CHARACTERIZATION OF THE STRUCTURE OF THE L178H MUTANT OF
APOLIPOPROTEIN A-I
Megan Jessemyn Schifer Cochran
B.A., Denison University, 2005
THESIS
Submitted in partial satisfaction of
the requirements for the degree of
MASTER OF SCIENCE
in
CHEMISTRY
(Biochemistry)
at
CALIFORNIA STATE UNIVERSITY, SACRAMENTO
SUMMER
2012
© 2012
Megan Jessemyn Schifer Cochran
ALL RIGHTS RESERVED
ii
CHARACTERIZATION OF THE STRUCTURE OF THE L178H MUTANT OF
APOLIPOPROTEIN A-I
A Thesis
by
Megan Jessemyn Schifer Cochran
Approved by:
__________________________________, Committee Chair
Linda M. Roberts
__________________________________, Second Reader
Thomas J. Savage
__________________________________, Third Reader
Roy W. Dixon
____________________________
Date
iii
Student: Megan Jessemyn Schifer Cochran
I certify that this student has met the requirements for format contained in the
University format manual, and that this thesis is suitable for shelving in the Library
and credit is to be awarded for the thesis.
___________________________, Graduate Coordinator
Susan Crawford
Department of Chemistry
iv
___________________
Date
Abstract
of
CHARACTERIZATION OF THE STRUCTURE OF THE L178H MUTANT OF
APOLIPOPROTEIN A-I
by
Megan Jessemyn Schifer Cochran
The top two leading causes of death worldwide in 2004 (stroke and ischemic
heart attack) are both cardiovascular diseases (CVD). High-density lipoprotein and its
major protein apolipoprotein A-I (apoA-I, 243 residues) are each inversely associated
with risk of CVD and therefore believed to have cardio-protective properties.
However, fibrillar apoA-I has been found in arterial plaque, indicating that
fibrillogenesis of apoA-I may actually contribute to the development of CVD.
Fourteen fibril forming variants of apoA-I are known whose mutations cluster in two
regions of the protein's N-terminal helical domain, the "inside" cluster (residues 26107) and the "outside" cluster (residues 154-178). A mutant from the "inside" cluster,
G26R, was previously characterized by members of our research group (Lagerstedt,
J.O., Cavigiolio, G., Roberts, L.M., Hong, H.S., Jin, L.W., Fitzgerald, P.G., Oda,
M.N., Voss, J.C. 2007. Biochemistry 46, 9693-9699). The G26R protein was found
to exhibit an increase in beta-strand structure, N-terminal protease sensitivity and
hydrophobic surface area compared to wild-type apoA-I. We proposed that the
v
alteration at position 26 disrupts the N-terminal helical bundle, destabilizing it and
promoting the formation of misfolded fibrils. To determine whether mutations in the
"outside" cluster produce similar effects on the protein, and therefore similar
misfolded fibrils, the structure of the full-length L178H variant was studied using
ANS binding, limited proteolysis, and intrinsic fluorescence quenching. In this thesis
work, it is shown that the L178H mutation results in increased N-terminal protease
sensitivity and hydrophobic surface area, but with no change in the nonpolar
environment of the protein's tryptophan residues (Petrlova, J., Duong, T., Cochran, M.,
Axelsson, A., Morgelin, M., Roberts, L.M., Lagerstedt, J.O. 2012 J. Lipid Res. 53,
390-398). Thus, mutations from the two clusters result in similar structural effects on
the protein. ANS binding was also used to analyze conformational changes over time
in protein incubated at either 4oC or 37oC. L178H exhibited earlier and more
substantial changes in hydrophobic surface area at both temperatures compared to
wild-type apoA-I, which likely reflects the increased propensity of L178H to form
fibrils. The effects of the L178H mutation are reviewed in the context of recent
structural models of apoA-I.
, Committee Chair
Linda M. Roberts
______________________
Date
vi
ACKNOWLEDGMENTS
First and foremost, I would like to thank my husband for his constant support and
encouragement. Whenever I was discouraged, you were there to support me. Thank
you for the many Saturday and Sunday lunches in the Union and the many weekend
nights spent at home, so I could get up early the next morning. I know you had to
sacrifice for me, and you never complained, instead always being my biggest
supporter. Thank you.
If not for Linda, I wouldn’t be here today. You let me work in your lab on the
weekends to accommodate my schedule and put up with many calls and questions
while at home with your family. You have been patient with my questions, flexible
with my projects, and helpful with my thesis, especially from across the country.
Thank you.
I would like to thank my parents for not protesting when I wanted to move across the
country for my Masters and my marriage. Both were worth it. Thank you.
To Aunt Sara, for sparking my interest in science in the first place, thank you. Who
knows what I would be today if not for you and your class? I’ll never forget that,
“Chemistry is everywhere.” Thank you.
To the other students in Dr. Roberts’ lab over the years, and there have been too many
of you to name, thank you. For being there to talk, to vent, to discuss problems, to
babysit me on the weekends, and to curse protein, it’s been quite an interesting seven
years. Thank you.
vii
TABLE OF CONTENTS
Page
Acknowledgments ...................................................................................................... vii
List of Tables ............................................................................................................... ix
List of Figures............................................................................................................... x
INTRODUCTION…. ................................................................................................... 1
MATERIALS AND METHODS ............................................................................... 38
RESULTS. .................................................................................................................. 46
DISCUSSION............................................................................................................. 65
CONCLUSION .......................................................................................................... 75
References ................................................................................................................. 77
viii
vii
i
LIST OF TABLES
Tables
Page
1.
Comparison of models of the lipid-free tertiary structure of apo A-I…..….....15
2.
Summary of the apo A-I mutants found to date..………………………...…...34
3.
Comparison of proteolytic cleavage fragment sequences ………………...….54
4.
Comparison of estimated t ½ of ANS binding at various temperatures….........59
5.
Comparison of Stern-Volmer constant, Ksv, of protein at various
temperatures ……..……………………………………….………..................64
6.
Comparison of relative fluorescence intensities of apo A-I proteins at
485 nm.……...…………………..…………………………………….............66
ix
LIST OF FIGURES
Figures
Page
1.
The arterial overview of the development of atherosclerosis ………………... 5
2.
Mechanism of reverse cholesterol transport compared to forward transport…..8
3.
The role of apo A-I in reverse cholesterol transport ……………………….….9
4.
Structural organization of apolipoprotein A-I gene .…………………………11
5.
Comparison of secondary structures of lipid-free apo A-I determined
using HD-X and EPR…...............................…….………………………..…..14
6.
Proposed model for lipid-free apo A-I ……….....……………………………17
7.
Ribbon model of the first full length all-atom model of lipid-free apo A-I…..18
8.
Comparison of apo A-I structure determined using EPR of the C-terminal
domain and the full length structure…………………................………….....20
9.
The crystal structure of 1-184 apo A-I ………...……………………………..21
10.
Stereoviews of 1-43 apo A-I as a monomer, dimer, and tetramer……….….24
11.
Models of lipid-bound apo A-I on discoidal HDL …………………………...26
12.
The initial lipid-binding step of apo A-I mediated through helix 9 and 10 .... 27
13.
Generic -sheet amyloid fibril structure, picture of actual -sheet amyloid
fibrils, and a model of an-helical fibril supported by an electron
micrograph .......................................................................................................30
14.
Mechanisms of amyloid fibril formation……………………………………..31
15.
Dimeric structure of apo A-I based on the apo A-I 1-43 structure……….... 35
16.
L178H pilot expression ………………………………………………………47
17.
Metal-ion affinity chromatography of L178H apo A-I ……………………....48
18.
Analysis of proteolytic cleavage upon extended incubation…...……………. 50
19.
Limited proteolysis of WT and L178H apo A-I ……...…………………...... 53
20.
ANS binding to WT and L178H apo A-I...…………………………………..57
21.
Percent loss of ANS binding at 485 nm in L178H and WT over 28 days…....58
22.
Percent loss of intrinsic fluorescence of apo A-I proteins………...………….61
23.
Stern-Volmer plot of intrinsic fluorescence quenching of L178H apo A-I at
day 2 ………………………………………………………………………….63
x
24.
Representation of chymotryptic hot spots in wild type, G26R, L178H apo A-I,
and N-terminal deletion mutants………………………………….….......…...68
25.
Position of amyloidogenic mutations in structural models of apo A-I……….70
26.
Summary of important changes in WT and L178H apo A-I at 4ºC and 37ºC
over 28 days because of helix ………………………………………………..74
xi
1
INTRODUCTION
According to the World Health Organization (WHO), the top two leading causes
of death worldwide in 2004 were two different cardiovascular diseases (CVD), ischemic
heart disease and cerebrovascular disease, which together accounted for 19.9% of all
deaths (145). That percentage is projected to increase to 26.3 % of all deaths worldwide
from CVD in the year 2030. Another CVD, hypertensive heart disease, is projected to
increase from 14th leading cause of death to the seventh. All CVDs together, including
rheumatic heart disease, coronary artery disease (CAD), and others, caused 30.5 % of
deaths worldwide in 2008 according to WHO and 34.2 % of deaths in the United States
that same year (146). CVD is a leading target of medical and pharmaceutical
intervention, and understanding the mechanisms and risk factors underlying CVD is
critical in developing effective prevention and treatment.
Lipoproteins have been used for decades to predict risk of CVD. Lipoproteins
are molecules consisting of a layer of protein surrounding a lipid core that carry
cholesterol and lipids to the liver and other body tissues. Lipoproteins vary by size,
shape, protein content and density, and each is associated with CVD in a different way.
The two most commonly known lipoproteins are high density lipoprotein (HDL, or
"good" cholesterol) and low density lipoprotein (LDL, or "bad" cholesterol). The
beneficial effects of HDL are well documented. For example, a 2-3% decreased risk of
CAD is associated with an increased concentration of only 1 mg/dL blood plasma highdensity lipoprotein (HDL) (normal HDL blood level is > 40 mg/dL) (3). Conversely,
2
increased levels of low-density lipoprotein (LDL) are associated with an increased risk
of CVD (3). An early survey known as the Framingham study showed that only a weak
association exists for increased LDL levels and increased risk for CAD (122). Despite
that, for over thirty years, levels of LDL and total cholesterol (TC) have been used to
predict risk of CVD (135). In the Framinghan study, it was shown that low HDL levels
are a major risk factor for each main manifestation of CAD, such as angina pectoris
(chest pain), acute myocardial infarction (heart attack), and heart failure. Overall, HDL
levels had a significant inverse correlation association with heart disease when other
standard risk factors were taken into consideration (122).
Roughly 70% of the protein in HDL is comprised by apolipoprotein A-I (apo AI), a flexible protein that is part of the apolipoprotein family, which includes many
related proteins such as apo A, apo B, apo C, apo D, apo E, apo H, and apo M (1, 2, 84,
174). Within the past few years, it has been discovered that apolipoprotein A-I levels are
as accurate as HDL levels when used for prediction of cardiac events (4, 5). It has been
suggested that apo A-I levels are a better predictor of cardiovascular events than LDL
levels (4). And more recently, the ratio of apo B/apo A-I was suggested to be a better
indicator of the risk of CVD than total cholesterol/HDL which has been used by
physicians for decades (189). Apo A-I's significance is reflected in the high prevalence
of premature CAD exhibited in people with a mutation in the apo A-I gene that leads to
severe HDL deficiency (79).
3
Apart from gender and age, the main predictor of CAD in a group of low-risk
patients studied in 2001 was blood serum apo A-I level which is known to vary with age
in females, but not males (91). In the INTERHEART study, it was determined that the
strongest risk factors of heart attack were current smoking and apo B/apo A-I ratio and
these results are the same for all ages, sexes, and regions of the world (93). To further
underscore the importance of blood apo A-I levels, premature atherosclerosis, an
inflammatory disease of the arteries, is found in the majority of patients with severe
HDL and apo A-I deficiencies; it has been identified in people as young as 30 years old
(3, 79) In fact, it may be initiated by low levels of apo A-I in otherwise low-risk patients
(91).
Atherosclerosis
The main cause of CAD is atherosclerosis which is often referred to as
hardening of the arteries. It is an inflammatory disease of the arteries which can lead to
plaque formation (8, 104). Unstable plaques can cause narrowing of the arteries
(stenosis), restriction in blood supply (ischemia), or a complete blockage (infarction)
(81, 104). As atherosclerosis progresses, atherosclerotic lesions (atheroma) form. The
initiation, propagation, and activation of these lesions in atherosclerosis occur largely
because of immune mechanisms, in conjunction with metabolic risk factors (104). In
fact, the process of atherosclerosis can be characterized by different types of arterial
lesions that represent different stages in the inflammatory response. Such lesions may
be present anytime in a person’s life with the earliest type of lesion, the purely
4
inflammatory fatty streak, sometimes occurring in infancy and childhood (81). These
streaks can completely disappear, especially when occurring in childhood, or progress to
form full-on atherosclerotic lesions (104).
As with any other inflammatory response, an “injury” or other event must cause
atherosclerosis. It is hypothesized that the “injury” for atherosclerosis could be elevated
or modified LDL levels, free radicals caused by smoking, stress, or diabetes, genetic
modifications, infectious microorganisms, or other unknown factors (81, 112, 123).
Once the arterial injury occurs, the body tries to compensate to restore equilibrium in
the arterial endothelium. These responses include increased endothelial permeability and
adhesiveness, particularly to leukocytes and platelets, increased procoagulant properties
in the endothelium, and formation of vasoactive molecules, inflammatory cytokines, and
growth factors (81, 104). If this response is successful, the process can be reversed and
the “injury” can disappear, as in the case of fatty streaks in children. However, if the
response is unsuccessful, by not neutralizing or removing what caused the injury in the
first place, the response itself can continue indefinitely (81).
Continued inflammation then results in up-regulation of leukocyte and
endothelial adhesion molecules, which can stimulate recruitment and adherence of
monocytes and T cells at lesion-prone sites in the endothelium, such as the vascular side
of the aortic valve, which is a critical part in the process of plaque formation (123).
Then, as they are recruited, lipid-associated monocytes and macrophages (foam cells)
together with T-lymphocytes form fatty streaks, the precursor to atherosclerotic lesions
5
(87, 57, 104). Next smooth muscle cells migrate and proliferate, then mixing with
inflamed regions to form intermediate lesions. These lesions can then thicken the artery
wall, for which it compensates by dilating the artery, called remodeling (177) (Figure 1).
Figure 1. The arterial overview of the development of atherosclerosis (177).
With cell accumulation and lesion progression, fibrous tissues tend to cover the
lesion with a fibrous cap for protection of the lumen which may then form a necrotic
core as a result of apoptosis and necrosis. The lesions continue to expand and eventually
the artery cannot dilate anymore and the pathway ends up being blocked, altering the
blood flow. At sites covering the lesion, the fibrous cap can rupture enabling the blood
coming in contact with the plaque to coagulate. Platelets activated as a result of this
6
coagulation then lead to thrombus (blood clot) formation (81) (Figure 1). If the
thrombus blocks a vessel, an acute myocardial infarction can occur, or alternately, the
thrombus could also be reabsorbed. The healing response triggered by thrombosis can
again stimulate smooth muscle migration, proliferation and extracellular matrix
synthesis resulting in an even thicker fibrous cap (Figure 1). These thicker plaques may
then be less susceptible to rupture and thrombosis, but instead can cause severe chest
pain related to stenosis and ischemia.
Apo A-I in Atherosclerosis
Apo A-I influences development of atherosclerosis in many ways. First, it acts as
an anti-inflammatory agent by preventing monocyte adherence to endothelial cells and
their subsequent activation, which can prevent the entire process of atherosclerosis (10,
11). It can also stabilize antiatherogenic antioxidants like human paraoxonase/
arylesterase (9). By inhibiting procoagulant activity in erythrocytes, apo A-I protects
against blood clots (12). Apo A-I can also bind and protect against lipopolysaccharides
(LPS or endotoxin) by preventing an LPS-bound complex from binding a receptor on
monocytes/ macrophages and thereby preventing initiation of the cytokine release found
in atherosclerosis (52). This binding can also prevent the endotoxin-caused acceleration
of atherosclerosis (179).
Reverse Cholesterol Transport
One of the most studied anti-atherogenic properties of apo A-I is reverse
cholesterol transport (RCT) in which apo A-I promotes active transfer of excess
7
cholesterol from peripheral cells, particularly macrophage foam cells, to HDL and then
to the liver for catabolism (13,14, 139) (Figure 2). Other than the obvious importance
RCT plays in preventing atherosclerosis by cholesterol removal, it may also relate to
inflammation. Recently, inflammation has been shown to retard and impair (sometimes
by selective attenuation) RCT in vivo at multiple places along the pathway which has
led to the possibility that impaired RCT may be a link between low grade inflammation
and atherosclerosis (185). The reverse cholesterol transport pathway proceeds in four
steps: cholesterol efflux from cells in peripheral tissues, lecithin: cholesterol acyl
transferase (LCAT) -mediated esterification of HDL-associated cholesterol, receptormediated transfer of the ester to the liver, and clearance from the body in the liver for
secretion in bile and feces (14, 185).
8
Figure 2. Mechanism of reverse cholesterol transport compared to forward
transport. FC, free cholesterol; CE, cholesteryl ester; VLDL, very low density
lipoprotein; HDL, high density lipoprotein. Arrows indicate the direction of net
transport (14).
Apo A-I plays a critical role in RCT (Figure 3). First, lipid-free apo A-I
stimulates efflux of phospholipids (PL) and cholesterol in macrophages and other nonhepatic tissues by binding ATP binding cassette A1 (ABCA1, also called CERP)
transport protein (67, 174). Apo A-I’s amphipathic α-helices bind to a hydrophobic site
on ABCA1 thereby forming a time, temperature, and concentration dependent complex
with it (98, 155). To form the complex, a helical segment of the protein inserts into an
adjacent region of the plasma membrane bilayer, composed of free cholesterol (FC) and
PL in a perturbed lipid domain created by ABCA1 activity (67, 98, 155). The initial
9
lipidation of apo-A-I, primarily by plasma membrane PL, leads to the formation of
"lipid-poor” apo A-I (14, 97, 98, 155, 174).
Figure 3. The role of apo A-I (labeled as A-I) in reverse cholesterol transport (165).
CE, cholesteryl ester. FC, free cholesterol.
Continued acquisition of lipid converts “lipid poor” apo A-I to discoidal (also
called nascent) HDL. While the structure of synthetic discoidal HDL has been
thoroughly investigated (described below), the structure of lipid-poor apo A-I is not
completely understood. FC in discoidal HDL is converted to cholesteryl esters (CE) and
lyso-phosophatidylcholines by LCAT on the surface of HDL by a transesterification
reaction involving an Asp-His-Ser catalytic triad in the active site of LCAT (14, 65).
The esterification results in migration of CEs into the particle interior which leads to
formation of large spherical HDL particles. Spherical HDL then transports the
cholesterol to the liver (13-15) where apo A-I (as part of HDL) interacts with scavengerreceptor BI (SR-BI), causing the liver to uptake the cholesterol into cells (16, 110).
Much of the cholesterol is then converted to bile acid and excreted.
10
HDL and apo A-I are prime candidates for further study to be used in CVD
prevention and treatment. Because apo-A-I comprises most of the protein in HDL,
elucidating its structure is essential in determining HDL function. However, progress
toward understanding HDL and its function in preventing CVD has been slow due to a
lack of high resolution structural and structure-related functional information for both
HDL and its major protein, apo A-I.
