Caspase Biochemistry Practical Exam Flashcards
what action do proteolytic enzymes take?
hydrolyse peptide bonds
exothermic
give the four classes of proteolytic enzymes
serine proteases
cysteine proteases
aspartyl proteases
metalloproteases
what are the 6 broad categories of protease function?
nutrition invasion evasion adhesion processing signalling
what sort of specificity must occur for protein binding to take place?
substrate must demonstrate genomertical and physical complementarity to enzyme
for each protease, state which sort of AA it prefers to hydrolyse:
a) serine protease
b) chymotrypsin
c) trypsin
d) elastase
a) position R-(n-1)
b) bulky, aromatic
c) + charged
d) small, non-charged
describe serine proteases
- first protease class identified
- i.e. digestive enzyme trypsin
- active site has 3 residues; Ser, His and Asp (sequentially far apart but structurally close)
- substrate specificity can vary
which type of amino acid residue does chymotrypsin prefer?
an aromatic side chain on reside where α-carbon is part of peptide bond to be cleaved
which type of amino acid residue does trypsin prefer?
positively chrgaed Lys or Arg
name some other common ser proteases
elastase collegenase keratinase trypsin plasmin thrombin
give some examples of cysteine proteases
papain: well-studied plant protease
cathepsins: lysosomal proteases
caspases: cys asp proteases involved in apoptosis
calpains: Ca-activated proteases that cleave IC proteins involved in cell motility and adhesion
what is the catalytic mechanism of a cysteine protease?
cys sulfhydryl group
deprotonation of cys SH by adjacent His residue followed by nucleophilic attack of Cys S on peptide carbonyl carbon
thioester linking new carboxy-terminus to Cys thiol is intermediate of reaction (comparable to acyl-enzyme intermediate of Ser protease)
how is cysteine protease mechanism of papain different to chymotryspin?
similar but extra step required
Cys S not electronegative enough to disrupt O double bond
activated by His = tetrahedron 1 intermediate complex
(see lec slide 7 for more details)
what are the aspartyl proteases?
smallest class of human proteases
include digestive enzymes pepsin, lysosomal cathepsin D
renin (kidney enzyme)
HIV-protease
how do all aspartyl proteases operate?
two Asp residues at active site
two Asps work together as general acid-base catalysts
what structure do most aspartic proteases have?
a tertiary structure consisting of two lobes (N- and C- terminal) with approximate two-fold symmetry
how does the initial reaction of an Asp protease work?
one Asp accepts a proton from an actve site H₂O
which attacks carbonyl carbon of peptide linkage
simultaneously, other Asp donates a proton to oxygen of peptide carbonyl group
what is HIV-1 protease?
asp protease which cleaves polyprotin products of HIV genome
what does function of the HIV-1 protease imitate?
mammalian asp protease function
what is the structure of HIV-1 protease?
99 AAs for virus binding to form heterodimer
activates ability of protease to cleave substrate
simpler than mammalian heterodimer which is genetically economical
what is the life cycle of HIV?
- attachment of virus at CD4 receptor and chemokine co-receptors CXCR4 or CCR5
- viral fusion and uncoating
3-5. HIV reverse transciptase makes single DNA copy of viral RNA and makes another to form double-stranded viral DNA - migration to nucleus
7-8. integration of viral DNA into cellular DNA by enzyme integrase
9-11. transcription and RNA processing
12-13. protein synthesis - protease cleaves polyppetides into functional HI proteins and virion assembles
- virion budding
- virion maturation
what processing is required to produce mature HIV proteins?
the gag-pol polyrpitein must be cleaved by another protease
what can protease inhibitors be used as?
AIDS drugs
HIV therapy
how can HIV-1 protease inhibitors be used to prevent HIV production?
during HIV reproduction cycle, specific protease needed to process GAG and POL polyproteins into mature HIV components
if HIV-1 protease can be selectively inhibited, new HIV particles cannot form
if protease activity missing, non-infectous HIV formed
HIV protease inhibitors specific to HIV protease as it differs from human protease
are any protease inhibitors in use?
several marketed as HIV drugs
what is dolutegravir?
integrase inhibitor
effective antiviral for HIV
in HIV life cycle, cleaves polyprotein precursors that will generate core proteins and enzymes of mature virions
how does dolutegravir differ from reverse transcriptase (RT) inhibotors?
inhibits HIV protease by affecting cell directly - halts production of infectious virions
which seven protease inhibitors have been approved by the US FDA?
saquinavir indinavir ritonavir nelfinavir amprenavir lopinavir-ritonavir atazanavir
what are zinc metalloproteases?
include digestive enzymes, carboxypeptidases, matrix metalloproteases, lysosomal proteases
some involved in ECM degradation and tissue remodelling
what do some MMPs have a role in and how?
cell signalling
can release cell surface bound cytokines or growth factors
where have increases MMP activity been found?
in cancers
what are MMPs central to?
tumour invasion
angiogenesis
metastasis
how many members in the MMP family?
