O H U M A N H E M AT O P O I E T I C C E L L S
CXCR4 and CCR5 are the main chemokine receptors
for HIV-1 entry into cells, and blocking these receptors
limits entry of the virus. Naturally occurring polymorphisms of the gene for CCR5 indicate that disruption
of the gene provides protection from viruses that use
CCR5 to gain entry. Because polymorphisms are present in healthy persons, the use of genetic intervention
strategies that prevent or limit expression of CCR5
may provide protection from initial infection and limit
the spread of virus. With C.F. Barbas, Department of
Molecular Biology, we showed that intracellular expression of a CCR5-specific single-chain antibody (intrabody) efficiently disrupted expression of CCR5 on the
T-cell surface and protected cells from HIV-1 infection.
Moreover, we found that human stem cells expressing
the CCR5-intrabody develop into T cells and that the
decreased expression of CCR5 protected cells against
HIV-1 challenge and imparted a survival advantage in
the presence of HIV-1 infection.
In current studies, we are disrupting the function of
viruses that use either the CXCR4 or the CCR5 receptor for entry, the so-called R5X4 viruses. To accomplish
our goals, we are using combination vectors that genetically target chemokine receptors and viral and cellular
pathways critical for viral entry and replication.
R E G U L AT I O N O F M Y E L O I D D E V E L O P M E N T B Y P U . 1
AND DMP1
PU.1, a member of the Ets family of transcription
factors, is expressed solely in hematopoietic cells and
is necessary for directing myeloid development and for
regulating genes required for monocyte/macrophage and
neutrophil function. PU.1 has 3 major domains: the
transactivation, PEST, and Ets/DNA-binding domains.
PU.1 interacts with other transcription factors, and
domains of PU.1 have been implicated in its function.
266 MOLECUL AR AND EXPERIMENTAL MEDICINE
2006
THE SCRIPPS RESEARCH INSTITUTE
Myeloid development is controlled by temporal gene
expression of PU.1 and interactions among specific
transcription factors. We are addressing which PU.1
domains regulate myeloid lineage–specific commitment,
differentiation, and function. To determine which transcription factors interact with PU.1 and direct myeloid
development, we use a strategy in which the gene for
PU.1 is expressed only under certain conditions and a
proteomics approach. These studies are enabling us to
identify gene programs regulated by PU.1.
Cancer often originates from inactivation and/or
deregulation of the control of gene expression. The transcription factor DMP1 positively regulates expression of
human p14ARF and CD13/aminopeptidase N, thus playing a role in cell-cycle control, differentiation, and function of hematopoietic and nonhematopoietic cells. The
tumor suppressor ARF is critical for positive regulation
of p53, which in turn controls cellular proliferation and
modulates apoptosis. We have identified 2 novel and
developmentally expressed human DMP1 splice variants, β and γ. We found that the β variant functions as
a dominant-negative regulator of the originally reported
DMP1 protein. Currently, we are investigating the molecular and biological roles of the various isoforms in the
development of normal and leukemic cells.
DIVISION OF EXPERIMENTAL
PATHOLOGY
PUBLICATIONS
Britschgi, C., Rizzi, M., Grob, T.J., Tschan, M.P., Hügli, B., Reddy, V.A., Andres,
A.-C., Torbett, B.E., Tobler, A., Fey, M.F. Identification of the p53 family-responsive element in the promoter region of the tumor suppressor gene hypermethylated
in cancer 1. Oncogene 25:2030, 2006.
Suppression of Cytotoxic
T Lymphocyte Function by
PD-1–PD-L1 Interactions
After Antigen Recognition of
Hepatitis B Virus in the Liver
Cirulli, V., Zalatan, J., McMaster, M., Prinsen, R., Salomon, D.R., Ricordi, C.,
Torbett, B.E., Meda, P., Crisa, L. The class I HLA repertoire of pancreatic islets
comprises the nonclassical class 1b antigen HLA-G. Diabetes 55:1214, 2006.
Heaslet, H., Kutilek, V.D., Morris, G.M., Lin, Y.-C., Elder, J.H., Torbett, B.E.,
Stout, D.C. Structural insights into the mechanisms of drug resistance in HIV-1
protease NL4-3. J. Mol. Biol. 4:967, 2006.
Swan, C.H., Bühler, B., Tschan, M.P., Barbas, C.F. III, Torbett, B.E. T-cell protection
and enrichment through lentiviral CCR5 intrabody gene delivery. Gene Ther., in press.
Swan, C.H., Torbett, B.E. Can gene delivery close the door to HIV-1 entry after
escape? J. Med. Primatol., in press.
Francis V. Chisari, M.D., Division Head
Molecular Biology of Hepatitis B
and C Viruses and the Immune
Response to Their Antigens
epatitis B and C viruses are noncytopathic
DNA and RNA viruses that cause acute and
chronic hepatitis and hepatocellular carcinoma. More than 500 million persons worldwide are
chronically infected, and more than 2 million persons
die of these infections every year. The focus of our
research is to unravel the life cycle of these viruses,
discover the roles played by the innate and adaptive
immune responses in the control of the infections, and
elucidate the mechanisms responsible for viral clearance and disease pathogenesis. Our goal is to devise
novel strategies to prevent and cure these infections.
H
H. Maier, M. Isogawa, F.V. Chisari
roduction of IFN-γ by cytotoxic T lymphocytes
(CTLs) specific for hepatitis B virus (HBV) is
rapidly induced when the lymphocytes enter the
liver of mice transgenic for HBV and then is rapidly
suppressed, despite the continued presence of antigen. Suppression of IFN-γ production by the CTLs
coincides with the upregulation of PD-1, a cell-surface
signaling molecule known to inhibit T-cell function. To
determine whether PD-1 plays a role in the functional
suppression of IFN-γ secretion by CTLs, we treated
HBV transgenic mice with blocking antibodies specific
for PD-L1, the most widely expressed PD-1 ligand, and
adoptively transferred HBV-specific CTLs to the treated
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MOLECUL AR AND EXPERIMENTAL MEDICINE
mice. Treatment with antibodies to PD-L1 resulted in
an increase in the frequency of intrahepatic HBV-specific CTLs producing IFN-γ and enhanced clearance of
HBV from the liver.
These results indicate that interactions between
PD-1 and PD-L1 contribute to the suppression of IFN-γ
secretion after antigen recognition in the liver. Blockade of inhibitory pathways such as PD-1–PD-L1 may
prevent viral persistence and chronic infection in
instances in which the CTL response is suppressed
by this mechanism.
2006
THE SCRIPPS RESEARCH INSTITUTE
267
totropic virus infections in vivo, the events could explain
the propensity of hepatotropic viruses to persist.
Effect of the Size of the
Viral Inoculum on the Course
and Outcome of Hepatitis B
Virus Infection
S.F. Wieland, S. Asabe, K. Purton, R. Purcell,* F.V. Chisari
Impact of Intrahepatic Antigen
Recognition on Priming of the
CD8+ T-Cell Response in T-Cell
Receptor Transgenic Mice
M. Isogawa, F.V. Chisari
ecause our findings that interactions between
PD-1 and PD-L1 suppress the function of cytotoxic T lymphocytes specific for hepatitis B virus
(HBV) after antigen recognition in the liver reflect the
impact of antigen recognition by effector/memory cells,
the findings may not reflect what occurs in an immunologically naive host during HBV infection. To define the
immunologic events that occur during priming of HBVspecific T cells, we generated T-cell receptor (TCR) transgenic mice that have CD8+ T cells specific for the HBV
core and envelope proteins.
Preliminary results indicated that naive T cells
respond to HBV quite differently than do antigen-experienced effector/memory T cells. When naive T cells
from TCR transgenic mice were transferred into HBV
transgenic mice, the transferred cells proliferated in the
liver and caused liver disease but did not inhibit HBV
replication because they could not produce IFN-γ upon
antigen recognition. In contrast, the naive HBV-specific
CD8+ T cells produced IFN-γ after they were transferred
into HBV transgenic mice when the mice were systemically infected with recombinant vaccinia virus expressing
the corresponding HBV antigens.
These results suggest that when naive CD8+ T cells
recognize antigen exclusively in the liver, their proliferative and cytolytic programs are induced without initiating
cytokine production and, therefore, without suppressing viral replication. If similar events occur during hepa-
B
* National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
o monitor the influence of viral dose on the kinetics of viral spread and the course of infection,
we infected 6 chimpanzees with serial 1000fold dilutions of a monoclonal preparation of hepatitis
B virus (HBV). We observed a strict dose response in
the kinetics of viral spread in 4 HBV-naive animals
infected with 1010, 107, 104, 101 and 100 genome
equivalents of HBV. Unexpectedly, the course of infection in animals receiving 101 and 100 genome equivalents was greatly prolonged compared with the course
in recipients of larger doses, and peak viral titers were
comparable to the peak titer in the animal that received
1010 genome equivalents.
These counterintuitive results suggest that a lowdose HBV inoculum favors the development of persistent
infection, presumably because the low level of viral
antigens present in the inoculum does not prime an
adequate adaptive immune response. Consequently,
naive T cells make initial contact with HBV antigen in
the liver, a situation that results in partial activation of
the cells and failure to produce antiviral cytokines that
control the infection. Experiments designed to test this
hypothesis are in progress.
T
Coevolution of Virus and Host
During Persistent Hepatitis C
Virus Infection
J. Zhong, P. Gastaminza, J. Chung, G. Cheng, M. Isogawa,
F.V. Chisari
T
he virologic and cellular consequences of persistent hepatitis C virus (HCV) infection have been
elusive because of the lack of the requisite experi-
268 MOLECUL AR AND EXPERIMENTAL MEDICINE
mental systems to study HCV infections. We recently
established the conditions required to persistently infect
Huh-7 cells derived from a human hepatoma in vitro
with an HCV genotype 2a infectious molecular clone.
Persistent in vitro infection is characterized by the selection of viral variants that have accelerated expansion
kinetics, higher peak viral titers, and greater buoyant
densities than do wild-type virus.
Sequencing analysis revealed the evolution of a
single adaptive mutation in the HCV E2 envelope protein
that was largely responsible for the variant phenotype.
Furthermore, as the virus became more aggressive, cells
emerged that were resistant to infection, with escape
mechanisms operative at the levels of viral entry, HCV
RNA replication, or both. Collectively, these results
reveal the existence of coevolutionary events during
persistent HCV infection that favor survival of both
virus and host.
Antiviral Peptides That Prevent
Hepatitis C Virus Infection
G. Cheng, P. Gastaminza, A. Montero,* M.R. Ghadiri,*
F.V. Chisari
* Department of Chemistry, Scripps Research
sing a hepatitis C virus (HCV) in vitro infection
system, we identified several HCV-derived synthetic peptides that inhibit HCV infection by at
least 90% at nontoxic concentrations. The most potent
peptide, an 18-mer that contains the sequence of the
putative membrane anchor domain of the HCV NS5A
protein, and the peptide’s D-amino acid congener completely and permanently blocked HCV infection with no
cellular toxic effects. The peptide appears to inactivate
HCV extracellularly; it has no effect on HCV replicon
RNA replication and it must be added to cells together
with virus to prevent the infection.
