apoptosis 3

What is Apoptosis?

Apoptosis is a form of programmed cell death that occurs in multicellular organisms. It is a highly regulated and controlled process crucial for normal development, tissue homeostasis, and defense against disease.

Why is Apoptosis important?

Apoptosis is essential for:

1. Embryonic development: Sculpting tissues and eliminating unwanted cells (e.g., webbing between fingers).
2. Tissue homeostasis: Maintaining a constant cell population in tissues (balancing cell proliferation and death).
3. Immune system: Eliminating self-reactive lymphocytes and virus-infected cells.
4. Damage control: Removing irreparably damaged or abnormal cells (e.g., cancerous cells, DNA-damaged cells).

What are the morphological hallmarks of Apoptosis?

Key morphological changes include:

1. Cell shrinkage.
2. Chromatin condensation (pyknosis).
3. Nuclear fragmentation (karyorrhexis).
4. Formation of apoptotic bodies.
5. Absence of inflammation.

How does Apoptosis differ from Necrosis?

Apoptosis is distinct from necrosis:

• Apoptosis: Programmed, controlled, no inflammation, cell shrinks, forms apoptotic bodies, phagocytosed.
• Necrosis: Uncontrolled, passive, causes inflammation, cell swells and lyses, releases intracellular contents.

What are the key molecular players in Apoptosis?

The central executioners of apoptosis are a family of proteases called caspases.

What are Caspases?

Caspases (cysteine-aspartic proteases or cysteine aspartases) are a family of proteases that play essential roles in apoptosis and inflammation. They are synthesized as inactive pro-caspases and activated by proteolytic cleavage.

Name the two main types of Caspases involved in Apoptosis.

1. Initiator caspases: Caspase-8, Caspase-9, Caspase-10.
2. Effector (executioner) caspases: Caspase-3, Caspase-6, Caspase-7.

What is the role of Initiator Caspases?

Initiator caspases (e.g., Caspase-8, -9, -10) are activated by apoptotic signals and then cleave and activate effector caspases.

What is the role of Effector Caspases?

Effector caspases (e.g., Caspase-3, -6, -7) cleave various cellular substrates, leading to the systematic dismantling of the cell during apoptosis.

Name some cellular substrates cleaved by effector caspases.

Effector caspases cleave:

1. Nuclear lamins (leading to nuclear fragmentation).
2. Inhibitor of Caspase Activated DNase (ICAD), releasing CAD.
3. Cytoskeletal proteins (leading to cell shrinkage and blebbing).
4. DNA repair enzymes.

What are the two main pathways of Apoptosis?

1. Extrinsic (Death Receptor) Pathway.
2. Intrinsic (Mitochondrial) Pathway.

Describe the Extrinsic Pathway of Apoptosis.

The extrinsic pathway is initiated by extracellular signals (death ligands) binding to cell-surface death receptors. This binding triggers the assembly of a death-inducing signaling complex (DISC), leading to the activation of initiator caspases.

Name some Death Receptors and their ligands.

1. Fas (CD95) receptor binds Fas Ligand (FasL).
2. TNF Receptor 1 (TNFR1) binds TNF- \alpha.
3. TRAIL Receptors (DR4, DR5) bind TRAIL (TNF-related apoptosis-inducing ligand).

What is the Death-Inducing Signaling Complex (DISC)?

The DISC is a multiprotein complex formed after death ligand binding to death receptors. It consists of the death receptor, adaptor proteins (e.g., FADD), and initiator caspases (e.g., pro-caspase-8).

How is Caspase-8 activated in the Extrinsic Pathway?

Within the DISC, multiple pro-caspase-8 molecules are brought into close proximity, leading to their autocatalytic activation.

How does the Extrinsic Pathway directly activate effector caspases?

Activated Caspase-8 directly cleaves and activates effector caspases (e.g., Caspase-3, Caspase-7), initiating the execution phase of apoptosis.

What is the role of Bid in the Extrinsic Pathway?