24
which proteins can MMPs degrade and (except for MT MMPs) how are they secreted?
variety of ECM proteins
latent proenzymes
what cells are most MMPs secreted by and for?
stromal cells i.e. fibroblasts and macrophages in reponse to signals to tumour cells
what is MMP actiivty controlled by?
TIMP presence (broad spectrum inhibotor proteins that form complexes with MMPs)
what is the importance of TIMP and MMP level?
for invasive behaviour - higher TIMP levels may decrease metastatic potential
what potential do MMPs have in cancer therapy?
synthetic MMPs being developed as anti-metastatic therapies
what do MMPs have involvement in multiple steps of?
tumour invasion
what intersect in cancer and what does this demonstrate?
multiple protease pathways
the role of MMPs and other proteases
how are MMPs initially produced and how are they activated?
as pro-enzymes
by proteolysis/proteolytic cascades (important PTM)
what does understanding protease mechanisms enable?
drug design
how are most proteases synthesised and subsequently activated?
as larger pre-proteins
cleaved during activation to remove inhibitory segment
in some cases, what can the activation of proteases otherwise involve?
dissociation of an inhibitory protein
when may protease activation occur?
after a protease is delivered to a particular cell component or the extracellular milieu
how are caspases involved in the initiation of apoptosis activated by?
interaction with large complexes apoptosomes (scaffolding and activating proteins)
how are protease inhibitors used in healthy cell balance?
endogenous inhibitors to balance protein production/ degradation
what is the general mechanism of action for protease inhibitors?
proteins with domains that enter/block a protease active site to prevent substrate access
what are IAPs and how do they work?
inhibitors of apoptosis
proteins that block apoptosis by binding to and inhibiting caspases
the apoptosis-stimulating protein Smac antagonises the effect of IAPs on caspases
what are TIMPs and how do they work once secreted by cells?
inhibitors of metalloproteases
domain of the inhibitor protein interacts with the catalytic Zn++
what are cystatins and how do they work?
inhibitors of lysosomal cathepsins
some (also called stefins) are found in cytosol, and other in extracellular space
protect cells against cathepsins that may escape from lysosymes
describe the interaction of TIMP with MMP
N-terminal region of TIMP-1 interacting with active site of MMP
TIMP blocks active site of MMP, so it cannot degrade its substrates
what is the evidence that TIMPs contribute to tumor metastasis?
injection of recombinant TIMP-1 reduces experimental metastasis
antisense TIMP-1 increases experimental and spontaneous metastasis
transfection of cells with TIMP-1 and TIMP-2 reduces experimental metastasis
what happens to synthetic MMP inhibitors in experimental metastasis?
they are also inhibited
how much of the human genome is made up of protease degradome?
1.7%
second largest group after ubiquitin-ligase (larger than kinase family)
what are the five classes of human degradome enzymes?
inhibitor threonine serine metallo cysteine aspartic (smaller family)
(further subdivided by role)
where does selective protein degradation occur?
in the proteasome
large protein complex in nucleus and cytosol of eukaryotic cells
how are the four regions of a proteasome categorised?
2 α
2 β
give four essential processes regulated by proteasomal degradation
cell division
apoptosis
differentiation
development
what does a proteasome contain?
2 each of 14 different polypeptides:
7 α-type proteins form each of the two α rings at the end of the cylinder
7 β-type proteins form each of the 2 central β rings
what does the 20S proteasome core complex enclose?
a cavity with 3 compartments joined by narrow passageways
describe the activities that each of the 3 β subunits of different substrate specificity are associated with.
- catalytic β-subunit has a chymotrypsin-like activity with a preference for tyrosine/phenylalanine at P1 (peptide carbonyl position)
- trypsin-like activity with a preference for arginine/lysine at P1 position
- post-glutamyl activity with preference for glutamate or other acidic residue at the P1 position
what are the five main stages of proteasomal protein degradation?