Apolipoprotein A-I Structure
Exon Structure
The apo A-I gene resides on the long arm of chromosome 11 in a multi-gene
cluster of about 22 kilobases with apo C-III, apo A-IV, and apo A-V (30, 31, 50). The
gene structure of apo A-I is composed of four exons separated by three introns,
removed during splicing, similar to most other human apolipoproteins (Figure 4A).
The first exon is untranslated. The latter three-fourths of Exon 2 codes for signal
peptides. Exon 3 codes for signal peptides, the prosegment which is cleaved before
becoming mature apo A-I, and amino acids 1-43 in the mature apo A-I. Exon 4 codes
for amino acids 44-243 in apo A-I (1).
11
Figure 4. A. Structural organization of apolipoprotein A-I gene. Boxes represent
exons. Open bars represent untranslated regions. Shaded bars represent the signal
sequence region. Checked region represents the prosegment. Solid bars represent
regions coding mature protein. Horizontal lines represent introns. The numbers below
represent the sizes of introns or exons in number of nucleotides. Drawing is not to
scale (50, 61). B. Placement of amphipathic helices of apo A-I according to analysis by
SAD. Exon 3 (1-43) is labeled G* and Exon 4 helices (44-243) are labeled H1 through
H10 (212).
Lipid-free protein structure
Primary and secondary structure
Although lipid-free apo A-I constitutes less than 10% of plasma apo A-I, it has
received intense study because of its importance in reverse cholesterol transport. It is
important to understand the lipid-free structure because this form initiates lipidbinding, may interact with cellular receptors such as ABCAI, and is likely to be the
form from which amyloid structure develops. Apo A-I is synthesized in the liver and
small intestine in the form of preproapo A-I which has 267 residues (50, 119, 120,
125). The first eighteen amino acids form a hydrophobic signal sequence peptide
which undergoes co-translational cleavage by signal peptidase of the endoplasmic
reticulum to yield the 249-residue proapo A-I (119, 125). The remaining six amino
acids are cleaved extra-cellularly by a protease to generate mature plasma apo A-I
12
(119, 120, 125). The amino acid sequence of mature human apo A-I contains only 18
of the 20 different amino acids (127), lacking Ile and Cys.
Apo A-I (as well as many other apolipoproteins) contains a series of highly
homologous repeating amphipathic helices; an amphipathic helix contains polar and
nonpolar faces oriented opposite each other along the helix’s long axis (151). The apo
A-I sequence was analyzed using computer programs developed to identify different
classes of amphipathic helices by the Segrest group (73). These studies identified
within residues 44-243 ten contiguous 11- and 22- residue amphipathic helices (which
are often numbered 1-10), punctuated by proline residues similar to other
apolipoproteins (Figure 4B) (1, 147, 152). Helices 3 and 9 are comprised of 11
residues while the other eight are 22-residues long (147).
While sequence analysis has been helpful in understanding the role of the
amphipathic helix in apo A-I structure and function, the protein’s dynamic nature has
made it difficult to obtain definitive structural information for the lipid-free protein.
This is evidenced by the conflicting structural details emerging from a variety of
methods. The structure of lipid-free apo A-I has been studied by limited proteolysis,
circular dichroism (CD), X-ray crystallography, electron paramagnetic resonance
spectroscopy (EPR), Forster resonance energy transfer (FRET) studies, hydrogen
deuterium exchange (HD-X), nuclear magnetic resonance (NMR), chemical cross
linking, and mass spectrometry (2, 33, 36, 39, 70, 149, 193, 212, 224). In spite of
intensive study using these methods, it has been difficult to firmly establish either the
13
secondary or the tertiary structure of the lipid-free protein. For example, while CD
measurements consistently indicate lipid-free apo A-I has a helical content of 50-60 %,
the placement of helices has not been conclusively established (33, 41, 70).
Furthermore, the existence of a small but significant amount of beta structure has been
proposed by some (36, 39, 224) but disputed by others (193). By CD, about 8% of the
protein has beta structure which could be important in the development of amyloid
structure in some variants and under some conditions (33, 36, 39, 40, 41, 70, 224).
One of the more recent and promising techniques for measuring the secondary
structure of lipid-free apo A-I is EPR spectroscopy using spin-labeled protein. The
motion of the label at different positions along the sequence can reveal, through its
periodicity, the secondary structure of a segment of protein. This information, coupled
with the solvent accessibility of the spin label to different quenchers, reveals whether a
sequence of residues is helical, beta structure, or neither. EPR analysis of apo A-I
revealed the positions of α helices (residues 6-34 and 50-98), β strands (residues 4049), or areas without either (residues 35-39), which are assumed to be random coils
(36, 39, 224). In addition to secondary structure, tertiary structure can be determined
when EPR is used with dipolar coupling, discussed below (36).
HD-X coupled with mass spectrometry was also used in secondary structure
analysis. In this technique, hydrogen is replaced by deuterium in the amides of the
protein backbone. After this exchange is carried out, the protein is cleaved at various
sites resulting in overlapping peptide fragments which are then separated by HPLC.
14
The amount of exchange that occurred can then be determined by analysis of the
peptide fragment using mass spectrometry because the deuterium is heavier than the
hydrogen. That information can be used to determine solvent accessibility and
therefore protein secondary structure. In this analysis, 48 ± 3% of lipid-free apo A-I
was determined to have helical structure, which is in the range reported using CD
studies (193).
The location of helix according to HD-X and EPR can be compared (Figure 5).
The helical content of the EPR and HD-X structures is similar, but the locations are
quite different. The helical placements differ the most in the C-terminus. In addition
the EPR study places β-strands in both termini whereas the HD-X study has none. This
is a particularly important controversy to resolve because the EPR authors implicate βstrand structure in critical apo A-I functions, including lipid-binding. In addition,
establishing β-strand structure, if any, is critical for understanding amyloid formation.
This diagram shows the H-DX determined helical regions appear to be the most in line
with those predicted by Rogers, et. al in 1998 using limited proteolysis (70, 224)
Figure 5. Comparison of secondary structures of lipid-free apo A-I determined
using HD-X and EPR. Light gray boxes with border represent α helices, dark grey
boxes represent β strands, and light grey borderless boxes represent random coil/ β
turn. Arrows represent sites displaying intermolecular interaction (224).
15
Tertiary structure
A number of models of lipid-free apo A-I tertiary structure have been proposed
using data from a variety of techniques, including limited proteolysis (69, 70), FRET
(149), X-ray crystallography (212), chemical cross-linking coupled with mass
spectrometry (40), and EPR (36, 39, 224). Collectively, these studies suggest the lipidfree protein has two domains, an amino-terminal helix bundle from residues 1-190 and
a more loosely folded and less structured, smaller carboxy-terminal domain with some
helix and/or beta-strand structure. The various models that have been proposed are
summarized in table 1.
Table 1. Comparison of models of the lipid-free tertiary structure of apo A-I
Model
Main Features
Techniques
Borhani, et al Proposed to represent lipidCrystallization
bound conformation (44-243)
Rogers, et al Model proposes helix bundle Denaturation, analytical
from 1-190, unstructured
ultra centrifugation,
carboxy- terminal domain
limited proteolysis
Brouillette, et Supports globular helixSpin labeling, FRET
al
bundle and not helical
hairpin
Davidson, et
First all- atom model,
Cross linking chemistry,
al
depicting a helical bundle
high resolution mass
structure with termini close spectrometry, molecular
to each other
modeling
Voss, et al Dynamic solution structure,
Spin label EPR
contains beta-sheet
spectroscopy
Mei, et al
Half-circle two-domain
Crystallization
dimer structure of helical
repeats, truncated protein
(1-184).
References
7 (1997)
70 (1998)
149 (2005)
40 (2005)
36, 39, 224
(2007, 2012)
212 (2011)
16
Limited proteolysis involves the addition of a protease to the protein and analysis of
the cleavage pattern that occurs. It is assumed the protein is loosely folded and/or has a
flexible structure at the cleavage positions (206). Several limited proteolysis
experiments have been performed on apo A-I using different proteases. Together, these
studies reveal a major cleavage point around residue 190 (Y192 when chymotrypsin is
used, E191 when S. aureus V8 is used) which is the junction between proposed amino
and carboxy-terminal domains (69). A series of studies on full-length and terminal
deletion mutants employed limited proteolysis along with stability analysis and
analytical centrifugation to develop one of the first models of lipid-free apo A-I
(Figure 6). While proposed more than a decade ago, many aspects of this model are
upheld by recent research using a variety of techniques. In it, an equilibrium exists
between a bundle of six helices and a helical hairpin with five helices. In both models,
the carboxy terminus is unstructured.
17
Figure 6. Proposed model for lipid-free apo A-I. This is presented in equilibrium
between helical bundle (A) and helical hairpin (B) (70). Cylinders represent helices.
The carboxy and amino termini are identified as C and N respectively. Solid circles
represent the locations of methionine residues. Open arrow heads represent the
positions of cleavage in methionine-reduced apo A-I. Dashed lines represent probable
secondary structure which may or may not be helices (70).
FRET was used to distinguish between the helical bundle and the helical
hairpin conformations (149). FRET measures the energy transfer between two
fluorophores, in this case tryptophan and the external fluorescent probe (AEDANS,
aminoethylaminonaphthalene-1-sulfonic acid). The energy transfer is distance
dependent. The distance between the tryptophan and the probe would be quite different
in a helical bundle conformation than in a helical hairpin. The FRET data supports a
discrete helical bundle conformation for lipid-free apo A-I comprising residues 1-186
18
as part of a single folded domain. The FRET data does not support the existence of a
helical hairpin in solution.
In 2005, the first all-atom full-length apo A-I structure was proposed based on
cross-linking of lysine residues combined with high resolution mass spectrometry
(Figure 7) (40). After chemical cross-linking, the sample was digested with trypsin, the
digests were separated by liquid chromatography, and their masses were determined by
mass spectrometry. These masses were used to identify the various cross-links that
were then put into a molecular homology model that was built using proteins with >
30% sequence homology. The resulting model is a helical bundle structure with both
termini close to each other as was also proposed in the model based on FRET analysis
(Figure 7).
Figure 7. Ribbon model of the first full length all-atom model of lipid-free apo A-I
(40). Spirals represent helices, lines represent coils.
19
This model does uphold previously proposed structures with its helical bundle
and its unstructured carboxy-terminus. However, the authors of this study point out the
limitations of this technique due to the flexible nature of apo A-I in solution. Because
of the dynamic nature of apo A-I, this model may represent one of several solution
structures. Despite this limitation, the technique does identify residues close enough to
each other for a long enough time period to cross link and these points of contact
should be considered in developing models of the protein.
EPR spectroscopy has been used to develop a tertiary structure model of lipidfree apo A-I based on side-chain mobility, quencher/ solvent accessibility, and dipole
coupling of spin-labeled apo A-I (39, 224) (Figure 8). β–strands are located from
residues 20-25, 120-129, 150-158, and 214-220 and helices are located in residues 1419, 26-51, 55-85, 92-98, 102-115, 130-148, 159-188, 200-205, and 224-231. The β–
strands are found anti-parallel to each other and could possible exist in one β–sheet
while the helices are found in an amphipathic helical bundle.
20
Figure 8. Comparison of apo A-I structure determined using EPR of the Cterminal domain (panel A) and the full length structure (39, 224). A, The model
contains the tertiary structure of the C-terminal domain according to EPR. B, the “betaclasp” model of lipid-free apo A-I consisting of four anti-parallel beta-strands
represented by arrows (maybe in a single sheet) surrounded by an amphipathic helical
bundle with each helix represented by a spiral. The model is on the left and the
schematic design is on the right (224).
A crystal structure of residues 1-184 has been recently published (Figure 9)
(212). The structure is composed of a half-circle dimer with a backbone of two
elongated antiparallel proline-kinked monomers composed of two helices each. The Nterminal domain of one molecule forms a four-helix bundle with the carboxy-terminal
region of the other molecule. The center of the molecule is flexible and the bundles
may unfold to bind lipid. This crystal structure is a good resolution, at 2.2 angstroms,
but it has a helical content of approximately 80%, much higher than has been predicted
using various other methods. This may be due to the nature of the crystallization
21
process or to the fact that the protein’s unstructured region (by solution methods) is
deleted (residues 185-243).
Figure 9. The crystal structure of 1-184 apo A-I. A, The overall structure of the
monomer of 1-184 apo A-I. B, The model shows the dimerization of the two
monomers (212).
Though much progress has been made in determining the structure of apo A-I
in recent years, an all-atom model that agrees with all experimental data is still lacking.
It has not been possible to firmly establish even the secondary structure of the protein.
In particular, the various methods employed vary greatly in their proposed secondary
structure for the carboxy-terminus. The high helix content indicated by crystallization
is probably not reflective of the solution structure. However, the two solution methods
(EPR and HD-X) show little agreement for the secondary structure in the carboxyterminus. The various techniques reach closer agreement for the amino-terminal
domain where extensive helix is postulated, particularly in the first 100 residues.
However, the exact position of helical segments remains in dispute. As mentioned
22
before, it is difficult to identify an exact structure due to the dynamic nature of the
protein.
In summary, while the structure of apo A-I has yet to be definitively
determined, some aspects are consistently revealed by a variety of methods. The
secondary structure of lipid-free of apo A-I in solution has been found by multiple
methods to be approximately 50-55% α helical (33, 70, 36, 193). The protein has two
domains, with a helix bundle and possibly a small segment of beta strand located in the
N-terminal domain while the C-terminus is either a combination of helix, sheet, and
random coil or simply random coil, and is less structured than the N-terminus. In most
models, the N- and C- termini are close to each other. This may be important in the
self-association properties apo A-I possesses and could be a factor in amyloid
formation, as discussed further below.
Lipid Bound Structure
Nascent HDL
When lipid free apo A-I has lipid added to it, it forms a lipid-protein complex
with a discoidal shape and is referred to as nascent discoidal HDL (or preβ-2 HDL)
which is a critical intermediate in the formation of spherical HDL (131, 225).
Discoidal HDL is typically 10 nm (or 100 Å) in diameter but its size can vary
depending upon its protein content and lipid composition (96, 174, 225). Most HDL
particles consist of either two or four molecules of apo A-I. The size and shape of the
23
apo A-I monomers make it suitable for wrapping around the particle perimeter
(discoidal or spherical), which can have diameters between 80 and 120 Å (190, 212).
A low-resolution crystal structure of ∆1-43 discoidal apo A-I was determined
in 1997 which led to a model of lipid-bound protein (7). The structure contains four
apo A-I molecules arranged in an anti-parallel fashion. The monomer structure within
the tetramer contains a long alpha helix punctuated by kinks caused by proline
residues, leading to a sharply curved horseshoe, or an almost closed circle (Figure 10
A). The monomer’s dimensions are 125 x 80 x 40 Å, but the curve of the ∆1-43
molecule is such that the N- and the C- termini are only 23 Å apart (7).
24
Figure 10. Stereoviews of Δ1-43 apo A-I as a monomer in A, a dimer in B, and a
tetramer in C. The N- and C- termini are labeled (7).
The monomer then self-associates to form a tightly associated antiparallel
dimer (Figure 10 B) overlapping enough to form a closed elliptical ring instead of an
open horseshoe. Because of its antiparallel association and the length of the helices,
25
helix 1 (using the helix numbering applied by the Segrest group, referring to the 10
amphipathic α -helices identified by their computer programs, figure 4B) on one
monomer overlaps with helix 9 on the second monomer. The two helix 5’s from each
monomer overlap. Two apo A-I dimers associate to form a tetramer ring that is 135 x
90 Å on the outside with an inner diameter of 95 x 50 Å (Figure 10 C). The formation
of tetramers is probably due to the absence of lipid in the structure because the dimers
orient to sequester their hydrophobic faces from exposure to water (7).
This appears to be a good model of lipid-bound protein for several reasons.
First, ∆1-43 discoidal apo A-I exhibits similar lipid-binding properties to those of
native apo A-I as determined by measuring surface activity, lipid association of 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine vesicles, and lipid association with
plasma lipoproteins (68). Second, the dimensions of the dimer are close to those that
would be needed to wrap around a lipid disc of approximately 100 nm. Based on the
∆1-43 structure, the Segrest group proposed a computational model that retained some
features of the crystal structure but added modifications to improve the wrapping of the
protein around a lipid-disc (90).
The Segrest model, also referred to as the double belt model, consists of two
antiparallel apo A-I molecules that extend with the long axes of the helices parallel to
the plane of the lipid bilayer (187, 190). Experimental data has since been found to
support this model including cross-linking studies coupled with mass spectrometry, IR
experiments, EPR, and FRET studies (2, 131, 160). These experiments led to models
26
which are essentially minor variations of the double belt model, except for the proposal
of a hairpin structure where two monomers fold on themselves and are then wrapped
around the disc. This would again give an antiparallel association of amphipathic
helices (Figure 11). The spherical model of HDL is less well-developed than discoidal
HDL, but presumably contains overlapping antiparallel helices (7).
Figure 11. Models of lipid-bound apo A-I on discoidal HDL including (A) the
double belt and (B) the hairpin (32).
Stability and Lipid Binding
Conformational Stability
The N-terminus is required to maintain the lipid-free structure because the
helix-bundle structure is lost when residues 1-43 are removed, presumably due to
disruption of the N-terminal helix bundle. This deletion may also cause loss of a
stabilizing interaction between N- and C- termini (76, 175). Experimental evidence
including CD, chemical denaturation, and fluorescence spectroscopy of mutants shows
that the C- and N-terminal regions are in close proximity, supporting an interaction
between the C-terminus and the N-terminal helix bundle (76, 175). It has been
suggested that disrupting the interaction between the termini is important for lipidbinding (36, 39, 70, 175). Because the C-terminus is relatively flexible and has a high
27
affinity for lipid, this interaction may be relatively easy to disrupt when lipid is present
(36, 39, 76, 175).
Figure 12. The initial lipid-binding step of apo A-I mediated through helix 9 and
10. The biding of helices 9 and 10 is triggered by presence of lipid. This releases helix
1 for binding, following by the remaining helices (33, 187).
Lipid Binding
The C-terminal domain has been found to be critical in driving apo A-I to bind
lipid with high affinity (80, 147, 154). The helices of highest lipid affinity, 9 and 10,
bind lipid first. The helices with the next highest affinity, 1 and 2, bind next, followed
by the binding of the rest of the apo A-I helices to the lipid (Figure 12) (80, 147). Not
only does the C-terminus serve as a site of initial lipid contact, but it also is a source of
energy to drive the process of lipid association (39). It is proposed that both
intermolecular as well as intramolecular interhelical interactions play a part in this
binding (147, 154). While helices 9, 10, 1, and 2 have the highest lipid affinity, they
28
aren’t the only helices with any lipid affinity. Even when the extreme N- and C-termini
are cleaved, the remaining residues (44-186) can still bind lipid (132). However, in
normal apo A-I, it is likely that the presence of lipid triggers disruption of the N-C
interaction.
It is believed that, in addition to an intramolecular interaction, intermolecular
interaction is prevalent and leads to aggregated lipid-free protein. The physiological
relevance of this, if any, is only now being investigated. It is suggested apo A-I selfassociation is important for key steps in reverse cholesterol transport such as lipid
binding (226). However, intermolecular interaction presents an opportunity for
undesired aggregation and the possible development of amyloid protein.
Amyloid Apo A-I
Some proteins aggregate in an organized or semi-organized fashion to produce
an amyloid conformation. Amyloid was originally defined based on the ability of
protein aggregates to bind to dyes applied to histological specimens. Now, it usually
refers to aggregated protein that has a cross-beta structure (discussed below). However,
even this application of the term is becoming outdated as polymorphisms are being
discovered in amyloid protein associated with different conditions including
Alzheimer’s, Huntington’s, Parkinson’s, and cataracts. It is becoming increasingly
clear that most proteins can form amyloid or some kind of fibril structure under the
right conditions (95). In recent years, a number of amyloidogenic variants of apo A-I
29
have been identified (141). These proteins form amyloid deposits in various tissues
throughout the body including nerves, skin, heart, liver, kidney, testicles and larynx.