In some cell types (Type II cells), activated Caspase-8 cleaves Bid to tBid (truncated Bid). tBid then translocates to mitochondria and activates the intrinsic pathway, amplifying the apoptotic signal.

Describe the Intrinsic Pathway of Apoptosis.

The intrinsic pathway is triggered by intracellular stress signals (e.g., DNA damage, growth factor withdrawal, ER stress) that lead to mitochondrial outer membrane permeabilization (MOMP) and the release of pro-apoptotic factors into the cytoplasm.

What is the central event in the Intrinsic Pathway?

Mitochondrial Outer Membrane Permeabilization (MOMP), which allows the release of pro-apoptotic proteins from the intermembrane space into the cytosol.

Name a key pro-apoptotic factor released from mitochondria.

Cytochrome c is a crucial pro-apoptotic factor released from the mitochondrial intermembrane space.

What happens after Cytochrome c is released into the cytosol?

Cytochrome c binds to Apaf-1 (Apoptotic Protease Activating Factor-1), which then oligomerizes to form the Apoptosome.

What is the Apoptosome?

The apoptosome is a large protein complex formed by Apaf-1 and Cytochrome c. It serves as a platform for the activation of initiator caspase-9.

How is Caspase-9 activated in the Intrinsic Pathway?

Within the apoptosome, pro-caspase-9 molecules are brought into proximity, leading to their autocatalytic activation.

How does the Intrinsic Pathway activate effector caspases?

Activated Caspase-9 cleaves and activates effector Caspases (e.g., Caspase-3, Caspase-7), triggering the execution phase of apoptosis.

What is the role of the Bcl-2 protein family in Apoptosis?

The Bcl-2 family proteins are key regulators of the intrinsic pathway, controlling mitochondrial outer membrane permeabilization (MOMP). They can be pro-apoptotic or anti-apoptotic.

Name some Anti-Apoptotic Bcl-2 family proteins.

Bcl-2, Bcl- \text{X}_{\text{L}}, Mcl-1, Bcl-w, A1.

What is the function of Anti-Apoptotic Bcl-2 proteins?

They prevent MOMP by binding to and inhibiting pro-apoptotic Bcl-2 family proteins (e.g., Bax, Bak).

Name some Pro-Apoptotic Bcl-2 family proteins (Effector BH123 proteins).

Bax, Bak.

What is the function of Pro-Apoptotic Bcl-2 proteins (Bax, Bak)?

Upon activation, Bax and Bak oligomerize in the mitochondrial outer membrane, forming pores or channels that lead to MOMP and the release of pro-apoptotic factors.

Name some Pro-Apoptotic Bcl-2 family proteins (BH3-only proteins).

Bad, Bid, Bim, Puma, Noxa.

What is the function of BH3-only proteins?

BH3-only proteins are crucial initiators of the intrinsic pathway. They are activated by various stress signals and promote MOMP by inhibiting anti-apoptotic Bcl-2 proteins and/or directly activating Bax/Bak.

How do BH3-only proteins induce MOMP?

They can either directly activate Bax and Bak (e.g., Bim, Bid, Puma) or indirectly activate them by binding to and neutralizing anti-apoptotic Bcl-2 proteins (e.g., Bad, Noxa).

Which protein inhibits Caspase-8 in the extrinsic pathway, especially in Type I cells?

FLIP (FLICE-like Inhibitory Protein) can bind to FADD and pro-caspase-8 in the DISC, preventing pro-caspase-8 activation.

What is IAPs and their role in Apoptosis?

IAPs (Inhibitors of Apoptosis Proteins) are a family of proteins that directly bind to and inhibit active caspases (e.g., Caspase-3, -7, -9), thereby suppressing apoptosis.

How is the action of IAPs regulated?

IAPs are themselves inhibited by mitochondrial proteins (e.g., Smac/DIABLO, Omi/HtrA2) released during MOMP. These proteins bind to IAPs, relieving caspase inhibition.

What role does DNA damage play in Apoptosis?

