Download Caspase 3

Toad Lilies
Elephant Frog
Cell- cell interaction
Structures in the B7/CD28
family. Structures are
modeled on the crystal
determinations. Loops have
been added to one end of
the IgV domains to
emphasize the orientation
of the CDR-like loops and
their interaction with ligand
or lack thereof.
EPSPs: excitatory post-synaptic potentials
IPSPs: inhibitory post-synaptic potentials
What kinds of hormone are there?
Known Hormonal Classes
• Proteins & peptides
chemcases.com/olestra/
images/insulin.jpg
• Lipids (steroids, eicosanoids)
• Amino acid derived
(thyronines, neurotransmitters)
chem.pdx.edu/~wamserc/
ChemWorkshops/ gifs/W25_1.gif
• Gases (NO, CO)
website.lineone.net/~dave.cushman/
epinephrine.gif
What is a hormone receptor?
Hormone Receptors are cellular proteins that
bind with high affinity to hormones & are
altered in shape & function by binding; they
exist in limited numbers.
Binding to hormone is noncovalent &
reversible.
Hormone binding will alter binding to other
cellular proteins & may activate any receptor
protein enzyme actions.
What are the main types of receptors?
Membrane Receptors
Imbedded in target cell membrane; integral
proteins/
glycoproteins; penetrate through membrane
For protein & charged hormones (peptides or
neurotransmitters)
3 major groups: Serpentine = 7 transmembrane
domains, Growth factor/cytokine = 1
transmembrane domain, Ion channels
What are the main types of receptors?
Nuclear Receptors
Nuclear proteins that usually act in pairs & bind to
specific Hormone Recognition Elements (HREs) =
sequences on the DNA in the promoter regions of
target genes
For small, hydrophobic molecules (steroids,
thyroid hormones)
G – G protein, GTP – guanosine triphosphate, PLC - phospholipase C, PLD phospholipase D, PLA2 - phospholipase A2, AC – adenylyl cyclase, IP3 –
inositoltriphosphate, DG – diacylglycerol, cAMP – cyclic adenosine monophosphate,
FFA – free fatty acids, PKC – protein kinase C, PKA – protein kinase A, PKCaM – Ca2+ and
calmodulin dependent protein kinase, PP – phosphoprotein phosphatase
Signal transduction: Adenyl cyclase system
Gs/i/o/x – G proteins, CaM – calmodulin, GTP – guanosine triphosphate, ATP –
adenosine triphosphate, ADP – adenosine diphosphate, cAMP – cyclic adenosine
monophosphate, 5´-AMP - 5´-adenosine monophosphate, PKA – protein kinase A,
PKC – protein kinase C
Signal transduction: Phosphoinositide system
PI-PLC - phospholipase C specific for phosphoinositides, PIP2 - phosphatidylinositol4,5-biphosphate, IP3 - inositol-1,4,5-triphosphate; DG - diacylglycerol; PKC – protein
kinase C
AR – adrenoceptor, G – G protein, PI-PLC – phosphoinositide specific
phospholipase C, IP3 – inositoltriphosphate, DG – diacylglycerol, CaM –
calmodulin, AC – adenylyl cyclase, PKC – protein kinase C
Gs/i/o/x – G proteins, CaM – calmodulin, GTP – guanosine triphosphate,
ATP – adenosine triphosphate, ADP – adenosine diphosphate, cAMP –
cyclic adenosine monophosphate, 5´-AMP - 5´-adenosine
monophosphate, PKA – protein kinase A, PKC – protein kinase C
GC – guanylyl cyclase, CaM – calmodulin, NO – nitric oxide, nNOS – nitric
oxide synthase, ATP – adenosine triphosphate, cGMP – cyclic guanosine
monophosphate
Phosphatidylinositol–3 kinase (PI-3 kinase)
PI-3 kinase
Cell
survival
Cytoskeletal
rearrangements
(Yao and Cooper, 1995)
•
Models of estrogen action. In the
“classical” pathway of estrogen action
(i), estrogen or other selective
estrogen receptor modulators (SERMs)
bind to the estrogen receptor (ER), a
ligand-activated transcription factor
that regulates transcription of target
genes in the nucleus by binding to
estrogen response element (ERE)
regulatory sequences in target genes
and recruiting coregulatory proteins
(CoRegs) such as coactivators. Rapid
or “nongenomic” effects of estrogen
may also occur through the ER
located in or adjacent to the plasma
membrane (ii), which may require the
presence of “adaptor” proteins, which
target the ER to the membrane.
Activation of the membrane ER leads
to a rapid change in cellular signaling
molecules and stimulation of kinase
activity, which in turn may affect
transcription. Lastly, other non-ER
membrane-associated estrogenbinding proteins (EBPs) may also
trigger an intracellular response (iii).
