Caspases Flashcards
The most consistent risk factor for developing a neurodegenerative disorder is
increasing AGE
Aging begins when
development ends
Damage from aging
- 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)
Adapting to aging
- chaperons
- growth factors
- anti-apoptotic proteins
- anti-oxidant enzymes
- neurogenesis
- growth of axons and dendrites
- synaptogenesis
- DNA repair
- neural plasticity
Why doesn’t all aging lead to NDDs
because the body can adapt to damage caused by aging
Damage is balanced by plasticity and adaptation
Neural plasticity in aging
older people use different circuits than young people for the same task, adaptation due to damaged circuits with age
WHy aging causes NDDs
When damage is increased and repair/plasticity is decreased due to either genetic or environmental factors
leads to accelerated cell death
AGe-dependent effects of neurotoxin proteins: mHTT
- 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
Mechanisms of neurodegeneration
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
T/F: only neurons are implicated in NDD
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
T/F: transplanting neural stem cells works over the long term
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
Best therapeutic approaches for NDDs and why
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.
Caspases and NDDS: old view
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
Caspases and NDD: current view
Recent info suggests that caspases are active prior to and independent of apoptosis
therefore their inhibition may work earlier and help increase neuronal function
Caspases
- 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
Initiator caspases: role
are usually activated by specific cellular or extracellular stimuli
cleave and activator effector caspase
Effector caspases: role
execute the apoptotic program by cleaving several intracellular proteins
Inflammatory caspases: role
are involved in activation of cytokines and modulation of immunity
Inflammatory caspases (numbers)
caspase 1
Initiator caspases (numbers)
caspases 8 and 9
Effector caspases (numbers)
caspases 3 & 6
Caspase structure
Cys residue in active is essential for protease activity
Caspase binding
Asp in P1 position is essential cleavage
Note: P1 is the AA immediately prior to the cleavage site
Caspase binding specificity
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
Activation of caspases
For initiator caspases: dimerization is sufficient for activation.
Executioner caspases: require proteolytic removal of the prodomain, and cleavage into large and small subunits
Caspases are expressed as…
- 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
Extrinsic pathway
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
In some cases of extrinsic pathway
Cas 8 can cleave BID –> truncated BID (tBID) can promote mitochondrial cytochrome C release –> activates cas 9
similar to the intrinsic pathway events
Intrinsic pathway: crucial event
mit outer memb permeabililzation controlled by the BCL-2 family of proteins
Intrinsic pathway
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
How do we make sure apoptosis is prevented when we don’t want it
Cas 3 and 7 (effector caspases) are blocked in normal conditions due to their binding by XIAP –> prevents activation without cellular stress
How do we remove the ‘break’ on apoptosis
Omi/HTRA2 and Smac/Diablo bind to XIAP during cellular stress to remove the ‘break’ on apoptosis
Once effector caspases are activated
once activated caspases (3, 6, 7) orchestrate the dismantling of cell structures through cleavage of specific substrates
Dismantling of cell structure
- by effector caspases
- through cleavage of various substrates: ICAD,
ICAD
when cleaved releases an active DNASase molecule that cleaves DNA –> DNA fragmentation
Nuclear fragmentation
by caspase-mediated cleavage of nuclear envelope proteins
Phosphatidylserine (PS)
- 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
Cytoskeleton contraction in apoptosis
proteolysis of cytoskeleton regulator –> contraction of actin cytoskeleton –> plasma memb blebbling
T/F caspases only apply to neurodegeneration and not development
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.
