Mol Bio lecture 5.pdf

1.Compare and contrast necrosis, apoptosis, and autophagy ◦List the main steps of each process in order where applicable Necrosis, apoptosis, and autophagy are distinct processes of cell death or survival, each with specific characteristics, triggers, and consequences for the cell and surrounding ti...

1.Compare and contrast necrosis, apoptosis, and autophagy ◦List the main steps of each process in order where applicable Necrosis, apoptosis, and autophagy are distinct processes of cell death or survival, each with specific characteristics, triggers, and consequences for the cell and surrounding tissue. Necrosis Necrosis is an uncontrolled form of cell death typically caused by factors such as injury, infection, or extreme stress. It is characterized by cell swelling, membrane rupture, and the release of intracellular contents into the extracellular space. This release triggers an inflammatory response, which can cause damage to surrounding tissues. Necrosis often results from a lack of oxygen (ischemia), toxins, or trauma and is generally harmful to the organism because of the subsequent tissue damage and inflammation. Key features of necrosis: Uncontrolled and passive process Triggered by external factors like injury or infection Leads to cell swelling and rupture Releases cellular contents into the extracellular space Causes inflammation and tissue damage Apoptosis Apoptosis, often called programmed cell death, is a highly regulated process that allows the body to remove damaged, unnecessary, or harmful cells without causing inflammation. Apoptosis can be initiated by intrinsic signals (internal damage like DNA mutations) or extrinsic signals (external death signals from neighboring cells). During apoptosis, the cell undergoes controlled shrinkage, chromatin condensation, DNA fragmentation, and formation of apoptotic bodies, which are then engulfed by phagocytic cells. The process of apoptosis involves specific molecular pathways: Intrinsic pathway: Triggered by internal signals such as DNA damage, leading to mitochondrial outer membrane permeabilization and the release of pro-apoptotic factors like cytochrome c. The  apoptosome forms, activating caspase-9 and initiating the caspase cascade, which dismantles the cell. Extrinsic pathway: Triggered by death ligands binding to cell surface receptors (e.g., Fas receptor). This activates caspase-8, which in turn activates downstream executioner caspases, leading to the degradation of cellular components. Key features of apoptosis: Regulated and energy-dependent Can be triggered by internal (intrinsic) or external (extrinsic) signals Involves caspases, which degrade cellular components Results in cell shrinkage and formation of apoptotic bodies Does not cause inflammation, as apoptotic bodies are phagocytosed Autophagy Autophagy is a survival mechanism in which the cell degrades and recycles its own damaged organelles or misfolded proteins to maintain homeostasis, especially under stress conditions like nutrient deprivation. It is not a form of cell death but a process that can delay apoptosis by promoting cellular repair. Autophagy begins with the formation of autophagosomes, which engulf damaged components and fuse with lysosomes for degradation. While autophagy promotes cell survival, excessive or dysregulated autophagy can lead to cell death, sometimes referred to as "autophagic cell death." Unlike apoptosis, autophagy does not involve caspases and does not result in DNA fragmentation. Instead, it helps maintain cellular function by recycling cellular components and providing nutrients during stress. Key features of autophagy: A regulated survival process, not inherently a death mechanism Engages in the degradation of damaged organelles and proteins Can help delay apoptosis by promoting cell repair Involves the formation of autophagosomes and lysosomal degradation Does not cause inflammation Comparative Summary Necrosis is an uncontrolled process resulting from external injury, leading to cell rupture, release of cellular contents, and inflammation.  