All amyloid fibrils have a long rigid structure in common, while other aspects
of amyloid fibrils such as their secondary structure and length can differ greatly
depending on the protein from which they’re formed (Figure 13). Initially it was
believed that fibrils could only be formed from β –sheets, but it has more recently been
determined α-helical fibrils exist as well. Many protofilaments that form fibrils have a
cross-β structure, meaning the β-sheets in the protofilament, and therefore the fibril,
run perpendicular to the fibrillar axis (49, 88) (Figure 13 B).
30
Figure 13. Generic β-sheet amyloid fibril structure, picture of actual β-sheet
amyloid fibrils, and a model of an α-helical fibril supported by an electron
micrograph. A, β-sheets make up the four (shown here) protofilaments. The β-sheets
run perpendicular to the fibrillar axis (49). B, an atomic force microscope image of
bundle of fibrils of the immunoglobulin light chain variable domain (47). C, a model of
an α-helical fibril in which a bar represents a helix. The helix is then flipped vertically
and horizontally, polymerized, and twisted to form α-helical protofilaments where the
helical axis is actual perpendicular to the fibrillar axis (195). The proposed structure is
supported by an electron micrograph of the protofilament.
The formation process of this long rigid amyloid structure has been
investigated (Figure 14). One possible explanation for the process of amyloid
formation is that after proteins are synthesized on the ribosome, they then begin to
31
form some type of secondary structure, whether random coil, α-helix, or β-sheets, and
are referred to as partially folded (47). These partially folded molecules can then
aggregate under certain conditions and have either monomers or protein aggregates
(also called protofibrils) added to them to form protofilaments which are units, 2-5 nm
in diameter (47, 88). Typically, mature amyloid fibrils consist of 2-6 protofilaments
associated together, sometimes twisted, to form fibrils about 4-13 nm in diameter (47).
Figure 14. Mechanisms of amyloid fibril formation (88). An additional step not
shown in the figure occurs between the β-structured aggregates and the amyloid fibrils
called the protofilament.
32
The initial instigator of amyloid formation is unknown but may involve a pH
change, a temperature change, or other environmental factors. A very delicate balance
exists in the protein’s environment to form the correct conformation as opposed to a
fibrillogenic, sometimes fatal, one. Once formed, one fibril’s existence can greatly
increase amyloid formation because it is accelerated when seeded (88). In other words,
the first amyloid fibril takes the most time to form and each subsequent fibril forms
more quickly, similar to the growing of a crystal (95).
Once a fibril is formed, it is generally linked to disease. Yet, it is debated as to
how amyloids actually cause disease. One debate is whether the fibrillar aggregates or
their precursors, oligomers and protofibrils, cause disease (95). One study supporting
the protofibrils hypothesis states that because no correlation exists between number of
amyloid fibrils present at autopsy and severity of Alzheimer’s or Parkinson’s disease,
perhaps protofibrils cause disease by their toxic, pore-like morphology (44). Studies
have been performed to test this hypothesis where amyloid fibrils were injected in the
brains of mice. Some of those mice then began to exhibit amyloid accumulation and
symptoms similar to Alzheimer’s Disease, insinuating amyloid fibrils, not their
precursors, are actually the cause of the disease (158).
Apo A-I’s fibrils are often associated with disease. Apo A-I fibrils are caused
by different mutations but they can also be formed by WT apo A-I under the right
conditions (141, 227).
33
Apo A-I Amyloidogenic Mutants
Four different apolipoproteins have been found to be amyloidogenic in vivo
(aposerum AA, A-I, A-II, and A-IV), a fifth is amyloidogenic in vitro (C-II), and a
sixth exists in amyloid deposits as a minor component (E) (141). Mutations in apo A-I
have been discovered for more than 30 years (Table 2). According to a 2008 paper,
over 70 mutations have been found so far (129). Some of these mutants cause
amyloidosis and some have an effect on HDL and apo A-I levels, but these are not
mutually exclusive. Many mutants reported have caused amyloidosis with, or without,
CVD (Table 2). Because of the role of apo A-I in HDL production, mutations in the
apo A-I gene often disrupt both apo A-I and HDL production (133, 129).
The first four amyloidogenic apo A-I mutations discovered involved an
addition of a positive charge (113) (Table 2). At one point, this charge change was
thought to be a reason for the amyloid formation. Eventually, additional mutations
were found with similar introduction of positive charges without forming amyloids and
amyloidogenic mutations were discovered without a difference in charge (53). One
possible explanation for why some mutations cause amyloid formation in apo A-I and
others don’t is that amyloidogenic mutations alter the interaction between the C- and
N- terminal domains. This disruption leaves an unstable partially folded N-terminal
helix bundle, which appears to be important in fibril formation (175).
34
Table 2. Summary of the apo A-I mutants found to date (37, 55).
Mutation
Fibril Location
Arg173Cys
(Milano)
None
Other Notes
Hypertriglyceridemia and
decreased
HDL without atherosclerosis
Gly26Arg Nerves, Kidney, 1st report of apo A-I
(Iowa)
Liver
amyloidosis; low HDL and apo
A-I, neuropathy, +1 charge
difference between amino acids
Leu60Arg
Kidney
Non-neuropathy amyloidosis
Del.60-71;
Liver
Non-neuropathy amyloidosis
Ins Val/Thr
and low HDL and apo A-I levels
Del. 70-72
Kidney
Extensive deposits in liver and
spleen
but no adverse clinical effects
Leu90Pro
Skin
Cardiomyopathy; 1st neutralneutral substitution
Leu174Ser
Heart, Joints,
1st time fibril fragment doesn’t
Nerves
include mutation
Arg173Pro Skin, Heart, Larynx Amyloidosis, decreased LCAT
activation
Leu178His Heart, Larynx, Skin 1st time fibrils have full length
apo A-I and TTR fragments
Ala175Pro Visceral, Larynx Infertility
Leu178Pro
Leu75Pro
Larynx, Heart,
Abdominal Fat
Testicles, Liver
Premature CAD and increased
risk of CAD complications,
Infertility, enlarged testicles,
hypo-gonadism, macroorchidism
Year
Reference
Discovered
1980
133, 83,
130
1988
126, 128,
113, 143
1992
1996
153
114
1998
72
1999
53, 71
1999
54, 181
1999
15, 92
2000
43
2002
82
2004
134, 181
2008
62
Amyloidogenic apo A-I mutations tend to be located in two clusters within the
protein sequence, both involving the amino terminal helix bundle domain (46). The
first cluster spans residues 26-107 (Table 2) and contains “inside” mutations because
they occur mainly within the fragments of protein (1-90) found in deposits in vivo
35
(229). These mutations tend to lead to formation of apo A-I amyloid in the liver and
kidneys. Of these mutations, only the G26R mutation is associated with decrease in
HDL. Mutations are also found in a small cluster from residues 154-178 (the “outside”
mutations) and tend to lead to amyloid formation in the larynx, heart, and skin.
Although the two clusters are separated in the sequence, they are in pretty close
proximity in lipid-bound apo A-I (Figure 15).
Figure 15. Dimeric structure of apo A-I based on the apo A-I Δ1-43 structure. In
dark grey are side chains of residues mutated in amyloidosis when the mutations are
outside V93. The light grey dots are mutated residues found within the peptide 1-93.
The bright white dot represents Val 93, which is a common site of cleavage in
fragments (141).
One amyloidogenic mutant associated with cardiac and larynx amyloidosis and,
less frequently, with skin lesions is L178H in which the leucine residue at position 178
has been mutated to a histidine residue, replacing a bulky nonpolar residue with a polar
and weakly basic one (43). Interestingly, another mutation occurs at the same position
but involves proline instead of histidine and has a completely different clinical
36
manifestation (134). This mutation still leads to laryngeal amyloidosis like L178H, but
also has been associated with amyloids in the heart (181). L178P was assumed to be
associated with systemic amyloids when they were found even in abdominal fat (181).
The L178P mutation has been known to cause endothelial dysfunction, changes in
arterial wall thickness, premature CAD and increases risk of CAD complications,
which is very different from L178H (134).
The L178H mutant is unique in that, when it was discovered in 2000, it was the
first amyloid apo A-I mutant whose fibrils contained full-length protein (43). Other
mutants found prior to this produced amyloid deposits containing only amino-terminal
fragments from residue 1 to about 90, commonly 1-93. It was thought at one time that
mutations had to occur within the amino-terminal 90 residues to produce an amyloid
conformation, but this was shown to be false in 1999 when L174S was found to
produce the 1-93 fragment (54, 113). It is now clear that mutations in both of the
clusters can lead to amyloid containing the ~ 90 residue N- terminal fragment as the
fragment has been found in the amyloid stemming from multiple apo A-I mutations
(92, 53, 126, 124, 43).
Our group, in collaboration with researchers at UC Davis, Children’s Hospital,
Oakland Research Institute, and Lund University in Sweden, published the first
structural analysis of an amyloid variant from the 1-90 cluster, G26R. In it, the G26R
mutation was found to have decreased lipid-binding ability when compared to wild
37
type apo A-I, amino-terminal protease sensitivity, and increased β strand secondary
structure.
FOCUS OF THIS RESEARCH
A number of amyloidogenic mutations in apo A-I have been identified which
cluster in two regions of the protein. Furthermore, in addition to amyloid produced by
mutation, WT apo A-I protein has been found in some amyloid plaques. Because
plaques are an early step in many CVD, it is important to elucidate the mechanism of
amyloid formation to try to understand and prevent plaque formation and therefore
CVD. One full-length apo A-I amyloidogenic mutant has already been studied, G26R,
and the sum of its structural differences. This mutation is inside the 1-100 cluster. In
this work, apo A-I with a mutation in the 173-178 region, L178H is characterized using
ANS binding, limited proteolysis, and intrinsic fluorescence studies. In this work, it is
shown that the L178H mutations, like G26R, lead to increased amino-terminal protease
susceptibility and increased hydrophobic surface area. This suggests the two proteins
have a similar tertiary structure that may promote amyloid fibril formation.
38
MATERIALS AND METHODS
Recombinant Protein Production
Transformation
Dr. John Voss (University of California, Davis) generously provided both WT
and L178H apo A-I as 6X-His TAG L178H cDNA fusions in the pNFXex plasmid
(109). The plasmid was transformed into One Shot® Top10F’ (for long-term plasmid
storage) and BL21(DE3)pLysS (for expression) E. coli chemically competent cells
(both Invitrogen). Cells and DNA were thawed on ice and aliquotted into pre-cooled
sterile centrifuge tubes with 1 µL DNA (or water for blank) and 20 µL desired cells.
Mixtures were stirred gently with pipet tip and incubated on ice for five minutes.
Mixtures were then heated at 42° C for 45 seconds and put back on ice for 2 minutes.
180 µL of sterile room temperature SOC media (a nutrient rich media used for protein
culture) was added and samples were put in a shaking 37° C incubator at 250 rpm for
45 minutes.
Fifty µL of the transformation mixtures were plated on a LB plate with 50µL of
50 mg/mL ampicillin (AMP) to select for transformants bearing the ampR gene. The
plates with BL21(DE3)pLysS E. coli competent cells also had 34 µL of 34 mg/mL
chloramphenicol (CAM) to select for retention of the pLysS plasmid. The plates were
incubated at 37º overnight. Individual colonies were then chosen for expression.
39
Inoculation and Expression
Four 125 mL Erlenmeyer flasks containing 50 mL of sterile LB media, pH 7.2,
35 µg/mL CAM, and 50µg/mL AMP were inoculated with 1 colony from an agar plate
or from a frozen glycerol stock made of 8% sterile glycerol stored at -80º C. The
inoculated flask was then incubated at 37º C overnight for 12-16 hours while shaking
at 250 rpm. Four one L flasks with 500 mL of sterile LB media, 35 µg/mL CAM, and
50µg/mL AMP were then inoculated with the entire contents of the overnight culture
and grown at 37ºC while shaking at 250 rpm until the OD600nm was greater than 0.6,
usually 4-6 hours. Then, 500 µL of 0.5 M Isopropyl-β-D-thiogalactopyranoside (IPTG)
was added to induce the expression of apo A-I. After the addition of IPTG, flasks were
incubated again at 37ºC shaking at 250 rpm for three hours. The cells were then
pelleted by centrifugation in an SLA-1500 rotor at 8000 RPM for 15 minutes at 4º C.
Cell pellets were frozen in an acetone/dry ice bath and stored at -20º C until ready for
purification.
Protein Purification
L178H cell pellets were thawed in an ice bath. Then 500 µL protease inhibitor
cocktail p2714 (Sigma), 10 mL phosphate buffered saline (PBS, 0.02 M sodium
phosphate, 0.15 M NaCl, pH 7.2), and one mL 1% triton-x-100 were added to each
pellet before shaking over ice for 20 minutes. Pellets were then lysed using sonication
at 60% maximum in six separate 30-seconds bursts with 60 second breaks on ice in
40
between bursts. The resulting solution was centrifuged at 27,000 * g for 20 minutes at
4º C. The pellets were discarded and the resulting supernatant was filtered through a
0.45 µm syringe filter. The total volume of clarified cell lysate was determined and
guanidine and imidazole were added to make the final solution 3M guanidine and 40
mM imidazole.
A 10 cm x 1 cm chromatography column was loaded with 10 mL His·Bind®
resin (Novagen) and allowed to settle to 5 mL final bed volume. The column was then
rinsed with 20 mL deionized water, charged with 30 mL charge buffer (400 mM
NiSO4) and washed with 30 mL binding buffer (1X PBS, 3 M guanidine, 40 mM
imidazole). The crude cell extract was then loaded onto the column and the flow
through was collected in bulk except for the last 1.5 mL which was collected
separately. The column was then washed with 70 mL binding buffer with the first
sixteen 1.5 mL fractions collected separately and the rest of the buffer collected in
bulk. The protein was then eluted from the column with 35 mL elution buffer (1X
PBS, 500 mM imidazole) supplemented with 100 µL protease inhibitor cocktail p8849
(Sigma) and the first 13 1.5 mL fractions were collected for analysis with the rest of
the sample collected in bulk. The column was then stripped using 36 mL strip buffer
(0.5M NaCl, 100 mM ethylenediaminetetraacetic acid (EDTA), 20 mM Tris-HCl, pH
8.0) with the first 1.5 mL collected separately and the rest collected in bulk.
To determine protein concentration, protein was usually diluted 1:1 or 1:10 in 6
M guanidine and an absorbance spectrum was collected. The absorbance at 280 nm
41
was taken in triplicate samples and averaged and then inserted into the Beer-Lambert
Law (A=εbc) to determine protein concentration using 1.13 cm2/mg as the apo A-I
extinction coefficient and 1 cm as the path length (33). The purity of the protein was
determined by SDS-PAGE on 0.75 mm 15% Tris-Glycine sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS PAGE) gels (Jule Inc.) or 8-16% Precise
(Pierce) gels. Low weight molecular weight markers previously mixed with 5x sample
buffer (Bio Rad) were used as standard. Samples were prepared by combining 10 µL
sample of a fraction with 10 µL 5x sample buffer. Gels were run at 120 V at 4° C until
the leading edge was right above the bottom of the gel rig, approximately 90 minutes.
Gels were then stained in coomassie blue for at least 30 minutes followed by destain
overnight.
Structural Analysis
Limited Proteolysis
Limited proteolysis experiments were performed on both WT and L178H apo
A-I. Before proteolysis, protease inhibitor was dialyzed out of the sample into PBS for
3 hours. Then between 5 and 40 µg of protein were digested at 37ºC with a 1:2000
(wt:wt) ratio of chymotrypsin to apo A-I protein for times ranging from 1 minute to 4
hours. Reactions were quenched with 5µL protease inhibitor cocktail cat. # p8849
(Sigma). Five to 15 µL 5X sample buffer were then added to digests before freezing at
-20° C. Resulting digests were later analyzed by SDS-PAGE.
42
To obtain sufficient proteolyzed fragments for sequencing, selected limited
proteolysis digests were separated on SDS-PAGE gels along with pre-stained
standards, then transferred via electroblotting to a PVDF membrane. The blotting
apparatus was layered top to bottom as follows: cathode, two pieces Beckman blotter
paper soaked in Towbin solution (0.025M tris, 0.192M glycine, 20% Methanol, pH
8.0), gel briefly soaked in Towbin, 0.45 µm pore size Millipore polyvinylidene
fluoride (PVDF) membrane soaked in methanol and then Towbin, two more Towbinsoaked blotting papers, anode. The blotter paper and PVDF membrane were cut to the
exact size of the gel before soaking in appropriate solutions. Electroblotting was
performed using a TE70 blotting apparatus (Hoefer Scientific) at 30 V at 4° C for 90
minutes. Transferred proteins were visualized by staining the PVDF membrane with
in Coomassie blue for 2 minutes followed by destaining in methanol/acetic acid for 10
minutes. Membrane was then air dried before bands of interest were excised and sent
for sequencing.
Protein fragment size was determined by measuring migration distances of
proteolytic fragments on three similar limited proteolysis gels from three different
batches of protein (i.e. protein produced in three different expressions). The retention
factor (Rf) was determined by dividing the migration distance by the length of the gel,
measured from bottom of well to dye front. A standard curve was created by plotting
the Rf vs. log of the molecular weights of the known standards. The molecular weights
of the fragments were then predicted from their Rf using the regression line equation.
43
The Rf and the molecular weights of the uncleaved protein was similarly determined.
The calculated molecular weight of the fragment was subtracted from the calculated
molecular weight of the full-length protein to determine the size with the small
fragment removed by proteolysis. Because the intact protein’s calculated molecular
weight was not exactly the known molecular weight of 28.1 kDa but 30 kDa instead,
the size of the larger proteolysis fragment was established by subtracting the smaller
fragment from 28 kDa. For example, if a fragment’s calculated molecular weight was
24.3 kDa, the 24.3 was subtracted from 30 kDa. The difference of 5.7 was then
subtracted from 28 so the molecular weight of this fragment would be reported as 22.3
kDa. This calculated molecular weight was then divided by 115 (the average molecular
weight of an amino acid) to calculate the number of residues in each fragment . Since
the N-terminal sequence of the fragment was known from Edman sequencing, the
residue at the cleavage site and its place in the protein (residue number) was known as
well. The number of residues in the fragment were then added to the residue number of
the N-terminal cleavage site and the C-terminus was determined for all three gels.
Since proteases cleave at specific residues and chymotrypsin cleaves on the carboxyl
side of tyrosine, tryptophan, and phenylalanine, the sites of cleavage with residue
specificity closest to the c-terminus fragments were assumed to be the correct
positions.
44
ANS Binding
ANS (1-anilinonaphthalene-8-sulfonate) (Sigma-Aldrich) binding experiments
were performed on solutions containing 50 µg/mL protein and 250 µM ANS (>100
molar excess) in a 2 mL final volume (68). Bovine serum album (BSA) was used as a
positive control (50 µg/mL BSA and 250 µM ANS with a 2 mL final volume). Data
were collected on a Shimadzu RF- 5301 PC Spectrofluorophotometer. ANS
fluorescence was obtained as an emission spectrum from 410-560 nm with 395 nm
excitation wavelength using excitation and emission slit widths of 5 nm and 3 nm,
respectively. All samples were run in triplicate on 10 protein preps, though only 8 were
used due to undesired protein cleavage.
