The Extracellular Matrix
November 19, 2015
Jeff Miner, Ph.D.
Renal Division
7717 Wohl Clinic
362-8235
minerj@wustl.edu
All multicellular animals have ECM.
Why do all multicellular animals have ECM?
• Acts as structural support to maintain cell organization and integrity
(endothelial tubes of the cardiovascular system; mucosal lining of gut;
skeletal muscle fiber integrity)
• Compartmentalizes tissues (pancreas: islets vs. exocrine component;
skin: epidermis vs. dermis)
• Provides hardness to bone and teeth (collagen fibrils become
mineralized/calcified)
• Presents information to adjacent cells:
• Inherent signals (e.g., RGD motif in fibronectin)
• Bound signals (BMP7, TGF, FGF, SHH, etc.)
• Serves as a highway for cell migration during development (neural
crest migration), in normal tissue maintenance (intestinal mucosa),
and in injury or disease (wound healing and cancer)
Types of ECMs
• Basement membrane (basal lamina)
• Epithelia, endothelia, muscle, fat, nerves
• Elastic fibers
• Skin, lung, large blood vessels
• Stromal or interstitial matrix
• Bone, tooth, and cartilage
• Tendon and ligament
Generalizations
• Most ECM proteins are large, modular,
multidomain glycosylated or glycanated proteins
• Some domains recur in
different ECM proteins
•
•
•
•
•
•
Fibronectin type III repeats
Immunoglobulin repeats
EGF-like repeats
Laminin Globular (LG) domain
others
Exon shuffling the likely mechanism
Perlecan
The Major Basement Membrane Proteins
α1α1α2
LM-511
Perlecan
Major BM Proteins
• Can individually polymerize to form a network
• Laminin
• Collagen IV
• Linkage, regulatory, other functions
• Perlecan, Nidogen, Agrin
• Glucoseaminoglycan (GAG) side chains
• - impart negative charge to the BM
Basement Membranes
• In general, basement membranes appear very similar
to each other by EM.
• But all are not alike!
• There is a wealth of molecular and functional
heterogeneity among basement membranes, due
primarily to isoform variations of basement
membrane components.
Basement Membranes are Involved in a
Multitude of Biological Processes
• Cell proliferation, differentiation, and migration
• Cell polarization and organization, as well as
maintenance of tissue structure
• Separation of epithelia from the underlying
stroma/mesenchyme/interstitium, which contains
a non-basement membrane matrix
• Kidney glomerular filtration (barrier between the
bloodstream and the urinary space)
Laminin
Heterotrimers are composed of
one a , one b, and one c chain.
• Major glycoprotein of basement membranes—
it’s required!
• Chains are evolutionarily related.
• 15 heterotrimers described to date.
• Alpha chains are unique
• contain a C-terminal laminin globular “LG”
domain, ~100 kDa
LM-521
Laminin Trimers Polymerize
• Laminin chains assemble into
trimers in the ER and are
secreted as trimers into the
extracellular space.
• Full-sized laminin trimers can
self-polymerize into a
macromolecular network
through short arm-short arm
interactions.
• The a chain LG domain on the
long arm is left free for
interactions with cellular
receptors.
Receptor-mediated Assembly
Involves LG domains and receptors on the surface of cells.
Results in laminin polymerization and signal transduction.
Sulfated Proteoglycans
• Have protein cores with large
glycosaminoglycan (GAG) side chains (from 1
to >100) attached to serines
• Some PGs contain heparan sulfate
• Perlecan, Agrin, Collagen XVIII
(endostatin)
• Others contain chondroitin, keratan or
dermatan sulfate
• GAG chains are responsible for most of the
biological properties of proteoglycans and
provide negative charge to basement
membranes
• Hydrated
• Enriched in cartilage (lubrication)
Proteases Release Anti-Cancer Peptides
Cleavage of Matrix proteins to peptides
Laminin
cleavages
MMP = Matrix Metalloproteinase
MT-MMP = Membrane-Tethered MMP
From Zent and Pozzi, 2005
The Collagens
•
•
•
•
The most ubiquitous structural protein. A
triple helical protein containing peptide chains
with repeating Gly-Xaa-Yaa (usually Pro)
triplets.
The triple helix forms through the association
of three related polypeptides ( -chains)
forming a coiled coil, with the side chain of
every third residue directed towards the center
of the superhelix. Steric constraints dictate
that the center of the helix be occupied only by
Glycine residues.