Consistent with this hypothesis, the peptide rapidly
increases the permeability of cholesterol-phospholipid
liposomes in a dye-release assay, suggesting that it could
destabilize or otherwise compromise the integrity of the
viral membrane. N- and C-terminal truncation of the
peptide indicates that full antiviral and liposome dyerelease activities are retained when 4 amino acids are
removed from its C terminus and that both activities
are lost when the peptide is shortened any further. Further analysis of the antiviral activity of this peptide and
the remaining 12 antiviral peptides is under way.
U
2006
THE SCRIPPS RESEARCH INSTITUTE
Cooperativity and Cholesterol
Dependence of CD81 and
Scavenger Receptor B Type I in
the Initiation of Hepatitis C
Virus Infection
S.B. Kapadia, T. Baumert,* J. McKeating,** F.V. Chisari
* University of Freiburg, Freiburg, Germany
** University of Birmingham, Birmingham, England
sing surrogate models of hepatitis C virus (HCV)
infection, we identified several cellular proteins
as possible receptors for entry of HCV into cells.
Among the proteins, the tetraspanin CD81 and scavenger
receptor B type I, both of which localize to specialized
plasma membrane domains enriched in cholesterol, may
be key players in HCV entry. Using our in vitro HCV
infection system, we showed that CD81 and scavenger
receptor B type I are both required for authentic HCV
infection in vitro, that the 2 proteins function cooperatively to initiate HCV entry, and that CD81-mediated
HCV entry depends, in part, on membrane cholesterol.
U
Establishment of Hepatitis C
Virus Infection in Highly
Differentiated, Growth-Arrested
Hepatocyte Cell Lines In Vitro
B. Sainz, F.V. Chisari
hen actively dividing, poorly differentiated
cells derived from a human hepatoma (Huh7 cells) are cultured in the presence of 1%
dimethyl sulfoxide, which induces the differentiation
of primary hepatocytes in vitro, the cells become cytologically differentiated and transition into a nondividing state and the induction of hepatocyte-specific genes.
We recently showed that these cells are highly permissive for hepatitis C virus infection and that these cultures
can be persistently infected. Because hepatitis C virus
naturally replicates in highly differentiated nondividing
human hepatocytes, this system may more accurately
mimic the cellular conditions under which the virus
replicates in vivo than do models based on poorly differentiated, rapidly dividing cell lines.
W
MOLECUL AR AND EXPERIMENTAL MEDICINE
PUBLICATIONS
Bui, H.H., Sidney, J., Peters, B., Sathiamurthy, M., Sinichi, A., Purton, K.A.,
Mothé, B.R., Chisari, F.V., Watkins, D.I., Sette, A. Automated generation and
evaluation of specific MHC binding predictive tools: ARB matrix applications.
Immunogenetics 57:304, 2005.
Cheng, G., Zhong, J., Chisari, F.V. Inhibition of dsRNA-induced signaling in hepatitis C virus-infected cells by NS3 protease-dependent and -independent mechanisms. Proc. Natl. Acad. Sci. U. S. A. 103:8499, 2006.
Dryden, K.A., Wieland, S.F., Whitten-Bauer, C., Gerin, J.L., Chisari, F.V., Yeager,
M. Native hepatitis B virions and capsids visualized by electron cryo-microscopy.
Mol. Cell, in press.
Gilbert, R.J.C., Beales, L., Blond, D., Simon, M.N., Lin, B.Y., Chisari, F.V., Stuart, D.I., Rowlands, D.J. Hepatitis B small surface antigen particles are octahedral. Proc. Natl. Acad. Sci. U. S. A. 102:14783, 2005.
Guidotti, L.G., Chisari, F.V. Immunobiology and pathogenesis of viral hepatitis. In:
Mechanisms of Disease. Abbas, A.K., Downing, J.R., Kumar, V. (Eds.). Annual
Reviews, Palo Alto, CA, 2006, p. 23. Annual Review of Pathology; Vol. 1.
Iannacone, M., Sitia, G., Isogawa, M., Marchese, P., Castro, M.G., Lowenstein,
P.R., Chisari, F.V., Ruggeri, Z.M., Guidotti, L.G. Platelets mediate cytotoxic T lymphocyte-induced liver damage. Nat. Med. 11:1167, 2005.
Isogawa, M., Robek, M.D., Furuichi, Y., Chisari, F.V. Toll-like receptor signaling
inhibits hepatitis B virus replication in vivo. J. Virol. 79:7269, 2005.
Kimura, K., Moriwaki, H., Nagaki, M., Saio, M., Nakamoto, Y., Naito, M.,
Kuwata, K., Chisari, F.V. Pathogenic role of B Cells in anti-CD40-induced necroinflammatory liver disease. Am. J. Pathol. 168:786, 2006.
Murray, J., Wieland, S.F., Purcell, R.H., Chisari, F.V. Dynamics of hepatitis B virus
clearance in chimpanzees. Proc. Natl. Acad. Sci. U. S. A. 102:17780, 2005.
Zhong, J., Gastaminza, P., Cheng, G., Kapadia, S., Kato, T., Burton, D.R.,
Wieland, S.F., Uprichard, S.L., Wakita, T., Chisari, F.V. Robust hepatitis C virus
infection in vitro. Proc. Natl. Acad. Sci. U. S. A. 102:9294, 2005.
Role of Platelets in Hemorrhagic
Lymphocytic Choriomeningitis
Virus Infection in Mice
L.G. Guidotti, M. Iannacone, G. Sitia, J.K. Whitmire,*
P. Marchese, F.V. Chisari, Z.M. Ruggeri
* Molecular and Integrative Neurosciences Department, Scripps Research
e recently showed that an initial immuneinduced inflammatory response to viral antigens expressed within the liver results in
changes of the vessel wall that promote adhesion and
activation of platelets. In turn, platelet adhesion and activation favor the exit of virus-specific cytotoxic T lymphocytes (CTLs) from the bloodstream and accumulation
of the lymphocytes within the infected organ. These
studies indicate that platelets play an unexpected and
important role in the pathogenesis of CTL-mediated antiviral activity and liver damage. To investigate the contribution of platelets in the control of an acute viral
infection that involves multiple tissues and organs, we
W
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THE SCRIPPS RESEARCH INSTITUTE
269
injected different isolates of lymphocytic choriomeningitis virus (LCMV) into adult immunocompetent mice
that had or had not been depleted of platelets.
In the mice not depleted of platelets, despite a
marked thrombocytopenia (80% reduction in normal
platelet counts) associated with platelet dysfunction,
acute infection with LCMV resulted in a mild hemorrhage
(20% decrease in hematocrit and presence of fecal
blood) and rapid clearance of the infection. Depletion
of platelets (98% reduction of normal platelet counts),
but not treatment with an anticoagulant, resulted in a
severe hemorrhage (80% decrease in hematocrit) that
was often lethal and involved mostly the skin. Lethal
hemorrhage required IFN-α/β–dependent signaling but
was independent of CTL-induced immunopathologic
changes or TNF-α–mediated responses. Platelet-depleted
mice that survived for up to 5–6 days after LCMV
infection had reduced CTL numbers in the infected
organs and did not clear the virus. Platelet transfusion
prevented death, ameliorated hemorrhage, restored CTL
responses, and cleared LCMV, indicating that platelets
protect mice from lethal hemorrhagic LCMV infection
and mediate viral clearance. The results also suggest
that similar events may happen in patients infected
with arenaviruses such as Lassa virus or Junin virus,
in which thrombocytopenia, platelet dysfunction, and
exceptionally high levels of circulating IFN-α/β have
been associated with hemorrhage, impaired cellular
immunity, lack of viral clearance, and death.
PUBLICATIONS
Guidotti, L.G., Chisari, F.V. Immunobiology and pathogenesis of viral hepatitis.
Annu. Rev. Pathol. Mech. Dis. 1:23, 2006.
Iannacone, M., Sitia, G., Guidotti, L.G. Pathogenetic and antiviral immune
responses against hepatitis B virus. Future Virol. 1:189, 2006.
Iannacone, M., Sitia, G., Isogawa, M., Marchese, P., Castro, M.G., Lowenstein,
P.R., Chisari, F.V., Ruggeri, Z.M., Guidotti, L.G. Platelets mediate cytotoxic T lymphocyte-induced liver damage. Nat. Med. 11:1167, 2005.
Sitia, G., De Bona, A., Bagaglio, S., Paties, C., Uberti-Foppa, C., Guidotti, L.G.,
Lazzarin, A., Morsica, G. HIV/HCV co-infected patients naive to antiretroviral therapy show higher intrahepatic levels of CD3, IFN-γ and TNF-α mRNAs when compared to either co-infected patients treated with ART or HCV mono-infected
patients. Antivir. Ther., in press.
270 MOLECUL AR AND EXPERIMENTAL MEDICINE
DIVISION
OF
HEMATOLOGY
Ernest Beutler, M.D., Division Head
Studies in Human
Genetic Disease
E. Beutler, K. Crain, J. Flanagan, T. Gelbart, P. Lee, H. Peng,
J. Truksa, J. Waalen, L. Wang, C. West
ur epidemiologic studies of hemochromatosis
have greatly expanded our understanding of the
phenotype of homozygotes for the common
C282Y mutation in HFE, the gene responsible for the
most common form of primary hemochromatosis. Our
finding that only about 1% of C282Y homozygotes manifested the disease was so counter to common belief
that it was regarded with great skepticism—even hostility. This reaction was not surprising, because it had
been suggested, on the basis of clinical impressions,
that the penetrance of the disease phenotype was as
high as 95% among men. However, our results have
now been amply confirmed by many other studies, and
this finding has had a major impact on attitudes regarding the appropriateness of general population screening for this disorder.
Moreover, longitudinal studies of our HFE C282Y
homozygous patients have shown a remarkable lack of
clinical progression, even among patients with markedly
elevated serum ferritin levels and abnormal liver function tests. The DNA samples we collected in the course
of these studies also provided us and others with a valuable resource. The 30,000 plus DNA samples have made
it possible to perform large studies examining the relationship between genetic polymorphisms and a variety
of diseases. The samples have enabled studies of genetic
factors, including those that may affect aging, hypertension, drug addiction, obesity, arthritis, atherosclerosis, amyloidosis, and lung infection. In addition, the
laboratory data that we gathered made it possible for
us to establish more robust standards for the diagnosis of anemia than had been previously available and
have shown that the prevalence of unexplained anemia among the elderly is not nearly as high as had
been suggested.
We continue to construct model systems to allow us
to better understand how body iron content is regulated.
O
2006
THE SCRIPPS RESEARCH INSTITUTE
One of these is the measurement of iron absorption by
mice; we feed them radioactive iron and estimate the
amount of iron retained by using total body counting.
One attractive candidate regulator that we have studied is soluble transferrin receptor. Using the technique
of hydrodynamic transfection, we were able to greatly
increase the levels of this putative regulator in the
plasma of mice. Measuring iron absorption in these
animals, we showed that soluble transferrin receptor
had no influence on iron absorption.
