Apoptosis and Programmed Cell Death

Apoptosis: Programmed Cell Death

• Apoptosis is a natural and necessary mechanism for removing cells or tissues that are no longer needed, damaged, or infected.

• It is a crucial process during organismal development.

• In Class -

  • Too many cells dying or too much proliferation are not mutually exclusive. Bothe extremes bad

  • Ex. We are not born with interwebbed toes and fingers due to apoptosis. Natural process during development. Inhinted pathway leads to opposite

• Morphological signs of Apoptosis

• Blebbing of Plasma Membrane (PM): The cell membrane forms irregular bulges or blebs.

• Pyknosis: Nuclear condensation, leading to the shrinkage and increased density of the nucleus.

• Golgi Fragmentation: The Golgi apparatus breaks into smaller vesicles.

• DNA Fragmentation: The cell's DNA is cleaved into distinct fragments.

• In Class -

  • Necrosis Cells dying and other nearby cells are dying due to the release of signaling molecules that trigger a cascade of apoptosis in surrounding tissues.

  • Histone compaction occurs during apoptosis, leading to the condensation of chromatin and further facilitating the fragmentation of DNA. In contrast, apoptosis is characterized by a highly regulated process that involves cellular shrinkage, membrane blebbing, and ultimately the formation of apoptotic bodies, which are engulfed by neighboring cells or macrophages, preventing inflammation.

Caspases: Mediators of Apoptosis

• Proteases: These enzymes play a crucial role in apoptosis by cleaving specific proteins to execute the cell death program, ensuring that the process occurs efficiently and without harm to surrounding tissues.

• Extensive DNA damage- P53, P21, activate procaspases, leading to the initiation of apoptosis as a response mechanism to prevent the propagation of damaged genetic material.

• Apoptosis is primarily mediated by a family of proteins known as Caspases.

• Caspases are Cysteine aspartic acid proteases.

• As cysteine proteases, they specifically cleave their substrate proteins after an aspartate residue.

Caspase Activation

• Caspases are initially produced in a catalytically inactive form called procaspases.

• Activation of caspases occurs through proteolytic cleavage of these procaspases.

• Initiator Caspases are present in the cell as inactive monomers and become activated through dimerization and cleavage.

Types of Caspases

1. Initiator Caspases: Their primary role is to cleave and activate the inactive forms of effector caspases.

2. Executioner Caspases: Once activated by initiator caspases, these directly cleave a wide number of crucial cellular proteins, leading to the characteristic features of apoptosis.

Consequence of Caspase Activation

• A significant consequence of caspase activation is the cleavage of ICAD (Inhibitor of Caspase Activated DNAse).

• The cleavage of ICAD releases CAD (Caspase Activated DNAse).

• CAD is an endonuclease that subsequently enters the nucleus and cleaves DNA, contributing to the DNA fragmentation observed during apoptosis.

• Pan caspase inhibitor treatment can prevent apoptosis by inhibiting the activation of all caspases, thereby blocking the cleavage of ICAD and subsequent DNA fragmentation associated with this process. Takes external drugging of a cell

Apoptotic Pathways

Extrinsic Pathway (Death Receptor Pathway)

• This pathway is initiated by signaling through death receptors located on the cell surface.

• These receptors belong to the TNF (Tumor Necrosis Factor) receptor family.

• Death receptors are characterized by three main domains:

  • An extracellular domain that binds to specific death ligands.

  • A transmembrane domain anchoring the receptor in the cell membrane.

  • An intracellular death domain essential for signaling.

• Both the death receptors and their corresponding ligands exist as homotrimers.

• Mechanism:

  • Upon ligand binding, death receptors recruit adaptor proteins, such as FADD (Fas-associated death domain protein).

  • This recruitment leads to the formation of a multi-protein complex called the Death Inducing Signaling Complex (DISC).

  • Within the DISC, initiator Caspase (specifically Caspase 8) is activated.