Intrinsic Fluorescence
The intrinsic fluorescence of the protein was monitored over time. Data were
collected on the Shimadzu RF- 5301 PC Spectrofluorophotometer. Fluorescence was
obtained as an emission spectrum from 310-400 nm with a 295 nm excitation
wavelength and slit widths of 5 nm (excitation) and 3 nm (emission). Intrinsic
fluorescence quenching experiments were carried out on protein at 0.02 mg/mL
concentration. KI was added from 2M stock (with 1mM Na2S2O3 to prevent oxidation
of I-) to give final concentrations varying from 0.01M to 0.5M. KCl was added to keep
the total ionic strength 0.5M. The Stern Volmer constant, Ksv, was determined using
the equation Fo/F = 1 + Ksv (Q) where Fo and F are the fluorescence intensity at 331 nm
45
in the absence and presence of quencher, respectively, and Q is the concentration of I-.
All samples were run in triplicate on separate protein preps.
46
RESULTS
L178H Expression and Purification
To conduct a structural analysis of L178H apo A-I, it was first necessary to
obtain the pure protein in high yield. The L178H cDNA in a pNFXex plasmid (109)
was obtained from the laboratory of Dr. John Voss at UC Davis. It was transformed
into One Shot® Top10F’ cells because of their excellent transformation efficiency and
for production of DNA for sequence analysis. For protein expression, the plasmid was
transformed into BL21(DE3)pLysS cells which have an IPTG-inducible gene for T7
RNA polymerase that can also lower the expression of background genes while not
interfering with the level of expression of the target IPTG-inducible gene. To
determine the optimal expression conditions, a pilot expression of L178H was
performed in which the temperature of incubation and the length of time after
induction with IPTG was varied. Cell lysates were prepared by sonication of cell
pellets resuspended with Triton x100 and guanidine. Protein expression was evaluated
by analyzing aliquots of cell lysate by SDS-PAGE. Incubating the cells at 37° C for
three hours after IPTG induction produced the most protein (Figure 16).
47
Figure 16. L178H pilot expression. Arrow indicates migration position of apo A-I
(29kDa). Lanes are: 1, molecular weight markers; 2-5, incubation at 25ºC for 0, 1, 2,
and 3 hours after IPTG induction; 6-9, incubation at 37º C for 0, 1, 2, and 3 hours after
IPTG induction.
Large scale expressions using approximately 2 L of media were then carried
out to obtain sufficient protein for structural analysis. Cell lysates from these
experiments were loaded onto a nickel ion affinity column and eluted by increasing
imidazole concentration from 40 mM to 500 mM. Aliquots of column fractions were
subjected to SDS-PAGE to detect the elution of L178H protein, which was usually
most abundant in fractions 2-6 (Figure 17A).
48
Figure 17. Metal-ion affinity chromatography of L178H apo A-I. A, Example of
protein not cleaved at time of purification. Lanes as follows: 1, molecular weight
marker; 2, flow through; 3-5, binding fractions 4, 7, and 10; 6-10, elution fractions 2,
4, 6, 8, and 10. Solid arrow designates L178H apo A-I. B, Example of protein cleaved
at time of purification. Lanes as follows: 1, molecular weight marker; 2, flow through;
3-5, binding fractions 2-4; 6, elution fraction 1. Hollow arrow designates cleaved apo
A-I.
In some purifications, the protein was cleaved (Figure 17 B). This is a known
problem due to the high protease susceptibility of apo A-I (38,69). To minimize nonspecific proteolysis, protease inhibitor several times more concentrated than
recommended was used throughout the purification process. In some cases, guanidine
was added to the cell lysate to both prevent protein aggregation and to help denature
residual proteases. However, addition of guanidine did not significantly reduce
nonspecific proteolysis (data not shown).
To minimize proteolytic degradation over time, the protein was lyophilized and
stored at -80º C following purification. However, the resolubilized protein frequently
exhibited extensive proteolysis and was highly insoluble. Therefore, lyophilization was
not used in subsequent preparations.
49
The proteolysis problem was exacerbated by long incubations carried out to
examine conformational changes over time (up to one month). Protein that was intact
at time of purification was sometimes found to be cleaved days or weeks later. To
establish the quality of the protein for structural analysis, aliquots were taken
periodically throughout a typical 28-day incubation period and analyzed by SDSPAGE to determine whether the protein was still intact at the end of the study (Figure
18). Sometimes protein remained intact through 28 days and sometimes it didn’t; the
basis of this inconsistency is unclear. The degree of degradation was also determined,
not surprisingly, to be temperature dependent. In one experiment, the integrity of the
protein was monitored over 28 days at three different temperatures. The protein
remained intact at 4° C but was degraded almost completely by day four when
incubated at 25 and 37° C. All the experiments reported in this thesis are from protein
that was demonstrated to be fully intact by SDS-PAGE analysis.
50
Figure 18. Analysis of proteolytic cleavage upon extended incubation. Lanes are as
follows: 1, molecular weight markers; 2 – 4, protein incubated for 4 days after
purification and incubated at 4º C, 25º C, and 37º C, respectively; 5-7, day 7 after
purification incubated at 4º C, 25º C, and 37º C, respectively.
Structural Analysis of L178H Apo A-I
The structure of lipid-free apo A-I has been extensively studied, both as plasma
apo A-I isolated from HDL as well as WT apo A-I produced in various prokaryotic and
eukaryotic expression systems. Due to the dynamic nature of this protein, a complete
understanding of its structure remains elusive. However, some consistently observed
features include about 55% -helix content, organization of residues 1-190 into an Nterminal helix bundle with a more loosely structured C-terminal domain (residues 191243), and a small, but significant amount of -strand structure within the N- and Ctermini.
51
There are two clusters of amyloid mutations in apo A-I, both within the Nterminal helix bundle formed from residues 1-190. One cluster occurs within residues
26-107 ("inside" mutations) and the other in residues 154-178 ("outside" mutations). In
conjunction with John Voss’ group at UC Davis and Michael Oda at Children’s
Hospital Oakland Research Institute, we characterized a mutant in the first cluster,
G26R, and found it causes some of the helices in the N-terminus to convert to random
coil and beta-strand structures, leading to the formation of fibrils (38). In addition, the
G26R protein has a much higher sensitivity to protease digestion in the amino
terminus, presumably due to increased β-strand structure beyond residue 26. Here, we
analyzed the effect of a mutation in the second cluster, L178H, on apo A-I structure
using limited proteolysis, ANS binding, and intrinsic fluorescence.
Limited Proteolysis
Limited proteolysis involves the addition of a small amount of protease to a
protein for a short time. Initial cleavage sites indicate positions in the protein that have
increased flexibility (206) or loosely folded structure, and thus more accessibility to
the protease. Despite lipid-free apo A-I’s susceptibility to cleavage compared to typical
globular proteins, limited proteolysis can still be used to examine the protein’s
structure because the protease is added in very small amounts; for apolipoproteins, a
ratio of about 1:2000 (w/w) is used compared to 1:50 or 1:20 (depending upon the
protease used) for lipid-bound apo A-I (69). Previous studies have employed limited
proteolysis to explore the structure of wild type lipid-free apo A-I (38, 69). Two
52
proteases, chymotrypsin and Staphylococcus aureus V8 protease, were used to show
that major cleavage occurs near residue 190 with additional minor cleavage occurring
in the middle of the protein and the extreme carboxy-terminus. The same pattern of
cleavage was observed by others using chymotrypsin and elastase (211). We also
employed limited proteolysis to detect structural changes resulting from the G26R
mutation. In contrast to wild type, digestion with either Staphylococcus aureus V8
protease or chymotrypsin produced cleavage primarily in the N-terminus of the protein
(Y18, E 34, and F57).
Here, limited proteolysis was performed to examine the effect of the L178H
mutation on the structure of apo A-I. WT and L178H were each treated with a 2000:1
(w/w) protein to chymotrypsin ratio. Samples were incubated with enzyme for periods
ranging from one minute to four hours and the resulting digests were analyzed by SDSPAGE (Figure 19, Table 3). The cleavage pattern of wild type protein appeared very
similar to that reported in previous studies (38, 69). Based on N-terminal sequencing
and size analysis of the proteolytic fragment, the major cleavage product corresponds
to the fragment D1- Y192 (WTb) and the most prominent minor product corresponds
to the fragment D1-F229 (WTa) (38).
53
Figure 19. Limited proteolysis of A. WT and B. L178H apo A-I. Both A and B,
lanes as follows: 1, molecular weight markers; 2-8, 0, 1, 5, 30, 60, 120, and 240
minutes of exposure to chymotrypsin. Bands that were sequenced are indicated by
arrows in B. The fragments indicated by the arrows are (top to bottom), LHa, LHb,
LHc.
The cleavage pattern of L178H is superficially similar to that exhibited by WT
with bands close in size to intact protein constituting the major fragments. Both
54
L178H and WT apo A-I exhibit cleavage patterns which show cleavage occurring at or
before one hour and very little full length protein remaining after 4 hours. However,
several differences are evident: there are more bands close in size to intact protein with
the one closest in size (LHa) appearing slightly sooner in L178H relative to WT, and a
greater abundance of smaller fragments in L178H. Sequence analysis reveals that the
three bands LHa, b, and c are all produced by cleavage in the N-terminus (Table 3).
Table 3. Comparison of proteolytic cleavage fragment sequences.
Band Name
Sequence1
Fragment
ID2
Number of
Residues
MW
Estimation in
kDa
LH a
VDVLK
V19-D243
224
24.9
LH b
DRVKD
D9-Y192
184
21.8
LH c
SKLRE
S58-E235
177
20.4
WT a
MHHHH
M1-F2253
224
NR4
WT b
MHHHH
M1-Y1923
191
NR4
Iowa a
VDVLK
V19-D2433
224
NR4
Iowa b
SKLRE
S58-D2433
185
NR4
1, Edman analysis of Western Blot fragment. 2, Determined from Nterminal sequence and molecular weight estimation. 3, reference 38. 4 Not
Reported.
Two bands, here numbered one and two, were excised from a PVDF blot of a
gel containing protein digested for 30 minutes. Edman sequencing revealed two
distinct sequences within the first band. The first sequence, LHa, was VDVLK
55
indicating cleavage at Y-18. The second sequence, LHb, was DRVKD, indicating
cleavage at W-8. Two sequences were also found in the second band. One was the
LHb sequence found in the first band. The second sequence found in band two, LHc,
was SKLRE which indicates cleavage at F-57. Overall, the cleavage pattern of L178H
is much more similar to that of G26R than to wild-type apo A-I, with major cleavage
occurring in the N-terminus of the protein.
Amyloid structure in proteins develops over time. To determine timedependent changes in structure, limited proteolysis was conducted on samples
incubated for different periods of time. A monomeric concentration of protein was
used to prevent dimers of L178H from forming. Limited proteolysis was performed on
both WT and L178H protein stored at 4º C, 25º C and 37º C for one month. Samples
incubated at 25º C and 37º C degraded and could not be evaluated. For both WT and
L178H protein stored at 4º C, the proteolysis pattern at day 28 appeared the same as
day 0 indicating little or no change in protease susceptibility at this temperature. For
L178H protein stored at 25º C and 37º C, the proteolysis pattern at day 2 appears the
same as day 0. However, by day 7, protein had degraded which prevented analysis of
structures brought on by the mutation at these incubation temperatures.
ANS Binding
The hydrophobic binding of ANS has been used to evaluate changes in protein
tertiary structure (200, 201); ANS exhibits increased fluorescence intensity and a blue
shift in the emission wavelength from approximately 500 nm to between 460 and 470
56
nm when introduced into a nonpolar environment (176). ANS binds to proteins by both
its nonpolar anilinonaphthalene and negatively charged sulfonate groups. Despite the
ability to bind via its negative charge, it is generally understood that ANS is a probe
for hydrophobic environments (176, 200). It has frequently been used to probe the
molten globule state of proteins because ANS binds to the molten globule state but not
to fully folded or to unfolded proteins (176). As applied to apolipoproteins, ANS
binding has been used to report tertiary structure changes or changes in lipid-binding
sites (68, 175, 202). Since neither the complete tertiary structure nor specific lipidbinding sites in apo A-I are fully known, ANS data is mostly qualitative in nature and
is used to follow changes in structure.
To analyze the effect of the L178H mutation on the hydrophobic surface
exposure of apo A-I, the ANS binding of L178H and WT apo A-I were compared. At
day 0, the intensity of the fluorescence of ANS bound to L178H apo A-I is more than
three times higher than ANS bound to the same concentration of wild type apo A-I
(Figure 20). The maximum emission wavelength of ANS bound to WT is 484 nm
compared to 473 nm for L178H, a blue shift of 11 nm from WT (Figure 20).
Therefore, L178H apo A-I has a greater exposure of hydrophobic surface than wild
type apo A-I. A similar increase in ANS binding was previously observed for the
G26R protein (38).
57
Figure 20. ANS binding to WT and L178H apo A-I. The standard deviation of both
WT (triangles) and L178H (squares)at 480 nm was ±8 arbitrary fluorescence units. The
ANS binding collected over a period of time was compared relative to the day 0 value.
ANS binding was used to assess amyloid formation over time because as
amyloids are formed, hydrophobic surface exposure may be expected to change. The
fluorescence intensity of ANS binding of protein incubated at 4º C and 37º C from day
0 to day 28 was determined. The undesired proteolysis previously described leading to
degradation of protein was again observed in this experiment. Protein stored at 4º C
typically stayed intact over the entire 28-day incubation period while protein incubated
at 37º C over 28 days degraded in some experiments, but not others. Only experiments
involving intact protein as demonstrated by SDS-PAGE analysis are shown here
58
except for experiments repeated at 37ºC where fluorescence results are similar to those
of experiments where SDS-PAGE showed no degradation.
A
B
Figure 21. Percent loss of ANS binding at 485 nm in L178H and WT over 28 days.
Data was collected from multiple L178H protein samples. A. 4ºC and B. 37ºC. A. ◆
LH sample 5; ■ LH sample 6; X LH sample 16; ▲ LH sample 18; * WT protein. B. ■
LH sample 14; ▲ LH sample 16; ◆ LH sample 18; *WT protein.
59
At 4º C, ANS binding of wild type protein remained constant over the 28-day
period of incubation (Figure 21). In contrast, L178H protein exhibited a large decrease
in ANS binding ability between days 14 and 21, with an approximate t1/2 of 18 days.
This suggests that a structural change, possibly formation of either pre-fibrillar
oligomers or fibrils, was exhibited by L178H that limited the exposure of the ANS
binding site around day 18 (Table 4). In contrast to incubation at 4º C, ANS binding by
WT protein decreased at 37ºC. The decrease was largest between days 2 and 6 with an
estimated t1/2 of 4 days, reaching a maximum decrease of 50% that persisted for an
additional two weeks. L178H protein incubated at 37º C had its largest decrease in
ANS binding ability a few days later, approximately double that of WT protein (Table
4). Four curves representing four different protein purifications are shown in Figure
21B. The curves have different shapes, which precludes accurate curve-fitting.
However, visual examination of the two curves with the longest incubation times (LH
sample 14 and 18) yield an estimated t1/2 of 10 days. In these same samples, the final
ANS fluorescence intensity was higher for WT than for L178H indicating a greater
loss of hydrophobic surface area in L178H.
Table 4. Comparison of estimated t1/2 of ANS
binding at various temperatures.
Protein
4º C
37º C
L178H
18 days
10 days
WT
No change
4 days
60
These results indicate that WT and L178H apo A-I incubated at 37º C exhibit
loss of ANS binding, and that this occurs at a more rapid rate at 37º C for L178H than
at 4ºC. Although this data does not report on fibril formation, it does show that
structural changes occur in either protein incubated at 37º C, leading to reduced
exposure of hydrophobic surfaces over time.
Quenching of Intrinsic Fluorescence
Apo A-I contains tryptophans at positions 8, 50, 72, and 100. Since all the
tryptophans are within the first 100 amino acids of the protein, intrinsic fluorescence
reports on the structure of the N-terminal helix bundle domain, (residues 1-190). To
analyze structural changes in the two proteins over time, the intrinsic tryptophan
fluorescence of L178H was measured at 4º C and 37º C and of WT at 4º C over a 2-4
week period (Figure 22). The maximum fluorescence intensity at 331 nm was plotted
versus time of incubation.
61
Figure 22. Percent loss of intrinsic fluorescence of apo A-I proteins. ◆ (diamond)
LH sample 19 at 4º C and ▲ LH sample 19 at 37º C. ■ LH sample 20 at 4ºC and X
LH sample 20 at 37º C. *WT sample 3 at 4º C. All data reported here had SDS-PAGE
gel evidence to prove the protein was intact at time of data collection.
The intrinsic fluorescence of both the L178H and WT proteins is unchanged
when incubated at 4º C over a 28-day period (Figure 22). However, incubation at 37º C
led to a 60% decrease in intrinsic fluorescence of L178H between days 2 and 7 and an
80% decrease by day 14. It appears to be mostly quenched by day 7 with little
additional change at day 14. These data indicate the L178H protein undergoes
structural changes between days 2 and 7 that result in quenching of tryptophan
fluorescence.
62
To further investigate the tryptophan environment of the two proteins, iodide, a
tryptophan fluorescence quencher, was used. It was added in various concentrations to
the incubated protein and the resulting fluorescence was measured. A Stern-Volmer
plot was made in which the initial fluorescence divided by the quenched fluorescence
is plotted versus the concentration of the quencher. The slope is the Stern-Volmer
constant, KSV. A straight line is expected if the protein is homogenously emitting and
only has one fluorophore (Figure 23). Fluorescence of most proteins is heterogeneous
because of their multiple tryptophan residues. Since multiple tryptophan molecules
usually exist in different environments, it is expected that quenching tryptophan in apo
A-I would not necessarily give a straight trendline in Stern-Volmer plots.
63
Figure 23. Stern-Volmer plot of intrinsic fluorescence quenching of L178H apo AI at day 2. L178H was studied at 4º C ◆ and 37º C ■ and WT was at day 0 ▲ at 4º
C.
The KSV of WT at 4º C and L178H at 37º C are both 1.5, whereas the KSV of
L178H at 4º C is 1.7, the same as a double truncation apo A-I mutant containing
residues 44-186 (Table 5). The calculated KSV of WT in this experiment is the same as
that reported elsewhere (132). For the WT protein, the trendline is the least linear with
an R2 value of 0.88 whereas the R2 values of the trendlines of the L178H protein
incubated at 4º C at 37º C are 0.97 and 0.99.
64
Table 5. Comparison of Stern-Volmer constant, Ksv, of protein
at various temperatures.
Protein
Ksv
R2
L178H, 4º C
1.7
0.97
L178H, 37º C
1.5
0.99
WT
1.5
0.88
WT1
1.5
Apo A-I (44-186) 1
1.7
Human Plasma2
1.4
1, reference 132. 2, Roberts, L.M. unpublished observations.
65
DISCUSSION
Amyloidogenic mutations in human apo A-I cluster in two regions within the
amino-terminal helix bundle from residues 26-107 (termed "inside" mutations) and
154-178 (termed "outside" mutations). The effects of a mutation from the 1-90 cluster,
G26R, were investigated in a collaboration between the laboratory of John Voss at UC
Davis and our group (38). This mutation leads to increased susceptibility to
proteolysis in the N-terminus, increased hydrophobic surface area, and increased strand content in the N-terminus. The goal of this research was to determine, through
analysis of L178H apo A-I, if mutations from the two clusters lead to similar structural
effects. The overall conclusion from this research is that L178H mutation produces
structural changes similar to those observed for G26R. This suggests there may be a
common underlying mechanism for initiating fibril formation among mutations from
the two clusters.