•
Risk factors can be
distinguished in terms of
their ability to cause breast
cancer directly, through
genetic damage, or by
altering hormonal
metabolism. Vulnerability
factors prolong the
duration of breast cell
growth, while contributing
factors can distort
hormone levels.
•
Bifunctional pathways to
breast cancer. Abbreviations:
E2, 17ß-estradiol; E1, estrone;
OHE1, hydroxyestrone; ER,
estrogen receptor. In the
bifunctional pathway, the E2
metabolites affect cell
proliferation and breast
cancer development either
directly via receptorindependent mechanisms
involving structural/functional
alterations in DNA, or
indirectly via receptordependent mechanisms
involving phenotypic growth
regulation. Both mechanisms
eventually upregulate
aberrant proliferation and
development of breast cancer
(37).
Figure 19-34 Fluctuating Levels of Mitotic Cyclin and MPF
During the Cell Cycle
Figure 19-38 Role of the Rb Protein in Cell Cycle Control
Figure 19-39 Role of the p53 Protein in Responding to DNA
Damage
Apoptosis
• Apoptosis (1972)
– Greek word “falling off”
• Built-in (programmed)
mechanism)
• or self-destructionsuicide
• Type of programmed
cell death based upon
morphological features
Programmed cell death during development. Programmed cell death is involved in
forming structures such as the digits of the hand (a), deleting structures such as nearly all
of an insect's larval components (b), controlling cell numbers in, for example, the nervous
system (c) and eliminating abnormal cells such as those that harbour mutations (d).
Studies on the
development of the
nervous system showed
that in the process of
assembling sensory
fields, neurons are
eliminated by orderly
cell death in order to
tailor sensory input to
environmental stimuli
(elimination or
transplantation of limbs
as key examples).
Apoptosis plays in an important role in
normal developmental processes
Jacobson et al (1997) Cell, Vol. 88, 347–
A cancer cell (mauve) undergoing apoptosis
Comparison of cell death by necrosis and apoptosis
Apoptosis
Cell directed "suicide"
Volume loss
Membrane blebbing
Chromatin condensation
Cytochrome C
Caspase
DNA fragmentation
Apoptotic bodies
Non inflammation
Known Apoptotic Stimuli
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Withdrawal of NGF
Etoposide
Actinomycin D
UV radiation
Staurosporin
Enforced m-Myc expression
Glucocorticoids
Physiological Relevance of Apoptosis
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Embryonic Development
Regulation of/by Immune System
Negative Selection
CTL Killing (eg. Immune surveillance, viral infections)
Terminating Active Immune Response
Tight Regulation of Cell Number (eg. BM, GI, Uterus, Skin)
Compensatory Response to Cell Stress
Intrinsic Pathway (e.g. GF removal, XRT, Chemo.)
Extrinsic Pathway (e.g. FAS.Ag, TNF-R Activation)
Senescence (ageing)
Evidence Needed to Verify the Occurrence
of Apoptosis
• Morphological changes
– Formation of apoptotic bodies and blebbing
• DNA fragmentation
– Genomic DNA: Ladder appearance
– Positive TUNEL*
• Effectiveness of endonuclease inhibitors and
protein synthesis inhibition
• Annexin V positive labeling
•
TUNEL- Terminal UDP Nick-End labeling
Morphology of Apoptosis
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–
–
–
–
–
cell shrinkage
extracellular exposure of phosphatidylserine
shows boiling and blebbing
chromatin condenses*-most characteristic feature
DNA is degraded into oligonucleosomal fragments
disassembly into apoptotic bodies
• membrane bound, contains portions of the nucleus and
various organelles
– phagocytosis by neighboring cells
Does not elicit inflammation-hallmark of
apoptosis
mitochondria
Caspase-3 activation via tumor necrosis factor (TNF) family receptors (for
example, Fas), FADD (Fas-activated death domain protein) and caspase-8
represents the extrinsic pathway (blue), whereas caspase-3 activation via the
mitochondrial release of cytochrome c and Apaf-1–mediated processing of caspase9 represents the intrinsic pathway (red)3.
For clarity, not all of the players are shown. Procaspase-3 is shown as a PAC-1–
sensitive dormant single-chain precursor with an N-terminal prodomain (Pro).
During apoptosis, caspase-3 assembles as an active p17-p12 heterotetramer after
proteolytic processing between the p17 and p12 subunits (at Asp175) and removal
of the prodomain2.
PAC-1 is proposed to regulate the Asp-Asp-Asp (DDD) safety catch at amino acids
179–181 in procaspase-3, consequently inducing a conformational change that
leads to proteolytic processing into the active p17 and p12 subunits1.
Cys163 is the catalytic cysteine in the active site of caspase-3; the sequence shown
illustrates its proximity to the DDD safety catch and DDM motif. Although
caspase-7 (not shown) is believed to be a downstream caspase, its position relative
to caspase-3 in apoptosis pathways is unclear.