Severe DNA damage can trigger the intrinsic apoptotic pathway by activating p53, which in turn induces the expression of pro-apoptotic BH3-only proteins (e.g., Puma, Noxa).

What is p53's role in Apoptosis?

p53, a tumor suppressor protein, orchestrates responses to cellular stress like DNA damage. It can induce cell cycle arrest or, if damage is irreparable, trigger apoptosis primarily by upregulating pro-apoptotic Bcl-2 family members like Puma and Noxa.

What happens to apoptotic bodies after they are formed?

Apoptotic bodies are rapidly recognized and engulfed by phagocytes (e.g., macrophages, neighboring cells) through specific 'eat me' signals on their surface, preventing inflammation.

Name some 'eat me' signals on apoptotic cells.

The most well-known 'eat me' signal is phosphatidylserine (PS), which normally resides on the inner leaflet of the plasma membrane but is flipped to the outer leaflet during apoptosis.

What is the role of Caspase-Activated DNase (CAD)?

CAD is normally inhibited by ICAD. Upon caspase activation, ICAD is cleaved, releasing active CAD, which translocates to the nucleus and cleaves DNA into characteristic internucleosomal fragments (e.g., 180-200 bp multimers).

What is anoikis?

Anoikis is a specific form of apoptosis triggered by the detachment of anchorage-dependent cells from the extracellular matrix. It prevents cells from surviving in inappropriate locations.

How is Apoptosis involved in cancer?

Defects in apoptotic pathways are a hallmark of cancer. Cancer cells often develop mechanisms to evade apoptosis, leading to uncontrolled proliferation and resistance to treatment. This can involve overexpression of anti-apoptotic proteins (e.g., Bcl-2) or mutations in pro-apoptotic proteins (e.g., p53).

How is Apoptosis involved in neurodegenerative diseases?

Excessive or inappropriate apoptosis can contribute to neuronal loss in neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's disease.

What is the concept of 'decoy receptors' in the extrinsic pathway?

Decoy receptors (e.g., DcR1, DcR2 for TRAIL) are receptors that can bind death ligands but lack an intracellular death domain, thus failing to transmit an apoptotic signal. They can compete with functional death receptors, thereby inhibiting apoptosis.

What is 'autophagy' and how does it relate to apoptosis?

Autophagy is a cellular process involving the degradation and recycling of cellular components. While primarily a survival mechanism, under certain conditions, it can either promote or inhibit apoptosis, and there's often crosstalk between the two pathways.

What is the role of endoplasmic reticulum (ER) stress in Apoptosis?

Severe or prolonged ER stress can activate the intrinsic apoptotic pathway. The unfolded protein response (UPR), initially a survival mechanism, can shift to promoting apoptosis if protein misfolding is overwhelming, often involving pro-apoptotic BH3-only proteins.

Why is mitochondrial integrity crucial for cellular life?

Mitochondria are the primary sites of ATP production. Loss of mitochondrial integrity (e.g., during MOMP) not only releases pro-apoptotic factors but also compromises cellular energy production, contributing to cell death.

What is the significance of the balance between Bcl-2 and Bax/Bak?

The ratio of anti-apoptotic (e.g., Bcl-2) to pro-apoptotic (e.g., Bax, Bak, BH3-only proteins) Bcl-2 family members determines whether a cell will undergo apoptosis via the intrinsic pathway. A shift towards pro-apoptotic factors favors apoptosis.

How does growth factor withdrawal trigger apoptosis?

Growth factor withdrawal deprives cells of essential survival signals. This often leads to a decrease in the expression of anti-apoptotic proteins (like Bcl- \text{X}_{\text{L}}) and/or an increase in pro-apoptotic BH3-only proteins, tipping the balance towards apoptosis.

What is the clinical relevance of targeting apoptotic pathways?

Targeting apoptotic pathways is a major strategy in cancer therapy. For example, drugs can activate apoptosis in cancer cells by inhibiting anti-apoptotic Bcl-2 proteins (e.g., Venetoclax) or by enhancing death receptor signaling.

