Apoptosis 2 week 6

How are BH3-only proteins regulated in unstressed cells?

They are kept inactive by being sequestered through interactions with other cellular proteins. For example Bim is sequestered to the microtubule cytoskeleton via the dynein motor complex and Bmf is sequestered to the actin cytoskeleton via the myosin V motor complex. Stress signals cause them to be released.

What is the specificity of BH3-only protein activation?

Different cellular stresses activate specific BH3-only proteins which then inhibit a specific subset of pro-survival Bcl-2 proteins. For example genotoxic damage (via p53) activates Noxa and Puma, while cytokine deprivation activates Bim and Hrk.

Describe the Extrinsic (Death Receptor) Pathway.

1. A Death Ligand (like FasL) binds to its corresponding Death Receptor (like Fas) on the cell surface.

2. The receptors trimerize causing their intracellular Death Domains (DD) to recruit an adaptor protein like FADD.

3. The Death Effector Domain (DED) of FADD then recruits pro-Caspase 8.

4. Bringing multiple pro-Caspase 8 molecules into close proximity within this DISC (Death-Inducing Signaling Complex) allows them to auto-activate.

5. Active Caspase 8 then directly cleaves and activates effector caspases (like Caspase-3) triggering apoptosis.

What is the role of Bid in linking the two apoptotic pathways?

Active Caspase 8 from the extrinsic pathway can cleave the BH3-only protein Bid into a truncated form tBid. tBid then translocates to the mitochondria to inhibit pro-survival Bcl-2 proteins thereby activating the intrinsic pathway and amplifying the death signal.

Name three ways p53 activates apoptosis.

1. Intrinsic Pathway: p53 is a direct transcription factor for pro-apoptotic BH3-only proteins like Puma and Noxa and also for the effector Bax.

2. Extrinsic Pathway: p53 can increase the transcription of the Fas death receptor making the cell more sensitive to death ligand-induced apoptosis.

3. Survival Signal Inhibition: p53 can transcribe IGFBP-3 which sequesters IGF-1/2 thereby inhibiting the pro-survival PI3K/Akt signaling pathway.

How does the PI3K/Akt pathway inhibit apoptosis?

When activated by survival signals (like IGF) Akt/PKB phosphorylates and inactivates several pro-apoptotic targets. This includes the BH3-only protein Bad (preventing it from inhibiting Bcl-2) and Caspase 9 (preventing its activation). It also leads to the activation of NF-κB a transcription factor for anti-apoptotic proteins like IAPs.

How do cancer cells evade death receptor signaling?

1. FLIP Expression: Cancer cells can express FLIP a protein that mimics pro-Caspase 8 but lacks a catalytic domain. FLIP competes for binding to the DISC preventing the recruitment and activation of actual Caspase 8.

2. Decoy Receptors (DcR): Cancer cells can express decoy receptors (like DcR3) that look like death receptors but lack an intracellular death domain. They bind to and sequester death ligands preventing them from activating functional receptors.

What is Dynamic BH3 Profiling?

A laboratory technique used to predict how a patient's cancer cells will respond to chemotherapy. It involves exposing permeabilized cancer cells to various BH3 peptides to measure how "primed" their mitochondria are to undergo apoptosis which can guide clinical decisions.

What is Venetoclax (ABT-199)?

A BH3-mimetic drug that is approved for treating certain leukemias (CLL/SLL). It acts as a specific inhibitor of the pro-survival protein Bcl-2 freeing pro-apoptotic proteins to activate Bax/Bak and trigger apoptosis in cancer cells that are dependent on Bcl-2 for survival.

BH3-only Protein Regulation (Unstressed)

Proteins like Bim and Bmf are kept inactive by being sequestered to cellular structures (Bim to microtubules via dynein; Bmf to actin cytoskeleton via myosin V).

Bim

A BH3-only protein activated by signals like cytokine deprivation or chemotherapy (e.g., Taxol); mediates lymphocyte apoptosis.

Bmf

A BH3-only protein activated by signals like loss of cell-ECM interaction (anoikis) by being released from the actin cytoskeleton.

Extrinsic Pathway (Death Receptor Pathway)

Apoptotic pathway initiated by an extracellular Death Ligand binding to a Death Receptor on the cell surface.

Death Receptor

Cell surface receptors containing an intracellular Death Domain (DD) (e.g. Fas. TNF. TRAIL receptors) that trimerize upon ligand binding.

Death Ligand (e.g. FasL. TNF)

Extracellular signaling molecule that binds to its corresponding death receptor to trigger the extrinsic pathway.

FADD (Fas-Associated Death Domain)

An adaptor protein recruited to the Death Domain of the receptor; it uses its Death Effector Domain (DED) to recruit pro-Caspase 8.

DISC (Death-Inducing Signaling Complex)

The complex formed at the receptor where FADD recruits multiple pro-Caspase 8 molecules leading to their auto-activation (auto-proteolysis).

Caspase 8

The initiator caspase of the extrinsic pathway; once activated in the DISC it directly cleaves and activates Caspase 3/6/7.

Bid

A BH3-only protein that is the key link between the extrinsic and intrinsic pathways.

tBid (Truncated Bid)

The active form of Bid created when Caspase 8 cleaves Bid; tBid moves to the mitochondria to activate Bax/Bak and amplify the death signal.

FLIP

A protein expressed by some cancer cells that mimics pro-Caspase 8 but lacks a catalytic domain; it binds to the DISC and prevents the activation of real Caspase 8 thereby evading apoptosis.

Decoy Receptors (DcR)

Non-signaling receptors (e.g., DcR3) expressed by cancer cells that bind to and sequester Death Ligands, preventing them from activating functional Death Receptors.

PI3K/Akt Pathway (Survival)

A major anti-apoptotic signaling pathway; activated Akt/PKB inhibits apoptosis by phosphorylating and inactivating pro-apoptotic proteins like Bad and Caspase 9.

NF-κB

A transcription factor activated by the PI3K/Akt pathway that transcribes genes for anti-apoptotic proteins including IAPs.

p53 (Apoptosis Activation)

Can promote apoptosis through the intrinsic pathway (by transcribing Puma, Noxa, Bax) and the extrinsic pathway (by transcribing the Fas death receptor).

IGFBP-3 (Insulin-like Growth Factor-Binding Protein 3)

A protein transcribed by p53 that sequesters pro-survival factors (IGF-1/2) inhibiting the PI3K/Akt pathway.

Dynamic BH3 Profiling

A functional assay that uses BH3 peptides to measure how "primed" a cell's mitochondria are for apoptosis used to predict chemoresistance/sensitivity.

Venetoclax (ABT-199)

A BH3-mimetic drug that is a specific inhibitor of the pro-survival protein Bcl-2; used therapeutically in certain Bcl-2-dependent leukemias.