Many Proline and Lysine residues are
enzymatically converted to hydroxyproline and
hydroxylysine.
~28 distinct collagen types; each is assigned a
Roman numeral that generally delineates the
chronological order in which the collagens
were isolated/characterized.
Collagen IV Network
Trimers (aka protomers)
associate with each other,
four at the N-terminus and
two at the C-terminus
(hexamer), to form a chicken
wire-like network that
provides strength and
flexibility to the basement
membrane.
Fibrillar Collagens (I, II, III, V)
• Connective tissue proteins that
provide tensile strength
• Triple helix, composed of three a
chains
• Glycine at every third position (GlyX-Y)
• High proline content
• Hydroxylation required for proper
folding and secretion
• Found in bone, skin, tendons,
cartilage, arteries
Biosynthesis of Fibril-forming Collagens
Prolyl hydroxylases
Lysyl hydroxylase
Glycosyltransferases
Procollagen N- and Cproteinases
Lysyl oxidase
Adapted from: Keilty, Hopkinson, Grant. In: Connective Tissue and
Its Inheritable Disorders, Wiley-Liss, 1993.
Collagen Crosslinking
•
•
•
Once formed, collagen fibrils are greatly strengthened by covalent crosslinks that form
between the constituent collagen molecules.
The first step in crosslink formation is the deamination by the enzyme lysyl oxidase of
specific lysine and hydroxylysine side chains to form reactive aldehyde groups.
The aldehydes then form covalent bonds with each other or with other lysine or
hydroxylysine residues.
Collagen Crosslinking
•
•
If crosslinking is inhibited (Lysyl
hydroxylase mutations; vitamin C
deficiency), collagenous tissues
become fragile, and structures such as
skin, tendons, and blood vessels tend
to tear. There are also many bone
manifestations of under-crosslinked
collagen.
Hydroxylation of specific lysines
governs the nature of the cross-link
formed, which affects the
biomechanical properties of the tissue.
Collagen is especially highly
crosslinked in the Achilles tendon,
where tensile strength is crucial.
Scurvy
• Liver spots on skin, spongy gums,
bleeding from mucous membranes,
immobility, depression
• Caused by Vitamin C deficiency
• Ascorbate is required for prolyl
hydroxylase and lysyl hydroxylase
activities
• Acquired disease of fibrillar collagen
Illustration from Man-of-War by Stephen Biesty (Dorling-Kindersley, NY, 1993)
Bone is Composed of Mineralized
Type I Collagen Fibrils
Bone is 70%
mineral and 30%
protein, mostly
collagen
Mineral is Dahllite,
similar to
hydroxyapatite
(contains calcium,
phosphate,
carbonate)
Different Types of Mutations in Collagen I a Chain Genes
Cause Different Disease Severities
Gene
location mutation
Syndrome
COL1A1
17q22
Null alleles
OI type I
Partial deletions; C-terminal
substitutions
OI type II
N-terminal substitutions
OI types I, III or IV
Deletion of exon 6
EDS type VII
COL1A2
7q22.1
Splice mutations; exon deletions OI type I
C-terminal mutations
OI type II, IV
N-terminal substitutions
OI type III
Deletion of exon 6
EDS type VII
Osteogenesis Imperfecta
(brittle bone disease)
Clinical:
Ranges in severity from mild to perinatal lethal
bone fragility, short stature, bone deformities, teeth abnormalities,
gray-blue sclerae, hearing loss
Biochemical:
Reduced and/or abnormal Type I collagen
Molecular Genetics:
Mutations in either Type I collagen gene, COL1A1 or COL1A2, resulting
in haploinsufficiency or disruption of the triple helical domain
(dominant negative: glycine substitutions most common)
COL1 Haploinsufficiency (Dominant)
(α1)2α2
Byers P. Connective Tissue and Its Inheritable Disorders 1993, pp317-50.
Dominant Negative COL1 Mutations
½ of the
trimers are
abnormal
*
Gly subst. in COL4A2
*
Gly subst. in COL4A1
¾ of the
trimers are
abnormal
Byers P. Connective Tissue and Its Inheritable Disorders 1993, pp317-50.
Elastin and Elastic Fibers Exhibit
Rubber-Like Properties
•
•
•
Physiological importance lies in the unique
elastomeric properties of elastin. Found in tissues
in which reversible extensibility or deformability are
crucial, such as the major arterial vessels (esp.
aorta), the lung and the skin.
