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PMCOL 412 > Caspases > Flashcards

Caspases Flashcards

1
Q

The most consistent risk factor for developing a neurodegenerative disorder is

A

increasing AGE

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2
Q

Aging begins when

A

development ends

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3
Q

Damage from aging

A
  • Energy Dysregulation (insulin resistance, oxyradical production, glycation)
  • Molecular Damage (DNA oxidation/mutations, protein modifications, protein aggregation, decreased proteostatic mechanisms)
  • cell alterations (microglia activation, impaired detoxification, demyelination, impaired neurogenesis, synaptic dysfunction)
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4
Q

Adapting to aging

A
  • chaperons
  • growth factors
  • anti-apoptotic proteins
  • anti-oxidant enzymes
  • neurogenesis
  • growth of axons and dendrites
  • synaptogenesis
  • DNA repair
  • neural plasticity
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5
Q

Why doesn’t all aging lead to NDDs

A

because the body can adapt to damage caused by aging

Damage is balanced by plasticity and adaptation

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6
Q

Neural plasticity in aging

A

older people use different circuits than young people for the same task, adaptation due to damaged circuits with age

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7
Q

WHy aging causes NDDs

A

When damage is increased and repair/plasticity is decreased due to either genetic or environmental factors
leads to accelerated cell death

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8
Q

AGe-dependent effects of neurotoxin proteins: mHTT

A
  • mice striatums were injected with either mutant or normal HTT
  • mHTT caused neurodegeneration and the older the mouse the greater the loss
  • normal HTT caused no change and didn’t have different effects depending on age
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9
Q

Mechanisms of neurodegeneration

A

Failure in:

  • protein quality control: chaperones and proteasome (increased misfolding and aggregation)
  • autophagy-lysosome pathway
  • mitochondria metabolism: homeostasis and quality control (decreased energy + increased ROS –> no energy for proteostasis and increased damage)
  • excitotoxicity
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10
Q

T/F: only neurons are implicated in NDD

A

FALSE
degen occurs in all brain cells (incl. astrocytes, glia, oligodendrocytes)
- cytokine, etc. release activates glia
- impaired glial function can occur due to abberant proteins

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11
Q

T/F: transplanting neural stem cells works over the long term

A

FALSE
Short term: helpful, as neurons derived from these stem cells integrate and replace those lost
BUT over time as these neurons were exposed to a diseased environment, they will degen like their predecessors

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12
Q

Best therapeutic approaches for NDDs and why

A

Must target the cause of the disease (ex. the protein itself–mHTT, alpha-syn) b/c the damage occurs through many different pathways that attacking one pathway won’t effectively alter disease course.

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13
Q

Caspases and NDDS: old view

A

cell death occurs through caspase-mediated apoptosis

therefore blocking caspases will decrease cell death–but may be too late as it will not decrease cell dysfunction

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14
Q

Caspases and NDD: current view

A

Recent info suggests that caspases are active prior to and independent of apoptosis
therefore their inhibition may work earlier and help increase neuronal function

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15
Q

Caspases

A
  • cysteine aspartic acid-specific proteases
  • are proteases that have an essential Cys residue in their active site and require Asp at the substrate cleavage site.
  • Amino acids that occupy the P4 position are the most important determinant of individual caspases specificity
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16
Q

Initiator caspases: role

A

are usually activated by specific cellular or extracellular stimuli
cleave and activator effector caspase

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17
Q

Effector caspases: role

A

execute the apoptotic program by cleaving several intracellular proteins

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18
Q

Inflammatory caspases: role

A

are involved in activation of cytokines and modulation of immunity

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19
Q

Inflammatory caspases (numbers)

A

caspase 1

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20
Q

Initiator caspases (numbers)

A

caspases 8 and 9

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21
Q

Effector caspases (numbers)

A

caspases 3 & 6

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22
Q

Caspase structure

A

Cys residue in active is essential for protease activity

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23
Q

Caspase binding

A

Asp in P1 position is essential cleavage

Note: P1 is the AA immediately prior to the cleavage site

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24
Q

Caspase binding specificity

A

AAs upstream from the cleavage site (P1, P2, P3, P4) determine substrate specificity
P4 (4 AAs prior to cleavage site) is the single most important determinant of caspase specificity

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25
Q

Activation of caspases

A

For initiator caspases: dimerization is sufficient for activation.
Executioner caspases: require proteolytic removal of the prodomain, and cleavage into large and small subunits