The central regulator of iron absorption is hepcidin,
and how this 25 amino acid peptide is regulated by iron
remains a mystery, particularly since the iron regulation
can only be observed in whole animals but not in isolated hepatocytes. We have used the hydrodynamic
transfection method to introduce hepcidin promoter
luciferase constructs into whole mice to identify the
region of the hepcidin promoter that is regulated by iron.
Hepcidin is also regulated by inflammation. Using DNase
footprinting and electrophoretic mobility shift assays, we
have examined the regions of the hepcidin promoter that
are essential for regulation by inflammation.
Several proteins without any obvious connection to
iron metabolism apparently play major roles in iron homeostasis. For example, hemojuvelin, which is closely
related to retinal guidance molecules and most highly
expressed in skeletal muscle, is essential for regulating hepcidin expression. Bone morphogenic proteins
have proved to be potent stimulators of hepcidin transcription; again, their relationship to iron homeostasis
was entirely unexpected. We are attempting to unravel
these relationships.
In collaboration with Jeff Kelly, Department of Chemistry, we have established a transgenic mouse model to
express a mutant human glucocerebrosidase that is the
most common cause of Gaucher disease. Our purpose
is to use this animal as a model to study the effectiveness, in vivo, of chaperone compounds that have been
shown by researchers in Dr. Kelly’s laboratory to increase
the amount of enzyme made by cultured cells producing
this mutant enzyme. These studies could lead to a better
treatment for this inherited glycolipid storage disorder.
PUBLICATIONS
Barton, J.C., Lee, P.L. Disparate phenotypic expression of ALAS2 R452H (nt
1407 G→A) in two brothers, one with severe sideroblastic anemia and iron overload, hepatic cirrhosis, and hepatocellular carcinoma. Blood Cells Mol. Dis.
36:342, 2006.
Barton, J.C., Lee, P.L., Bertoli, L.F., Beutler, E. Iron overload in an African American woman with SS hemoglobinopathy and a promoter mutation in the X-linked
erythroid-specific 5-aminolevulinate synthase (ALAS2) gene. Blood Cells Mol. Dis.
34:226, 2005.
MOLECUL AR AND EXPERIMENTAL MEDICINE
Barton, J.C., Lee, P.L., West, C., Bottomley, S.S. Iron overload and prolonged
ingestion of iron supplements: clinical features and mutation analysis of hemochromatosis-associated genes in four cases. Am. J. Hematol., in press.
Beutler, E. Gaucher disease: multiple lessons from a single gene disorder. Acta
Paediatr. Suppl. 95:103, 2006.
2006
THE SCRIPPS RESEARCH INSTITUTE
Waalen, J., Beutler, E. Hereditary hemochromatosis: screening and management.
Curr. Hematol. Rep. 5:34, 2006.
Zimran, A., Elstein, D., Beutler, E. Low-dose therapy trumps high-dose therapy
again in the treatment of Gaucher disease. Blood, in press.
Beutler, E. Hemochromatosis. In: Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine. Ganten, D., Ruckpaul, K. (Eds.). Springer, New
York, in press.
Cell Death in the Heart
Beutler, E. Hemochromatosis: genetics and pathophysiology. Annu. Rev. Med.
57:331, 2006.
R.A. Gottlieb, N. Brady, A. Cartier, Å.B. Gustafsson,
Beutler, E. Lysosomal storage diseases: natural history and ethical and economic
aspects. Mol. Genet. Metab., in press.
271
A. Hamacher-Brady, C. Huang, D. Kubli, M.R. Sayen,
P. Wentworth, Jr.,* M. Yeager,** H. Rosen***
Beutler, E. Red blood cell enzymopathies. In: Clinical Hematology. Young, N.S.,
Gerson, S.L., High, K.A. (Eds.). Mosby, St. Louis, 2006, p. 308.
* Department of Chemistry, Scripps Research
Beutler, E. Screening for hemochromatosis. Bloodmed Exclusives. September 28,
2005. Available at: http://www.BloodMed.com.
*** Department of Immunology, Scripps Research
** Department of Cell Biology, Scripps Research
R E G U L AT I O N O F C E L L D E AT H PAT H WAY S I N
Beutler, E. The treatment of Gaucher disease in countries with limited health care
resources. Ind. J. Hum. Genet. 11:121, 2005.
Beutler, E., Gelbart, T., Scott, C.R. Hematologically important mutations: Gaucher
disease. Blood Cells Mol. Dis. 35:355, 2005.
Beutler, E., Waalen, J. The definition of anemia: what is the lower limit of normal
of the blood hemoglobin concentration? Blood 107:1747, 2006.\
Beutler, E., Waalen, J., Gelbart, T. Chronic inflammation does not appear to modify the homozygous hereditary hemochromatosis phenotype. Blood Cells Mol. Dis.
35:326, 2005.
Lee, P., Promrat, K., Mallette, C., Flynn, M., Beutler, E. A juvenile hemochromatosis patient homozygous for a novel deletion of cDNA nucleotide 81 of hemojuvelin. Acta Haematol. 115:123, 2006.
Lee, P.L., Barton, J.C. Hemochromatosis and severe iron overload associated with
compound heterozygosity for TFR2 R455Q and two novel mutations TFR2 R396X
and G792R. Acta Haematol. 115:102, 2006.
Lee, P.L., Barton, J.C., Rao, S.V., Acton, R.T., Adler, B.K., Beutler, E. Three kinships with ALAS2 P520L (c. 1559 C→T) mutation, two in association with severe
iron overload, and one with sideroblastic anemia and severe iron overload. Blood
Cells Mol. Dis. 36:292, 2006.
Lee, P.L., Beutler, E. Hemochromatosis. In: Handbook of Molecular Diagnostics.
Grody, W.W., Nakamura, R.M., Strom, C. (Eds.). Elsevier, San Diego, in press.
Lee, P.L., West, C., Crain, K., Wang, L. Genetic polymorphisms and susceptibility
to lung disease. J. Negat. Results BioMed. 5:5, 2006.
Noel, N., Flanagan, J., Bajo, M.J.R., Kalko, S.G., del Mar Mañú, M., Fuster, J.L.G.,
Perez de Ossa, P., Carreras, J., Beutler, E., Vives-Corrons, J.-L. Two new phosphoglycerate kinase mutations associated with chronic haemolytic anaemia and neurological dysfunction in two patients from Spain. Br. J. Haematol. 132:523, 2006.
Sawkar, A.R., Adamski-Werner, S.L., Cheng, W.C., Wong, C.-H., Beutler, E., Zimmer, K.P., Kelly, J.W. Gaucher disease-associated glucocerebrosidases show mutation-dependent chemical chaperoning profiles. Chem. Biol. 12:1235, 2005.
Sipe, J.C., Arbour, N., Gerber, A., Beutler, E. Reduced endocannabinoid immune
modulation by a common cannabinoid 2 (CB2) receptor gene polymorphism: possible risk for autoimmune disorders. J. Leukoc. Biol. 78:231, 2005.
Waalen, J., Beutler, E. Beware of multiple comparisons: a study of symptoms
associated with mutations of the HFE hemochromatosis gene. Clin. Chim. Acta
361:128, 2005.
Waalen, J., Beutler, E. Effect of correcting transferrin saturation for body mass
index in HFE C282Y homozygotes. J. Hepatol. 44:433, 2006.
Waalen, J., Beutler, E. Gaucher disease as a model for an orphan disease. In:
Gaucher Disease. Futerman, T., Zimran, A. (Eds.). CRC Press, Boca Raton, FL,
in press.
MYOCARDIAL ISCHEMIA AND REPERFUSION
yocardial infarctions result in the death of half
a million persons in the United States each
year. We are interested in understanding the
molecular events that commit cells to a death program
after ischemia and reperfusion in the heart. Although
ischemia itself is deleterious because of energy depletion, further damage ensues upon reperfusion, when a
burst of reactive oxygen species is produced and when
apoptosis, or programmed cell death, is activated in
vulnerable cells. Because apoptosis is a tightly regulated
program, it may be possible to interfere with the process
and salvage cardiac cells. In addition to apoptosis, cells
can die via necrosis, which has generally been regarded
as an unregulated process that can occur after exposure to high levels of oxidants. However, recent evidence suggests that so-called necrotic cell death may
also be subject to biochemical regulation. We are
defining the biochemical events of cell death in the
heart, both apoptosis and necrosis, to identify potential therapeutic targets to mitigate reperfusion injury.
Using isolated perfused rat hearts subjected to
global ischemia and reperfusion, we found that calpain
is activated during reperfusion, leading to cleavage of
Bid, a proapoptotic member of the Bcl-2 family of antiapoptotic proteins. Bid targets the mitochondria, resulting in energetic failure and release of proapoptotic factors.
The protein apoptosis repressor with caspase recruitment domain is expressed at high levels in cardiac and
skeletal muscle and is strongly protective against cell
death mediated by oxidative stress. We have shown
that this protein interacts with Bax, another proapoptotic protein, to prevent apoptosis through the mitochondrial pathway.
M
272 MOLECUL AR AND EXPERIMENTAL MEDICINE
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THE SCRIPPS RESEARCH INSTITUTE
Cyclophilin D, a protein found in the inner membrane of mitochondria, regulates the mitochondrial
permeability transition pore. In collaboration with
J. Molkentin, Children’s Hospital Medical Center, Cincinnati, Ohio, we are examining the mitochondrial
alterations mediated by deletion and overexpression
of cyclophilin D.
R E G U L AT I O N A N D S I G N I F I C A N C E O F A U T O P H A G Y
IN THE MYOCARDIUM
Autophagy is a cell-autonomous mechanism to
remove damaged or unwanted organelles (Fig. 1). Using
high-resolution fluorescence microscopy with 3-dimensional deconvolution to image live cells expressing the
autophagy marker LC3-GFP (Fig. 2), we evaluated the
upregulation of autophagy in HL-1 cardiomyocytes
subjected to starvation or simulated ischemia and reperF i g . 2 . HL-1 cells were transfected with LC3-GFP and imaged
after simulated ischemia and 90 minutes of reperfusion (sI/R) or
incubation in normoxic (but nutrient-limited) buffer (KH) for the
same amount of time. The punctate structures indicate the formation of autophagosomal vesicles (AVs). The level of autophagic
activity (flux) was revealed by treating cells with inhibitors (+ i) to
prevent lysosomal fusion, resulting in the accumulation of AVs. The
bar graph quantifies the upregulation of autophagy by starvation
and sI/R and the impairment of flux by sI/R.