Elastin is characterized by a high index of
hydrophobicity (90% of all the amino acid residues
are nonpolar). One-third of the amino acid residues
are glycine with a preponderance of the nonpolar
amino acids Ala, Val, Leu, and Ile. As in collagen,
one-ninth of the residues are proline (but with very
little hydroxylation).
Early in development, the elastic fibers consists of
microfibrils, which define fiber location and
morphology. Over time, tropoelastin accumulates
within the bed of microfibrils.
Elastic Fiber Biogenesis
•
Elastic fibers are very complex, difficult to
repair structures
•
There are two morphologically
distinguishable components
•
•
•
Microfibrils
Elastin
Assembly follows a well-defined sequence
of events:
1.
2.
3.
Assembly of microfibrils
Association of tropoelastin aggregates with
microfibrils
Crosslinking of tropoelastins with each other
by lysyl oxidase to form polymers
Shifren and Mecham, 2006
Major steps underlying the assembly
of microfibrils and elastic fibers
Crosslinking
Ramirez, F. et al. Physiol. Genomics 19: 151-154 2004;
doi:10.1152/physiolgenomics.00092.2004
Copyright ©2004 American Physiological Society
Microfibril Components: ~30
• Fibrillin--three forms
• Microfibril-associated glycoproteins
(MAGPs)--two forms
• Latent TGFb Binding Proteins (LTBPs)-four forms
• Proteoglycans, MFAPs, Fibulins,
Emilins, Collagens, Decorin, et al.
Fibrillin-1
Pro
RGD
Fibrillin-2
Gly
RGD
Fibrillins
Fibrillin-3
P/G
RGD
RGD
LTBP-1
Fibrillin-1
EGF
Pro
Fibrillin-2
Gly
Fibrillin-3
RGD
LTBP-2
RGD
LTBP-3
P/G
EGF--Ca Binding
8-Cys (CCC)
Hybrid (CC)
Unique
Glycosylation (potential)
RGD
RGD
LTBP-4
LTBP-1
RGD
EGF
Binding
• Large glycoproteins (~350 kDa) whose primaryEGF--Ca
structures
are
8-Cys (CCC)
LTBP-2
dominated by Ca++ binding EGF domains (cbEGF)
in the presence
Hybrid that,
(CC)
Unique
ofLTBP-3
Ca2+, adopt a rodlike structure
Glycosylation (potential)
RGD
• Limited
intracellular assembly may occur, but microfibril assembly
LTBP-4
initiates at the cell surface after secretion, perhaps with the help of
cellular receptors
RGD
Latent TGFb Binding Proteins
• Members of the fibrillin superfamily
• Maintain TGFb in the inactive state by forming the
“large latent complex”
• TGFb – secreted signaling protein
• Promotes the expression of ECM
Marfan Syndrome
• Caused by dominant Fibrillin-1
(FBN1) mutations
• Haploinsufficiency is the culprit
• Deficiency of elastin-associated
microfibrils
• Syndrome seems to result from
increased TGFb signaling, because
there are not enough microfibrils
present to bind TGFβ (and its
associated proteins) to keep it
inactive.
Cell-Matrix Interactions
November 24, 2015
Jeff Miner, Ph.D.
Renal Division
7717 Wohl Clinic
362-8235
minerj@wustl.edu
Twitter: @JeffMinerPhD
Fibronectin
• A glycoprotein associated with many extracellular
matrices and present in plasma/serum
• Alternative splicing generates many isoforms that
heterodimerize covalently via S-S bonding
• Fibroblasts make it, assemble it, stick to it, and
respond to it
• FN harbors the “RGD” motif (in domain III-10) that
serves as a ligand for various integrins, especially
a5b1
• Fn-/- mouse embryos die at E8.5 due to defects in
the vasculature and in heart development
Mao and Schwarzbauer, Matrix Biol. 2005
Fibronectin and Branching Morphogenesis
Sakai et al., Nature 2003
Fibronectin and Branching Morphogenesis
Inhibiting FN expression
with siRNA reduces
branching
Adding FN promotes
branching
Sakai et al., Nature 2003
Integrins Direct FN Fibril Formation
Secreted compact
soluble FN binds
integrin
FN binding induces
reorganization of actin
and signaling
Cell contractility leads to
changes in FN
conformation, exposing
FN interaction domains
and allowing fibril
formation
Mao and Schwarzbauer, Matrix Biol. 2005
Integrins
• Large family of transmembrane receptors
for extracellular matrix and cell surface
proteins.