Functional homologues of caspases and caspase regulators across species are
indicated by the same colour.
Caspase-9 in mammals and Dronc in the fruitfly Drosophila melanogaster are
initiator caspases, whereas caspase-3 and -7 in mammals and Drice in fruitflies
belong to the class of effector caspases.
CED-3 (cell-death abnormality-3) in the nematode worm Caenorhabditis elegans
functions both as an initiator and effector caspase.
The inhibitor of apoptosis (IAP) proteins suppress apoptosis by negatively
regulating the caspases, whereas SMAC (second mitochondria-derived activator
of caspases)/DIABLO (direct IAP-binding protein with low pI) in mammals and
the RHG proteins Reaper, Hid, Grim and Sickle in fruitflies can remove the
IAP-mediated negative regulation of caspases.
AIF, apoptosis-inducing factor;
APAF1, apoptotic-protease-activating factor-1;
Cyt c, cytochrome c;
EndoG, endonuclease G;
HTRA2, high-temperature-requirement protein A2.
Role of Caspases
• Effectors (cell disassembly) (caspases 2,3,6,7)
and initiators (caspases 8,9)
• 14 identified mammalian caspases- 12 in humans
• Cysteine protease that has an absolute
requirement
requirement for cleavage after
aspartic acid
• High specificity for which proteins are digested
– PARP (116 kDa) nuclear polymerase that repairs DNA is
cleaved by caspase 7.
Types of Caspases
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Nedd2/Ich-1/caspase2
YAMA/CPP32/apopain/caspase-3- most characterized
TX/Ich-2/ICErelII/caspase-4
TY/ICErelIII/caspase-5
Mch2/caspase-6
ICE-LAP-3/Mch-3/CMH1/caspase-7
FLICE/MACH/caspase-8-most proximally activated caspase
ICE-LAP-6/caspase-9
Mch-4/FLICE 2/caspase-10
Ich3/caspase-11
Caspases (cysteine aspartic acid-specific proteases) are highly
specific proteases that cleave their substrates after specific
tetrapeptide motifs (P4-P3-P2-P1) where P1 is an Asp residue.
The caspase family can be subdivided into initiators, which are
able to auto-activate and initiate the proteolytic processing of
other caspases, and effectors, which are activated by other
caspase molecules. The effector caspases cleave the vast
majority of substrates during apoptosis.
All caspases have a similar domain structure comprising a propeptide followed by a large and a small subunit (see figure).
The pro-peptide can be of variable length and, in the case of
initiator caspases, can be used to recruit the enzyme to
activation scaffolds such as the APAF1 apoptosome. Two
distinct, but structurally related, pro-peptides have been
identified; the caspase recruitment domain (CARD) and the
death effector domain (DED), and these domains typically
facilitate interaction with proteins that contain the same motifs.
Caspase activation is usually initiated through proteolytic
processing of the caspase between the large and small subunits
to form a heterodimer. This processing event rearranges the
caspase active site into the active conformation. Caspases
typically function as heterotetramers, which are formed
through dimerization of two caspase heterodimers. Initiator
caspases exist as monomers in healthy cells, whereas effector
caspases are present as pre-formed dimers.
Not all mammalian caspases participate in apoptosis. For
example, caspase-1, caspase-4, caspase-5 and caspase-12 are
activated during innate immune responses and are involved in
the regulation of inflammatory cytokine processing (for
example, IL1
and IL18). Interestingly, caspase-12 is
expressed as a truncated, catalytically inactive protein in most
humans (caspase-12S*). However, a subset of individuals of
African descent express full-length caspase-12 (caspase-12L*)
and these individuals appear to be more susceptible to
inflammatory diseases. To date,
400 substrates for the
mammalian caspases have been identified, but the significance
of many of these cleavage events remains obscure.
Caspase 3
• Caspase 3 (CPP32/apopain/YAMA)
– shares similarity to CED 3.
– Protein substrates include:
• PARP (poly ADP ribose) polymerase
• PKC
• sterol-regulatory element-binding protein
• DNA dependent protein kinase (DNA repair)
• U1-associated 70 kDa protein (mRNA splicing)
• MEKK
• DNA fragmentation factor- cytosolic factor that induces
nuclear fragmentation
•
DNA damage-apoptosis model structure. The diagram depicts the general
structure of the model. The model contains mathematical equations describing the
individual protein-protein interactions and catalytic reactions for over 80 species that
fall within the broad outlines shown. The equations are solved in specialised software
including MatLab, and are run multiple times to simulate multi-cell environments.
p53 Dependent Apoptotic Pathways
Schematic representation of the
p53-dependent apoptotic
pathways by transcriptional
activation of BAX, PUMA and
APAF-1.
Frill-necked Lizard
Squid lizard