F i g . 1 . Autophagy in simulated ischemia and reperfusion. Induction of autophagy requires activity of Beclin1 and its interacting
partner, class III phosphatidylinositol-3′-kinase (PI3-K; hVps34),
resulting in the generation of phosphatidylinositol-3′-phosphate, and it
is negatively regulated by class I PI3-K through mTOR. Formation of
the phagophore requires conjugation of Atg12 to lysine 130 of Atg5
as a prerequisite for recruiting LC3-II. Sequestration of cytoplasmic
material can be nonspecific or selective; mechanisms that may govern selectivity are incompletely understood. In order to accomplish
degradation of the autophagosome and its cargo, the autophagosome
is then transported to and fuses with the acidic lysosome, generating
the autophagolysosome. Within the autophagolysosome, lysosomal
proteases degrade the inner autophagosomal membrane and cargo.
During ischemia, autophagy is inhibited at the level of autophagosome formation. Upon reperfusion, autophagy partially recovers, with
submaximal induction and impaired degradation. Enhancing autophagic flux is protective against simulated ischemia-reperfusion injury.
fusion. We found that autophagy is upregulated in
nutrient-limited conditions, but this response is completely suppressed during simulated ischemia and only
partially recovers during reperfusion. Nevertheless, the
induction of autophagy is part of a cytoprotective
response to ischemia-reperfusion injury. We hypothesize that autophagy removes damaged proapoptotic
mitochondria, thereby averting apoptosis. Mitochondria must fragment into small spherules before they
can be engulfed by an autophagosome. We are now
exploring the control of autophagy in the heart and
examining the conditions that lead to fragmentation
and removal of mitochondria via autophagy.
C H A R A C T E R I Z AT I O N O F T H E R A P E U T I C A G E N T S
FOR ISCHEMIA-REPERFUSION INJURY
We made the serendipitous discovery that chloramphenicol and other inhibitors of cytochrome P450
monooxygenases reduce ischemia-reperfusion injury
in the heart. These drugs are protective even when
administered after ischemia, suggesting that they may
have therapeutic potential in the treatment of myocardial infarction. Cytochrome P450 monooxygenases in
the heart metabolize arachidonic acid to eicosanoids
that regulate contractility and vasomotor tone. Some
P450 enzymes are also potent sources of superoxide,
MOLECUL AR AND EXPERIMENTAL MEDICINE
which may contribute to reperfusion injury. We are
investigating the basis for the protective effect of P450
inhibition. We are focusing on the downstream signal
transduction events such as activation of calpain and
p38 MAP kinase.
Analogs of sphingosine-1-phosphate (S1P) are being
developed as immunomodulators. We have studied the
effects on the heart of S1P and synthetic receptor-selective agonists. We found that an agonist selective for S1P 1
greatly exacerbated reperfusion arrhythmias. Receptor-selective agonists will require further evaluation for
safety in clinical trials in humans.
ROLE OF BNIP3 IN THE MYOCARDIUM
Bnip3 is a member of the “BH3-only” subfamily of
proapoptotic Bcl-2 proteins and is localized primarily
to the mitochondria in cardiomyocytes. We found that
Bnip3 contributes to ischemia-reperfusion injury and
that overexpression of Bnip3 leads to mitochondrial
dysfunction and cell death in cardiac myocytes. We also
found that Bnip3 induces extensive fragmentation of the
mitochondrial network (Fig. 3), which coincides with
F i g . 3 . Overexpression of Bnip3 causes fragmentation of the
mitochondrial network. HL-1 cardiac myocytes were cotransfected
with Mito-dsRed-2 (to label mitochondria) and pcDNA3.1 or
Bnip3, and fluorescent images were obtained 48 hours later.
a dramatic upregulation of autophagy. 3-Dimensional
deconvolution rendering of fluorescent images revealed
fragmented mitochondria inside autophagosomes. Inhibition of the formation of autophagosomes resulted in
increased Bnip3-mediated cell death, supporting the
notion that autophagy might serve as a protective
response by sequestering damaged mitochondria. Currently, we are elucidating the molecular mechanism by
which Bnip3 mediates mitochondrial dysfunction and
cell death. We are also interested in identifying proteins
that interact with Bnip3 in the heart and in determining the functional significance of this interaction.
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PUBLICATIONS
Brady, N.R., Hamacher-Brady, A., Gottlieb, R.A. Proapoptotic BCL-2 family members and mitochondrial dysfunction during ischemia/reperfusion injury, a study
employing cardiac HL-1 cells and GFP biosensors. Biochim. Biophys. Acta, in press.
Brady, N.R., Hamacher-Brady, A., Westerhoff, H.V., Gottlieb, R.A. A wave of reactive oxygen species (ROS)-induced ROS release in a sea of excitable mitochondria.
Antioxid. Redox Signal., in press.
Gustafsson, Å.B., Gottlieb, R.A., Granville, D.J. TAT-mediated protein transduction: delivering biologically active proteins to the heart. Methods Mol. Med.
112:81, 2005.
Hamacher-Brady, A., Brady, N.R., Logue, S.E., Sayen, M.R., Jinno, M., Kirshenbaum, L.A., Gottlieb, R.A., Gustafsson, Å.B. Response to myocardial ischemia/
reperfusion injury involves Bnip3 and autophagy. Cell Death Differ., in press.
Genetics of the Endogenous
Cannabinoid System
J.C. Sipe, A. Gerber
e focus on the genetics of the endogenous
cannabinoid system and the role of genetic
abnormalities in this system in human diseases. Several common human neurobehavioral disorders, such as drug addiction, obesity, anxiety, and
chronic pain, most likely are related to malfunction of
endocannabinoids, chemicals known as the brain’s own
marijuana. We recently found a link between autoimmune diseases such as multiple sclerosis and abnormalities of endocannabinoid function in the immune system.
Since our discovery in 2002 of a naturally occurring
human mutation, P129T, in fatty acid amide hydrolase,
the main enzyme that controls levels of endocannabinoid signaling in the nervous system, we have focused
on disorders in humans that appear to be associated
with genetic variations. In collaboration with B.F. Cravatt,
Department of Cell Biology, we found that the P129T
mutation results in significantly reduced cellular activity
and expression of fatty acid amide hydrolase. Previously, we were the first investigators to show a significant association between the P129T mutation and
overweight and obesity, suggesting that this variation
may be an important risk factor for these major public
health problems.
More recently, we collected new information on the
influence of P129T in human disorders of reward and
craving, such as drug abuse and addiction. Collaborative
studies with several drug abuse centers resulted in more
detailed data on the P129T variation. The mutation was
not a risk factor for marijuana dependence in marijuana
users, but it was linked to abuse of sedative drugs. In
a study of heroin addicts done in collaboration with
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274 MOLECUL AR AND EXPERIMENTAL MEDICINE
colleagues in New York City, we found no increased risk
for heroin addiction in patients with the P129T mutation. However, in a study of subjects with abuse of
several different types of drugs, we identified several
mutations linked to P129T. We calculated that this
mutation in humans is an ancient one that arose more
than 100,000 years ago in Africa and was carried into
all racial groups after the African Diaspora. In collaboration with colleagues at the National Institute on Drug
Abuse, Baltimore, Maryland, we confirmed our earlier
findings that the P129T mutation is associated with
multiple drugs of addiction.
PUBLICATIONS
Flanagan, J., Gerber, A.L., Cadet, J.L., Beutler, E., Sipe, J.C. The fatty acid amide
hydrolase 385 A/A (P129T) variant: haplotype analysis of an ancient missense
mutation and confirmation of risk for drug abuse. Hum. Genet., in press.
Proudnikov, D., Sipe, J.C., Barral, S., Ott, J., LaForge, S., Kreek, M.J. The 385 A
allele coding the low activity fatty acid amide hydrolase may be associated with
reduced vulnerability to heroin addiction in African-American males. Neurosci.
Lett., in press.
Tyndale, R.F., Payne, J.I., Gerber, A.L., Sipe, J.C. The fatty acid amide hydrolase
C385A (P129T) missense mutation in THC users: studies of drug use and dependence in Caucasians. Pharmacogenet. Genomics, in press.
DIVISION
OF
MOLECULAR ONCOLOGY
Thomas F. Deuel, M.D., Division Head
Pleiotrophin: A Cytokine With
Critical Roles in Growth and in
Development and Progression
of Human Neoplasms
T.F. Deuel, Y. Chang, L. Ezquerra-Ruiz, G. Herradon,
P. Perez-Pinera, W. Zhang
e recently identified and cloned pleiotrophin,
an 18-kD cytokine with diverse roles in normal growth and in the development, differentiation, and progression of malignant tumors. Pleiotrophin
signals through a unique mechanism; it inactivates the
receptor protein tyrosine phosphatase (RPTP)β/ζ. Through
inactivation of RPTPβ/ζ, pleiotrophin increases levels
of tyrosine phosphorylation of the substrates of RPTPβ/ζ
due to the continued activities of unknown tyrosine
kinases that phosphorylate the same sites that normally
are dephosphorylated by RPTPβ/ζ in cells not stimulated
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with pleiotrophin. Substrates of RPTPβ/ζ that we have
discovered include β-catenin, β-adducin, Fyn, anaplastic lymphoma kinase (ALK), and TrkA, the receptor of
nerve growth factor. Increased tyrosine phosphorylation
of these substrates in pleiotrophin-stimulated cells leads
to disruption of adherent junction complexes and loss
of homophilic cell-cell adhesion, suggesting the importance of pleiotrophin signaling in the regulation of these
vital cell functions.
We recently discovered that pleiotrophin signaling
critically regulates the renin-angiotensin biosynthesis
pathway and the catecholamine biosynthesis pathway
and that pleiotrophin stimulates synthesis of a specific
cohort of collagens and elastin. Pleiotrophin also is a
potent angiogenic factor that stimulates growth of new
blood vessels when injected into ischemic myocardium.
Ptn, the gene for pleiotrophin, is also a proto-oncogene and is constitutively expressed in many human
malignant cancers. In all cases studied, pleiotrophin
signaling was essential to maintain the transformed
phenotype of the cancers. Inappropriate pleiotrophin
signaling thus is a potent promoter of tumor progression. We found that constitutive pleiotrophin signaling
in malignant cells stimulates an epithelial-mesenchmal
transition, loss of cell-cell adhesion, gain of a motile
phenotype, degradation of cadherins, and expression
of different integrins at the cell surface. In vivo, pleiotrophin stimulates tumor angiogenesis, extensive remodeling
of the microenvironment and induction of carcinomaassociated fibroblasts, morphologic changes of the carcinoma cell itself to a more aggressive phenotype, and
a more rapid and aggressive growth of the tumors.
Our long-range goals are to expand knowledge of
this unique pleiotrophin–RPTPβ/ζ signaling pathway,
to use pleiotrophin as a therapeutic agent to stimulate
angiogenesis, and to target the pleiotrophin signaling
pathway as a tool for treating progression of human
neoplasms with constitutive expression of Ptn.
SIGNALING
In the past year, we identified ALK as a substrate
of RPTP β/ζ. ALK is a receptor-type transmembrane
tyrosine kinase with a unique mechanism of activation.
Inactivation of the tyrosine phosphatase activity of
RPTP β/ζ is responsible for ALK activation; the kinase
is not activated directly by a cytokine acting through a
cell-surface receptor.