• Consist of an a and a b subunit, both with
a single-pass transmembrane domain.
• 16 different a chains and 8 different b
chains associate to form 22 distinct
heterodimers.
• Cytoplasmic tails of both a and b chains
mediate cell signaling events in response
to ligand binding.
Integrins
• Some integrins bind to a specific site on
matrix proteins, such as Arg-Gly-Asp
(RGD), which is found in fibronectin,
vitronectin, tenascin, et al.
• Ligand binding absolutely requires divalent
cation** (Mg++ or Ca++)
• As mechanotransducers, integrins link
the extracellular matrix to the force
generating actin-myosin cytoskeleton.
This both allows the cell to influence
the nature of the extracellular matrix,
and allows the ECM to influence cellular
architecture and behavior.
Integrins Need to be Activated
• Integrin adhesiveness can be
dynamically regulated through a
process termed inside-out signaling.
• Ligand binding transduces signals from
the cellular environment to the interior
of the cell through outside-in
signaling.
• Protein structure analyses have
provided insights into the mechanisms
whereby integrins become activated to
bind ligand and how ligand binding
translates to changes in intracellular
signaling.
Adair and Yeager, Meth. Enzymol. 2007
Model for Integrin Activation
• Involves a switchblade-like
motion when the headpiece
extends
• Downward movement of the a7helix leads to b subunit hybrid
domain swing out, separation of
the knees, and opening of the
headpiece for high affinity
ligand binding
• Activation can occur by PKC
stimulation, GPCR activation, or
binding of proteins such as talin
to the b subunit tail.
• A delicate equilibrium among
the different conformation states
exists.
Anoikis
(Greek for Homelessness)
• Apoptosis induced by
inadequate or
inappropriate cell/matrix
interactions.
• Resistance to anoikis can
lead to metastasis of
epithelium-derived cancer
cells (carcinomas).
Receptors for the Basement Membrane
• Cells are thought to recognize the basement
membrane through receptors that interact with specific
basement membrane components, primarily with
laminin.
• Integrins
• Dystroglycan
• Binding of receptors to the basement membrane can
result in signal transduction and alterations in cell
behavior.
Laminin-Binding Integrins
• a3b1, a6b1, a7b1, and a6b4
• They are found on the surface of many
epithelial (a3 and a6), endothelial (a3, α6),
and muscle (a7) cells.
• They bind primarily to laminin α chains and
demonstrate some specificity.
• Their activities are modulated by members of
the tetraspanin family of 4-pass
transmembrane proteins
• CD9, CD81, CD151
Tetraspanin
Hemidesmosome Assembly vs. Disassembly
• The binding of integrin a6b4 to
plectin plays a central role in HD
assembly. Disrupting the
association between these two
proteins, through serine/threonine
phosphorylation of the b4
cytoplasmic domain (perhaps by
PKC and PKA), is a critical event in
the disassembly of HDs.
• De-phosphorylation of residues
distal to the plectin binding domain
leads to unfolding of the tail,
exposing the binding site for plectin.
• EGF signaling can lead to
phosphorylation of integrin b4 and
HD disassembly.
Discoidin Domain Receptors (DDRs)
Bind fibrillar and BM collagens
 Members of the transmembrane RTK family.
 Two distinct family members: DDR1 and
DDR2
 DDR1: epithelial cells in lung, kidney, colon,
and brain
 DDR2: mesenchymal cells including
fibroblasts, myofibroblasts, smooth muscle,
and skeletal muscle
 The N-terminal DDR discoidin domains are
homologous to discoidin I, a secreted protein
from the slime mold Dictyostelium discoideum
 DDR1 binds to all known collagens, whereas
DDR2 binds to fibrillar collagens
Mechanism of
Activation
• Slow activation process vs. other
RTKs
• Receptors exist as dimers even
before ligand stimulation.
• Collagen stimulation induces rapid
aggregation and internalization of
the receptor
Dystroglycan
• Highly glycosylated
• Dystroglycan is involved in and perhaps necessary for
laminin polymerization at the surface of some cells
• Laminin polymerization initiates basement membrane
formation (certain cell types).