- substrate with poly-ubiquitin chain and protease binding
- unfolding
- de-ubiquitylation
- translocation and hydrolysis
- release
what is apoptosis?
the physiological process of cell auto-destruction (“cell suicide”)
central to cell development and disease
explain some of the biochemistry of the process of apoptosis?
tightly-regulated
active and energy-dependent
has recognisable morphological characteristics
cell debris cleared by phagocytosis
what does no inflammatory response in tissues mean?
no tissue damage
give examples of where apoptosis plays a role
development of interdigital spaces (otherwise webbed hands and feet)
immune system regulation - controls number of WBCs
where can accelerated apoptosis be found?
infertility
immunodeficiency
acute/chronic degenerative disease
what is inhibition of apoptosis characteristic of?
cancer and immune cells reponsible for autoimmunity
what is evasion of apoptosis characteristic of?
a hallmark of cancer (hence a good anticancer drug target)
give three examples of conditions that result in activation of the apoptotic pathway.
DNA damage
inflammation
uncontrolled proliferation
compare apoptosis and necrosis
in apoptosis:
- DNA interchromosomal cleavage but necrosis random degradation
- nuclear chromosomal margination but in necrosis pykonosis
- membrane integrity persists until late but necrosis compromised early
- mitochondria appear normal but in necrosis swollen
- no inflammation but in necrosis there is
- individual cells but in necrosis multiple cells
- cell volume decreased but in necrosis increases early
- apoptotic bodies involved but in necrosis cell lysis instead
give an overview of the biochemical stages of apoptosis
- microvilli contract, intracellular junctions break, chromatin begins to condense
- cell shrinks, chromatin condenses around cell periphery
- cell blobs violently, chromatin condensation continues
- cell fragments into membrane-enclosed apoptotic bodies
- apoptotic bodies phagocytosed by neighbouring cells and roving macrophages
(no inflammation - cell contents never released into environment)
why, as biochemists, are we interested in the mechanistic stages of apoptosis?
because it may reveal therapeutic targets for intervention - either promoting or inhibiting apoptotic pathway
give an overview of the two apoptotic pathways
- extrinsic pathway stimulated by death-inducing (proinflammatory) signals produced by cytotoxic T-cells from IS in response to damaged/infected cells
- intrinsic pathway signals DNA damage, growth factor or cytokine deprivation (in immune cells can act as survival or growth signals)
- caspases cleave cellular components required for normal cell function (esp. nuclear/cytoskeletal proteins) so cells shrink and bleb
- targets are hundreds of intracellular proteins
what is the intrinsic pathway of apoptosis triggered in response to and what does involve the release of?
DNA damage, upregulated oncogenes, deprivation of growth factors, excess Ca2+ oxidants, microtubule-targeting drugs and other types of severe cell stress
involves release of pro-apoptotic factors from mitochondria
give an overview of the stages of the intrinsic pathway of apoptosis
- p53-mediated activation of pro-apoptotic BCL2 family proteins, including PUMA, NOXA, BAX and BAK, leads to release of cyt c and SMAC/DIABLO from mitochondria
- Cyt c binds APAF1, forming apoptosome that activates protease caspase 9
- Cas 9 activates downstream caspases 3, 6, and 7, leading to apoptosis
- SMAC/DIABLO, a pro-apoptotic protein, directly interacts with inhibitor of apoptosis (IAP) proteins, preventing them from attenuating apoptosis
- p53 activates intrinsic pathway through transcriptional upregulation of pro-apoptotic members of Bcl-2 protein family (i.e. PUMA and BAX)
- pro-apoptotic Bcl-2 family members cause release of Cyt c from mitochondria, which forms apoptosome with Cas 9
- Cas 9 activates effector caspase cascade inducing final stages of apoptosis
how do members of the anti-apoptotic BCL2 family (BCL2, BCLXL, and MCL1) regulate the mitochondria-initiated caspase activation pathway?
by preventing release of Cyt c in presence of apoptotic stimuli
When is tumour suppressor inactivated by mutations?
in more than half of human cancers
what can failure of cancer cells to enter apoptosis through p53 mutations result in?
resistance to conventional anticancer therapies (chemotherapy and radiotherapy)
describe the three subsets of regulation of mitochondria by BCL-2 family proteins
- anti-apoptotic (e.g. Bcl-2) inhibit apoptosis
- pro-apoptotic (e.g. BAX) promotes apoptosis
- BH₃-only proteins; upregulated by apoptotic stimulus then activate BAX and BAK
what is BAX regulated by?