We showed that pleiotrophin activates the tyrosine
kinase activity of ALK in pleiotrophin-stimulated cells
and that the activated ALK kinase phosphorylates
MOLECUL AR AND EXPERIMENTAL MEDICINE
β-catenin. In addition, we found that the site phosphorylated in β-catenin by ALK is a site recognized
and dephosphorylated by RPTP β/ζ. This tyrosine
phosphorylation site in β-catenin is potentially important, because when it is phosphorylated in pleiotrophin-stimulated cells, it disrupts the association of
β-catenin with N-cadherin needed for cells to adhere
to each other. Because disruption of homophilic cellcell adhesion is characteristic of highly malignant cells
that express Ptn, our data suggest that one mechanism
through which pleiotrophin stimulates a more aggressive phenotype in malignant cells is disruption of normal cytoskeletal architecture. In collaboration with
J.R. Yates, Department of Cell Biology, we are using
mass spectrometry to identify the sites of tyrosine phosphorylation in β-catenin and in ALK that are phosphorylated in pleiotrophin-stimulated cells.
In PC12 cells, stimulation with pleiotrophin activates TrkA. The cessation of growth and progression of
neurite outgrowth in PC12 cells stimulated with pleiotrophin are same as the cessation and progression of
neurite outgrowth that occur in PC12 cells stimulated
with nerve growth factor. We found that PC12 cells
require pleiotrophin to survive and that pleiotrophin
acts through an autocrine mechanism. The activation
of TrkA requires phosphorylation of the same tyrosine
that is phosphorylated in pleiotrophin-stimulated cells.
Thus, the mechanism of activation of TrkA is the same
as the mechanism of activation of ALK, suggesting that
the regulation of different tyrosine kinase receptors by
pleiotrophin may be a unique mechanism of maintaining the trophism of cells.
ANGIOGENESIS
We found that expression of Ptn is upregulated in
developing microvasculature, macrophages, and astrocytes after acute ischemic brain injury and that pleiotrophin directly injected into ischemic myocardium induces
formation of functional neovasculature in vivo, including
stimulating growth of new capillaries and arterioles that
functionally interconnect with existent coronary vascular
systems. Furthermore, in other studies, we showed that
reversal of endogenous pleiotrophin signaling in human
glioblastoma cells, via introduction of a dominant-negative
Ptn gene, reverses both the malignant and the angiogenic phenotypes of these cells in vivo.
These findings indicate that pleiotrophin is an angiogenic factor in vivo and that constitutive signaling of
the endogenous pleiotrophin in cancer cells is sufficient
to initiate tumor angiogenesis and aggressive tumor
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275
growth. We are now studying the mechanisms and pathways of pleiotrophin signaling that lead to angiogenesis in both in vitro and in vivo models.
BREAST CANCER
To extend our studies on pleiotrophin in neoplasia,
we used a dominant-negative Ptn and found that it
reversed the malignant phenotype of human breast cancer cells in vitro and in vivo. Currently, we are determining the mechanisms by which pleiotrophin signaling
stimulates a malignant state in human breast cancer
cells. In vitro, we identified reciprocal signaling between
breast cancer cells that express an activated Ptn and
activated stromal fibroblasts. We have now shown that
through reciprocal cross talk pleiotrophin secreted from
human breast cancer cells cocultured with NIH 3T3
cells sharply upregulates protein kinase C δ and matrix
metalloproteinase 9 in both the NIH 3T3 cells and the
human breast cancer cells. Furthermore, the upregulation of both protein kinase C δ and matrix metalloproteinase 9 in both cells depends entirely on secretion of
pleiotrophin from the breast cancer cell.
To further test the relevance of pleiotrophin in promoting the growth of malignant breast cancers in vivo,
we used bitransgenic mice predisposed to breast cancer. We found that constitutive pleiotrophin signaling
driven by the mouse mammary tumor virus promoter,
which directs genes to mammary gland cells for expression, does not induce breast cancer in mice; inappropriate expression of pleiotrophin alone is insufficient to
induce breast cancer. However, inappropriate expression of pleiotrophin cooperates with signals driven by
polyoma middle T antigen to accelerate the growth of
mouse breast cancers and initiate formation of new
blood vessels in the tumors.
In ongoing collaborative studies with Z.-Y. Wang,
Creighton University, Omaha, Nebraska, we identified and
partially characterized a novel form of the estrogen receptor. The significance of this finding is under investigation.
PUBLICATIONS
Ezquerra, L., Herradon, G., Nguyen, T., Silos-Santiago, I., Deuel, T.F. Midkine is a
potent regulator of the catecholamine biosynthesis pathway in mouse aorta. Life
Sci., in press.
Wang, Z.Y., Zhang, X.T., Shen, P., Loggie, B.W., Chang, Y.C., Deuel, T.F. A novel
variant of estrogen receptor α, hER-α36: tranduction of estrogen- and antiestrogendependent membrane-initiated mitogenic signaling. Proc. Natl. Acad. Sci. U. S. A.,
in press.
Zhang, N., Zhong, R., Perez-Pinera, P., Herradon, G., Ezquerra, L., Wang, Z.Y.,
Deuel, T.F. Identification of the angiogenesis signaling domain in pleiotrophin
defines a mechanism of the angiogenic switch. Biochem. Biophys. Res. Commun.
343:653, 2006.
276 MOLECUL AR AND EXPERIMENTAL MEDICINE
The S-Phase Checkpoint in
Mammalian Cells
X. Wu, E. Olson, E. Liu, A. Lee
enome instability is a hallmark of the malignant
phenotype and a driving force for tumorigenesis. S phase is genetically the most vulnerably
period of the cell cycle. In this phase, DNA must be
replicated faithfully in a timely fashion, and the entire
genome must be duplicated exactly once per cell cycle.
Errors or lesions generated spontaneously or in response
to damaging environmental injuries must be repaired to
maintain genome stability. The S-phase checkpoint
monitors S-phase progression. It inhibits ongoing replication as soon as DNA damage is detected, allowing
time for DNA repair.
In one area of our research, we focus on a diseaselinked protein complex termed Mre11/Rad50/Nbs1
(MRN). Mutations in the genes NBS1 and Mre11 lead
to the Nijmegen breakage syndrome and ataxia telangiectasia–like disorder, respectively. Cells derived from
patients with Nijmegen breakage syndrome or ataxia
telangiectasia–like disorder undergo radioresistant DNA
synthesis, failing to suppress DNA replication in response
to ionizing radiation. How MRN affects DNA replication to control the S-phase checkpoint, however, is
not clear.
We observed that MRN directly interacts with replication protein A (RPA) and that this interaction is needed
for MRN to correctly localize to replication centers. Abolishing the interaction of Mre11 with RPA leads to pronounced radioresistant DNA synthesis. We also found
that the interaction of MRN and RPA is required for suppressing the initiation of replication upon DNA damage.
This suppression depends on the recruitment of MRN to
sites near the origins of chromosomal replication in S
phase by a direct interaction with RPA. These studies
indicate that in response to DNA damage MRN acts
directly at sites proximal to the origins of chromosomal
replication to inhibit initiation of DNA replication, thereby
providing an important mechanism underlying the intraS-phase checkpoint in mammalian cells.
The second focus of our research is understanding
how DNA replication is controlled so that DNA is replicated once and only once per cell cycle. Rereplication of
the genome, or even a segment of it, could lead to
genome instability. One key to the control of initiation
of DNA replication is the tightly regulated assembly of
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prereplication complexes at replication origins. Consistent with this finding, overexpression of the replication
licensing factor Cdt1 induces rereplication in certain
tumor cell lines. We found that when the licensing
control is impaired by Cdt1 overexpression, the S-phase
checkpoint is activated and rereplication is inhibited
through the ataxia telangiectasia–mutated and Rad3related pathway. Our findings suggest that when the
licensing control is compromised in mammalian cells,
the S-phase checkpoint provides another protection
mechanism to prevent DNA rereplication.
DIVISION
OF
ONCOVIROLOGY
Peter K. Vogt, Ph.D., Division Head
Molecular Genetics of Cancer
P.K. Vogt, A. Bader, D. Bai, K. Bower, I. Dang, A. Denley,
A. Galkin, M. Gymnopoulos, H. Jiang, S. Kang, U. Karst,
M. Scheerer, J. Shi, L. Zhao
he focus of our research is molecular mechanisms
of carcinogenesis. We study viral and cellular
oncoproteins and tumor suppressors, defining
their functions in oncogenesis and identifying molecular
targets for therapeutic intervention. In high-throughput
screens, we look for small molecules that can interact
with these targets and inhibit or reverse oncogenic
cellular transformation.
T
O N C O G E N I C T R A N S F O R M AT I O N
Oncogenic transformation of cells requires changes
in gene activities, regulated at the level of transcription,
translation, or posttranslational modification. These
changes result in a gain of function for specific growthpromoting genes and a loss of function for growthattenuating genes.
P H O S P H AT I D Y L I N O S I T O L - 3 ′ - K I N A S E A S A N O N C O G E N E A N D C A N C E R TA R G E T
Phosphatidylinositol-3′-kinase (PI3K) is a lipid
kinase. It generates phosphatidylinositol 3,4,5-trisphosphate, an important second messenger molecule that
sets in motion complex growth-promoting signaling
chains in the cell. Gain of function in PI3K-dependent
signaling is common in cancer and has 3 principal
causes: amplification of the gene PIK3CA, which codes
for the catalytic subunit p110α of class I PI3K; point
mutations in PIK3CA; and loss of function in PTEN,
MOLECUL AR AND EXPERIMENTAL MEDICINE
the antagonist of PI3K that functions as an important
tumor suppressor. Increased PI3K activity is a critical
determinant of the oncogenic cellular phenotype. When
fused to a sequence that mediates membrane localization, p110α functions as a retroviral oncoprotein, inducing transformation in cell culture and tumors in animals.
Frequently occurring human cancers, such as breast
and colorectal cancers, have a high incidence of mutations in PIK3CA. The mutations are not randomly distributed over the gene but are concentrated in 3 major
hot spots along the coding sequence. This finding suggests that they are selected for and that they confer a
growth advantage to the cell.* The mutations are somatic
and cancer specific. We have shown that these mutations induce a gain of enzymatic function and that they
activate the PI3K signals in the cell.
The mutant PI3K proteins can transform cells in culture and induce tumors in animals. These tumors and the
cells transformed in culture are highly sensitive to the
TOR inhibitor rapamycin. TOR is a protein kinase and a
component of the PI3K signal chain. Mutated PI3K is a
highly attractive cancer target. The mutated protein is
essential for the oncogenic phenotype of the tumor cell.
The mutations do not occur in normal tissue. Because
PI3K is an enzyme, it can be readily manipulated with
small molecules, and a gain of function is more easily
corrected than a loss of function. We have started a program to identify small-molecule inhibitors that are specific for cancer-derived mutants of the enzyme.