• Dystroglycan KO embryonic stem cells cannot
assemble soluble laminin at the cell surface
Dystroglycan Function Requires
Extensive Glycosylation
• DG isolated from certain muscular dystrophy
patients or mice does not bind a DG antibody
with an epitope dependent on glycosylation
• This DG also shows reduced binding to laminin
• Six glycosylation enzymes are mutated in human
muscular dystrophies (called
“dystroglycanopathies”)
• The protein core of DG has little receptor
function on its own; glycosylation is critical!
• MD is a disease characterized by defective
muscle cell/matrix interactions.
Martin, P. T. Glycobiology 2003 13:55R-66R
Indirect Promoters of Muscle
Pathology in Muscular Dystrophy
A polymorphism/mutation in LTBP4
impacts disease in a mouse model of
muscular dystrophy.
Genetic modifiers of disease
(different backgrounds)
Polymorphisms in human LTBP4
impacts disease in patients with
Duchenne’s muscular dystrophy.
Heydemann et al., J. Clin. Invest. 2009
Basement Membrane Proteins Regulate
Mammary Cell Gene Expression:
Streuli et al,
J. Cell Biol. 1991
What is the Active EHS Matrix Component?
Which Receptors Recognize It?
• Dystroglycan and integrins
cooperate to organize laminin,
transduce the information from
the ECM, induce cell
polarization, and activate
expression of milk proteins.
Weir et al., J. Cell Sci. 2006
Intracellular Protein
Degradation
Chris Weihl MD/PhD
weihlc@neuro.wustl.edu
Department of Neurology
Consequence of impaired protein
degradation
• Protein aggregates
• Ubiquitinated inclusions
• Vacuolation (impairments in autophagy)
• Damaged organelles
• Secondary impairment in other cellular processes
• Cell Death
• Underlying pathogenesis of degenerative disorders
(neurodegeneration, muscle degeneration, liver
degeneration, lung disease, aging)
Protein Degradation
(regulated process)
Turnover of protein is NOT constant
Half lives of proteins vary from minutes to infinity
“Normal” proteins – 100-200 hrs
Short-lived proteins
regulatory proteins
enzymes that catalyze committed steps
transcription factors
Long-lived proteins
Special cases (structural proteins, crystallins)
Protein Degradation
• May depend on tissue distribution
Example: Lactic Acid Dehydrogenase
Tissue
Half-life
Heart
1.6 days
Muscle
31 days
Liver
16 days
• Protein degradation is a regulated process
Example: Acetyl CoA carboxylase
Nutritional state Half-life
Fed
48 hours
Fasted
18 hours
Protein Degradation
 Ubiquitin/Proteasome Pathway
80-90%
Most intracellular proteins
• Lysosomal / Autophagosomal /
Endosomal processes
10-20%
Extracellular proteins
Cell organelles
Some intracellular proteins
UBIQUITIN
 Small peptide that is a “TAG”
 76 amino acids
 C-terminal glycine - isopeptide
bond with the e-amino group of
lysine residues on the substrate
 Attached as monoubiquitin or
polyubiquitin chains
G
K
Ubiquitination of proteins is a FOUR-step process
 First, Ubiquitin is activated by forming
a link to “enzyme 1” (E1 ubiquitin
ligase).
 Then, ubiquitin is transferred to one
of several types of “enzyme 2” (E2).
 Then, “enzyme 3” (E3) catalizes the
transfer of ubiquitin from E2 to a Lys
e-amino group of the “condemned”
protein. Where specificity occurs.
 Lastly, molecules of Ubiquitin are
commonly conjugated to the protein to
be degraded by E3s & E4s (chain
AMP
The UPS is enormous!
The UPS is enormous!
