Overview of Caspase Function

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Overview of Caspase Function
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1010 cells are produced every day in a healthy
adult human. The same number of cells must be
removed to maintain homeostasis. This occurs by
Apoptosis and/or Autophagy
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Too Much vs. Too Little Cell Death

• Too much
• Unrestricted cell death
• Neurodegenerative disorders
• Alzheimers
• Parkinsons
• Autoimmune disorders
• Diabetes
• Arthritis
• Other
• Cardiovascular Disease
• AIDS
• Too little
• Unrestricted cell growth
• Cancer

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Apoptosis Is A Type Of Cell Death That Is A
Normal Part of Development

• Formation of independent digits
• Animal development
• Development of the brain
• Reproductive organs

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Apoptosis is carried out by a group of proteases,
called caspases, that dismantle the cell.

• The dismantled cell is packaged into apoptotic
  bodies and phagocytized by macrophages or
  surrounding tissue.
• The word Caspase is an acronym for cysteinyl
  aspartate-specific protease.

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Cellular Proteins Are Cleaved by Caspases All
aspects of the cell are disrupted, dismantled and
packaged
Protein kinases - PKC and many others -
CaMKIV - MEKK1 - Akt1
Cytoskeleton proteins - actin, fodrin Junction
proteins - b-catenin
Signal transduction proteins - Interleukins
- RasGAP kinase modulator - GDI -
Phospholipase C-g1 - SREBP-1 and -2
Cell cycle and proliferation - p21Cip1/Waf1
- p27Kip1 - pRB - CDC27 and nedd4
DNases - CAD/ICAD
Caspase-3
Nuclear structural proteins - Lamin A and B
- Lamin B receptor - Nuclear mitotic apparatus
protein (NuMa) RNA-binding proteins and RNPs
- U1 RNP
DNA metabolism and repair - PARP -
DNA-dependent protein kinases - DNA
topoisomerase II
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As shown in the next slide

• There are fourteen caspases.
• Eleven caspases are found in humans.
• Caspases can be separated into three groups
  based on their function
• Inflammatory caspases
• Initiator caspases
• Effector caspases
• The latter two groups function in apoptosis.
• Caspases are derived from single polypeptide
  chains that consist of N-terminal pro-domains,
  large subunit, intersubunit linker, and small
  subunit.

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Intersubunit
Linker
Prodomain
Group III
1
Large
Small
Caspase 6
293
24
194
179
Effectors of Apoptosis
Caspase 7
Large
Small
1
303
24
198
207
Group II Caspases Asp-Glu-X-Asp
Caspase 3
Large
Small
1
277
181
29
175
Caspase 2
Large
Small
CARD
1
435
16
331
108
153
316
Initiation of Apoptosis
Caspase 9
Large
Small
CARD
1
416
Group III Caspases (Leu/Val)-Glu-X-Asp
331
2
92
139
315
Caspase 8
DED
DED
Large
Small
1
479
99
176
217
374
79
385
Caspase 10
Large
Small
DED
DED
478
1
18
99
113
189
220
372
CARD
Caspase 5
1
Large
Small
44
418
132
311
331
Inflammatory Response
Caspase 4
Large
Small
CARD
1
377
Group I Caspases Trp-Glu-His-Asp
270
2
90
290
Caspase 1
Large
Small
CARD
1
404
2
91
120
298
317
Recognition sequences
Caspase 14
Large
Small
Unknown
1
242
16
153
MacKenzie,S. Clark, A.C. (2007), in press.
Small numbers refer to amino acids in each protein
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As shown in the next slide

• Caspases must be activated to function.
• Caspases are active only as dimers.
• The caspase cascade is responsible for
  activation of caspases
• In the extrinsic pathway
• Death receptors bind external ligands and
  transmit the death signal into the cell.
• Initiator caspases bind to specialized scaffolds
  and are activated.
• In the intrinsic pathway
• Cytochrome c is released from the mitochondria
  and binds to Apaf-1 to form the apoptosome.
• The apoptosome activates procaspase-9.
• Activated initiator caspases activate the
  effector procaspases -3, -6 and -7.

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As shown in the next slide

• Dimerization is a critical event in caspase
  maturation.
• Monomeric caspases have no enzymatic activity.
• One important question is why are the effector
  caspases stable dimers in the cell but have
  little activity?
• A related question is why are the initiator
  caspases stable monomers, but when they form the
  dimer via CARD-CARD interactions in the death
  scaffolds, why does the dimer exhibit relatively
  high activity?
• These two questions are critical to caspase
  function and regulatioin.

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S
S
L
L
Effector Caspases (e.g. caspase-3)
S
S
L
L
Initiator / Inflammatory Caspases (e.g. caspase-1)
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As shown in the next two slides

• It is known that the active site loops of the
  procaspase rearrange upon maturation.
• The protein is cleaved in the intersubunit
  linker.
• This cleavage releases two constrained active
  site loops and allows new interactions to occur
  between L4, L2 and L2.
• Therefore, the active site is comprised of
  interactions between the two monomers, which
  explains why the monomer is not enzymatically
  active.

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Active Site Loop Rearrangements During Activation
Procaspase
Mature Caspase
D175
L3
L2
L4
L1
L2
inhibitor
D175
Chai et al. (2001) Cell 107, 399 Riedl et al.
(2001) PNAS 98, 14790.
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Procaspase
Mature Caspase
W206
L3
L2
L4
L4
R207
L3
W206
R207
L2
L1
L1
H121
C163
R164
C163
H121
R164
W214
Chai et al. (2001) Cell 107, 399 Riedl et al.
(2001) PNAS 98, 14790.
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As shown in the next slide

• We have focused on interactions in the dimer
  interface and their function in active site
  formation as well as dimerization.

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Cys264 - Met268 Ile265 - Ser267 Val266 -
Val266 Ser267 - Ile265 Met268 - Cys264
Dimer interface
Mature caspase-3
Procaspase-3
Cys264 - Met268 Ile265 - Ser267 Glu266 -
Glu266 Ser267 - Ile265 Met268 - Cys264
Cys264 - Met268 Ile265 - Ser267 His266 -
His266 Ser267 - Ile265 Met268 - Cys264
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http//www.nearingzero.net/
One goal of caspase research is to learn to
selectively manipulate the activity of caspases,
either to activate or inactivate the enzymes,
depending on the disease under study.