Part I Structural Effects
ANS binding is substantially increased in L178H compared to WT as
evidenced by a 3-fold increase in the intensity and a 4 nm blue shift (figure 20)
indicating more hydrophobic surfaces for binding to ANS in L178H than in WT.
G26R exhibited a similar, if somewhat lower, increase in ANS binding (Table 6).
Thus, both the G26R and L178H mutations lead to an increase in accessible
hydrophobic surface area.
66
Table 6. Comparison of relative fluorescence intensities of apo
A-I proteins at 485 nm.
WT
G26R
L178H
1.0
1.91
3.4
1, Hilt, S. and Roberts, L.M. unpublished observations.
Studies on apo A-I with single-site mutations engineered into the protein for
structural analysis show that mutations in the N-terminal helix bundle domain (1-190)
generally lead to an increase in ANS binding whereas those that occur in the
unstructured C-terminal domain lead to a decrease in ANS binding. The ANS
fluorescence in the presence of N- terminal single site mutants of apo A-I averaged
about 1.5 times that of WT apo A-I with an average of a 4 nm blue shift in wavelength
of maximum fluorescence (103). In contrast, the maximum fluorescence intensities of
ANS bound to C-terminal single site mutants were about 0.5 that of WT showing that
those mutations decreased the exposed hydrophobic surfaces of the molecule.
Combining an N-terminal mutation at Y18P with one of more C-terminal mutations
increased the intensities back to 1.5 that of WT (180).
ANS binding was also studied in truncation mutants. When the entire Cterminus (187-243) was removed from apo A-I, the intensity of the mutants was
approximately half that of WT apo A-I (33, 70). Combining a similar carboxy-terminal
truncation with a Y18P mutation increased the ANS fluorescence intensity to 1.5 that
of WT (180).
67
Given these results, it is likely that both G26R and L178H mutations alter
interactions within the N-terminal helix bundle that lead to increased hydrophobic
surface exposure and therefore, increased accessibility to ANS. Increased hydrophobic
surface exposure may be a requirement to initiate fibril formation (234).
Disruption of the helix bundle by both mutations is further supported by the
limited proteolysis results. Both G26R and L178H led to an increase in N-terminal
protease susceptibility, with major cleavage occurring at Y18 and F57 for both
proteins. This is in contrast to WT protein, where the major cleavage occurs between
the two domains (at Y192 when chymotrypsin is used) (Figure 24). An additional
cleavage at W8 was detected in L178H, which may indicate more accessibility in
L178H vs G26R. However, an exhaustive analysis of minor and major cleavage
products of both proteins using LC-MS would be needed to determine if this is a
significant difference.
G26R and L178H are both point mutations that result in N-terminal protease
sensitivity. N-terminal truncations, which would similarly disrupt the helix bundle,
also exhibit N-terminal protease susceptibility. Two truncations have been studied by
our group, Δ1-23 (184) and Δ1-43, whose crystal structure was used to develop models
of lipid bound protein (7). All four of these mutations, G26R, L178H, Δ1-23, and Δ143, have a cleavage site at F-57 showing that perturbations to the helical domain all
result in similar protease susceptibility.
68
Cleavage also occurred in the C-terminal domain of L178H as evidenced by
fragments containing the intact N-terminal sequence. Based on molecular mass, the
cleavage positions are most likely Y192 and F229, but this would need to be confirmed
by mass spectrometry. Thus, the C-terminal domain retains a protease susceptibility in
L178H and G26R similar to that of WT (38) and the main effect of these mutations is
within the N-terminal helix bundle domain.
Figure 24. Representation of chymotryptic hot spots in wild type, G26R, L178H
apo A-I, and N-terminal deletion mutants. (38, 69, 184) Arrows signify cleavage
sites determined by sequence analysis and estimated mass of proteolysis fragments.
69
While it is clear from both ANS binding and limited proteolysis data that
L178H disrupts the helix bundle domain, intrinsic fluorescence experiments show the
tryptophan environment remains relatively nonpolar. L178H compared to WT protein
had a similar wavelength of maximum intensity and exhibited only a very slightly
increased accessibility to quenching by iodide (Table 5). Since the quenching of
peptide fluorescence by iodide can yield Ksv values of 20 M-1 or more and range from
about 2 to about 9 M-1 for proteins in the presence of urea, an increase from 1.5 to 1.7
is essentially insignificant and the tryptophans of L178H remain in a non-polar
environment, despite alterations to the helix bundle (230, 231).
Structural models can be used to examine the locations of these two mutations
and how, despite their separation into two clusters, they might produce similar effects.
The different models of lipid-free apo A-I were described in the introduction. Here,
we refer to the two most recent models, the EPR model of full-length apo A-I
published by Lagerstedt and co-workers (224) and the crystal structure of 187-243
apo A-I published by Mei and Atkinson (229). Each model has some drawbacks. The
EPR model does not agree with all of the experimental data for apo A-I, particularly in
the placement of beta-strands, while the crystal structure is from a C-terminal
truncation mutant. However, both models are consistent with some features of apo A-I
in solution determined by a variety of methods by different groups of researchers, as
discussed in the Introduction, and can be used as a reference point for interpreting
structural changes brought about by mutation. In the crystal structure of the dimeric C-
70
terminal truncated mutant (Figure 25), residues 26 and 178 are very near each other
spatially.
Figure 25. Position of amyloidogenic mutations in structural models of apo A-I.
A and B, Crystal structure of 185-243 (229). The amyloidogenic mutation sites and
neighboring residues are shown as stick figures. C, EPR model of full-length apo A-I.
L178H is represented as a sphere and G26R by a light grey arrow (228).
The dimeric structure serves as a model for the monomeric structure if it is assumed
the monomer folds back on itself to produce the same inter-helical interactions that are
in the dimer (229). According to the Atkinson group, the bundle-opening effect
produced by G26R is caused by the loss of a flexible glycine in the structure and by
replacement of an attractive interaction with a positively charged residue causing a
repulsive interaction. The Atkinson group postulates that L178H destabilizes the
bundle by adding a polar residue in the middle of the structure therefore disrupting the
packing at the bottom of the bundle. Based on the position of amyloidogenic
mutations within the crystal structure, these researchers further hypothesize that the
general effect of amyloidogenic mutations is to disrupt the salt-bridge interactions in
71
the helical bundle increasing the solvent exposure which renders the protein more
susceptible to protease cleavage. Experiments performed in this thesis support this
theory as both L178H and G26R increased protease accessibility within the N-terminal
helix bundle.
The EPR model proposed by Lagerstedt et al, developed from the full-length
protein in solution, resembles the crystal structure of 185-243 apo A-I in that it
contains a helix bundle and extensive helix-helix interactions involving numerous salt
bridges. However, its topology is quite different and it contains about 18% beta-strand
structure. The authors of this model do not address the relationship of their model to
the two clusters of amyloidogenic mutations. However, we note here that this model
also places G26 and L178 in close proximity (Figure 25C). Therefore, the structural
similarity of G26R and L178H are consistent with a close spatial location of these
residues in two disparate models of apo A-I.
In conclusion, the thesis research shows that the L178H mutation, like the
G26R mutation, produces a disruption to the N-terminal helix bundle. Such disruption,
particularly when it involves increased hydrophobic surface exposure, is likely to lead
to increased aggregation, and from there, to the development of fibrillar, and possibly
amyloid, structure.
Part II. Development of Aggregated Structure
The changes occurring in WT and L178H apo A-I over time presented in this
thesis and elsewhere by our group (228) are summarized in Figure 26. ANS binding
72
of both WT and L178H decreased upon incubation at 37oC with the t1/2 of WT
occurring earlier (4 days vs 10 days) but to a lesser extent than L178H. ANS binding
also decreased in L178H but not WT when incubated at 4oC. In general, a decrease in
ANS binding suggests loss of hydrophobic binding sites, as might be brought about by
a conformational change. Because ANS binds both pre-fibrillar and fibrillar states of
proteins (200, 233), it is not possible to use this method alone to distinguish between
monomeric, pre-fibrillar, and fibrillar states. However, this data can be compared to
circular dichroism data obtained by our research group in collaboration with the
Lagerstedt group (228). Circular dichroism data show that L178H incubated at 37o C
acquires helical, rather than beta-strand, structure over time, with a sigmoidal function
that has a t1/2 of about 12 days. The transitional region in this curve may represent prefibrillar species that aggregate into helical fibrils by about the third week of incubation
at 37o C; samples incubated for 1 month showed clear evidence of fibril formation
where WT did not (228). The change in ANS binding for L178H incubated at 37o C
shows a similar t1/2 of about 10 days (Figure 21B).
WT also exhibited a sigmoidal loss in ANS binding when incubated at 37o C.
This occurred with a shorter t1/2 of 4 days, and a smaller decrease in intensity than in
L178H. No change occurred in the secondary structure of WT over a 28 day period
(228). WT apo A-I has been shown to form aggregated fibrils in vivo and after
oxidation (186) in vitro. Researchers investigating the aggregation of WT apo A-I
before and after oxidation reported no aggregation occurring for un-oxidized apo A-I
73
in a variety of solutions conditions based on Thioflavin T binding (ThT binds to crossbeta structure), light scattering, and electron microscopy. However, samples were
incubated only up to 96 hours for ThT binding and researchers did not report whether
data from light scattering and electron microscopy experiments were collected past day
0. Our results indicate that while ANS changes in WT are smaller than those in
L178H, their occurrence suggests structural changes that may lead to aggregation, and
further investigation into this behavior is needed.
Neither WT nor L178H incubated at 4o C exhibited change in intrinsic
fluorescence and WT fluorescence did not change at 37o C (Figure 22). However,
L178H exhibited a large decrease in fluorescence within the first week of incubation at
37o C. There aren't enough data points to fit these curves but given the trends, the t1/2
must occur by day 5. The magnitude of the intensity decrease suggests the
fluorescence change is not due to solvent exposure as unfolded L178H retains much of
its fluorescence (235). Instead, the fluorescence is specifically quenched by interaction
with a quencher being brought into close proximity. At least eight different amino acid
side chains (237) and even the peptide bond (236) are capable of quenching tryptophan
fluorescence. Therefore, identification of the quencher source requires more defined
structural information.
74
Figure 26. Summary of important changes in WT and L178H apo A-I at 4º C and
37º C over 28 days because of helix (228).
75
CONCLUSION
The structure of the L178H mutant of apo A-I was studied over a 28-day period
at both 4º C and 37º C by using ANS binding, intrinsic fluorescence, and limited
proteolysis experiments. Through those experiments, the L178H protein was observed
to undergo structural changes within the first two weeks of incubation at 37o C that
may be due to formation of pre-fibrillar oligomers and/or fibrils. These may result
from the increase in exposed hydrophobic surface area evidenced by increased ANS
binding in L178H compared to WT at day 0-2. An increase in hydrophobic surface
area is often associated with the beginning of fibril formation (47, 88, 234). Changes in
both ANS binding and intrinsic fluorescence were correlated with increased -helix
and the formation of helical fibrils. In addition, we have shown that L178H was found
to have different N-terminal protease susceptibility when compared to WT, but similar
to the amyloidogenic apo A-I mutant, G26R. This may be due to the nearness of these
residues to each other in the protein, as illustrated by two recent structural models of
apo A-I derived from different experimental methods.
In collaboration with John Voss and Jens Lagerstedt, our group discovered
L178H forms helical fibrils (228) while G26R forms beta-sheet fibrils (38). The
structural similarity of the proteins presented in this thesis does not provide an easy
explanation for this difference. Mutations from either cluster may disrupt the helix
bundle in a similar fashion, creating a looser, more exposed structure which can then
aggregate. Cluster position may dictate which fibril type then forms. Additional
76
studies on proteins with mutations from the two clusters is needed to determine if fibril
type is related to "inside" versus "outside" mutations.
77
REFERENCES
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
Brouillette, C.G., Anantharamaiah, G.M., Engler, J.A., and Borhani, D.W.
(2001) Structural models of human apolipoprotein A-I: a critical analysis
and review. Biochim. et Biophys. Acta 1531, 4-46.
Davidson, W.S., and Thompson, T.B. (2007) The structure of
apolipoprotein A-I in high density lipoproteins. J Biol. Chem. 282, 2224922253.
Gordon, D.J., Probstfield, J.L., Garrison, R.J., Neaton, J.D., Castelli, W.P.,
Knoke, J.D., Jacobs, Jr., D.R., Bangdiwala, S., and Tyroler, H.A. (1989)
High-density lipoprotein cholesterol and cardiovascular disease. Four
prospective American studies. Circulation 79, 8-15.
Walldius, G. and Jungner, I. (2004) Apolipoprotein B and apolipoprotein
A-I: risk indicators of coronary heart disease and targets for lipid-modifying
therapy. J Intern Med. 255, 188-205.
Khuseyinova, N. and Koenig, W. (2006) Apolipoprotein A-I and risk for
cardiovascular diseases. Curr. Sci. 8, 365-373.
Ajees, A.A., Anantharamaiah, G.M., Mishra, V.K., Hussain, M.M., and
Murthy, H.M.K. (2006) Crystal structure of human apolipoprotein A-I:
Insights into its protective effect against cardiovascular diseases. Prot. Natl.
Acad. Sci. USA 103, 2126-2131.
Borhani D.W., Rogers, D.P., Engler, J.A., and Brouillette, C.G. (1997)
Crystal structure of truncated human apolipoprotein A-I suggests a lipidbound conformation. Prot. Natl. Acad. Sci. USA 94, 12291-12296.
Attie, A.D. (2007). ABCA1: at the nexus of cholesterol, HDL and
atherosclerosis. TRENDS Biochem. Sci. 32, 172-179.
Sorenson, R.C., Bisgaier, C.L., Aviram, M., Hsu, C., Billecke, S., and La
Du, B.B. (1999) Human Serum/Peroxanase/Arylesterase’s Retained
Hydrophobic N-Terminal Leader Sequence Assoicates with HDLs by
Binding Phospholipids: Apolipoprotein A-I Stabilizes Activity.
Arterioscler. Thromb. Vasc. Biol. 19, 2214-2225.
Cockerill, G.W., Rye, K.A., Gamble, J.R., Vadas, M.A., and Barter, P.J.
(1995) High-Density Lipoproteins Inhibit Cytokine-Induced Expression of
Endothelial Cell Adhesion Molecules. Arterioscler. Thromb. Vasc. Biol. 15,
1987-1994.
Hyka, N., Dayer, J.M., Modoux, C., Kohno, T., Edwards III, C.K., RouxLombard, P., and Burger, D. (2001) Apolipoprotein A-I inhibits the
production of interleukin-1β and tumor necrosis factor-α by blocking
contact-mediated activation of monocytes by T lymphocytes. Blood 97,
2381-2389.
78
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(20)
(21)
(22)
(23)
(24)
(25)
Epand, R.M., Stafford, A., Leon, B., Lock, P.E., Tytler, E.M., Segrest, J.P.,
and Anantharamaiah, G.M. (1994) HDL and apolipoprotein A-I protect
erythrocytes against the generation of procoagulant activity. Arterioscler.
Thromb. 14, 1775-1783.
Curtiss, L.K., Valenta, D.T., Hime, N.J., Rye, K.A. (2006) What is so
special about apolipoprotein A-I in reverse cholesterol transport?
Arterioscler. Thromb. Vasc. Biol. 26, 12-19.
Fielding, C.J., and Fielding, P.E. (1995) Molecular physiology of reverse
cholesterol transport. J Lipid Res. 36, 211-228.
Frank, P.G., and Marcel, Y.L. (2000) Apolipoprotein A-I: structurefunction relationships. J Lipid Res. 41, 853-872.
Sacks, F.M. (2003) HDL Metabolism and its Anti-Atherosclerosis Effect.
Br. J Cardiol. 10, 297-304.
Babin, P.J. and Vernie, J.M. (1989) Plasma lipoproteins in fish. J Lipid Res.
30, 467-489.
Babin, P.J., Thisse, C., Durliat, M., Andre, M., Akimenko, M.A., and
Thisse, B. (1997) Both apolipoprotein E and A-I genes are present in
nonmammalian vertebrate and are highly expressed during embryonic
development. Proc. Natl. Acad. Sci. USA 94, 8622-8627.
Chong, S., Montello, G.E., Zhang, A., Cantor, E.J., Liao, W., Xu, M.Q.,
and Benner, J. (1998) Utilizing the C-terminal cleavage activity of a protein
splicing element to purify recombinant proteins in a single chromatographic
step. Nuc. Acid. Res. 26, 5109-5115.
Guyon, J.R., Steffen, L.S., Howell, M.H., Pusack, T.J., Lawrence, C., and
Kunkel, L.M. (2007) Modeling human muscle disease in zebrafish.
Biochim. Biophys. Acta. 1772, 205-215.
Gerhard, G.S. (2007) Small laboratory fish as models for aging research.
Ageing Res. Rev. 6, 64-72.
Berger, J., and Currie, P. (2007) The role of zebrafish in chemical genetics.
Curr. Med. Chem. 14, 2413-2420.
Haffter, P., Granato, M., Brand, M., Mullins, M.C., Hammerschmidt, M.,
Kane, D.A., Odenthal, J., van Eeden, F.J.M., Jiang, Y.J., Heisenberg, C.P.,
Kelsh, R.N., Furuntani-Seiki, M., Vogelsang, E., Beuchle, D., Schach, U.,
Fabian, C., and Nusselein-Volhard, C. (1996) The identification of genes
with unique and essential functions in the development of zebrafish, Danio
rerio. Development 123, 1-36.
Engeszer, R.E., Patterson, L.B., Rao, A.A., and Parichy, D.M. (2007)
Zebrafish in the wild: a review of natural history and new notes from the
wild. Zebrafish 4, 21-40.
LaVallie E.R., and McCoy, J.M. (1995) Gene fusion expression systems in
Escheria coli. Curr. Opin. Biotechnol. 6, 501-506.
79
(26)
(27)
(28)
(29)
(30)
(31)
(32)
(33)
(34)
(35)
(36)
Chong, S., Mersha, F.B., Comb, D.G., Scott, M.E., Landry, D., Vence,
L.M., Perler, F.B., Benner, J., Kucera, R.B., Hirvonen, C.A., Pelletier, J.J.,
Paulus, H., and Xu, M.Q. (1997) Single-column purification of free
recombinant proteins using a self-cleavable affinity tage derived from a
protein splicing element. Gene 192, 271-281.
Baroukh, N. Lopez, C.E., Saleh, M.C., Reclade, D., Vergnes, L., Ostos,
M.A., Fiette, L., Fruchart, J.C., Castro, G., Zakin, M. M., and Ochoa, A.
(2004) Expression and secretion of human apolipoprotein A-I in the heart.
FEBS Letters 557, 39-44.
Plump, A.S., Scott, C.J., and Breslow, J.L. (1994) Human apolipoprotein
A-I gene expression increases high density lipoprotein and suppresses
atherosclerosis in the apolipoprotein E-deficient mouse. Prot. Natl. Acad.
Sci. USA 91, 9607-9611.
Hughes, S.D., Verstuyft, J., and Rubin, E.M. (1997) HDL deficiency in
genetically engineered mice requires elevated LDL to accelerate
atherogenesis. Arterioscler. Thomb., Vasc. Biol. 17, 1725-1729.
Karathanasis, S.K. (1985) Apolipoprotein multigene family: tandem
organization of human apolipoprotein A1, CIII, and AIV genes. Prot. Natl.
Acad. Sci. USA 82, 6374-6378.