The genes of the UPS constitutes ~5% of the
The genes of the UPS constitutes ~5% of the genome
genome
 E1’s- 1-2 activating enzymes
• E1’s1-210-20
activating
enzymesenzymes
E2’sconjugating
• E2’s10-20
conjugating
enzymes
E3’s500-800
ubiquitin
ligase- drives specificity
• E3’s500-800
specificity
DUBs100ubiquitin
ubiquitin ligasespecificdrives
proteasesregulators of pathway
• DUBs100 ubiquitin specific proteases- regulators of pathway
 De-ubquitinases
PROTEASOME COMPONENTS
20S Proteasome
(Catalytic core, ATP independent)
19S Particle
(cap, recognizes ubiquitin tag,
deubiqutinase (Usp14), ATP dependent
(AAA ATPase, unfolds the protein)
26S
Proteasome
MURF/Atrogin – E3 ligase
Confer specificity for myosin
Knockout of Atrogin (E3) Rescues
atrophy
Dynamic regulation:
Proteasome inhibition increases deubiquitinase activity
Increased expression of deubiquitinase impairs protein degradation
Decrease steady-state levels of aggregate prone proteins in the absence of
DUB Usp14 (pharmacologic inhibitors are coming online)
Lee, BH et al
Nature 467:179-84
2010
Autophagy – lysosomal degradation
• Lysosomal degradation of proteins and organelles
• Occurs via three routes
• Macroautophagy
• Microautophagy (direct uptake of cellular debris via the
lysosome)
• Chaperone mediated autophagy (selective import of
substrates via Hsc70 and Lamp2a)
Direct
invagination of
cytosolic
components
Double layer
membrane
Direct insertion
of proteins into
lysosome
Macroautophagy
Lysosome
FOXO3
Beclin
ATG7
mTOR
ATG5-ATG12-ATG16L1
Induction
(Stress,
starvation,
etc)
Nucleation
Phagophore
Autophagosome
Sequestration
Trafficking
& Cargo loading
“Autophagic Flux”
Autolysosome
Fusion
Degradation
Complete loss of ATG5 leads to lethality
Tissue specific requirements of autophagy
• Degeneration of CNS tissue
• Hepatomegaly in Liver; Komatsu et al 2005
• Atrophy and weakness of skeletal muscle; Masiero et al 2009
• Pathologic similarities
• Ubiquitinated inclusions
• Aberrant mitochondria
• Oxidatively damaged protein
Basal Autophagy
• Autophagy has a “housekeeping” role in the
maintenance of cellular homeostasis
• Autophagy is responsible for the clearance of
ubiquitinated proteins
Selective Autophagy
• Aggregaphagy– p62/SQSTM1, Nbr1
• Mitophagy – Parkin, Nix
• Reticulophagy – endoplasmic reticulum
• Ribophagy – translating ribosomes
• Xenophagy – e.g. Salmonella via optineurin
• Lipophagy – autophagy mediated lipolysis
• Performed by an expanding group of ubiquitin
adaptors
LC3 on the autophagosome membrane
Via receptor, pull autophagic cargo into the
growing autophagosome
Ubiquitin adapter proteins
UBA and LIR domains
p62 as an autophagic tool
• p62 associates with ubiquitinated proteins and LC3
• p62 is an autophagic substrate
• Used to monitor autophagic degradration
• Autophagosome and its contents get degraded
Lysosomal inhibition
Proteasome inhibition
LC3 as an autophagic tool
LC3-I (18kD)
LC3-II (16kD)
(Soluble)
GFP-LC3
starved
Why do autophagosomes
accumulate?
• Upregulation of functional
autophagosomes
• Decrease in autophagosome degradation
or “autophagic flux”
• Phagophore closure
• Autophagosome-lysosome fusion
• Absence of functional lysosomes
Rapamycin as an inducer of
autophagy
 Immunosuppressant used to treat transplant
rejection
 Inhibits the mTOR pathway
 mTOR integrates extrinsic growth signals and cellular
nutrient status and energy state
 Active mTOR
 Protein synthesis and cell growth
 Inactive mTOR (rapamycin, mito damage, starvation)
 Inhibition of protein synthesis and increased autophagic
degradation of protein
TITLE P AGE I NTRODUC TION
THE P ROCESS
H ISTORIC AL L A N D M A R K S
N O N L I N E A R D EVEL O PM E N TAL P ROGRAM
AND
S IGNALING P ATHWAYS BCL-2 P ROTEINS P ORE F ORMING S TRUCTURE
I NITIATIN
T HE C ELL B IOLOGY OF A POPTOSIS
T O L IVE IS
TO
DIE – METALLICA (2007)
Paul H. Schlesinger
Department of Cell Biology and Physiology
Office McDonnell 401
Washington University Medical School
pschlesinger@wustl.edu
December 9, 2014
S CHLESINGER ( WUMS )
A PO PTO SIS
D ECEMBER 9, 2014
77 /
TITLE P AGE I NTRODUC TION
THE P ROCESS
S IGNALING P ATHWAYS BCL-2 P ROTEINS P ORE F ORMING S TRUCTURE
I NITIATIN
M OTIVATIONS FOR THE S TUDY OF C ELL D EATH
D RAMATIC , U NIVERSAL , I NTEGRATED
Apoptosis is characteristic of plants and metazoans≡animals
Allows for non-linear development – e.g. temporary and
scaffold type structures
Immense change in membrane structure during apoptosis,
but membrane integrity is maintained
Most cancers suppress apoptosis – different mutations
Many viruses suppress apoptosis
Apoptosis monitors the cell for stress