Apoptosis is a controlled, programmed cell death process, initiated by either internal or external signals, involving caspase activation, and resulting in non-inflammatory cell death. Autophagy is a regulated survival mechanism that helps maintain cellular homeostasis by recycling damaged cellular components, delaying apoptosis, and promoting repair. Key Differences Necrosis is an unplanned, uncontrolled form of cell death resulting from external injury, while apoptosis and autophagy are tightly regulated processes that are energy-dependent. Apoptosis leads to the removal of damaged or unnecessary cells in a clean, non-inflammatory manner, whereas necrosis triggers inflammation due to cell membrane rupture and the spilling of cellular contents. Autophagy is primarily a survival mechanism, allowing cells to recycle their components during stress, but excessive autophagy can also lead to cell death, blurring the line between survival and death. Key Similarities Apoptosis and autophagy are both highly regulated processes that contribute to cellular homeostasis. While necrosis is more chaotic and harmful, apoptosis and autophagy are adaptive processes that either maintain health (autophagy) or remove damaged cells (apoptosis). General Steps of Apoptosis 1. Initiation:  Intrinsic Pathway (Mitochondrial Pathway): Triggered by internal signals like DNA damage, oxidative stress, or growth factor withdrawal. Mitochondria release cytochrome c, which activates apoptotic protease-activating factor 1 (Apaf-1) and caspase-9. Extrinsic Pathway (Death Receptor Pathway): Triggered by external signals binding to death receptors (e.g., Fas or TNF receptors). This activates caspase-8. 2. Caspase Activation: Both pathways converge to activate caspases (cysteine-aspartic proteases), which are enzymes responsible for breaking down cellular components. Caspase-8 and caspase-9 initiate this process, leading to the activation of executioner caspases like caspase-3, -6, and -7. 3. Execution Phase: Activated caspases dismantle the cell by cleaving various proteins and breaking down the cytoskeleton, leading to cell shrinkage and chromatin condensation (pyknosis). DNA fragmentation occurs, and the nuclear envelope breaks down (karyorrhexis). 4. Formation of Apoptotic Bodies: The cell breaks apart into small, membrane-bound fragments called apoptotic bodies, containing cellular contents like organelles and chromatin. 5. Phagocytosis: Apoptotic bodies are recognized and engulfed by phagocytic cells, such as macrophages, without inducing an inflammatory response. ◦Match the activity to the process for each of the following: death domains, procaspase-3 and caspase-3, procaspase-8 and caspase-8, procaspase-9 and caspase-9, Bax, apoptosome, cyclophilin D, MPTP, cytochrome c, Apaf-1, and Bcl-2 Death Domains  Process: Extrinsic Pathway Activation Activity: These are protein interaction modules found in death receptors (like Fas or TNF receptors) that recruit adaptor proteins to form the death-inducing signaling complex (DISC), leading to the activation of procaspase-8. Procaspase-3 and Caspase-3 Process: Execution Phase of Apoptosis Activity: Procaspase-3 is the inactive precursor, which is cleaved to form caspase-3. Caspase-3 is a key executioner caspase that cleaves various cellular substrates, leading to apoptosis. Procaspase-8 and Caspase-8 Process: Extrinsic Pathway Activation Activity: Procaspase-8 is cleaved into caspase-8 as part of the extrinsic pathway upon activation by death receptors, initiating the apoptotic cascade. Procaspase-9 and Caspase-9 Process: Intrinsic Pathway Activation Activity: Procaspase-9 is activated in the intrinsic pathway by the apoptosome, which leads to the cleavage of procaspase-9 into active caspase-9, initiating the downstream cascade. Bax Process: Intrinsic Pathway/Mitochondrial Pathway Activity: Bax is a pro-apoptotic member of the Bcl-2 family. It promotes the release of cytochrome c from mitochondria by forming pores in the mitochondrial membrane. Apoptosome Process: Intrinsic Pathway Activation Activity: The apoptosome is a multi-protein complex formed by cytochrome c, Apaf-1, and procaspase-9. It activates procaspase-9, which initiates the caspase cascade. Cyclophilin D  Process: Mitochondrial Permeability Transition Pore (MPTP) Regulation Activity: Cyclophilin D is involved in regulating the mitochondrial permeability transition pore (MPTP), which can lead to mitochondrial swelling and cell death through necrosis or apoptosis. Mitochondrial Permeability Transition Pore (MPTP) Process: Mitochondrial Dysfunction Activity: The MPTP is a channel that forms in the mitochondrial membrane during stress, leading to loss of mitochondrial membrane potential, swelling, and release of pro-apoptotic factors like cytochrome c. Cytochrome c Process: Intrinsic Pathway Activation Activity: Released from mitochondria into the cytosol, cytochrome c binds Apaf-1, leading to apoptosome formation and activation of procaspase-9. Apaf-1 (Apoptotic Protease-Activating Factor-1) Process: Intrinsic Pathway Activation Activity: Apaf-1 binds cytochrome c to form the apoptosome, which activates procaspase-9, initiating the caspase cascade in the intrinsic pathway. Bcl-2 Process: Intrinsic Pathway/Mitochondrial Pathway Inhibition Activity: Bcl-2 is an anti-apoptotic protein that prevents apoptosis by inhibiting the release of cytochrome c from mitochondria, thereby blocking the intrinsic pathway. ◦Determine when each of the processes would be favored and inhibited (What conditions trigger or suppress each process?) 