Pennacchio, L.A., Olivier, M., Hubacek, J.A., Cohen, J.C., Cox, D.R.,
Fruchart, J.C., Krauss, R.M. and Rubin, E.M. (2001) An apolipoprotein
influencing triglycerides in humans and mice revealed by comparative
sequencing. Science 294, 169-173.
Martin, D. D. O., Budamagunta, M.S., Ryan, R.O., Voss, J.C., and Oda,
M.N. (2006) Apolipoprotein A-I Assumes a “Looped Belt” Conformation
on Reconstituted High Density Lipoprotein. J Biol. Chem. 281, 2041820426.
Rogers, D.P., Roberts, L.M., Lebowitz, J., Datta, G, Anantharamaiah, G.M.,
Engler, J.A., and Brouillette, C.G. (1998) The lipid-free structure of
apolipoprotein A-I: effects of amino terminal deletions. Biochemistry 37,
11714-11725.
Zhu, H.L., and Atkinson, D. (2007) Conformation and lipid binding of a Cterminal (198-243) peptide of human apolipoprotein A-I. Biochemistry 46,
1624-1634.
New England Biolabs, Inc. (2006) IMPACT-CN: Protein purification
system now featuring fusion to C- or N- terminus of the target protein.
Instruction Manual 2.1, 1-48.
Lagerstedt, J.O., Budamagunta, M.S., Oda, M.N., and Voss, J.C. (2007)
Electron Paramagnetic Resonance Spectroscopy of Site-diected Spin Labels
Reveals the Structural Heterogeneity in the N-terminal Domain of ApoA-I
in Solution. J. Biol. Chem. 282, 9143-9149.
80
(37)
(38)
(39)
(40)
(41)
(42)
(43)
(44)
(45)
(46)
(47)
(48)
(49)
Joy, T., Wang, J., Han, A., and Hegele, R.A. (2003) APOA1 related
Amyloidosis: A Case Report and Literature Review. Clin. Biochem. 36,
641-645.
Lagerstedt, J.O. Cavigiolio, G., Roberts, L.M., Hong, H.S., Jin, L.W.,
FitzGerald, P.G., Oda, M.N., and Voss, J.C. (2007) Mapping the Structural
Transition in an Amyloidogenic Apolipoprotein A-I. Biochemistry 46,
9693-9699.
Oda, M.N., Forte, T.M., Ryan, R.O., and Voss, J.C. (2003) The C-terminal
domain of apolipoprotein A-I contains a lipid-sensitive conformational
trigger. Nature Structural Biol. 10, 455-460.
Silva, R.A.G.D., Hilliard, G.M., Fang, J., Macha, S., and Davidson, W.S.
(2005) A Three-dimensional Molecular Model of Lipid-Free
Apolipoprotein A-I Determined by Cross-Linking/ Mass Spectroscopy and
Sequence Threading. Biochemsitry 44, 2759-2769.
Brubaker, G., Peng, D.Q., Somerlot, B., Abdollahian, D.J., and Smith, J.D.
(2006) Apolipoprotein A-I lysine modification: Effects on helical content,
lipid binding and cholesterol acceptor activity. Biochim. Biophys. Acta
1761, 64-72.
Edelstein, C. and Scanu, A.M. (1980) Effect of Guanidine Hydrochloride
on the Hydrodynamic and Thermodynamic Properties of Human
Apolipoprotein A-I in Solution. J. Biol. Chem. 255, 5747-5754.
Mendes de Sousa, M., Vital, C., Ostler, D., Fernandes, R., Pouget-Abadie,
J., Carles, D., and Saraiva, M.J. (2000) Apolipoprotein A-I and
Transthyretin as Components of Amyloid Fibrils in a Kindred with apoA1
Leu178His Amyloidosis. Am. J. Pathol. 156, 1911-1917.
Lashuel, H.A., Hartley, D., Petre, B.M., Walz, T., and Lansbury, Jr., P.T.
(2002) Amyloid pores from pathogenic mutations. Nature 418, 291.
Kelly, J.W. (1998) The alternative conformations of amyloidogenic
proteins and their multi-step assembly pathways. Curr. Opin. Struct. Biol.
8, 101-108.
Andreola, A., Bellotti, W., Giogetti, S., Mangione, P., Obici, L., Stoppini,
M., Torres, J., Monzani, E., Merlini, G., and Sunde, M. (2003)
Conformational Switching and Fibrillogenesis in the Amyloidogenic
Fragment of Apolipoprotein A-I. J. Biol. Chem. 278, 2444-2451.
Uversky, V.N., and Fink, A.L. (2004) Conformational Constriants for
Amyloid Fibrillation: The Importance of Being Unfolded. Biochim.
Biophys. Acta 1698, 131-154.
Hubbell, W.L., Cafiso, D.S., and Altenbach, C. (2000) Identifying
conformational changes with site-directed spin-labeling. Nature Struct.
Biol. 7, 735-739
Sunda, M., Serpell, L.C., Bartlam, M., Fraser, P.E., Pepys, M.B., and
Blake, C.C.F. (1997) Common Core Structure of Amyloid Fibrils by
Synchrotron X-Ray Diffraction. J. Mol. Biol. 273, 729-739.
81
(50)
(51)
(52)
(53)
(54)
(55)
(56)
(57)
(58)
(59)
(60)
(61)
Li, W.H., Tanimura, M., Luo, C.C., Datta, S., and Chan, L. (1988) The
apolipoprotein multigene family: biosynthesis, structure, structure-function
relationships, and evolution. J. Lipid Res. 29, 245-271.
American Heart Association (AHA). Heart Disease and Stroke Statistics
2008 Update: A Report from the American Heart Association Statistics
Committee and Stroke Statistics Subcommittee. Circulation 117, e25-e146.
Ma, J., Liao, X.L., Lou, B., and Wu, M.P. (2004) Role of Apolipoprotein
A1 in Protecting against Endotoxin Toxicity. Acta Biochim. Biophys.
Sinica 36, 419-424.
Hamidi Asl, L., Liepnieks, J.J., Hamidi Asl, K., Uemichi, T., Moulin, G.,
Desjoyaux, E., Loire, R., Delpech, M., Grateau, G., and Benson, M.D.
(1999) Hereditary Amyloid Cardiomyopathy Caused by a Variant
Apolipoprotein A1. Am. J. Pathol. 154, 221-227.
Obici, L., Bellotti, V., Mangione, P., Stoppini, M., Arbustini, E., Verga, L.,
Zorzoli, I., Anesi, E., Zanotti, G., Campana, C., Vigano, M., Merlini, G.
(1999) The New Apolipoprotein A-I Variant Leu174 → Ser Causes
Hereditary Cardiac Amyloidosis, and the Amyloid Fibrils Are Constituted
by the 93-Residue N-Terminal Polypeptide. Am. J. of Pathol. 155, 695-702.
Coriu, D., Dispenzieri, A., Stevens, F.J., Murphy, C.L., Wang, S., Weiss,
D.T., and Solomon, A. (2003) Hepatic Amyloidosis resulting from
deposition of the apolipoprotein A-I variant Leu75Pro. Amyloid 10, 15223.
Saito, H., Lund-Katz, S., and Phillips, M.C. (2004) Contributions of
Domain Structure and Lipid Interaction to the Functionality of
Exchangeable Human Apolipoproteins. Prog. Lipid Res. 43, 350-380.
Nievelstein-Post, P., Mottino, G., Fogelman, A., Frank, J. (1994) An
ultrastructural study of lipoprotein accumulation in cardiac valves of the
rabbit. Arterioscler. Thromb. Vasc. Biol. 14, 1151-1161.
Aiello, R.J., Brees, D., Bourassa, P.A., Royer, L., Lindsey, S., Coskran, T.,
Haghpassand, M., and Francone, O.L. (2002) Increased Atherosclerosis in
Hyperlipidemic Mice with Inactivation of ABCA1 in Macrophages.
Arterioscler. Thromb. Vasc. Biol. 22, 630-637.
Wang, N., and Tall, A.R. (2003) Regulation and Mechanisms of ATPBinding Cassette Transporter A1-Mediated Cellular Cholesterol Efflux.
Arterioscler. Thromb. Vasc. Biol. 23, 1178-1184.
Spagnoli, L.G., Bonanno, E., Sangiorgi, G., and Mauriello, A. (2007) Role
of Inflammation in Atherosclerosis. J. Nucl. Med. 48, 1800-1815.
Boguski, M.S., Birkenmeier, E.H., Eishourbagym N.A., Taylor, J.M., and
Gordon, J.L. (1986) Evolution of Apolipoproteins: Structure of the Rat Apo
A-IV Gene and Its Relationship to the Human Genes for Apo A-I, C-III,
and E. J. Biol. Chem. 261, 6398-6407.
82
(62)
(63)
(64)
(65)
(66)
(67)
(68)
(69)
(70)
(71)
(72)
Scalvini, T., Martini, P.R., Gambera, A., Tardanico, R., Biasi, L., Scolari,
F., Gregorini, G., and Rosei, E.A. (2008) Spermatogenic and Steroidogenic
Impairment of the Testicle Characterizes the Hereditary Leu75Pro
Apolipoprotein A-I Amyloidosis. J. Clin. Endocrin. Metab. Feb 19 (Epub
ahead of print).
Roberts, L.M. (2007) NIH Grant proposal for Apolipoprotein A-I Research.
Unpublished.
Engeszer, R.E., Patterson, L.B., Rao, A.A., and Parichym D.M. (2007)
Zebrafish in the Wild: A Review of Natural History and New Notes from
the Field. Zebrafish 4, 21-38.
Jonas, A. (2000) Lecithin cholesterol acyltransferase. Biochim. Biophys.
Acta 1529, 245-256.
Behling Agree, A.K., Tricerri, M.A., McGuire, K.A., Tian, S.M., Jonas, A.
(2002) Folding and Stability of the C-terminal Half of Apolipoprotein A-I
Examined with a Cys-specific Fluorescence Probe. Biochim. Biophys. Acta
1594, 286-296.
Yancey, P.G., Bielicki, J.K., Johnson, W.J., Lund-Katz, S., Palgunachari,
M.N., Anantharamaiah, G.M., Segrest, J.P., Phillips, M.C., and Rothblat,
G.H. (1995) Efflux of Cellular Cholesterol and Phospholipid to Lipid-Free
Apolipoproteins and Class A Amphipathic Peptides. Biochemistry 34,
7955-7965.
Rogers, D.P., Brouillette, C.G., Engler, J.A., Tendian, S.W., Roberts, L.,
Mishra, V.K., Anantharamaiah, G.M., Lund-Katz, S., Phillips, M.C., and
Ray, M. (1997) Truncation of the Amino Terminus of Human
Apolipoprotein A-I Substantially Alters Only the Lipid-Free Conformation.
Biochemistry 36, 288-300.
Roberts, L.M., Ray, M.J., Shih, T.W., Hayden, E., Reader, M.M., and
Brouillette, C.G. (1997) Structural Analysis of Apolipoprotein A-I: Limited
Proteolysis of Methionine-Reduced and –Oxidized Lipid-Free and LipidBound Human Apo A-I. Biochemistry 36, 7615- 7624.
Rogers, D.P., Roberts, L.M., Lebowitz, J., Engler, J.A., and Brouillette,
C.G. (1998) Structural Analysis of Apolipoprotein A-I: Effects of Aminoand Carboxy-Terminal Deletions on the Lipid-Free Structure. Biochemistry
37, 945-955.
Walsh. M.T. (1999) A Novel Amyloidogenic Variant of Apolipoprotein AI: Implications for a Conformational Change Leading to Cardiomyopathy.
Am. J Pathol. 154, 11-14.
Persey, M.R., Booth, D.R., Booth, S.E., van Zyl-Smit, R., Adams, B.K.,
Fattaar, A.B., Tennent, G.A., Hawkins, P.N., and Pepys, M.B. (1998)
Hereditary Nephropathic Systemic Amyloidosis Caused by a Novel Variant
Apolipoprotein A-I. Kidney Intl. 53, 276-281.
83
(73)
(74)
(75)
(76)
(77)
(78)
(79)
(80)
(81)
(82)
(83)
(84)
Jones, M.K., Anantharamaiah, G.M., and Segrest, J.P. (1992) Computer
Programs to Identify and Classify Amphipathic α Helical Domains. J Lipid
Res. 33, 287-296.
Natarajan, P., Forte, T.M., Chu, B., Phillips, M.C., Oram, J.F., and Bielicki,
J.K. (2004) Identifications of an Apolipoprotein A-I Structural Element
That Mediates Cellular Cholesterol Efflux and Stabilizes ATP Binding
Cassette Transport A1. J Biol. Chem. 279, 24044-24052.
Calabresi, L., Canavesi, M., Bernini, F., and Franceschini, G. (1999) Cell
Cholesterol Efflux to Reconstituted High-Density Lipoproteins Containing
the Apolipoprotein A-IMilano Dimer. Biochemistry 38, 16307-16314.
Gorshkova, I.N., Liadaki, K., Gursky, O., Atkinson, D., and Zannis, V.I.
(2000) Probing the Lipid-Free Structure and Stability of Apolipoprotein A-I
by Mutation. Biochemistry 39, 15910-15919.
Tricerri, M.A., Behling Agree, A.K., Sanchez, S.A., Bronski, J., and Jonas,
A. (2001) Arrangement of Apolipoprotein A-I in Reconstituted HighDensity Lipoprotein Disks: An Alternative Model Based on Fluorescence
Resonance Energy Transfer Experiments. Biochemistry 40, 5065-5074.
Fanh, Y., Gursky, O., and Atkinson, (2003) Lipid-Binding Studies of
Human Apolipoprotein A-I and Its Terminally Truncated Mutants.
Biochemistry 42, 13260-13268.
Ng, D.S., Vezina, C., Wolever, T.S., Kuksis, A., Hegele, R.A., and
Connelly, P.W. (1995) Apolipoprotein A-I Deficiency: Biochemical and
Metabolic Characteristics. Arterioscler., Thromb., Vasc. Biol. 15, 21572164.
Palgunachari, M.N., Mishra, V.K., Lund-Katz, S., Phillips, M.C., Adeyeye,
S.O., Alluri, S., Anantharamaiah, G.M., Segrest, J.P. (1996) Only the Two
End Helixes of Eight Tandem Amphipathic Helical Domains of Human
Apo A-I Have Significant Lipid Activity: Implications for HDL Assembly.
Aterioscler., Thromb., Vasc. Biol. 16, 328-338.
Ross, R. (1999) Atherosclerosis: An Inflammatory Disease. NEJM 340,
115-126.
Lachmann, H.J., Booth, D.R., Booth, S.E., and Bybee, A. (2002)
Misdiagnosis of Hereditary Amyloidosis as A1 (Primary) Amyloidosis.
NEJM 346, 1786-1791.
Cheung, M.C., Nichols, A.V., Blanche, P.J., Gong, E.L., Franceschini, G.,
and Sirtori, C.R. (1988) Characterization of A-I containing lipoproteins in
subjects with A-I Milano Variant. Biochim. Biophys. Acta 960, 73-82.
Alaupovic, P., Lee, D.M., and McConathy, W.J. (1972) Studies on the
Composition and Structure of Plasma Lipoproteins: Distribution of
Lipoprotein Families in Major Density Classes of Normal Human Plasma
Lipoproteins. Biochim. Biophys. Acta 260, 689-707.
84
(85)
(86)
(87)
(88)
(89)
(90)
(91)
(92)
(93)
(94)
(95)
(96)
(97)
Li, H., Reddick, R.L., and Maeda, N. (1993) Lack of Apo A-I Is Not
Associated with Increased Susceptibility to Atherosclerosis in Mice.
Arterioscler. Thromb. 13, 1814-1821.
Moore, R.E., Kawashiri, M.A., Kitajima, K., Secreto, A., Millar, J.S.,
Pratico, D., and Rader, D.J. (2003) Apolipoprotein A-I Deficiency Results
in Markedly Increased Atherosclerosis in Mice Lacking the LDL Receptor.
Arterioscler. Thromb. Vasc. Biol. 23, 1914-1920.
Blankenberg, S., Barbaux, S., and Triet, L. (2003) Adhesion Molecules and
Atherosclerosis. Atheroscler. 170, 191-203.
Chiti, F., and Dobson, C.M. (2006) Protein Misfolding, Functional
Amyloid, and Human Disease. Annu. Rev. Biochem. 75, 333-366.
Kontush, A., and Chapman, A.J. (2006) Functionally Defective HighDensity Lipoprotein: A New Therapeutic Target at the Crossroads of
Dyslipidemia, Inflammation, and Atherosclerosis. Pharmacol. Rev. 58, 342374.
Bergeron, J., Frank, P.G., Emmanuel, F., Latta, M., Zhao, Y., Sparks, D.L.,
Rassart, E., Denefle, P., and Marcel, Y.L. (1997) Characterization of human
apolipoprotein A-I expressed in Escherichia coli. Biochim. Biophys. Acta
1344, 139-152.
Francis, M.C., Frohlich, J.J. (2001) Coronary Artery Disease in Patients at
Low Risk- Apolipoprotein A-I As an Independent Risk Factor. Atheroscler.
155, 165-170.
Asl, K.H., Liepnieks, J.J., Nakamura, M., Parker, F., and Benson, M.D.
(1999) A Novel Apolipoprotein A-I Variant, Arg173 Pro, Associated with
Cardiac and Cutaneous Amyloidosis. Biochem. Biophys. Res. Comm. 257,
584-588.
Yusuf, S., Hawken, S., Ounpuu, S., Dans, T., Avezum, A., Lanas, F.,
McQueen, M., Budaj, A., Pais, P., Varigos, J., and Lisheng, L. (2004)
Effect of Potentially Modifiable Risk Factors Associated with Myocardial
Infarction in 52 Countries (the INTERHEART Study): Case-Control Study.
Lancet 364, 937-952.
Falk, R.H., Comenzo, R.L., and Skinner, M. (1997) The Systemic
Amyloidoses. NEJM 337, 898-910.
Dobson, C.M (2001) The Structural Basis of Protein Folding and Its Links
with Human Disease. Philosoph. Trans. Biol. Sci. 356, 133-145.
Koppaka, V. (2001) Structural Studies of Discoidal Lipoprotein A-I. Cell.
Mol. Life Sci. 58, 885-893.
Denis, M., Haidar, B., Marcil, M., Bouvier, M., Krimbou, L., and Genest,
Jr., J. (2004) Molecular and Cellular Physiology of Apolipoprotein A-I
Lipidation by the ATP-binding Cassette Transporter A1 (ABCA1). J. Biol.
Chem. 279, 7384-7394.
85
(98)
(99)
(100)
(101)
(102)
(103)
(104)
(105)
(106)
(107)
(108)
(109)
(110)
Fitzgerald, M.L., Morris, A.L., Chroni, A., Mendez, A.J., Zannis, V.I., and
Freeman, M.W. (2004) ABCA1 and Amphipathic Apolipoproteins Form
High-Affinity Molecular Complexes Required for Cholesterol Efflux. J.
Lipid Res. 45, 287-294.
Han, J.M., Jeong, T.S., Lee. W.S., Choi, I., and Cho, K.H. (2005) Structural
and Functional Properties of V156K and A158E Mutants of Apolipoprotein
A-I in the Lipid-Free and Lipid-Bound States. J. Lipid Res. 46, 589-596.
Gross, E., Peng, D.Q., Hazen, S.L., and Smith, J.D. (2006) A Novel
Folding Intermediate State for Apolipoprotein A-I: Role of the Amino and
Carboxy Termini. Biophys. J. 90, 1362-1370.