Past a threshold – programmed cell death
S CHLESINGER ( WUMS )
A PO PTO SIS
D ECEMBER 9, 2014
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TITLE P AGE I NTRODUC TION
C LASSIFIC ATION
OF
THE P ROCESS
S IGNALING P ATHWAYS BCL-2 P ROTEINS P ORE F ORMING S TRUCTURE
I NITIATIN
C ELLULAR D EATH
H OW C ELLS A CHIEVE M ORTALITY
Apoptosis (Programed Cell Death)
Necrosis – cell loses control of environment, parts of it are genetically
programmed (e.g. autophagy)
Autophagy – see Weihl lecture
Senescence – telomeric shortening, genotoxic
damage
S CHLESINGER ( WUMS )
A PO PTO SIS
D ECEMBER 9, 2014
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TITLE P AGE I NTRODUC TION
C LASSIFIC ATION
OF
THE P ROCESS
S IGNALING P ATHWAYS BCL-2 P ROTEINS P ORE F ORMING S TRUCTURE
I NITIATIN
C ELLULAR D EATH
Cellular death can
be Initiated by:
Stress
Death Receptors
DNA Damage
Cell Infection
S CHLESINGER ( WUMS )
A PO PTO SIS
D ECEMBER 9, 2014
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Nuclear changes associated with Cell Death
Pyknosis – Nuclear Shrinkage
Karyorrhexis – Nuclear/Chromatin Fragmentation
Karyolysis – Nuclear/Chromatin Dissolution
Mitochondrial Permeability Transition
Apoptosis occurs at the Outer Mitochondrial Membrane
Inner mitochondrial membrane is impermeable to protons
Loss of this permeability barrier occurs through a series of events
Impairment in ATP production
Swelling of mitochondria and rupture of OMM -> release of CytC
TITLE P AGE I NTRODUC TION
THE P ROCESS
S IGNALING P ATHWAYS BCL-2 P ROTEINS P ORE F ORMING S TRUCTURE
I NITIATIN
MORPHOLOGICAL
A POPTOSIS : M ORPHOLOGY
Morphological Progression
Retain Membrane Barriers
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TITLE P AGE I NTRODUC TION
THE P ROCESS
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MORPHOLOGICAL
A POPTOSIS : M ORPHOLOGY
Apoptotic Cells Shrink
Intact Membranes — Volume Reduction — Membrane Channel Activity
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TITLE P AGE I NTRODUC TION
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S IGNALING P ATHWAYS BCL-2 P ROTEINS P ORE F ORMING S TRUCTURE
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MORPHOLOGICAL
A POPTOSIS : M ORPHOLOGY
Phagocytosis
phosphotidylserine as a signal for phagocytosis
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APOPTOSIS
Initiation
Genetic
Clonal Selection
Development
Stress
Death Decision
Execution
Cytochrome
c – releasedc from IMM
Cytochrome
Membrane Packaging
Caspases
Nucleases
APOPTOSIS
Initiation
Genetic
Clonal Selection
Development
Stress
?
Death Decision
Execution
Cytochrome c
Membrane Packaging
Caspases
Nucleases
ATP Dependence
TITLE P AGE I NTRODUC TION
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CE,FLY ,M OUSE
A POPTOSIS C HANGE A CROSS C HORDATA
Apotosis is different across species, but same basic rheostat mechanism:
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TITLE P AGE I NTRODUC TION
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CE,FLY ,M OUSE
A POPTOSIS C HANGE A CROSS C HORDATA
Apotosis is different across species, but same basic rheostat mechanism:
Commonalities:
Proteins with Caspase activation and recruitment domains (CARD): Ced4, , dark, Apaf-1
Caspase activation requires mitochondrial membranes and soluble
proteins
The combined protein-protein and protein-membrane interactions are
critical to the regulation of apoptosis
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TITLE P AGE I NTRODUC TION
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M ODES O F A POPTOS IS
E XTRINSIC P ATHWAY OF A POPTOSIS
Engage Cell Surface “Death”
Receptor
- Activates Caspase 8, initiator
caspase
- Cleaves BID
- Causes Mito to
release CytC
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TITLE P AGE I NTRODUC TION
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M ODES O F A POPTOS IS
I NTRINSIC P ATHWAY OF A POPTOSIS
Consensus Intracelluar Stress
Sensing
Conformational change in Bcl2
family proteins (BAX, BAK)
BAX and BAK and/or Mito
permeability transition cause CytC
release
(point of no return)
Caspase 9 activation (Intrinsic
pathway is “Caspase independent”)
BID is an activator of BAX (Changes
its conformation)
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TITLE P AGE I NTRODUC TION
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C ASPASES
E XECUTION BY C ASPASES
cysteine-aspartic-acid-proteases Regulatory and signalling proteins
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D IRECT G ENETIC A N D A POPTOSIS C ONTROL
T UMOR S UPPRESSOR P53
p53 is a tumor suppressor
found in the nucleus and
cytosol.