p53
under healthy conditions, what is the mechanism for Bcl-2 family protein regulation of apoptosis?
anti-apoptotic proteins such as bcl-2 inhibit pro-apoptotic proteins BAX and BAK
apoptotic stimuli often upregulate BH₃ proteins which overcome anti-apoptotic function, allowing release of BAX and BAK which can then oligomerise which open ion channels in mitochondrial membrane
this causes permeabilisation which allows Cyt c, SMAC, and Omi to be released from mitochondria into cytosol
some cancer cells can avoid apoptosis even if mitochondrial membrane becomes permeable
what interactions occur between Bcl-2 pro- and anti-apoptotic members?
anti-apoptotic protein, BCL-XL is inhibited by binding of pro-apoptotic BH₃-only protein in groove between BH₁ and BH₃
which two things should you note about BH₃-only proteins at a glance?
MAP kinases are also present so phosphorylation can regulate BH₃ proteins too
histone deacetylase inhibitors can change expression levels
describe apoptosome assembling
conformational change occurs in Apaf-1 when cyt-C is released from mitochondria
BH₁₃ domains are 9-16 AAs present in pro-apoptotic proteins whereas anti-apoptotic Bcl-2 family members contain BH₁₄ domains
describe the molecular structure of cytochrome C
heptamer (7 chains)
blue helices in middle bind and activate procaspase to caspase 9
caspase 9 activates procaspases 3, 6 and 7
where is the apoptosome assembled?
in cytosplasm where cyt c is released from mitchondria and binds to Apaf-1
Describe activation of the extrinsic pathway
key functional pro-apoptotic receptors DR49 and DR510 are cell-surface TM receptors expressed in broad range of malignancies
endogenous Apo2L/TRAIL is protein capable of triggering apoptosis in variety of tumor cells, regardless of p53 status
Apo2L/TRAIL does not induce detectable apoptosis in normal tissue cell types including epithelial, endothelial, fibroblastic, SM and astrocytic cells and hematopoietic stem cells
procaspase 8 and 10 are self-cleaved to form activated caspase 8 and 10, and released from DISC into cytoplasm
describe the convergence of intrinsic and extrinsic pathways
extrinsic pathway activated caspase 8 leads to activation of BH₃ interacting-domain death agonist (BID) – a BH₃ only protein which activateds and oligomerises BAD and BAK causing intrinsic pathways to become activated
describe caspase recognition of substrate
recognise tetrapeptide sequence and hydrolyse peptide bond after Asp in substrate
what are the three caspase subtypes and their sequence preference (sp)?
inflammatory caspases (1,4,5) sp: WEHD initiator caspases (2, 8, 9, 10) sp: DEHD; LEXD executioner saspases (6, 3, 7) sp: VEHD; DEVD
what is the function of an executioner caspase?
breakdown cell structures through cleavage of specific substrates:
- actin cytoskeleton
- lamins
- golgi
- translation apparatus
give examples of diseases that can occur from insufficient apoptosis
cancer
leukemia
autoimmune lymphoproliferative syndrome (ALPS)
microbial disease (pathogen)
give examples of diseases that can occur from excessive apoptosis
amyotrophic lateral sclerosis (ALS) AIDS (excessive apoptosis of T-helper cells) Parkinson's disease Huntington's disease cardiac ischaemia cerebral ischaemia microbial disease (host)
describe motoneurone disease (ALS)
progressive and specific loss of motor neurons in brain
familial and sporadic forms
mutation in superoxide dismutase (SOD1) in majority of patients
prolonged period of neuronal caspase-1 activity in ALS mice
progression: elevated caspase-1 and -3 mRNA
what was found with IV injection of broad caspase inhibitor (zVAD-fmk)?
found to be neuroprotective and extended survival of ALS mice
ALS mice bearing which transgene survived longer than ALS mice?
Bcl-2 transgene
what is found in the spinal cord of patients with ALS?
elevated caspase-1 and -3 activity
what causes dysregulation of the intrinsic apoptoticpathway in cancer cells upstream from the mitochondria?
mutations of upstream proteins e.g. p53, PTEN, Akt, Ras
what causes dysregulation of the intrinsic apoptoticpathway in cancer cells at the mitochondria?
expression and activity of Bcl-2 family members
blockade of >15% mitochondrial permeability response (MOMP)
what causes dysregulation of the intrinsic apoptoticpathway in cancer cells downstream from the mitochondria?
elevated expression of inhibitors of apoptosis proteins (IAPs)
epigenetic silencing of Apaf-1, caspase-3 deletion
inactivating caspase mutations
caspase-independent mechanisms e.g. AIF (apoptosis-inducing factor)
in 50% of cancers, there is upregulation of which protein?
