HUANG - LECTURE 5 Flashcards
what are ways in which MOMP is regulated
Bax/Bak: Dimerization (form both homodimer or heterodimer 🡪 Activate MOMP.
BCL-2, BCL-XL, MCL-1: Inhibit Bax/Bak dimerization.
Anti-apoptotic Family
BH3-only proteins (e.g, tBid, Noxa, PUMA):
Inhibit anti-apoptotic BCL-2 family.
Promote Bax/Bak activation.
Pro-Apoptotic family
Genome Structure:
BCL-2 family proteins share the BH1 - 3 domains and form a hydrophobic core.
BAK/BAX and some of the BH3-only proteins (Noxa, PUMA) are P53 targets
P53: Tumor suppressor, which induces apoptosis of cancer cell
what is the activation mechanism of MOMP
In resting state: Bax is cytosolic, Bak is membrane-bound.
Upon Apoptotic signal:
Induce the localization of Bax to the outer membrane of mitochondria
Expose the BH3 domain, which is stimulated by the BH3-only activator (e.g. tBid)
tBid binds to the hydrophobic group of the Bak and stimulates the conformational change (release the helix – BH3 domain)
After the conformational change, tBid leaves
Dimerization of Bax/Bak via BH3 domain + hydrophobic groove.
Oligomerization → pore formation → cytochrome C release.
what is the inhibition mechanism of MOMP
BCL-2 Anti-Apoptotic family also has the BH3 domain, which will bind into the hydrophobic group of the Bak/Bax, preventing dimerization.
BH3-only protein can also bind and inhibit the BCL-2 family.
how do cancer cells evade apoptosis
Upregulation of anti-apoptotic proteins:
Downregulation of pro-apoptotic proteins: BH3-only proteins (e.g., tBid, Puma, Noxa), Bax, Bak.
obatoclax
Obatoclax: BCL-XL/MCL-1 inhibitor.
Induces apoptosis by releasing inhibition on Bax/Bak
Experimental with Obatoclax:
4–8 hours: Cytochrome C release observed.
8–12 hours: Caspase-9 and caspase-3 cleavage; PARP cleavage confirms apoptosis.
Cyto C is released first and lead to the cleavage of Caspase 9.
ABT-737
Targets both BCL-2 and BCL-XL by binding their hydrophobic groove.
MCL-1 binding pocket is structurally different 🡪 does not inhibit it completely
Cancer cells upregulate MCL-1 to evade apoptosis when treated with ABT-737.
eIF4A inhibitors
MCL-1 mRNA has a highly structured 5′ UTR → requires strong helicase activity for translation.
Translation is dependent on eIF4A (a helicase in the eIF4F complex).
Natural compounds isolated from algae and plants.
Inhibit translation of MCL-1 and other oncogenes with structured 5′ UTRs.
Result: ↓ MCL-1 protein 🡪 ↑ apoptosis in cancer cells.
what are IAPs
inhibitors of apoptosis proteins
Examples: XIAP, cIAP, survivin.
Some binds to and Inhibit caspases directly
Some IAPs act as E3 ubiquitin ligases:
Promote proteasomal degradation of caspases.
IAPs can also be inhibited
how are IAPs themselves inhibited
Inhibitors of IAPs: HtrA2, Smac (Double negative becomes positive)
SMAC and HTRA2 inhibit IAPs → IAPs no longer inhibit caspases → caspases activated → apoptosis proceeds.
Upon Apoptotic stimuli, SMAC and Htra2 release
SMAC:
Binds to IAPs like XIAP.
Prevents XIAP from inhibiting caspases.
Functions via competitive binding—sequesters IAPs away from caspases.
Htra2:
A serine protease.
Binds to IAPs and degrades them.
how are apoptotic genes post translationally regulated and an example
Alternative Splicing: Example: Bcl-XI vs. Bcl-XS:
Bcl-Xl (long isoform): Anti-Apoptotic
Contains BH1 domain.
Binds Bax/Bak to inhibit apoptosis.
Bcl-XS (short isoform): Pro-Apoptotic
Lacks BH1 domain → cannot bind Bax/Bak.
Can form heterodimers with Bcl-Xl → sequesters it/keep it away from Bak/Bax.
Results in free Bax/Bak → promotes MOMP → apoptosis.
example of caspase 9 regulation by splicing
Caspase-9a: Pro-Apoptotic
Full-length isoform.
Contains catalytic domain
Caspase-9b: Anti-Apoptotic
Splice variant lacking catalytic domain.
Dominant-negative effect:
Caspase-9 recruited by the apoptosome
Caspase-9b (without catalytic domain) binds apoptosome, preventing the recognition of full-length Caspase 9b by the apoptosome 🡪 preventing the cleavage of it
Competes without downstream cleavage activity.
Overall inhibits apoptosis.