Class I PI3K occurs in 4 isoforms encoded by different genes. Cancer-specific mutations have been found
only in the α isoform. We investigated the oncogenic
potential of the β, γ, and δ isoforms and discovered that
these isoforms are oncogenic as wild-type proteins,
inducing transformation in cell cultures. In contrast, the
α isoform is nononcogenic as wild-type protein and
requires a gain-of-function mutation to become transforming. The oncogenicity of the wild-type non-α isoforms of PI3K raises the possibility that these isoforms
could be involved in human cancer. Increased levels of
expression of non-α isoforms have been found in specific
cancers, and the role of the isoforms in determining the
oncogenic phenotype deserves further study.
S M A L L - M O L E C U L E R E G U L AT O R S O F T H E
MYC NETWORK
Myc is a transcriptional regulator that can strongly
stimulate cell proliferation. Increased levels and enhanced
function of Myc are common in cancer. They result from
gene amplification, elevated levels of transcription, and
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277
activated translation. A correlation exists between the
gain of function in Myc, tumor grade, and poor prognosis, suggesting that Myc plays an important role in the
causation and progression of cancer.
To function as a transcription factor, Myc must form
a dimer with another protein, Max. The structure of
the Myc-Max dimerization interface is known; single
amino acid substitutions at critical sites can break or
stabilize dimerization. In collaboration with D.L. Boger
and K.D. Janda, Department of Chemistry, we have
isolated several small molecules that interfere with the
dimerization of Myc and Max. As a consequence, these
molecules also prevent Myc DNA binding, Myc-dependent transcriptional activation, and Myc-induced oncogenic transformation.
The Myc-Max dimer belongs to a complex network
that includes activators as well as repressors of transcription. All of the activators and repressors function
as dimers with the Max protein, making Max the common denominator of the network.* Max is also the only
component of the network that can form homodimers,
albeit weak and transcriptionally inactive homodimers.
Small molecules that specifically stabilize the Max
homodimer would make this essential partner unavailable for heterodimerization and for transcriptional regulatory activities. Such compounds would downregulate
the entire network.
We have used the software program Autodock to
identify small molecules that bind preferentially to the
Max homodimer and enhance its stability. Two of these
compounds also effectively inhibit Myc-induced oncogenic transformation in cell culture. The principle of
downregulating the Myc network by stabilizing the Max
homodimer was also validated by genetic experiments.
A mutant of Max was generated that formed homodimers
of enhanced stability with the wild-type proteins. Expression of this Max mutant made cells resistant to Mycinduced transformation by keeping Max in homodimers,
unavailable for dimerization with Myc. We are currently performing additional screens for small-molecule
stabilizers of Max and will analyze their effects on Mycdependent transcription and oncogenic transformation.
PUBLICATIONS
Bader, A.G., Kang, S., Vogt, P.K. Cancer-specific mutations in PIK3CA are oncogenic in vivo. Proc. Natl. Acad. Sci. U. S. A. 103:1475, 2006.
Bader, A.G., Kang, S., Zhao, L., Vogt, P.K. Oncogenic PI3K deregulates transcription and translation. Nat. Rev. Cancer 5:921, 2005.
Bader, A.G., Vogt, P.K. Leucine zipper transcription factors: bZIP proteins. In:
Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine. Ganten, D., Ruckpaul, K. (Eds.). Springer, New York, in press.*
278 MOLECUL AR AND EXPERIMENTAL MEDICINE
Bader, A.G., Vogt, P.K. Protein synthesis and cancer. In: Nutritional Genomics:
Impact on Health and Disease. Brigelius-Flohé, R., Joost, H.-G. (Eds.). Wiley-VCH,
New York, 2006, p. 180.
Harada, J.N., Bower, K.E., Orth, A.P., Callaway, S., Nelson, C.G., Laris, C.,
Hogenesch, J.B., Vogt, P.K., Chanda, S.K. Identification of novel mammalian
growth regulatory factors by genome-scale quantitative image analysis. Genome
Res. 8:1136, 2005.
Kang, S., Denley, A., Vanhaesebroeck, B., Vogt, P.K. Oncogenic transformation
induced by the p110β, γ, and δ isoforms of class I phosphoinositide 3-kinase.
Proc. Natl. Acad. Sci. U. S. A. 103:1289, 2006.
Vogt, P.K., Bader, A.G. Jun: stealth, stability, and transformation. Mol. Cell
19:432, 2005.
Vogt, P.K., Bader, A.G. Oncogenes and proto-oncogenes: jun oncogenes. In: Encyclopedia of Respiratory Medicine. Laurent, G.J., Shapiro, S.D. (Eds.). Academic
Press/Elsevier, Philadelphia, 2006, p. 241.
Vogt, P.K., Bader, A.G., Kang, S. Phosphoinositide 3-kinase: from viral oncoprotein
to drug target. Virology 344:131, 2006.
Vogt, P.K., Bader, A.G., Kang, S.K. PI 3-kinases: hidden potentials revealed. Cell
Cycle 5:946, 2006.
Vogt, P.K., Kang, S.K. Kinase inhibitors: vice becomes virtue. Cancer Cell 9:327,
2006.
Xu, Y., Shi, J., Yamamoto, N., Moss, J.A., Vogt, P.K., Janda, K.D. A credit-card
library approach for disrupting protein-protein interactions. Bioorg. Med. Chem.
14:2660, 2006.
Molecular Mechanisms of
Leukemia Development and
Protein Modification by a
Ubiquitin-Like Modifier
D.-E. Zhang, O.A. Malakhova, L.F. Peterson, M. Yan,
A. Boyapati, J.-K. Luo, W. Zou, J.R. Biggs, J.-H. Kim,
E.-Y. Ahn, J. Wang, A.J. Okumura, F. Okumura, B. Yeung,
B. Abdulla, X. Yin, M.-C. Lo
AML1 AND ITS FUSION PROTEIN AML1-ETO IN
B L O O D C E L L D I F F E R E N T I AT I O N
cute myeloid leukemia is a major hematopoietic
malignant neoplasm characterized by the proliferation of a malignant clone of myeloid progenitor
cells. One of the most common targets of chromosomal
translocations that have been implicated in this neoplasm is the gene AML1 (RUNX1). The gene was isolated
through a study of t(8;21) chromosomal translocation;
the results revealed that the runt homology domain of
AML1 is fused to a gene termed ETO (MTG8) to form a
fusion protein called AML1-ETO. Subsequent studies
indicated that the protein AML1 is crucial for normal
hematopoiesis. We previously discovered that AML1
synergistically activates the expression of a critical
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myeloid gene, the gene for the M-SCF receptor, with 2
other important transcription factors, C/EBP and PU.1.
To study the effect of AML1-ETO on hematopoiesis,
we produced various mouse models in which wild-type
AML1 was replaced by AML1-ETO. Currently, we are
identifying cofactors involved in the synergy among
various transcription factors and in AML1-ETO–associated development of leukemia.
A NOVEL UBIQUITIN-SPECIFIC ENZYME, UBP43
In studying genes differentially expressed in AML1ETO mice, we isolated the gene for a novel enzyme
UBP43 (USP18), which belongs to a family of ubiquitin-specific proteases. Like phosphorylation and dephosphorylation, ubiquitylation and deubiquitylation are
mechanisms for protein modification. Recently, we
showed that UBP43 is the only currently known enzyme
that removes a ubiquitin-like modifier, ISG15, from
ISG15 conjugates. In mice that lacked the gene for
UBP43, UBP43-deficient bone marrow cells were
hypersensitive to treatment with type I interferon and
died via apoptosis in the presence of interferon. Most
important, in UBP43-deficient cells, interferon induced
a prolonged Stat1 tyrosine phosphorylation, DNA binding, and interferon-mediated gene activation. UBP43deficient mice are resistant to certain viral and bacterial
infections. Currently, we are analyzing molecular pathways affected by UBP43.
RO L E O F I S G 1 5 C O N J U G AT I O N I N I M M U N E
RESPONSES
The gene for ISG15 was originally cloned as a gene
highly upregulated by interferon and encodes a small
ubiquitin-like protein. Unlike ubiquitin and other ubiquitin-like modifiers, ISG15 is not present in lower
eukaryotes, such as yeast, indicating that it may be
associated with specialized functions in higher eukaryotic cells. Upon viral infection, bacterial infection, or
other stress stimulation, ISG15 can be detected in cells
both in free and in conjugated form (ISGylation). Using
high-throughput Western blot analysis, we identified 4
ISGylated proteins: Stat1, Jak1, Erk1, and PLCγ1. We
also found that Ubc8 is an ISG15-conjugating enzyme
and that Efp is an ISG15 ligase. Regulation of protein
ISGylation may provide valuable treatments to control
cell function and survival. We are using techniques
such as gene depletion, protein interaction, biochemical purification, and gene regulation to study the biological function of this interesting protein modification.
PUBLICATIONS
Biggs, J.R., Zhang, Y., Peterson, L.F., Garcia, M., Zhang, D.-E., Kraft, A.S. Phosphorylation of AML1/RUNX1 regulates its degradation and nuclear matrix association. Mol. Cancer Res. 3:391, 2005.
MOLECUL AR AND EXPERIMENTAL MEDICINE
Giannakopoulos, N.V., Luo, J.-K., Papov, V., Zou, W., Lenschow, D.J., Jacobs,
B.S., Borden, E.C., Li, J., Virgin, H.W., Zhang, D.-E. Proteomic identification of
proteins conjugated to ISG15 in mouse and human cells. Biochem. Biophys. Res.
Commun. 336:496, 2005.
Kim, K.I., Malakhova, O.A., Hoebe, K., Yan, M., Beutler, B., Zhang, D.-E.
Enhanced antibacterial potential in UBP43-deficient mice against Salmonella
typhimurium infection by up-regulating type I IFN signaling. J. Immunol. 175:847,
2005.
Kim, K.I., Yan, M., Malakhova, O.A., Luo, J.-K., Shen, M.-F., Zou, W., de la
Torre, J.C., Zhang, D.-E. Ube1L and protein ISGylation are not essential for α/β
interferon signaling. Mol. Cell. Biol. 26:472, 2006.
Kim, K.I., Zhang, D.-E. UBP43, an ISG15-specific deconjugating enzyme: expression, purification, and enzymatic assays. Methods Enzymol. 398:491, 2005.
Peterson, L.F., Boyapati, A., Ranganathan, V., Iwama, A., Tenen, D.G., Tsai, S.,
Zhang, D.-E. The hematopoietic transcription factor AML1 (RUNX1) is negatively
regulated by the cell cycle protein cyclin D3. Mol. Cell. Biol. 25:10205, 2005.
Zou, W., Papov, V., Malakhova, O.A., Kim, K.I., Dao, C.T., Li, J., Zhang, D.-E.
ISG15 modification of ubiquitin E2 Ubc13 disrupts its ability to form thioester
bond with ubiquitin. Biochem. Biophys. Res. Commun. 336:61, 2005.
Zou, W., Zhang, D.-E. The interferon-inducible ubiquitin-protein isopeptide ligase
(E3) EFP also functions as an ISG15 E3 ligase. J. Biol. Chem. 281:3989, 2006.