Intrinsic Pathway (Mitochondrial Pathway)

• The intrinsic pathway of apoptosis is also known as the mitochondrial pathway and is regulated by internal cellular signals, often related to cell stress or damage.

Regulation of Apoptosis by Bcl2 Family of Proteins

• The Bcl2 family of proteins plays a critical role in regulating apoptosis, particularly in the intrinsic pathway.

• The balance between pro-apoptotic (e.g., Bax, Bak) and anti-apoptotic (e.g., Bcl2, Bcl-Xl) Bcl2 family members ultimately determines whether a cell commits to apoptosis or not.

Convergence of Apoptotic Pathways

• Both the intrinsic and extrinsic apoptotic pathways eventually converge, often at the level of caspase activation, leading to a common execution phase involving executioner caspases.

Activation of Apoptosis by $p53$

• The tumor suppressor gene $p53$ is a key activator of apoptosis, particularly in response to DNA damage or cellular stress. It can initiate apoptosis through various mechanisms, including transcriptional upregulation of pro-apoptotic genes.

Anti-Apoptotic Strategies Used by Cancer Cells

Cancer cells often develop mechanisms to evade apoptosis, allowing them to survive and proliferate. These strategies include:

• Increase in Mdm2 expression: Mdm2 is a ligase that targets $p53$ for degradation, thus reducing $p53$-mediated apoptosis.

• Production of FLIP: FLIP (Fas-associated death domain-like interleukin-$1 \beta$-converting enzyme-like inhibitory protein) acts as a dominant-negative inhibitor of caspase activation in the DISC, thereby blocking the extrinsic apoptotic pathway.

• Increase in levels of anti-apoptotic proteins: Cancer cells may overexpress endogenous anti-apoptotic proteins such as IAP (Inhibitor of Apoptosis Proteins) and Bcl2, which directly inhibit caspase activity or prevent mitochondrial outer membrane permeabilization, respectively.

Here are 10 practice questions based on the provided material, designed to test your understanding from a professor's perspective:

1. Define apoptosis and elucidate its critical importance in processes such as organismal development and the elimination of damaged or infected cells. Provide a specific example to illustrate its developmental role.

2. Describe the characteristic morphological changes that occur in a cell undergoing apoptosis, including observations at the plasma membrane, nucleus, and Golgi apparatus. How do these apoptotic features fundamentally differ from those seen in necrosis, particularly concerning the cellular impact on surrounding tissues?

3. Caspases are central to the execution of apoptosis. Explain the nature of caspases, their inactive precursor form, and the general mechanism (e.g., proteolytic cleavage) by which they become activated to initiate and propagate the cell death program.

4. Differentiate between initiator caspases and executioner caspases, detailing their respective roles and hierarchical activation within the apoptotic signaling cascade. Furthermore, elaborate on the mechanism by which executioner caspases lead to DNA fragmentation, specifically mentioning ICAD and CAD.

5. Outline the entire sequence of events that constitutes the extrinsic (death receptor) pathway of apoptosis, commencing with ligand binding. Identify the key protein components involved, including the death receptors, adapter proteins, and the specific initiator caspase activated in this pathway.

6. The Bcl2 family of proteins critically regulates the intrinsic (mitochondrial) pathway of apoptosis. Discuss how the delicate balance between pro-apoptotic (e.g., Bax, Bak) and anti-apoptotic (e.g., Bcl2, Bcl-Xl) members of this family determines whether a cell commits to or is protected from apoptosis.

7. The tumor suppressor gene $p53$ is activated in response to various cellular stresses, including DNA damage. Explain the mechanisms by which $p53$ triggers apoptosis, emphasizing its role in transcriptional regulation of pro-apoptotic genes.

8. Cancer cells are notorious for their ability to evade programmed cell death. Describe at least three distinct strategies that cancer cells employ to circumvent apoptosis, thereby contributing to their survival and proliferation.

9. Specifically explain how an increased expression of Mdm2 or the production of FLIP by cancer cells contributes to their resistance to apoptosis. Detail the molecular targets and inhibitory mechanisms of each.