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26
Q

Caspases are expressed as…

A
  • inactive proenzyme (aka zymogen)
  • proenzyme is cleaved at caspase cleavage sequences (asp-X)
  • for executioners –> 2 large and 2 small subunits to form the active tetramer
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27
Q

Extrinsic pathway

A

Binding of FasL/TNFalpha to transmembrane death receptors –> recruitment of FADD that recruits + aggregates Cas 8 –> Cas8 autoprocessing and activation –> active Cas8 cleaves Cas7 + cas 3 –> cas 3 + 7 cleave cellular proteins –> apoptosis

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28
Q

In some cases of extrinsic pathway

A

Cas 8 can cleave BID –> truncated BID (tBID) can promote mitochondrial cytochrome C release –> activates cas 9

similar to the intrinsic pathway events

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29
Q

Intrinsic pathway: crucial event

A

mit outer memb permeabililzation controlled by the BCL-2 family of proteins

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30
Q

Intrinsic pathway

A

Cell stress activates BID/BIK/BIM etc (stress sensors) –> overcome antiapoptotic effects of BCL2 –> promotes oligomerization of BAX-BAK (pro-apoptotic proteins) taht form pores in the mit outer membrane –> allows cyt. c (and other proteins in it intermemb space) to leak into the cytosol –> cyt c release trigger apoptosome activation –> ca 9 activated –> ca 9 cleaves cas 3 and 7 –> cleavage of many cellular substartaes –> apoptosis

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31
Q

How do we make sure apoptosis is prevented when we don’t want it

A

Cas 3 and 7 (effector caspases) are blocked in normal conditions due to their binding by XIAP –> prevents activation without cellular stress

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32
Q

How do we remove the ‘break’ on apoptosis

A

Omi/HTRA2 and Smac/Diablo bind to XIAP during cellular stress to remove the ‘break’ on apoptosis

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33
Q

Once effector caspases are activated

A

once activated caspases (3, 6, 7) orchestrate the dismantling of cell structures through cleavage of specific substrates

34
Q

Dismantling of cell structure

A
  • by effector caspases

- through cleavage of various substrates: ICAD,

35
Q

ICAD

A

when cleaved releases an active DNASase molecule that cleaves DNA –> DNA fragmentation

36
Q

Nuclear fragmentation

A

by caspase-mediated cleavage of nuclear envelope proteins

37
Q

Phosphatidylserine (PS)

A
  • a phospholipid normally found in the inner leaflet of the plasma membrane but is flipped out during apoptosis and acts as an “eat me” signal to fibroblasts/macrophages/microglia for phagocytosis
38
Q

Cytoskeleton contraction in apoptosis

A

proteolysis of cytoskeleton regulator –> contraction of actin cytoskeleton –> plasma memb blebbling

39
Q

T/F caspases only apply to neurodegeneration and not development

A

FALSE
Caspases have a crucial role during neurodevelopment. Half of the neurons
generated during the development of the nervous system is eliminated by apoptosis to remodel brain structures and optimize synaptic connections.

40
Q

Deficiencies in these caspases etc. cause severe developmental abnormalities or death

A

Caspase 3, caspase 9, Apaf1 and cyt.c deficient mice die during embryonic or postnatal development and display severe brain abnormalities

41
Q

Non-apoptotic caspase-3 functions in neurons

A
  • Caspase 3 activity facilitates neurogenesis.
  • Involved in dendritic pruning.
  • Involved in synaptic plasticity.
  • Many proteins that have a role in synaptic plasticity are substrate of caspase 3.
  • Involved in learning and memory. Glutamate can induce caspase-3 activation without inducing cell death
42
Q

LTD occurs through

A

LTD occurs through internalization of AMPARs by GSK3-beta

43
Q

LTD

A

activity-dependent reduction in synaptic efficacy that normally lasts hrs or longer following a long patterned stimulus

44
Q

Reason for LTD

A
  • allow downregulation of synapses

- to prevent hitting a wall of max efficacy and preventing further learning

45
Q

LTD mechanism

A

NMDAR activation by glut –> calcium entry and activation of calcineurin –> active calciuneurin dephosphorylates (intiates) BAD –> BAX-BAK form pore in MIT –> cyt c release –> cas 9 activation –> cas 3 cleaves AKT –> removes break on GSK3-beta –> AMPAR endocytosis

46
Q

Cas 3 role in LTD: precise mechanism

A

AKT normally suppresses LTD by inhibiting GSK3-beta –> Cas 3 cleaves AKT –> GSK3-beta active –> AMPAR endocytosis

47
Q

Cas 3: basics

A

Caspase 3 stimulates AMPA receptors
internalization and long-term depression
(LTD).