DIVISION OF RHEUMATOLOGY
RESEARCH
W.M. Keck Autoimmune Disease Center
Joel N. Buxbaum, M.D., Division Head
Pathogenesis of Late-Onset
Genetic Diseases Related
to Abnormalities of
Protein Conformation
J.N. Buxbaum, N. Reixach, Z. Ye, L. Friske, M.J. Saraiva,*
N. Schork,** D. Jacobson,*** G. Gallo,**** C. Tagoe,*****
O. Suhr†
* Institute of Cell and Molecular Biology, Oporto, Portugal
** University of California, San Diego, California
*** Boston University School of Medicine, Boston, Massachusetts
**** NYU School of Medicine, New York, New York
***** Albert Einstein College of Medicine, Bronx, New York
†
Umeå University, Umeå, Sweden
e are studying the pathogenesis of a group of
hereditary human diseases, the transthyretin
amyloidoses, that are the result of protein
misfolding. The misfolded molecules aggregate and are
deposited in the heart, kidney, and peripheral nerves,
W
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279
producing organ-specific disease. We use 3 major
approaches: animals transgenic for the human protein
transthyretin, cell cultures to determine how the misfolded proteins injure their cellular targets, and genetic
epidemiology to identify potential disease carriers and
assess the effects of other hereditary and environmental factors on the disease.
Our studies of the clinical impact of the transthyretin
mutation Val122Ile, an allele carried by 3%–4% of
African Americans, have revealed that 10% of African
Americans more than 60 years old who have severe
heart failure are carriers of the amyloidogenic allele.
These data are consistent with our finding of an ageassociated decrease in the prevalence of the allele in
African Americans, which suggests that the allele has
a discrete mortality effect. Our projections, based on
prevalence and demographic data, indicate that at any
moment as many as 100,000 to 150,000 persons in
the United States may have this form of heart disease.
Population genotyping in various African locales suggests
an origin in West Africa with maintenance of the allele
in the United States via a founder effect in the original
slave population. Our current studies are designed to
precisely define the disease-producing effects of the
allele, which behaves as an autosomal dominant with
age-dependent penetrance, and the development of
signs and symptoms.
In collaboration with D.R. Salomon, Department of
Molecular and Experimental Medicine, we have continued our studies in transthyretin transgenic mice. We
are using microarray techniques to analyze the transcriptional patterns of (1) tissues that are the targets
for transthyretin deposition and (2) the hepatocytes that
synthesize transthyretin. We have identified groups of
genes that are associated with resistance to, or lack
of, deposition and have defined changes in the hepatic
site of transthyretin synthesis that seem to influence
whether or not deposition occurs in distant tissues. Our
current hypothesis is that the quality of the hepatic
response to a misfolded protein determines how much
abnormal conformer with fibril-forming potential gets
into the circulation. These results shed light on the recent
observations in recipients of normally functioning liver
transplants from donors with transthyretin mutations; the
recipients experienced tissue deposition of transthyretin
in a shorter time than was anticipated on the basis of
studies of persons who carry these mutations.
We have extended our tissue culture studies designed
to determine how oligomeric aggregates of transthyretin
280 MOLECUL AR AND EXPERIMENTAL MEDICINE
produce damage to heart and nerve cells. We have now
defined the pathways of transit of transthyretin in cells
that are either sensitive to or resistant to the toxic
effects of aggregated transthyretin and the pathways
taken by toxic and nontoxic proteins.
In collaboration with J. Kelly, Department of Chemistry, and investigators in Boston; Rochester, Minnesota;
London; Umeå, Sweden; Portugal; and Japan, we organized a clinical trial of diflunisal, a small molecule capable of stabilizing the native structure of transthyretin.
The proposal recently received funding from the National
Institutes of Health, and we have begun recruiting
patients for the trial.
In collaboration with J. Waalen, Department of
Molecular and Experimental Medicine, and T. Bartfai,
Molecular and Integrative Neurosciences Department,
we performed an epidemiologic analysis of the prevalence of various forms of arthritis in persons with
extreme obesity (i.e., body mass index >30). We found
that the prevalence of rheumatoid arthritis, but not
osteoarthritis, was reduced in persons who are extremely
obese. However, examination of other data sets indicates that once rheumatoid arthritis develops, it is
more severe in persons who are obese than in persons
who are thinner. These data suggest that adipose tissue may be a significant source of anti-inflammatory
cytokines, sufficient to suppress the initial development
of rheumatoid arthritis in the presence of an inflammatory trigger. Once the inflammatory threshold is
breached, proinflammatory cytokines derived from adipose tissue add to the total level of inflammation.
PUBLICATIONS
Buxbaum, J.N. Meeting report: VIth International Symposium on Familial Amyloidotic Polyneuropathy and Other Transthyretin Disorders and the Vth International
Workshop on Liver Transplantation in Familial Amyloidotic Polyneuropathy, August
24-26, 2005, La Jolla, California, USA. Amyloid, in press.
Buxbaum, J.N. Transthyretin and the transthyretin amyloidoses. In: Protein Misfolding, Aggregation and Conformational Diseases. Uversky, V.N., Fink, A.L. (Eds.).
Springer, New York, in press. Vol. 4 in Protein Reviews. Atassi, M.A. (Series Ed.).
Buxbaum, J.N. Treatment and prevention of the amyloidoses: can the lessons
learned be applied to sporadic inclusion-body myositis? Neurology 66(2 Suppl. 1):
S110, 2006.
Buxbaum, J.N., Jacobson, D.R., Tagoe, C., Alexander, A., Kitzman, D., Greenberg, B., Thaeemit-Chen, S., Lavori, P. Transthyretin V1221 in African Americans
with congestive heart failure. J. Am. Coll. Cardiol. 47:1724, 2006.
2006
THE SCRIPPS RESEARCH INSTITUTE
Oxidative Stress, Protein
Oxidation, and Disease
J.S. Friedman, R. Gabriel, F.M. Martin, J. Yi
e are investigating how loss of superoxide
dismutase 2 (SOD2) affects blood cells.
SOD2 deficiency in murine blood cells results
in an anemia that is similar to sideroblastic anemia
in humans. During the past year, we developed and published a novel method for purification of iron-overloaded
cells from SOD2-deficient mice. This method relies on
magnetic purification of iron-loaded cells (Fig. 1). We
W
F i g . 1 . Purification of SOD2 –/– siderocytes. A, Inset, iron-laden
SOD2 +/+ (left) and SOD2–/– (right) cells purified from red blood
cells (RBCs). Scale bar = 1 cm; CBF, column-bound fractions. Dot
plot shows a significant (>25-fold) enrichment of magnetic ironladen red blood cells purified from SOD2 –/– (n = 22) and SOD2+/+
(n = 18) cell suspensions. ***P < .001 by unpaired 2-tailed t
test.B, Perl stain of SOD2–/– magnetically purified siderocytes; inset
shows deposition of cellular iron. Original magnifications x63 and
x100, respectively.
have adapted the method to purify abnormal cells from
the bone marrow of patients with sideroblastic anemia, an advance that will facilitate both gene expression and proteomic analyses in this disease. Using this
method, we are analyzing protein oxidation and identifying protein components most closely associated with
magnetically susceptible iron, that is, biological iron
that is attracted to a magnet.
MOLECUL AR AND EXPERIMENTAL MEDICINE
One of our initial findings is that iron-positive subcellular fractions from SOD2-deficient cells or cells from
patients with sideroblastic anemia are enriched in mitochondria. We will use the magnetic purification method
to characterize mitochondria from patients with sideroblastic anemia for functional defects or mutations in
mitochondrial DNA that may play a role in pathogenesis
of the anemia or the related disorder myelodysplasia.
Because protein turnover is slow or absent in mature
red cells, SOD2-deficient cells and iron-loaded cells in
patients with sideroblastic anemia accumulate oxidatively
damaged protein. Using SOD2-deficient cells, we developed 2 novel methods for enriching and identifying oxidized proteins that can be used in 2-dimensional gel
electrophoresis. Use of these methods will allow more
detailed comparison of changes in oxidized proteins
that may accompany aging, inflammatory processes,
and neurodegenerative disease.
The first method involves the use of multiple fluorophores (e.g., Cy-2, Cy-3, Cy-5) that can form derivatives
of carbonylated proteins by using a hydrazide moiety.
Individual samples are labeled with distinct fluorophores
and then are combined for comparative 2-dimensional
gel analysis. The second method involves the use of a
biotin “hook” to obtain oxidized proteins from more complex protein mixtures. Using these techniques, we can
enrich, identify, and quantitatively compare oxidized
proteins in experimental samples. We think that these
techniques will be useful in probing the role of protein
oxidation in signal transduction and will help identify
specific oxidation-sensitive proteins important in the
pathogenesis of sideroblastic anemia and perhaps, more
generally, targets of oxidation in age-related degenerative disease.
PUBLICATIONS
Martin, F.M., Bydlon, G., Welsh, M.L., Friedman, J.S. A method for rapid mouse
siderocyte enrichment. Exp. Hematol. 33:1493, 2005.
Martin, F.M., Friedman, J.S. SOD2 deficiency anemia and RBC oxidative stress.
Antioxid. Redox Signal., in press.
Transcriptional Gene Silencing
by Small Interfering RNA
K.V. Morris, J. Han, J.P. Delacruz
R
NA interference via small interfering RNAs
(siRNAs) is a new experimental method for
knocking out (i.e., inactivating or suppressing)
2006
THE SCRIPPS RESEARCH INSTITUTE
281
genes. We wish to understand the mechanism of
siRNA-mediated transcriptional gene silencing (TGS)
in human cells and to use conditionally replicating lentiviral vectors to apply RNA interference to treat infection
with HIV type 1.
Currently, most investigators use siRNAs to target
and inactivate a particular gene transcript in a procedure termed posttranscriptional gene silencing. In
human cells, as in plants and the fission yeast Saccharomyces pombe, siRNAs mediate TGS by targeting the
gene promoter. The observation that siRNAs targeted
to a gene’s promoter can specifically silence that gene
were exciting, but the fundamental mechanism of this
silencing remained unknown. During the past year, we
have uncovered many of the mechanistic interactions
involved in siRNA-mediated TGS in human cells.
We found that siRNAs targeted to promoter regions
can cause silent modifications in chromatin, such as
methylation of histone 3, lysine 9, and lysine 27. We
also discovered that DNA methyltransferase 3A is
involved in a putative transcriptional silencing complex
that is directed to the targeted promoter by the antisense
strand of the siRNA. Moreover, the protein Argonaute 1
appears to be involved, possibly in unwinding the siRNAs
and presenting the antisense strand to the complex.
Although much has been learned about the putative
transcriptional silencing complex in human cells, it has
remained unclear whether siRNAs, specifically the
antisense strand of the siRNA, can recognize and bind
directly to DNA or to an uncharacterized RNA that overlaps the targeted promoter.