Genotoxic and oncogenic
stress that stabilizes p53
which transcriptionally
regulates cell-cycle arresting
and apoptosis genes.
The cytosolic p53 induces
apoptosis.
p53 has a BH3 interaction
site – Bcl2 family member?
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TITLE P AGE I NTRODUC TION
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H ISTORY
BCL-2 P ROTEINS
B-cell Lymphoma 2 Gene, BCL -2
The constitutitve expression of this protein resulted
from a gene translocation in chromosomes 14 and
18 of B-cell follicular lymphomas
Loss of Programmed Cell Death
Genetic analysis in C. elegans suggested effector (EGL-1) and
repressor (CED-9) functions
The biochemical basis of BCL -2 action was unknown
BCL -2
associated x-protein BAX
Proposed Neutralizatin Interaction
Homology and Interaction Now Defines a Family of ≈25 Proteins
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TITLE P AGE I NTRODUC TION
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H ISTORY
BCL-2 P ROTEINS
B-cell Lymphoma 2 Gene, BCL -2
Genetic analysis in C. elegans suggested effector (EGL-1) and
repressor (CED-9) functions
The biochemical basis of BCL -2 action was unknown
BCL -2
associated x-protein BAX
Isolated by immunoprecipitation of BCL-2, sequenced
and cloned
Significant, domain specifc homology with BCL-2
Proposed Neutralizatin Interaction
Homology and Interaction Now Defines a Family of ≈25 Proteins
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TITLE P AGE I NTRODUC TION
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S IGNALING P ATHWAYS BCL-2 P ROTEINS P ORE F ORMING S TRUCTURE
I NITIATIN
H ISTORY
BCL-2 P ROTEINS
B-cell Lymphoma 2 Gene, BCL -2
Genetic analysis in C. elegans suggested effector (EGL-1) and
repressor (CED-9) functions
The biochemical basis of BCL -2 action was unknown
BCL -2 associated x-protein BAX
Proposed Neutralizatin Interaction
BCL-2
BAX
overexpression prevents death
overexpression sensitizes to pro-apoptotic stress
Interaction is central to regulation
Homology and Interaction Now Defines a Family of ≈25 Proteins
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F OLD H OMOLOGY – BAX and Colicin
Structural homology to colicins
Colicins – bacterial toxins that make
pores in membranes
Homologus regions of colicins insert
to form channels
The oligomerize in target membranes
forming a large pore
The pore tranports a toxin protein
Molten globule structure
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T HE ROLE OF M ITOCHONDRIA IN A POPTOSIS
Cytochrome c movement into the cytoplasm results in apoptosis
Cytosolic cytochrome c activates the apoptosome, caspase 9, and
effector caspases
During apoptosis Bax translocates to the mitochondria
Bax oligomerizes in the mitochondria – larger, more
stable tetramer form allows CytC to leave
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PORE ACTIVATIO N
B AX A CTIVATION – occurs via BID, inhibited by Bcl-xl
Inactive Bax doesn’t bind to membranes
Bcl-xl (Bcl-x-long) inhibits the activation of Bax
Hypothesis – Bcl-xl changes the PM structure –
which inhibits the bax
Normalized Pore Activation
Bcl-xl binds BID strongly bind to each other
0.9
BCL
cBID (nM)
XL
-
0.6
50
50 100
50
50
0.3
seconds
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BH3-I NHIBITOR
S EQUESTRATION A N D D IRECT A CTIVATION
FCS – Fluorescence Correlation
Spectroscopy
7
Direct measurement of protein interactions at single molecule level
with statistical significance.
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