48
Q

LTD vs. apoptosis

A
  • Lower levels of Cas 3 activation and transient activation

- enough cas 3 activation for AKT cleavage but not enough for apoptosis

49
Q

Apoptosis and Neurodegenerative Diseases

A
  • Apoptosis is a common form of cell death occurring in ND (but not the only one)
  • Caspases are activated in many NDDs, before cell death occurs (and independently from it)
  • Caspase 3 and 6 can contribute to pathogenesis based on their nonapoptotic functions in modulation of learning and memory and protein cleavage, respectively
  • Inhibitors of caspases and apoptosis may be beneficial in the treatment of neurodegenerative disorders.
50
Q

Cas 3 in AD

A
  • Cas 3 is activated above normal levels in AD mice, notably after 3 months
  • Overactivation of Cas 3 at the synapse may lead to cognitive deficits in AD
51
Q

Caspase 6–unusual because of…

A
  • Caspase 6 is classified as an executioner caspase, however, it is also associated with inflammation.
  • Caspase 6 activation requires removal of the pro-domain of the zymogen and further cleavage to generate the large and the small subunit and expose the active site to the substrates
52
Q

Activation of cas 6

A
  • Can be activated by caspase 3 or 1, but can also self-activate
  • A certain level of activation is also
    possible without cleavage, through a conformational change in the zymogen, which makes the active site accessible.
53
Q

Cas 6 structure

A
  • It has been proposed that is in a dynamic equilibrium between a tetramer (off-state) and a dimer (on state).
  • dimers will self-cleave = be active
  • tetramer active site is hidden = inactive
54
Q

Cas 6 alpha vs beta

A

1) In the alpha-helical structure, a hydrophobic patch is exposed that helps with the initial recruitment of substrates
2) a region of cas 6 continuously interconvert from alpha-helical structure to a beta-strand structure
3) substrate binding stabilizes the beta-strand conformation, which in turn promotes enzyme activity

55
Q

Cas 6 modulation: therapy

A

alterations in beta/alpha structure of cas 6 can alter function:

  • stabilize beta structure = increase activity
  • stabilize alpha-helix = decreased activity
56
Q

Issues with therapeutic modulation of Cas 6 structure

A

alpha helix structure is needed to recruit substrates and stabilizes beta-strand
if we increase beta-strand without alpha strand = no stabilization or binding

57
Q

Increased Cas 6 in AD: old view

A
  • unclear why cas 6 is increased in AD
  • proposed that n-terminal fragment of APP (N-APP) generated by cleavage by beta-secretase or Abeta activate DR-6 receproe –> DR-6 receptor normally activates cas 6 for neuronal axon trimming etc. –> potential hijacking of these processes causing axonal degradation and cell death
58
Q

Cas 6 in AD:

A
  • Caspase 6 expression is upregulated in AD. BUT exact mechanism unknown
  • APP is a substrate for caspase 6. Cleavage by caspase 6 facilitates further production of Aβ
59
Q

Increased Cas 6 in AD: new view

A
  • cell death can occur without DR-6 –> suggests other mechanisms of cas 6 activation in AD
60
Q

Increased Cas 6 in AD: new view

A
  • cell death can occur without DR-6 –> suggests other mechanisms of cas 6 activation in AD
61
Q

Cas 6 in HD

A
  • Active caspase 6 is detected in HD brains
  • Caspase 6 cleaves mHTT generating a highly toxic fragment
  • mHTT 586 fragments binds to caspase 6 zymogen and promotes a conformation capable of substrate binding, also facilitating full caspase activation –> increased Cas 6 activity
62
Q

Cas 6 reisstant mice in HD

A

Caspase-6 resistant mice (C6R) generated by site-directed mutagenesis show decreased pathology compared to regular HD mice (HD53)

63
Q

Cas 6 drugs in HD

A

Drugs that affect caspase-6-mediated cleavage of mutant HTT may have therapeutic application to slow down HD
progression

64
Q

Targeting apoptosis in CANCER

A

Goal is to INCREASE APOPTOSIS of cancer cells
Main approaches:
- Compounds that inhibit Bcl-2
-Antisense oligonucleotides to decrease Bcl2 expression
-Small molecules that activate caspases
-Inhibitors of XIAP

65
Q

Targeting apoptosis in NDDs

A

Goal is to DECREASE APOPTOSIS

  • Caspase inhibitors (by Binding to the active site or Allosteric inhibitors)
  • Minocycline
66
Q

Minocycline

A

Second-generation, semi-synthetic tetracycline analog.