We now have direct evidence that a low-copy RNA
is transcribed through RNA polymerase II promoters for
6 different genes. These promoter-specific RNAs are
initially detected by the antisense strand of promoterdirected siRNA, and their expression is reduced along
with that of the corresponding promoter-expressed
mRNA. Additionally, when antisense phosphorothioate
oligodeoxynucleotides are used to block the siRNA target site in the promoter-specific RNA, siRNA-mediated
TGS is abrogated. These data suggest that low levels
of promoter-specific RNAs are present in RNA polymerase II promoters that act either in trans (Fig. 1A)
during transcription or in cis (Fig. 1B) as a local scaffolding recognition motif for the antisense strand of the
siRNA to bind the targeted promoter and mediate TGS.
PUBLICATIONS
Kawasaki, H., Taira, K., Morris, K.V. siRNA induced transcriptional gene silencing
in mammalian cells. Cell Cycle 4:442, 2005.
282 MOLECUL AR AND EXPERIMENTAL MEDICINE
2006
THE SCRIPPS RESEARCH INSTITUTE
Autoimmunity Induced by
Xenobiotics
K.M. Pollard, D. Cauvi, G. Cauvi
e focus on how interactions between the
environment and genetics affect induction of
autoimmune diseases. We use murine models
of systemic autoimmunity in which disease is elicited
by exposure to xenobiotics. An important aspect of our
research is a comparison of the similarities and differences between induced systemic autoimmunity and
idiopathic systemic autoimmunity, such as systemic
lupus erythematosus, in mice and humans.
W
F i g . 1 . Two models for siRNA-mediated TGS in human cells. A,
In the trans model, the promoter-specific RNA (pRNA) is recognized by
the antisense strand of the siRNA during RNA polymerase II
(RNAPII)–mediated transcription of the siRNA targeted promoter. The
antisense strand of the siRNA guides a putative transcriptional silenc-
ing complex (possibly composed of DNMT3A, Ago-1, HDAC-1,
and/or EZH2) to the targeted promoter where histone modifications
leading to initial gene silencing would occur. B, In the cis model, a
pRNA acts as a scaffolding by overlapping the entire targeted promoter, possibly as part of the local chromatin structure. The pRNA
then acts as a scaffolding for the antisense strand of the siRNA to
bind the targeted promoter along with the putative transcriptional
silencing complex or to recruit the complex.
Morris, K., Castanotto, D., Al-Kadhimi, Z., Jensen, M., Rossi, J.J., Cooper, L.J.N.
Enhancing siRNA effects in T cells for adoptive immunotherapy. Hematology
10:461, 2005.
Morris, K.V. siRNA-mediated transcriptional gene silencing: the potential mechanism and a possible role in the histone code. Cell Mol. Life Sci. 62:3057, 2005.
D O W N R E G U L AT I O N O F D E C AY - A C C E L E R AT I N G FA C T O R A N D A C T I VAT I O N O F C D 4 + T C E L L S I N
INDUCED SYSTEMIC AUTOIMMUNE DISEASE
Decay-accelerating factor (DAF/CD55) is a regulatory protein that protects cells from attack by autologous
complement proteins. DAF deficiency exacerbates autoimmunity, most likely by acting as a regulator of T-cell
immunity. Therefore, modulation of DAF expression on
T cells may contribute to the development of autoimmunity. To test this idea, we examined DAF expression
in murine mercury-induced autoimmunity.
In B10.S mice, which are susceptible to mercuryinduced autoimmunity, exposure to mercury resulted
in reduced expression of DAF mRNA (Fig. 1). Expression of the protein was reduced on CD4 + T cells, particularly those with an activated/memory phenotype
Morris, K.V. Therapeutic potential of siRNA-mediated transcriptional gene silencing. Biotechniques 40(Suppl.):S7, 2006.
Morris, K.V. VIR-496(VIRxSYS). Curr. Opin. Investig. Drugs 6:209, 2005.
Morris, K.V., Looney, D.J. Characterization of human immunodeficiency virus
(HIV)-2 vector mobilization by HIV-1. Hum. Gene Ther. 16:1463, 2005.
Morris, K.V., Rossi, J.J. Antiviral applications of RNAi. Curr. Opin. Mol. Ther.
8:115, 2006.
Morris, K.V., Rossi, J.J. Lentivirus-mediated RNA interference therapy for human
immunodeficiency virus type 1 infection. Hum. Gene Ther. 17:479, 2006.
Weinberg, M.S., Villeneuve, L.M., Ehsani, A., Amarzguioui, M., Aagaard, L.,
Chen, Z.X., Riggs, A.D., Rossi, J.J., Morris, K.V. The antisense strand of small
interfering RNAs directs histone methylation and transcriptional gene silencing in
human cells. RNA 12:256, 2006.
F i g . 1 . Exposure to mercury reduced the expression of DAF1
mRNA in the spleens of B10.S mice, which are susceptible to mercury-induced autoimmunity. DBA/2, B10.S, and NZB mice were
injected with mercury (filled bar) or phosphate-buffered saline
(open bar) twice a week for 4 weeks. Total RNA isolated from
spleens was analyzed for DAF1 mRNA. DAF1 levels are expressed
relative to cyclophilin A. Data are expressed as mean ± SEM, with
n = 4–5 mice per group. *P < .05.
MOLECUL AR AND EXPERIMENTAL MEDICINE
(CD44 hi) that accumulate as a result of exposure to
mercury. In contrast, DBA/2 and INF-γ–deficient B10.S
mice, which are resistant to mercury-induced autoimmunity, had no increase in the number of activated/
memory CD4 + T cells and no change in DAF expression after exposure to the metal. These findings suggest that development of autoimmunity is linked to a
reduction in DAF expression on an expanded or longlived population of activated/memory CD4+ T cells.
CONTROL OF CONSTITUTIVE EXPRESSION OF DAF
BY SP1
Modulation of murine DAF expression during the
development of autoimmunity suggests that DAF contributes to CD4+ T-cell activity, and this idea is supported by the hyperactivity of T cells in DAF-deficient
mice, as reported by others. Modulation of DAF expression could therefore be a critical regulatory mechanism
in both innate and adaptive immune responses. To
identify and characterize key transcriptional regulatory
elements that control DAF expression in mice, we cloned
a 2.5-kb fragment corresponding to the 5′ flanking region
of Daf1, the mouse gene for DAF.
Sequence analysis showed that the mouse Daf1
promoter lacks conventional TATA and CCAAT boxes
and has a high guanine-cytosine content. Rapid amplification of cDNA ends was used to identify 1 major and
2 minor transcription start sites 47, 20, and 17 bp
upstream of the translational codon. Positive and negative regulatory regions were identified by transiently
transfecting sequential 5′ deletion constructs of the 5′
flanking region into NIH/3T3, M12.4, and RAW264.7
cells. Mutational analyses of the promoter region combined with an enzyme-linked immunosorbent assay
specific for the transcription factor Sp1 indicated that
Sp1 is required for basal transcription and lipopolysaccharide-induced expression of Daf1. These findings
provide new information on the regulation of the mouse
Daf1 promoter and will facilitate further studies on
the expression of Daf1 during immune responses.
PUBLICATIONS
Cauvi, D.M., Cauvi, G., Pollard, K.M. Constitutive expression of murine decayaccelerating factor 1 (DAF1) is controlled by the transcription factor Sp1. J.
Immunol., in press.
2006
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283
Pollard, K.M., Hultman, P. Fibrillarin autoantibodies. In: Textbook of Autoantibodies.
Shoenfeld, Y., Meroni, P.L., Gershwin, M.E. (Eds.). Elsevier, Philadelphia, in press.
Expression and Function of the
High-Affinity Receptor for IgE
M.W. Robertson, Z. Wang, X. Tian
he high-affinity receptor for IgE (FcεRI) is a multisubunit membrane complex that is critically
involved in the pathology of the allergic response.
The receptor is highly expressed by mast cells and
basophils. Upon stimulation by IgE-antigen complexes,
these cells secrete histamine and other mediators of
hypersensitivity, leading to the clinical signs and symptoms of allergy. Our main focus is defining the molecular basis of FcεRI assembly and expression and the role
of FcεRI structure in initiating or propagating IgE-mediated cellular activation.
T
F C ε R I A S S E M B LY A N D T R A N S P O R T
One of our goals is to understand how FcεRI αγ2 and
αβγ2 isoforms assemble and traffic in cells. Recently, we
assessed the structural basis of transport of the α-chain
of the FcεRI from the endoplasmic reticulum. We found
that a previously defined endoplasmic reticulum retention signal located near the C terminus of the α-chain is
only weakly functional in regulating steady-state transport of the receptor. We also found that a membraneproximal dilysine sequence in the cytoplasmic domain of
the α-chain regulates transport, and we determined that
the new motif functions synergistically with the C-terminal retention signal to stringently retain the FcεRI subunit in the endoplasmic reticulum.
In another study, we investigated the structural basis
of the assembly of the αγ subunit, a process previously
thought to occur exclusively through interaction of the
subunit transmembrane domains. Our data revealed
that the cytoplasmic domain determinants of each
subunit contribute significantly to optimal αγ association and to FcεRI-dependent function in transfected
mast cells.
I N H I B I T I O N O F F C εR I - M E D I AT E D C E L L U L A R
A C T I VAT I O N B Y A M O N O C L O N A L A N T I B O D Y T O
Hultman, P., Taylor, A., Yang, J.M., Pollard, K.M. The effect of xenobiotic exposure
on spontaneous autoimmunity in (SWR x SJL)F1 hybrid mice. J. Toxicol. Environ.
Health A 69:505, 2006.
Lynes, M.A., Fontenot, A.P., Lawrence, D.A., Rosenspire, A.J., Pollard, K.M.
Gene expression influences on metal immunomodulation. Toxicol. Appl. Pharmacol.
210:9, 2006.
Pollard, K.M. (Ed.). Autoantibodies and Autoimmunity: Molecular Mechanisms in
Health and Disease. Wiley-VCH, New York, 2006.
THE FCεRI α-CHAIN
Previously, we showed that 5H5F8, a monoclonal
antibody to the α-chain of FcεRI, inhibits IgE-dependent activation in mast cells and basophils by a unique
mechanism that does not involve perturbation of the
IgE-binding site. The 5H5F8 epitope has been mapped
284 MOLECUL AR AND EXPERIMENTAL MEDICINE
to the linear membrane proximal region, and the epitope assignment has now been confirmed from the
crystallographic structure of a complex formed between
5H5F8 and a synthetic peptide representing the 5H5F8
epitope. We hypothesized that the membrane proximal
region of the FcεRI α-chain may be critically important
in initiating or propagating FcεRI-dependent signaling.
In recent mutagenesis studies, we found that replacing
the native membrane proximal region with a series of
different sequences significantly enhanced cell-surface
expression of the receptor but also resulted in loss of
FcεRI-dependent function. On the basis of these findings, we propose that the membrane proximal region
is a multifunctional site that plays a critical role in both
FcεRI cell transport and transmembrane signaling.
PUBLICATIONS
Cauvi, D.M., Tian, X., von Loehneysen, K., Robertson, M.W. Transport of the IgE
receptor α-chain is controlled by a multicomponent intracellular retention signal. J.
Biol. Chem. 281:10448, 2006.
2006
THE SCRIPPS RESEARCH INSTITUTE