10. A pan-caspase inhibitor can prevent apoptosis. Based on your understanding of caspase function and activation, explain the molecular rationale behind this treatment and how it effectively blocks the downstream events of the apoptotic pathway, such as DNA fragmentation.

Here are the answers to your practice questions, concise and comprehensive:

1. Apoptosis is a natural and necessary mechanism of programmed cell death for removing cells or tissues that are no longer needed, damaged, or infected. It is crucial during organismal development, for instance, in the formation of distinct fingers and toes by removing the interwebbed tissue we are born with.

2. Morphological signs of apoptosis include blebbing of the plasma membrane, pyknosis (nuclear condensation), Golgi fragmentation, and DNA fragmentation. These features differ from necrosis, where cells swell and burst, releasing signaling molecules that cause inflammation and death in surrounding tissues. Apoptosis involves cellular shrinkage, membrane blebbing, and formation of apoptotic bodies which are engulfed, preventing inflammation.

3. Caspases are cysteine aspartic acid proteases that are central to apoptosis. They are initially produced as catalytically inactive procaspases. Activation occurs through proteolytic cleavage of these procaspases, initiating and propagating the cell death program.

4. Initiator caspases (e.g., Caspase 8) are activated first (e.g., by dimerization and cleavage) and their primary role is to cleave and activate the inactive forms of executioner caspases. Executioner caspases, once activated, directly cleave numerous crucial cellular proteins, leading to apoptotic features. They cause DNA fragmentation by cleaving ICAD (Inhibitor of Caspase Activated DNAse), which releases CAD (Caspase Activated DNAse). CAD then enters the nucleus and cleaves DNA.

5. The extrinsic pathway begins with death ligands binding to specific death receptors (e.g., TNF receptor family) on the cell surface. These receptors, characterized by extracellular, transmembrane, and intracellular death domains, exist as homotrimers. Ligand binding recruits adaptor proteins like FADD, forming the Death Inducing Signaling Complex (DISC). Within the DISC, initiator Caspase 8 is activated.

6. The Bcl2 family of proteins regulates the intrinsic (mitochondrial) pathway. The balance between pro-apoptotic members (e.g., Bax, Bak) and anti-apoptotic members (e.g., Bcl2, Bcl-Xl) determines cell fate. An excess of pro-apoptotic proteins promotes mitochondrial outer membrane permeabilization, leading to apoptosis, while anti-apoptotic proteins inhibit this process, protecting the cell.

7. The tumor suppressor gene $p53$ is a key activator of apoptosis in response to DNA damage or cellular stress. It triggers apoptosis through various mechanisms, including the transcriptional upregulation of pro-apoptotic genes, which can activate procaspases.

8. Cancer cells employ several strategies to evade apoptosis, including:

   • Increasing Mdm2 expression, which targets $p53$ for degradation.

   • Producing FLIP, a dominant-negative inhibitor of caspase activation in the DISC.

   • Increasing levels of anti-apoptotic proteins like IAP (Inhibitor of Apoptosis Proteins) and Bcl2, which inhibit caspase activity or prevent mitochondrial outer membrane permeabilization.

9. Increased Mdm2 expression by cancer cells targets $p53$ for degradation, thus reducing $p53$-mediated apoptosis by decreasing the level of the pro-apoptotic tumor suppressor. Production of FLIP (Fas-associated death domain-like interleukin-$1 eta$-converting enzyme-like inhibitory protein) acts as a dominant-negative inhibitor of caspase activation within the DISC, thereby blocking the activation of initiator caspases (like Caspase 8) in the extrinsic apoptotic pathway.

10. A pan-caspase inhibitor prevents apoptosis by blocking the activation of all caspases. Caspases are essential for executing the cell death program, including the proteolytic cleavage of ICAD that leads to DNA fragmentation by CAD. By inhibiting caspases, downstream events such as the release of CAD and subsequent DNA cleavage are effectively blocked, thereby preventing the characteristic morphological and biochemical features of apoptosis.