Panagotopulos, S.E., Witting, S.R., Horace, E.M., Hui, D.Y., Maiorano,
J.N., and Davidson, W.S. (2002) The Role of Apolipoprotein A-I Helix 10
in Apolipoprotein-mediated Cholesterol Efflux via the ATP-Binding
Cassette Transporter ABCA1. J. Biol. Chem. 277, 39477-39484.
Martinez, L.O., Agerholm-Larsen, B., Wang, N., Chen, W., and Tall, A.R.
(2003) Phosphorylation of a Pest Sequence in ABCA1 Promotes Calpain
Degradation and Is Reversed by ApoA-I. J. Biol. Chem. 278, 37368-37374.
Gorshkova, I.N., Liu, T., Kan, H.Y., Chroni, A., Zannis, V.I., and Atkinson,
D. (2006) Structure and Stability of Apolipoprotein A-I in Solution and in
Discoidal High-Density Lipoprotein Probed by Double Charge Ablation
and Deletion Mutation. Biochemistry45, 1242-1254.
Hansson, G.K. (2005) Mechanisms of Disease: Inflammation,
Atherosclerosis, and Coronary Artery Disease. NEJM 352, 1685-1696.
Arakawa, R., and Yokoyama, S. (2002) Helical Protein Stabilize ATPbinding Cassette Transporter A1 by Protecting It from Thiol Proteasemediated Degradation. J. Biol. Chem. 277, 22426-22429.
Surewicz, W.K., Epand, R.M., Pownall, H.J., and Hu, S.W. (1986) Human
Apolipoprotein A-I Forms Thermally Stable Complexes with Anionic but
Not with Zwitterionic Phospholipids. J. Biol. Chem. 261, 16191-16197.
Schmidt, H. H.-J., Genschel, J., Haas, R., Buttner, C., and Manns, M.P.
(1997) Expression and Purification of Recombinant Humans
Apolipoprotein A-I in Chinese Hamster Ovary Cells. Prot. Expr. Purif. 10,
226-236.
Panagotopulos, S.E., Witting, S.R., Horace, E.M., Maiorano, J.N., and
Davidson, W.S. (2002) Bacterial Expression and Characterization of
Mature Apolipoprotein A-I. Prot. Expr. Purif. 25, 353-361.
Ryan, R.O., Forte, T.M., and Oda, M.N. (2003) Optimized Bacterial
Expression of Human Apolipoprotein A-I. Prot. Expr. Purif. 27, 98-103.
Action, S., Rigotti, A., Landschulz, K.T., Xu,S., Hobbs, H.H., and Krieger,
M. (1006) Identification of Scavenger Receptor SR-BI as a High-Denisty
Lipoprotein Receptor. Science 271, 518-520.
86
(111) Rigotti, A., Acton, S.L., and Krieger, M. (1995) The Class B Scavenger
Receptors SR-BI and CD36 Are Receptors for Anionic Phospholipids. J.
Biol. Chem. 270, 16221-16224.
(112) Kostner, K.M. (2004) Activation of the Complement System: A Crucial
Link Between Inflammation and Atherosclerosis? Euro. J. Clin. Invest. 34,
800-802.
(113) Genschel, J., Haas, R., Propsting, M.J., and Schmidt, H. H.-J. (1998)
Apolipoprotein A-I Induced Amyloidosis. FEBS Lett. 430, 145-149.
(114) Booth, D.R., Tan, S.Y., Booth, S.E>, Tennent, G.A., Hutchinson, W.L.,
Hsuan, J.J., Totty, N.F., Truong, O., Soutar, A.K., Hawkins, P.N.,
Bruguera, M., Caballeria, J., Sole, M., Campistol, J.M., and Pepys, M.B.
(1996) Hereditary Hepatic and Systemic Amyloidosis Caused by a New
Deletion/ Insertion Mutation in the Apolipoprotein A-I Gene. J. Clin.
Invest. 97, 2714-2721.
(115) Bojanovski, D., Gregg, R.E., Zech, L.A., Meng, M.S., Bishop, C., Ronan,
R., and Brewer, Jr., H.B. (1987) In Vivo Metabolism of Proapolipoprotein
A-I in Tangier Disease. J. Clin. Invest. 80, 1742-1747.
(116) Klon, A.E., Jones, M.K., Segrest, J.P., and Harvey, S.C. (2000) Molecular
Belt Models of Apolipoprotein A-I Paris and Milano Mutations. Biophys. J.
79, 1679-1685.
(117) Breslow, J.L., Ross, D., McPherson, J., Williams, H., Kurnit, D.,
Nussbaum, A.L., Karathanasis, S.K., and Zannis, V.I. (1982) Isolation and
Characterization of cDNA Clones for Human Apolipoprotein A-I. PNAS
79, 6861-6865.
(118) Wang, N., Silver, D.L., Thiele, C., and Tall, A.R. (2001) ATP-binding
Cassette Transporter A1 (ABCA1) Functions as a Cholesterol Efflux
Regulatory Protein. J. Biol. Chem. 276, 23742-23747.
(119) Zannis, V.I., Karathanasis, S.K., Forbes, G.M., and Breslow, J.L. (1986)
Intra-and Extracellular Modifications of Apolipoproteins. Methods
Enzymol. 128, 690-712.
(120) Law, S.W., Gray, G., and Brewer, Jr., H.B. (1983) cDNA Cloning of
Human Apo A-I: Amino Acid Sequence of Preproapo A-I. Biochem.
Biophys. Res. Comm. 112, 257-264.
(121) Luchoomun, J., Theret, N., Clavey, V., Duchateau, P., Rosseneu, M.,
Brasseur, R., Denefle, O., Fruchart, J.C., and Castro, G.R. (1994) Structural
Domain of Apolipoprotein A-I Involved in Its Interaction with Cells.
Biochim. Biophys. Acta 1212, 319-326.
(122) Gordon, T., Castelli, W.P., Hjortland, M.C., Kannel, W.B., and Dawber,
T.R. (1977) High Density Lipoprotein as a Protective Factor Against
Coronary Heart Disease: The Framingham Study. Am. J. Med.62, 707-714.
(123) Schwartz, C.J., Valente, A.J., and Sprague, E.A. (1993) A Modern View of
Atherogenesis. Am. J. Cardiol. 71, B9- B14.
87
(124) Murphy, C.L., Wang, S., Weaver, J., Gertz, M.A., Weiss, D.T., and
Solomon, A. (2004) Renal Apolipoprotein A-I Amyloidosis Associated
with a Novel Mutant Leu64Pro. Am. J. Kidney Dis. 44, 1103-1109.
(125) Brewer, Jr., H.B., Fairwell, T., Kay, L., Meng, M., Ronan, R., Law, S., and
Light, J.A. (1983) Human Plasma Proapo A-I: Isolation and AminoTerminal Sequence. Biochem. Biophys. Res. Comm.113, 626-632.
(126) Nichols, W.C., Dwulet, F.E., Liepnieks, J., and Benson, M.D. (1988)
Variant Apolipoprotein A-I as a Major Constituent of a Human Hereditary
Amyloid. Biochem. Biophys. Res. Comm. 156, 762-768.
(127) Brewer, Jr., H.B., Fairwell, T., LaRue, A., Ronan, R., Houser, a., and
Bronzert, T.J. (1978) The Amino Acid Sequence of Human Apo A-I, an
Apolipoprotein Isolated from High Density Lipoproteins. Biochem.
Biophys. Res. Comm. 80, 623-630.
(128) Nichols, W.C., Gregg, R.E., Brewer, Jr., H.B., and Benson, M.D. (1990) A
Mutation in Apolipoprotein A-I in the Iowa Type of Familial Amyloidotic
Polyneuropathy. Genomics 8, 318-323.
(129) Pisciotta, L., Fasano, T., Calabresi, L., Bellocchio, A., Fresa, R., Borrini,
C., Calandra, S., and Bertolini, S. (2008) A Novel Mutation of the
Apolipoprotein A-I Gene in a Family with Familial Combined
Hyperlipidemia. Atheroscler. 198, 145-151.
(130) Sirtori, C.R., Calabresi, L., Franceschini, G., Baldassarre, D., Amato, M.,
Johansson, J., Salvetta, M., Monteduro, C., Zulli, R., Muiesan, M.L., and
Agabiti-Rosei, E. (2001) Cardiovascular Status of Carriers of the
Apolipoprotein A-I Milano Mutant: The Limone sul Garda Study.
Circulation 103, 1949-1954.
(131) Gangani, R.A., Silva, D., Hilliard, G.M., Li, L., Segrest, J.P., and Davidson,
W.S. (2005) A Mass Spectrometric Determination of the Conformation of
Dimeric Apolipoprotein A-I in Discoidal High Density Lipoproteins.
Biochem. 44, 8600-8607.
(132) Beckstead, J.A., Block, B.L., Bielicki, J.K., Kay, C.M., Oda, M.N., and
Ryan, R.O. (2005) Combined N- and C-Terminal Truncation of Human
Apolipoprotein A-I Yields a Folded, Functional Central Domain.
Biochemistry, 44, 4591-4599.
(133) Franceschini, G., Sirtori, C.R., Capurso, A., Weisgraber, K.H., and Mahley,
R.W. (1980) A-IMilano Apolipoprotein: Decreased High Density Lipoprotein
Cholesterol Levels with Significant Lipoprotein Modifications and without
Clinical Atherosclerosis in an Italian Family. J. Clin. Invest. 66, 892-900.
(134) Hovingh, G.K., Brownlie, A., Bisoendial, R.J., Dube, M.P., Levels, J.H.M.,
Petersen, W., Dullaart, R.P.F., Stroes, E.S.G., Zwinderman, A.H., de Groot,
E., Hayden, M.R., Kuivenhoven, J.A., and Kastelein, J.J.P. (2004) A Novel
Apo A-I Mutation (L178P) Leads to Endothelial Dysfunction, Increased
Arterial Wall Thickness, and Premature Coronary Artery Disease. J. Am.
Coll. Cardiol. 44, 1429-1435.
88
(135) Krieger, M. (1998) The “Best” of Cholesterols, the “Worst” of
Cholesterols: A Tale of Two Receptors. PNAS 95, 4077-4080.
(136) Acton, S., Rigotti, A., Landschultz, K.T., Xu, S., Hobbs, H.H., and Krieger,
M. (1996) Identification of Scavenger Receptor SR-BI as a High Density
Lipoprotein Receptor. Science 271, 518-520.
(137) Rigotti, A., Trigatti, B.L., Penman, M., Rayburn, H., Herz, J., and Krieger,
M. (1997) A Targeted Mutation in the Murine Gene Encoding the High
Density Lipoprotein (HDL) Receptor Scavenger Receptor Class B Type I
Reveals Its Key Role in HDL Metabolism. PNAS 94, 12610-12615.
(138) Mishra, V.K., Anantharamaiah, G.M., Segrest, J.P., Palgunachari, M.N.,
Chaddha, M., Sham, S.W.S., and Krishna, N.R. (2006) Association of a
Model Class A (Apolipoprotein) Amphipathic α Helical Peptide with Lipid:
High Resolution NMR Studies of Peptide-Lipid Discoidal Complexes. J.
Biol. Chem. 281, 6511-6519.
(139) Glomset, J.A. (1968) The Plasma Lecithin: Cholesterol Acyltransferase
Reaction. J. Lipid. Res. 9, 155-167.
(140) Rocken, C., Tautenhahm, J., Buhling, F., Sachwitz, D., Vockler, S., Goette,
A., and Burger, T. (2006) Prevalence and Pathology of Amyloid in
Atherosclerotic Arteries. Arterioscler. Thromb. Vasc. Biol. 26, 676-677.
(141) Obici, L., Franceschini, G., Calabresi, L., Giogetti, S., Stoppini, M.,
Merlini, G., and Bellotti, V. (2006) Structure, Function, and Amyloidogenic
Propensity of Apolipoprotein A-I. Amyloid 13, 191-205.
(142) Liz, M.A., Gomes, C.M., Saraiva, M.J., and Sousa, M.M. (2001) Apo A-I
Cleaved by Transthyretin Has Reduced Ability to Promote Cholesterol
Efflux and Increased Amyloidogenicity. J. Lipid Res. 48, 2385-2395.
(143) Benson, M.D., and Kincaid, J.C. (2007) The Molecular Biology and
Clinical Features of Amyloid Neuropathy. Muscle Nerve 36, 411-423.
(144) Gursky, O., and Atkinson, D. (1996) Thermal Unfolding of Human HighDensity Apolipoprotein A-I: Implications for a Lipid-Free Molten Globular
State. PNAS 93, 2991-2995.
(145) 2004 World Health Statistics (2008) World Health Organization.
http://www.who.int/healthinfo/global_burden_disease/GBD_report_2004up
date_part2.pdf
(146) 2008 World Health Statistics. (2011). World Health Organization.
http://apps.who.int/ghodata/?vid=10012
(147) Mishra, V.K., Palgunachari, M.N., Datta, G., Phillips, M.C., Lund-Katz, S.,
Adeyeye, S.O., Segrest, J.P., and Anantharamaiah, G.M. (1998) Studies of
Synthetic Peptides of Human Apolipoprotein A-I Containing Tandem
Amphipathic α-Helixes. Biochem. 37, 10313-10324.
(148) Maiorano, J.N., Jandacek, R.J., Horace, E.M., and Davidson, W.S. (2004)
Identification and Structural Ramifications of a Hinge Domain in
Apolipoprotein A-I Discoidal High-Density Lipoproteins of Different Size.
Biochem. 43, 11717-11726.
89
(149) Brouillette, C.G., Dong, W.J., Yang, Z.W., Ray, M.J., Protasevich, I.I.,
Cheung, H.C., and Engler, J.A. (2005) Förster Resonance Energy Transfer
Measurements Are Consistent with a Helical Bundle Model for Lipid-Free
Apolipoprotein A-I. Biochem. 44, 16413-16425.
(150) Yui, Y., Aoyama, T., Morishita, H., Takahashi, M., Takatsu, Y., and
Kawai, C. (1988) Serum Prostacyclin Stabilizing Factor Is Identical to
Apolipoprotein A-I (Apo A-I). J. Clin. Invest. 82, 803-807.
(151) Segrest, J.P., Jones, M.K., De Loof, H., Brouillette, C.G.,
Venkatachalapathi, Y.V., and Anantharamaiah, G.M. (1992) The
Amphipathic Helix in the Exchangeable Apolipoproteins: A Review of
Secondary Structure and Function. J. Lipid Res. 33, 141-166.
(152) Segrests, J.P., De Loof. H., Dohlman, J.G., Brouillette, C.G., and
Anantharamaiah, G.M. (1990) Amphipathic Helix Motif: Classes and
Properties. Prot: Struct. Funct. Genet. 8, 103-117.
(153) Soutar, A.K., Hawkins, P.N., Vigushin, D.M., Tennent, G.A., Booth, S.E.,
Hutton, T., Nguyen, O., Totty, N.F., Feest, T.G., Hsuan, J.J., and Pepys,
M.B. (1992) Apolipoprotein A-I Mutation Arg-60 Causes Autosomal
Dominant Amyloidosis. Prot. Natl. Acad. Sci. 89, 7389-7393.
(154) Saito, H., Dhanasekaran, P., Nguyen, D., Deridder, E., Holvoet, P., LundKatz, S., and Phillips, M.C. (2004) α- Helix Formation Is Required for High
Affinity Binding of Human Apolipoprotein A-I to Lipids. J. Biol. Chem.
279, 20974-20981.
(155) Vedhachalam, C., Liu, L., Nickel, M., Dhanasekaran, P., Anantharamaiah,
G.M., Lund-Katz, S., Rothblat, G.H., and Phillips, M.C. (2004) Influence of
Apo A-I Strucutre on the ABCA-I –Mediated Efflux of Cellular Lipids. J.
Biol. Chem. 279, 49931-49939.
(156) Mishra, V.K., and Palgunachari, M.N. (1996) Interaction of Model Class
A1, Class A2, and Class Y Amphipathic Helical Peptides with Membranes.
Biochem. 35, 11210-11220.
(157) Saito, H., Dhanasekaran, P., Nguyen, D., Holvoet, O., Lund-Katz, S., and
Phillips, M.C. (2003) Domain Structure and Lipid Interaction in Human
Apolipoprotein A-I and E, a General Model. J. Biol. Chem. 278, 2322723232.
(158) Xu, F., Vitek, M.P., Colton, C.A., Previti, M.L., Gharkholonarehe, N.,
Davis, J., and Van Norstrand, W.E. (2008) Human Apolipoprotein E
Redistributes Fibrillar Amyloid Deposition in Tg-SwDI Mice. J. Neurosci.
28, 5312-5320.
(159) Booth, D.R., Tan, S.Y., Booth, S.E., Hsuan, J.J., Tottoy, N.F., Nguyen, O.,
Hutton, T., Vigushin, D.M., Tennent, G.A., Hutchinson, W.L., Thomson,
N., Soutar, A.K., Hawkins, P.N., and Pepys, M.B. (1995) A New
Apolipoprotein A-I Variant, Trp50Arg, Causes Hereditary Amyloidosis. Q.
J. Med. 88, 695-702.
90
(160) Panagotopulos, S.E., Horace, E.M., Maiorano, J.N., and Davidson, W.S.
(2001) Apolipoprotein A-I Adopts a Belt-Like Orientation in Reconstituted
High Density Lipoproteins. J. Biol. Chem. 276, 42965-42970.
(161) Vitella, L.B., and Scanu, A. (1976) Studies on Human Serum High Density
Lipoproteins. J. Biol. Chem. 251, 1131-1136.
(162) Ritter, M.C., and Scanu, A.M. (1977) Role of Apolipoprotein A-I in the
Structure of Human Serum High Density Lipoproteins. J. Biol. Chem. 252,
1208-1216.
(163) Datta., G., Epand, R.F., Epand, R.M., Chaddha, M., Kirksey, M.A., Garber,
D.W., Lund-Katz, S., Phillips, M.C., Hama, S., Navab, M., Fogelman,
A.M., Palgunachari, M.N., Segrest, J.P., and Anantharamaiah, G.M. (2004)
Aromatic Residue Position on the Nonpolar Face of Class A Amphipathic
Helical Peptides Determines Biological Activity. J. Biol. Chem. 279,
26509-26517.
(164) Roosbeek, S., Vanloo, B., Duverger, N., Caster, H., Breyne, J., De Beun, I.,
Patel, H., Vandekerckhove, J., Shoulders, C., Rosseneu, M., and Peelman,
F. (2001) Three Arginine Residues in Apolipoprotein A-I Are Critical for
Activation of Lecithin: Cholesterol Acyltransferase. J. Lipid Res. 42, 31-40.
(165) Http://www.lipidsonline.org. (2002) Baylor College of Medicine. Slide 7.
(166) Matulis, D., Baumann, C.G., Bloomfield, V.A., and Lovrien, R.E. (1999) 1Anilino-8-Naphthalene Sulfonate as a Protein Conformational Tightening
Agent. Biopolymers 49, 451-458.
(167) Guglielmi, F., Monti, D.M., Arciello, A., Torrassa, S., Cozzolino, F., Pucci,
P., Relini, A., and Piccoli, R. (2009) Enzymatically Active Fibrils
Generated by the Self-Assembly of the Apo A-I Fibrillogenic Domain
Functionalized with a Catalytic Moiety. Biomaterials 30, 829-835.
(168) Fowler, D.M., Koulov, A.V., Balch, W.E., and Kelly, J.W. (2007)
Functional Amyloid- From Bacteria to Humans. TRENDS Biochem. Sci.
32, 217-224.