It is highly lipophilic and penetrates the BBB

67
Q

Minocycline mode of action

A

+ Anti-apoptotic action
- Inhibits expression of caspase 1 and 3
- Inhibits mitochondria depolarization (that occurs during apoptosis)
- Increases expression of anti-apoptotic protein (Bcl-2)
+ Anti-inflammatory action
- Modulation of microglia and peripheral immune cells
+ Modulates protein aggregation
+ Inhibits the activity of matrix metalloproteinases (proteins that contribute to NDDs)

68
Q

Minocycline in clinical trials

A
  • One small clinical trials in HD patients showed improvement in treated patients
  • A second small trial did not confirm efficacy
  • A third larger trial was interrupted because of serious hyperpigmentation side effects occurring after 1 year of treatment
  • A large clinical trial for the use of minocycline in ALS was halted due to worsening of the conditions in patients treated with the drug
  • Seems to work in MS
69
Q

Minocycline in MS

A

Phase III trial in MS patients is ongoing.
A previous phase II trial in combination with Copaxone (glatiramer acetate) showed 60% reduction in new lesions in MS patients.

70
Q

Peptidomimetic caspase inhibitors

A
  • Bind the active site. Bulky, electrophilic or anionic

- include Z-VAD and IDN-6556

71
Q

Z-VAD

A
  • Pan-caspase inhibitor (inhibits many caspases)
  • BUT Highest affinity for caspase 3 and 7
  • Clinical development was discontinued because Z-VAD catabolites (mainly fluoroacetate) produce liver damage.
72
Q

IDN-6556

A
  • Peptidomimetic irreversible pan-caspase inhibitor.
  • Well tolerated in phase I and II clinical trial for liver transplantation and hepatites C infection. Developed by Pfizer.
73
Q

Small molecules caspase inhibitors: why and examples

A
  • Developed to overcome limitations of peptidomimetics

incl. VX-799, M-826

74
Q

VX-799

A
  • a small molecule pan-caspase inhibitor.

- In clinical trial for sepsis. Effective in preventing neuronal cell death in vitro.

75
Q

M-826

A
  • Reversible small molecule inhibitor of caspase 3 and 7.
  • Developed by Merck Frosst.
  • Decreases brain tissue damage in murine models of hypoxia-ischemia
76
Q

Specificity in Caspase inhibitor

A

In general, developing specific caspase inhibitors that target the active site has been proven to be difficult, because the active site is very conserved across caspases
Try allosteric inhibitors

77
Q

Genentech/UCSF team to target Cas 6

A

Genentech/UCSF team leads drug discovery efforts to develop Cas 6 inhibitors:

  • small molecules allosteric inhibitors
  • peptide allosteric inhibitors
78
Q

Compound 5

A
  • Compound 5 binds pro-caspase 6, but not the active enzyme, and blocks caspase 6 self-activation.
  • It is one of the first reversible nM-affinity ligands for the dimer interface site of any caspase family member.
  • The molecular characteristics of this compounds, which is neither electrophilic nor anionic, make it a much more attractive lead compound for clinical development.
79
Q

What makes Compound 5 a better drug than previous case inhibitor

A
  • allosteric inhibitor–binds to dimer interface which is less conserved across caspases –> specificity
  • neither electrophilic nor anionic –> better as a drug/ for drug delivery
80
Q

Pep419

A
  • a small peptide that binds caspase 6 and stabilizes/promotes tetramers formation through a novel allosteric mechanism.
  • promotes the tetrameric (i.e. inactive) form–acts as a non-competitive inhib of active cas 6
  • Highly specific to caspase-6
81
Q

How allosteric inhibitors were found for cas 6

A

High-throughput screen (>22K compounds
screened) with HTT as a substrate –>
Identification of a group of irreversible
allosteric C6 inhibitors that bind to the dimer
interface.

PMCOL 412 (35 decks)

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