Can Apoptosis be inhibited?

Yes, apoptosis can be inhibited naturally by various endogenous mechanisms, such as expression of anti-apoptotic Bcl-2 proteins, IAPs, or FLIP. It can also be pharmacologically inhibited in certain contexts to prevent cell death in diseases like neurodegeneration or ischemia.

What is the general sequence of events in Apoptosis?

1. Apoptotic stimulus.
2. Activation of initiator caspases.
3. Activation of effector caspases.
4. Cleavage of cellular substrates.
5. DNA fragmentation, chromatin condensation, cell shrinkage, blebbing.
6. Formation of apoptotic bodies.
7. Phagocytosis of apoptotic bodies.

What is the 'amplification loop' in apoptosis, particularly in Type II cells?

In Type II cells, activated Caspase-8 (from the extrinsic pathway) cleaves Bid to tBid. tBid then activates the intrinsic pathway by promoting MOMP, leading to Cytochrome c release and apoptosome formation. This amplifies the caspase cascade through Caspase-9.

How does the immune system use apoptosis?

The immune system uses apoptosis to:

• Remove virus-infected and cancerous cells (via cytotoxic T lymphocytes).
• Eliminate self-reactive lymphocytes during development (thymic education) to prevent autoimmunity.
• Resolve immune responses (deletion of effector lymphocytes after infection control).

What is the significance of the plasma membrane in apoptosis?

During apoptosis, the plasma membrane undergoes significant changes, including: blabbing, loss of microvilli, and exposure of 'eat me' signals like phosphatidylserine on the outer leaflet, serving as a signal for phagocytic clearance.

How can Apoptosis be detected experimentally?

Common methods include:

• DNA laddering: Detecting internucleosomal DNA fragmentation.
• TUNEL assay: Identifying DNA strand breaks.
• Caspase activity assays: Measuring active caspases.
• Flow cytometry: Analyzing phosphatidylserine exposure (Annexin V binding) and cell cycle changes.

What is a 'bleb' in the context of apoptosis?

A bleb is an irregular bulge in the plasma membrane of a cell undergoing apoptosis. It forms due to changes in the cytoskeleton and contributes to the fragmentation of the cell into apoptotic bodies.

What is the role of cytochrome c release beyond caspase activation?

While its primary role is to activate Apaf-1 and caspase-9, cytochrome c (and other mitochondrial proteins like Smac/DIABLO) also contributes to the apoptotic process by inhibiting IAPs, further promoting caspase activity.

How do cytotoxic T lymphocytes (CTLs) induce apoptosis in target cells?

CTLs recognize and bind to target cells (e.g., virus-infected cells) and release perforin and granzymes. Perforin forms pores in the target cell membrane, allowing granzymes (e.g., granzyme B, a serine protease) to enter and directly cleave and activate caspases or Bid, thereby initiating apoptosis.

What is the role of $\text{Ca}^{2+}$ in apoptosis?

Dysregulation of intracellular $\text{Ca}^{2+}$ homeostasis can induce apoptosis. High cytosolic $\text{Ca}^{2+}$ can activate $\text{Ca}^{2+}$-dependent enzymes (e.g., endonucleases, proteases) and contribute to mitochondrial dysfunction and the release of pro-apoptotic factors.

What is the link between reactive oxygen species (ROS) and apoptosis?

Excessive production of ROS can cause oxidative stress, leading to DNA damage, protein modification, and lipid peroxidation. This damage can activate both the intrinsic and extrinsic apoptotic pathways, often by increasing mitochondrial permeability or triggering ER stress.

How do viral infections interact with apoptosis?

Viruses can either induce apoptosis to facilitate their spread or inhibit host cell apoptosis to prolong their replication cycle. Many viruses encode proteins that target host apoptotic machinery (e.g., viral anti-apoptotic Bcl-2 homologs).

What is 'extrinsic' about the Extrinsic Pathway?

It is 'extrinsic' because it is initiated by signals (death ligands) from outside the cell, binding to specific receptors on the cell surface.