Deficiencies in these caspases etc. cause severe developmental abnormalities or death
Caspase 3, caspase 9, Apaf1 and cyt.c deficient mice die during embryonic or postnatal development and display severe brain abnormalities
Non-apoptotic caspase-3 functions in neurons
- 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
LTD occurs through
LTD occurs through internalization of AMPARs by GSK3-beta
LTD
activity-dependent reduction in synaptic efficacy that normally lasts hrs or longer following a long patterned stimulus
Reason for LTD
- allow downregulation of synapses
- to prevent hitting a wall of max efficacy and preventing further learning
LTD mechanism
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
Cas 3 role in LTD: precise mechanism
AKT normally suppresses LTD by inhibiting GSK3-beta –> Cas 3 cleaves AKT –> GSK3-beta active –> AMPAR endocytosis
Cas 3: basics
Caspase 3 stimulates AMPA receptors
internalization and long-term depression
(LTD).
LTD vs. apoptosis
- Lower levels of Cas 3 activation and transient activation
- enough cas 3 activation for AKT cleavage but not enough for apoptosis
Apoptosis and Neurodegenerative Diseases
- 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.
Cas 3 in AD
- 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
Caspase 6–unusual because of…
- 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
Activation of cas 6
- 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.
Cas 6 structure
- 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
Cas 6 alpha vs beta
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
Cas 6 modulation: therapy
alterations in beta/alpha structure of cas 6 can alter function:
- stabilize beta structure = increase activity
- stabilize alpha-helix = decreased activity
Issues with therapeutic modulation of Cas 6 structure
alpha helix structure is needed to recruit substrates and stabilizes beta-strand
if we increase beta-strand without alpha strand = no stabilization or binding
Increased Cas 6 in AD: old view
- 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
Cas 6 in AD:
- 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
Increased Cas 6 in AD: new view
- cell death can occur without DR-6 –> suggests other mechanisms of cas 6 activation in AD
Increased Cas 6 in AD: new view
- cell death can occur without DR-6 –> suggests other mechanisms of cas 6 activation in AD
Cas 6 in HD
- 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
Cas 6 reisstant mice in HD
Caspase-6 resistant mice (C6R) generated by site-directed mutagenesis show decreased pathology compared to regular HD mice (HD53)
Cas 6 drugs in HD
Drugs that affect caspase-6-mediated cleavage of mutant HTT may have therapeutic application to slow down HD
progression
Targeting apoptosis in CANCER
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
Targeting apoptosis in NDDs
Goal is to DECREASE APOPTOSIS
- Caspase inhibitors (by Binding to the active site or Allosteric inhibitors)
- Minocycline
Minocycline
Second-generation, semi-synthetic tetracycline analog.
It is highly lipophilic and penetrates the BBB
Minocycline mode of action
+ 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)
Minocycline in clinical trials
- 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
Minocycline in MS
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.
Peptidomimetic caspase inhibitors
- Bind the active site. Bulky, electrophilic or anionic
- include Z-VAD and IDN-6556
Z-VAD
- 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.
IDN-6556
- Peptidomimetic irreversible pan-caspase inhibitor.
- Well tolerated in phase I and II clinical trial for liver transplantation and hepatites C infection. Developed by Pfizer.
Small molecules caspase inhibitors: why and examples
- Developed to overcome limitations of peptidomimetics
incl. VX-799, M-826
VX-799
- a small molecule pan-caspase inhibitor.
- In clinical trial for sepsis. Effective in preventing neuronal cell death in vitro.
M-826
- Reversible small molecule inhibitor of caspase 3 and 7.
- Developed by Merck Frosst.
- Decreases brain tissue damage in murine models of hypoxia-ischemia
Specificity in Caspase inhibitor
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
Genentech/UCSF team to target Cas 6
Genentech/UCSF team leads drug discovery efforts to develop Cas 6 inhibitors:
- small molecules allosteric inhibitors
- peptide allosteric inhibitors
Compound 5
- 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.
What makes Compound 5 a better drug than previous case inhibitor
- allosteric inhibitor–binds to dimer interface which is less conserved across caspases –> specificity
- neither electrophilic nor anionic –> better as a drug/ for drug delivery
Pep419
- 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
How allosteric inhibitors were found for cas 6
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.