BCL2
the majority of anti-cancer drugs depend on which mechanisms to kill cancer cells?
BCL2/BAX mechanisms
which natural product has been approved by WHO and the FDA as an anti-cancer agent and how does it work?
curcumin (in turmeric)
inhibits bcl-2 and XIAP
promotes ability of mitochondrial outer membrane permeability
why are death receptor agonists a potentially attractive therapeutic target in the treatment of colorectal cancer?
FLIP is a multifunctional protein regulating apoptosis (via caspase 8)
upregulated in head and neck cancer and lymphomas correlating with poor prognosis
What is FADD?
Fas-associated with death domain (FADD)- like IL1b converting enzyme inhibitory protein
potently blocks TRAIL-mediated cell death by interfering with caspase 8 activation (want to know if we can down-regulate it)
how many variants are there of FLIP and what do two of them do?
three splice variants
FLIP (S) and FLIP (R) inhibit proteolysis of pro-caspase 8
what does FLIP also have a role in?
cell survival
p53
mutated or altered expression in many cancers
Akt
amplified in many solid tumors - phosphorylates Bad
PTEN
mutated or altered expression in cancers; regulates Akt and subsequent phosphorylation of Bad
CD-95/Fas
mutated and down-regulated in lymphoid and solid tumors
TRAIL-R1/R2
mutated in metastatic breast cancers
MDM2
over-expressed in some tumors - negative regulator of p53
NFkB
deregulated activity in many cancers; transcriptionally activates expression of anti-apoptotic members of Bcl-2 and IAP families
BAX
mutated or decreased expression in tumors
Bak
mutated or decreased expression in some tumors
Bcl2
frequently over-expressed in many tumors
leukaemia marker
Apaf-1
mutated and silenced in melanoma and leukemia
Caspase-8
gene silenced in neuroblastomas
IAPs
frequently over-expressed in cancer
down-regulation of XIAP induces apoptosis in tumors
FLIP
over-expressed in cancers
prevents activation of casp-8
How does FLIP inhibit caspases?
(A) death receptor‐mediated apoptosis. activation of cell death receptors (TRAIL‐R1/R2, or Fas) initiates formation of DISC and activation of extrinsic signalling pathways through FADD‐dependent activation of caspase‐8; this is inhibited by FLIP. short FLIP (S) and FLIP (R) inhibit conversion of pro-caspase to caspase 8 and block apoptosis mediated by TNFR1
(B) TNFα‐induced apoptotic and necroptotic cell death. Upon ligation, TNFR1 recruits TRADD, RIPK1, TRAF2 and cIAP1/2 to form TNFR1 Complex‐I, which activates NFκB. Deubiquitination of RIPK1 by CYLD leads to formation of cytosolic complex‐II, similar in composition to DISC into which procaspase‐8 can be recruited and activated; like DISC, regulated by FLIP in splice form‐specific manner. caspase‐8/FLIP(L) heterodimer cannot cleave procaspases‐3/7 or BID, but can cleave RIPK1; however, if activity repressed, RIPK1 forms necrosome with RIPK3, which activates MLKL leading to necroptosis
(C) regulatory mechanisms of FLIP; several protein kinases been reported to upregulate and/or phosphorylate FLIP, including CAMKII, PKC, PI3K and AKT. Moreover, in response to various cellular stresses, activation of JNK signalling can promote proteasome‐mediated FLIP degradation by E3 ubiquitin ligase ITCH. Alternatively, USP8, a specific FLIP(L) deubiquitinase, stabilizes FLIP(L) by preventing UPS mediated degradation
(D) Autophagy. FLIP suppresses autophagosome formation by two different mechanisms:
(i) inhibiting interaction between Bcl‐2 and Beclin as part of an enzymatic complex with caspase‐10 that cleaves autophagy‐inducing BCLAF1
(ii) bydirect interaction with Atg3 thereby inhibiting autophagosome formation
which extracellular proteases may be involved in cancer formation?
serine proteases (plasmin, urokinase, prostate-specific antigen or PSA)
cathepsins (cathepsin B, cathepsin L, cathepsin D)
ADAMs
MMPs (gelatinasesm stromelysins, collagenasesm MT-MMPs, matrilysin)