RNA Stability and Regulation of Caspase-9 Expression
HuR (RNA-binding protein):
Binds to caspase-9 mRNA and stabilizes it, promoting protein expression.
Enhances apoptosis by increasing levels of caspase-9.
Northern Blot:
Use of actinomycin D (transcription inhibitor) + Northern blot probes to track RNA decay.
GFP-only control: caspase-9 mRNA degrades faster.
GFP-HuR fusion expression stabilizes caspase-9 mRNA → slower decay.
Caspase-3 and -7 cleave HuR during apoptosis.
Cleaved HuR enhances the stabilization of caspase-9 mRNA even more than full-length.
Positive feedback loop: Caspase activation 🡪 HuR cleavage 🡪 more stable caspase – 9mRNA 🡪 more caspase 9 protein
Full-length HuR stabilizes survivin mRNA
Survivin inhibits apoptosis by inhibiting caspase
translational regulation of apoptosis via eIF4G
eIF4G Cleavage by Caspases:
During early apoptosis, caspase-3.7 cleaves eIF4G.
eIF4G is critical for cap-dependent translation.
Cleavage halts most general protein synthesis.
translational regulation of apoptosis via IRES
Selective Translation via IRES:
IRES = Internal Ribosome Entry Site.
Allows cap-independent translation of mRNAs like:
XIAP (anti-apoptotic).
Outcome Based on Duration of Stress:
Short-term stress (a): Lead to Cell survival
XIAP synthesis dominates → apoptosis blocked.
Chronic exposure(b): induce Apoptosis
Caspase 3/7 can also cleave eIF4G homologs (DAP5, eIF4GI), promoting APAF1(component of apoptosome) translation →, enhancing caspase-9 activation → amplifying apoptosis.
p53 mediated apoptosis and regulation
P53: Transcription factor in the nucleus.
Induces expression of pro-apoptotic genes:
Bax, Puma, Noxa (BH3-only proteins).
In the cytosol, usually they are sequestered by BCL-2 families, making them inactive
Bh3 Only protein can bind to BCL-2, keep them away from p53
P53 can be activated and stimulate the formation of Bax/Bak dimer
P53 can also translocate into the mitochondria to promote mitochondria permeabilization (MOMP), leading to apoptosis.
Also subject to ubiquitination: Different lysine residues determine whether p53 is degraded or stabilized.
Effective Drug Combination Inducing Apoptosis
EGFR inhibitors: Erlotinib, Gefitinib, Cetuximab
BRAF inhibitor: PLX4032
MAPK (Ras → RAF → MEK → ERK) cascade is often hyperactivated in cancers like melanoma and colon cancer.
Mutations in BRAF are oncogenic and can be targeted with RAF inhibitors.
Inhibition of BRAF → suppression of MEK/ERK → feedback activation of EGFR (a receptor tyrosine kinase).
Increased upstream signaling (via EGFR) bypasses BRAF inhibition → reactivates downstream survival pathways.
But if Combine EGFR inhibitors + BRAF inhibitors.
Overcomes feedback resistance and induces effective apoptosis.
SWI/SNF Deficiency Cancers
SWI/SNF complex uses ATP to regulate chromatin accessibility.
SMARCA4: Inactivated by mutations
SMARCA2: epigenetically silenced (promoter repression).
They hydrolyze ATP (ATPase)
SCCOHT (Small Cell Carcinoma of the Ovary, Hypercalcemic Type):
Dual loss of SMARCA4 (mutated) and SMARCA2 (silenced).
Tumors show a complete lack of expression of both subunits (confirmed by IHC).
Also occurs in some non-small cell lung cancer.
Tumors with SMARCA4/2 loss → extremely resistant to chemotherapy.
poor survival
SMARCA4/2 role in regulation of intracellular Calcium Homeostasis
ITPR3 is essential for calcium flux from ER to mitochondria
Normal condition: transient Ca²⁺ release → mitochondrial respiration → ATP production.
Stress: mitochondrial calcium overload.
triggers inner mitochondrial membrane permeabilization, cytochrome C release, and caspase cascade activation.
Loss of SMARCA4 and SMARCA2 leads to reduced ITPR3 expression → reduced mitochondrial Ca²⁺ flux → resistance to apoptosis.
Cancer cells with loss of both are less responsive to apoptosis-inducing chemotherapy.
Epigenetic Reactivation of SMARCA2 to Restore Apoptosis
Although SMARCA2 is epigenetically silenced, its gene is intact.
The use of HDAC inhibitors (e.g., Quisinostat) can reactivate SMARCA2 expression.
Reactivated SMARCA2 restores ITPR3 → restores calcium flux → restores apoptotic sensitivity.
Western Blot: Quisinostat treatment induces SMARCA2 protein expression.
Mitochondrial calcium imaging:
Use of mitochondrial calcium probes and Mito Tracker.
The combination of Quisinostat + cisplatin leads to a high calcium signal in mitochondria.
A single drug shows minimal calcium flux.