(169) DiGaetano, S., Guglielmi, F., Arciello, A., Mangione, P., Monti, M.,
Pagnozzi, D., Raimondi, S., Giorgetti, S., Orru, S., Canale, C., Pucci, P.,
Dobsom, C.M., Bellotti, V., and Piccoli, R. (2006) Recombinant
Amyloidogenic Domain of Apo A-I: Analysis of Its Fibrillogenic Potential.
Biochem. Biophys. Res. Comm. 351, 223-238.
(170) Ishiguro, H., Yoshida, H., Major, A.M., Zhu, T., Babaev, V.R., Linton,
M.F., and Fazio, S. (2001) Retrovirus-mediated Expression of
Apolipoprotein A-I in the Macrophage Protects Against Atherosclerosis in
Vivo. J. Biol. Chem. 276, 36742-36748.
(171) Schaefer, E.J., Lamon-Fava, S., Ordovas, J.M., Cohn, S.D., Schaefer,
M.M., Castelli, W.P., and Wilson, P.W.F. (1994) Factors Associated with
Low and Elevated Plasma High Density Lipoprotein Cholesterol and
Apolipoprotein A-I Levels in the Framingham Offspring Study. J. Lipid
Res. 35, 871-882.
91
(172) Vinchurkar, M.S., Bricarello, D.A., Lagersedt, J.O., Buban, J.O., Stahlberg,
H., Oda, M.N., Voss, J.C., and Parikh, A.n. (2008) Bridging Across Length
Scales: Multi-Scale Ordering of Supported Lipid Bilayers via Lipoprotein
Self-assembly and Surface Patterning. J. Am. Chem. Soc. 130, 1116411169.
(173) Cavigiolio, G., Shao, B., Geier, E.G., Ren, G., Heinecke, J.W., and Oda,
M.N. (2008) The Interplay Between Size, Morphology, Stability, and
Functionality of High-Density Lipoprotein Subclasses. Biochem. 47, 47704779.
(174) Zannis, V.I., Chroni, A., and Krieger, M. (2006) Role of Apo A-I, ABCA1,
LCAT, and SR-BI in the Biogenesis of HDL. J. Mol. Med. 84, 276-294.
(175) Koyama, M., Tanaka, M., Dhanasekaran, P., Lund-Katz, S., Phillips, M.C.,
and Saito, H. (2009) Interaction Between the N- and C- Terminal Domains
Modulates the Stability and Lipid Binding of Apolipoprotein A-I. Biochem.
48, 2529-2537.
(176) Matulis, D., and Lovrien, R. (1998) 1-Anilino-8-Naphthalene Sulfonate
Anion-Protein Binding Depends Primarily on Ion Pair Formation. Biophys.
J. 74, 422-429.
(177) Libby, P. (2002) Inflammation in Atherosclerosis. Nature 420, 868-874.
(178) Gillmore, J.D., Stangou, A.J., Lachmann, H.J., Goodman, H.J.,
Wechalekar, A.D., Acheson, J., Tennent, G.A., Bybee, A., Gilbertson, J.,
Rowczenio, D., O’Grady, J.O., Heaton, N.D., Pepys, M.B., and Hawkins,
P.N. (2006) Organ Transplantation in Hereditary Apolipoprotein A-I
Amyloidosis. Am. J. Transplant. 6, 2342-2347.
(179) Reifenberg, K., Lehr, H.A., Fan, J., Koike, T., Wiese, E., Kupper, I.,
Sagban, T.A., Schaefer, S.C., Zahringer, U., Torzewski, M., Lackner, K.J.,
and Bhakdi, S. (2009) Endotoxin Accelerates Atherosclerosis Independent
of Complement Activation. Thrombosis Res. 123, 653-658.
(180) Tanaka, M., Dhanasekaran, P., Nguyen, D., Ohta, S., Lund-Katz, S.,
Phillips, M.C., and Saito, H. (2006) Contributions of N- and C- Terminal
Helical Segments to the Lipid-Free Structure and Lipid Interaction of
Apolipoprotein A-I. Biochemistry 45, 10351-10358.
(181) Hazenberg, A.J.C., Dikkers, F.G., Hawkins, P.N., Bijzet, J., Rowczenio, D.,
Gilbertson, J., Pasthumus, M.D., Leijsma, M.K., and Hazenberg, B.P.C.
(2009) Laryngeal Presentation of Systemic Apolipoprotein A-I-Derived
Amyloidosis. Laryngoscope 119, 608-615.
(182) Hellquist, H., Olofsson, J., Sokjer, H., and Odkvist, L.M. (1979)
Amyloidosis of the Larynx. Acta Otolaryngol. 88, 443-450.
(183) Semisotnov, G.V., Rodionova, N.A., Razgulyaev, O.I., Uversky, V.N.,
Gripas, A.F., and Gilmanshin, R.I. (1991) Study of the “Molten Globule”
Intermediate State in Protein Folding by a Hydrophobic Fluorescent Probe.
Biopolymers 31, 119-128.
92
(184) Randall, S. (2006) Cloning, Expression, Purification, and Characterization
of ∆1-23 Apolipoprotein A-I.
(185) McGillicuddy, F.C., de la Llera Moya, M., Hinkle, C.C., Joshi, M.R.,
Chiquoine, E.H., Billheimer, J.T., Rothblat, G.H., and Reilly, M.P.(2009)
Inflammation Impairs Reverse Cholesterol Transport In Vivo. Circulation
119, 1135-1145.
(186) Wong, Y.Q., Binger, K.J., Howlett, G.J., and Griffin, M.D.W. (2010)
Methionine Oxidation Induces Amyloid Fibril Formation by Full-Length
Apolipoprotein A-I. PNAS 107, 1977-1982.
(187) Klon, A.E., Segrest, J.P., and Harvey, S.C. (2002) Comparative Models for
Human Apolipoprotein A-I Bound to Lipid in Discoidal High-Density
Lipoprotein Particles. Biochemistry 41, 10895-10905.
(188) Eftink, M.R. (1991) Fluorescence Techniques for Studying Protein
Structure. Methods Biochem. Anal. 35, 127-205.
(189) Sniderman, A.D., Furberg, C.D., Keech, A., Roeters van Lennep, J.E.,
Frohlich, J., Junger, I., and Wallidus, G. (2003) Apolipoproteins Versus
Lipids as Indices of Coronary Risk and as Targets for Statin Treatment. The
Lancet 361, 777-780.
(190) Segrest, J.P., Jones, M.K., Klon, A.E., Sheldahl, C.J., Hellinger, M., De
Loof, H., and Harvery, S.C. (1999) A Detailed Molecular Belt Model for
Apolipoprotein A-I in Discoidal High Density Lipoprotein. J. Biol. Chem.
274, 31755-31758.
(191) Eftink, M.R., and Ghiron, C.A. (1981) Fluorescence Quenching Studies
with Proteins. Anal. Biochem. 114, 199-227.
(192) Gu, F., Jones, M.K., Chen, J., Patterson, J.C., Catte, A., Jerome, W.G., Li,
L., and Segrest, J.P. (2010) Structures of Discoidal High Density
Lipoproteins: A Combined Computational-Experimental Approach. J. Biol.
Chem. 285, 4652-4665.
(193) Chetty, P.S., Mayne, L., Lund-Katz, S., Stranz, D., Englander, S.W., and
Phillips, M.C. (2009) Helical Structure and Stability in Human
Apolipoprotein A-I by Hydrogen Exchange and Mass Spectrometry. PNAS
106, 19005-19010.
(194) Wu, Z., Gogonea, V., Lee, X., Wagner, M.A., Li, X.-M., Huang, Y.,
Undurti, A., May, R.P., Haertlein, M., Moulin, M., Zaccai, G., DiDonato,
J.A., and Hazen, S.L. (2009) Double Superhelix Model of High Density
Lipoprotein. J. Biol. Chem. 284, 36605-36619.
(195) Lazar, K.L., Miller-Auer, H., Getz, G.S., Orgel, J.P.R.O., and Meredith,
S.C. (2005) Helix-Turn-Helix Peptides That Form α-Helical Fibrils: Turn
Sequences Drive Fibril Structure. Biochemistry 44, 12681-12689.
93
(196) Park, K.-H., Shin, D.-G., Kim, J.-R., and Cho, K.-H. (2010) SenescenceRelated Truncation and Multimerization of Apolipoprotein A-I in HighDensity Lipoprotein With an Elevated Level of Advanced Glycated End
Products and Cholesteryl Ester Transfer Activity. J Gerontol A Biol Sci
Med Sci. 65A, 600-610.
(197) Wu, Z., Wagner, M.A., Zheng, L., Parks, J.S., Shy, J.M. 3rd., Smith, J.D.,
Gogonea, V., and Hazen, S.L. (2007) The Refined Structure of Nascent
HDL Reveals a Key Functional Domain for Particle Maturation and
Dysfunction. Nat. Struct. Mol. Biol. 14, 861-868.
(198) Tanaka, M., Koyama, M., Dhanasekaran, P., Nguyen, D., Nickel, M., LundKatz, S., Saito, H., and Phillips, M.C. (2008) Influence of Tertiary Structure
Domain Properties on the Functionality of Apolipoprotein A-I.
Biochemistry 47, 2172-2180.
(199) Gangani, R.A., Silva, D., Huang, R., Morris, J., Fang, J., Gracheva, E.O.,
Ren, G., Kontush, A., Jerome, W.G., Rye, K.-A., and Davidson, W.S.
(2008) Structure of Apolipoprotein A-I in Spherical High Density
Lipoproteins of Different Sizes. PNAS 105, 12176-12181.
(200) Bolognesi, B., Kumita, J.R., Barros, T.P., Esbjorner, E.K., Luheshi, L.M.,
Crowther, D.C., Wilson, M.R., Dobson, C.M., Favrin, G., and Yerbury, J.J.
(2010) ANS Binding Reveals Common Features of Cytotoxic Amyloid
Species. ACS Chemical Biology 5, 735-740.
(201) Ghahghaei, A., and Divsalar, A. (2010) The Effects of Molecular Crowding
on the Amyloid Fibril Formation of α-Lactalbumin and the Chaperone
Action of α-Casein. Protein J. 29, 257-264.
(202) Kalhor, H.R., Kamizi, M., Akbari, J., and Heydari, A. (2009) Inhibition of
Amyloid Formation by Ionic Liquids: Ionic Liquids Affecting Intermediate
Oligomers. Biomacromeolcules 10, 2468-2475.
(203) Marcon, G., Plakouts, G., Canale, C., Relini, A., Taddei, N., Dobson, C.M.,
Ramponi, G., and Chiti, F. (2005) Amyloid Formation from HypF-N under
Conditions in which the Protein Is Initially in Its Native State. J. Mol. Biol.
347, 323-335.
(204) Jones, M.K., Zhang, L., Catte, A., Li, L., Oda, M., Ren, G., and Segrest,
J.P. (2010) Assessment of the Validity of the Double Super Helix Model for
Reconstituted High Density Lipoproteins: A Combined ComputationalExperimental Approach. J. Biol. Chem. 285, 41161-41171.
(205) Lagerstedt, J.O., Cavigiolio, G., and Budamagunta, M.S., Pagani, I., Voss,
J.C., and M.N. Oda. (2011) Structure of Apolipoprotein A-I’s N-terminus
on Nascent High Density Lipoproteins. J. Biol. Chem. 286, 2966-2975.
(206) Fontana, A., de Laureto, P.P., Spolaore, B., Frare, E., Picotti, P., and
Zambonin, M. (2004) Probing Protein Structure by Limited Proteolysis.
Acta Biochim. Polonica 51, 299-321.
94
(207) Phillips, J.C., Wrigglers, W., Li, Z., Jonas, A., and Schulten, K. (1997)
Predicting the Structure of Apolipoprotein A-I in Reconstituted HighDensity Lipoprotein Disks. Biophys. J. 73, 2337-2346.
(208) Tanaka, M., Dhanasekaran, P., Nguyen, D., Nickel, M., Takechi, Y., LundKatz, S., Phillips, M.C., and Saito, H. (2010). Influence of N-terminal Helix
Bundle Stability on the Lipid-Binding Properties of Human Apolipoprotein
A-I. Biochim. Biophys. Acta 1811, 25-30.
(209) Jones, M.K., Gu, F., Catte, A., Li, L., and Segrest, J.P. (2011) “Sticky” and
“Promiscuous,” the Yin and Yang of Apolipoprotein A-I Termini in
Discoidal High-Density Lipoproteins: A Combined ComputationalExperimental Approach. Biochemistry 50, 2249-2263.
(210) Knopp, R.H. (2002) Risk Factors for Coronary Artery Disease in Women.
Am. J. Cardio. 89 (suppl), 28E-35E.
(211) Ji, Y., and Jonas, A. (1995) Properties of an N-terminal Protteolytic
Fragment of Apolipoprotein A-I in Solution and in Reconstituted High
Density Lipoproteins. J. Biol. Chem. 270, 11290-11297.
(212) Mei, X., and Atkinson, D. (2011) Crystal Structure of C-Terminal
Truncated Apolipoprotein A-I Reveals the Assembly of HDL by
Dimerization. J. Biol. Chem. 286, 38570-38582.
(213) Reimers, G.J., Jackson, C.L., Rickards, J., Chan, P.Y., Cohn, J.S., Rye, K.A., Barter, P.J., and Rodgers, K.J. (2011) Inhibition of Rupture of
Established Atherosclerotic Plagues by Treatment with Apolipoprotein A-I.
Cardiovasc. Res. 91, 37-44.
(214) Raimondi, S., Guglielmi, F., Giorgetti, S., Di Gaetano, S., Arciello, A.,
Monti, D.M., Relini, A., Nichino, Da., Doglia, S.M., Natalello, A., Pucci,
P., Mangione, P., Obici, L., Merlini, G., Stoppini, M., Robustelli, P.,
Tartaglia, G.G., Vendruscolo, M., Dobson, C.M., Piccoli, R., and Bellotti,
V. (2011) Effects of the Known Pathogenic Mutations on the Aggregation
Pathway of the Amyloidogenic Peptide of Apolipoprotein A-I. J. Mol. Biol.
407, 465-476.
(215) Monti, D.M., Guglielmi, F., Monti, M., Cozzolino, F., Torrassa, S., Relini,
A., Pucci, P., Arciello, A., and Piccoli, R. (2010) Effects of a Lipid
Environment on the Fibrillogenic Pathway of the N-terminal Polypeptide of
Human Apolipoprotein A-I, Responsible for In Vivo Amyloid Fibril
Formation. Eur Biophys. J. 39, 1289-1299.
(216) Teoh, C.L., Griffin, M.D.W., and Howlett, G.J. (2011) Apolipoproteins and
Amyloid Fibril Formation in Atherosclerosis. Protein Cell 2, 116-127.
(217) Borrell, B. (2009) Fraud Rocks Protein Community. Nature, 462, 970.
(218) Lehrer, S. (1971) Solute Perturbation of Protein Fluorescence. The
Quenching of the Tryptophyl Fluorescence of Model Compounds and of
Lysozyme by Iodide Ion. Biochemistry 10, 3254-3263.
95
(219) Eftink, M.R., and Ghiron, C.A. (1976) Exposure of Tryptophanyl Residues
in Proteins, Quantitative Determination by Fluorescence Quenching
Studies. Biochemistry 15, 672-680.
(220) Fersht, A.R. (1997) Nucleation Mechanisms in Protein Folding. Curr. Opin.
Struct. Biol. 7, 3-9.
(221) Kiss, R.S., Kay, C.M., and Ryan, R.O. (1999) Amphipathic α-Helix Bundle
Organization of Lipid-Free Chicken Apolipoprotein A-I. Biochemistry 38,
4327-4334.
(222) Talussot, Corinne, and Ponsin, G. (1994) The Transformation of Nascent
Disc Shaped High Density Lipoproteins into Mature Spherical High
Density Lipoproteins: Conformational Analysis of Apolipoprotein A-I in
Model Intermediate Particles. J. Theor. Biol. 169, 143-152.
(223) Pownall, H.J., and Smith, L.C. (1974) Interaction of ApolipoproteinAlanine and Apolipoprotein-Glutamine-I with Phosphatidylcholine. Effect
on Solute Quenching of Intrinsic Fluorescence. Biochem. 13, 2590-2593.
(224) Lagerstedt, J.O., Budamagunta, M.S., Liu, G.S., Devalle, N.C., and Oda,
M.N. (2012) The “Beta-Clasp” Model of Apolipoprotein A-I- A Lipid-Free
Solution Structure Determined by Electron Paramagnetic Resonance
Spectroscopy. Biochim. Biophys. Acta 1821, 448-455.
(225) Francis, G.A. (2010) The Complexity of HDL. Biochim. Biophys. Acta
1801, 1286-1293.
(226) Jayaraman, S., Abe-Dohmae, S., Yokomyama, S., and Cavigiolio, G.
(2011) Impact of Self-association on Function of Apolipoprotein A-I. J.
Biol. Chem. 286, 35610-35623.
(227) Zehender, F., Ziegler, A., Schoenfeld, H.J., and Seelig, J. (2012)
Thermodynamics of Protein Self-Association and Unfolding. The Case of
Apolipoprotein A-I. Biochemistry 51, 1269-1280.
(228) Petrlova, J., Duong, T., Cochran, M., Axelsson, A., Morgelin, M., Roberts,
L.M., and Lagerstedt, J.O. (2012) The Fibrillogenic L178H Variant of
Apolipoprotein A-I Forms Helical Fibrils. J. Lipid Res. 53, 390-398.
(229) Gursky, O., Mei, X., and Atkinson, D. (2012) The Crystal Structure of the
C- Terminal Truncated Apolipoprotein A-I Sheds Light on Amyloid
Formation by the N-Terminal Fragment. Biochem. 51, 10-18.
(230) Vos, E.P.P., Bokhove, M., Hesp, B.H., and Broos, J. (2009) Structure of the
Cytoplasmic Loop Between Putative Helices II and III of the Mannitol
Permease of Escherichia coli: A Tryptophan and 5-Fluorotryptophan
Spectroscopy Study. Biochem. 48, 5284-5290.
(231) Alston, R.W., Lasagna, M., and Grimsley, G.R., Scholtz, J.M., Reinhart,
G.D., and Pace, C.N. (2008) Peptide Sequence and Conformation Strongly
Influence Tryptophan Fluorescence. Biophys. J. 94, 2280-2287.
(232) Lindgren, M., Sorgjerd, K., and Hammarstrom, P. (2005) Detection and
Characterization of Aggregates, Prefibrillar Amyloidogenic Oligomers, and
Protofibrils Using Fluorescence Spectroscopy. Biophys. J. 88, 4200-4212.
96
(233) Lindgren, M. and Hammarstrom, P. (2010) Amyloidogenic Oligomers:
Spectroscopic Characterization of Amyloidogenic Protein States. FEBS J.
277, 1380-1388.
(234) Abelein, A., Bolognesi, B., Dobson, C.M., Graslund, A., and Lendel, C.
(2012) Hydrophobicity and Conformational Change as Mechanistic
Determinants for Nonspecific Modulators of Amyloid  Self-Assembly.
Biochem. 51, 126-137.
(235) Duong, T, Roberts, L.M. (2012) Unpublished Research.
(236) Adams, P.D., Chen, Y., Ma, K., Zagorski, M.G., Sonnichsen, F.D.,
McLaughlin, M.L., and Barkley, M.D.(2002) Intramolecular Quenching of
Tryptophan Fluorescence by the Peptide Bond in Cycling Hexapeptides. J.
Am. Chem. Soc. 124, 9278-9286.
(237) Chen, Y., and Barkley, M.D. (1998) Toward Understanding Tryptophan
Fluorescence in Proteins. Biochem. 37, 9976-9982.