2.Assess the consequences for changes in molecular processes associated with cell death in terms of cellular homeostasis and the ability to trigger or prevent necrosis, apoptosis, and autophagy ◦Identify the action for each of the following: death domains, procaspase-3 and caspase-3, procaspase-8 and caspase-8, procaspase-9 and caspase-9, Bax, apoptosome, cyclophilin D, MPTP, cytochrome c, Apaf-1, and Bcl-2 ◦Determine the direct and downstream consequences of changes in each of the previous components ◦Identify the consequence in terms of division or death for changes in each of the previous components Assessing Changes in Molecular Processes Associated with Cell Death The balance between cell survival and death is crucial for maintaining cellular homeostasis. Disruptions in cell death pathways (necrosis, apoptosis, autophagy) can lead to various consequences for health, such as cancer, neurodegenerative diseases, and immune disorders. Below is an assessment of the role of specific molecules in regulating these pathways, followed by the consequences of their alteration: 1. Death Domains (DDs) Action: Found in death receptors (e.g., Fas, TNF receptors), these domains recruit adaptor proteins (e.g., FADD) to initiate the extrinsic apoptotic pathway. Direct Consequence of Change: ○ Upregulation: Enhanced recruitment of procaspase-8, leading to more efficient induction of apoptosis. ○ Downregulation/Mutation: Inhibition of apoptosis initiation via the extrinsic pathway, promoting survival and possibly tumor development. Outcome: ○ Increased apoptosis or reduced apoptosis depending on the presence or absence of functional death domains. 2. Procaspase-3 and Caspase-3 Action: Procaspase-3 is cleaved into active caspase-3, the executioner caspase that cleaves cellular substrates leading to cell death. Direct Consequence of Change:  ○ Upregulation: Increased caspase-3 activation will enhance apoptosis, leading to more cell death. ○ Downregulation/Inhibition: Reduced caspase-3 activity will block apoptosis, potentially allowing damaged or cancerous cells to survive. Outcome: ○ Increased apoptosis with higher levels of caspase-3. ○ Increased survival with reduced caspase-3 activity. 3. Procaspase-8 and Caspase-8 Action: Initiator caspase in the extrinsic pathway, activated via death receptors. Caspase-8 activates downstream effector caspases like caspase-3. Direct Consequence of Change: ○ Upregulation: Leads to enhanced apoptosis via the extrinsic pathway. ○ Downregulation/Mutation: Leads to impaired apoptosis and potential for unchecked cell proliferation. Outcome: ○ Increased apoptosis with higher caspase-8 activity. ○ Reduced apoptosis with caspase-8 inhibition, potentially promoting tumorigenesis. 4. Procaspase-9 and Caspase-9 Action: Initiator caspase in the intrinsic (mitochondrial) pathway, activated by the apoptosome (Apaf-1, cytochrome c). Direct Consequence of Change: ○ Upregulation: Increased caspase-9 activity promotes apoptosis. ○ Downregulation/Mutation: Reduced activation will prevent apoptosis, contributing to cell survival in stressful conditions, such as in cancer. Outcome: ○ Increased apoptosis or blocked apoptosis, depending on caspase-9 activity levels. 5. Bax  Action: Pro-apoptotic Bcl-2 family member that promotes mitochondrial outer membrane permeabilization (MOMP), leading to cytochrome c release and apoptosis. Direct Consequence of Change: ○ Upregulation: More cytochrome c release, enhancing the intrinsic pathway of apoptosis. ○ Downregulation/Inhibition: Blocked cytochrome c release, leading to reduced apoptosis and potential for uncontrolled cell survival. Outcome: ○ Increased apoptosis when Bax is upregulated. ○ Reduced apoptosis with Bax inhibition, increasing survival and risk for cancer. 6. Apoptosome Action: Complex formed by Apaf-1, cytochrome c, and procaspase-9, initiating caspase-9 activation and apoptosis. Direct Consequence of Change: ○ Disruption of Formation: Prevents activation of caspase-9, blocking apoptosis. ○ Enhanced Formation: Accelerates apoptosis via the intrinsic pathway. Outcome: ○ Increased apoptosis with functional apoptosome formation. ○ Reduced apoptosis when formation is impaired, promoting survival of damaged cells. 7. Cyclophilin D Action: Regulates the mitochondrial permeability transition pore (MPTP), which can lead to mitochondrial depolarization and cell death (necrosis or apoptosis). Direct Consequence of Change: ○ Upregulation: Increased MPTP opening, leading to necrotic or apoptotic cell death. ○ Downregulation/Inhibition: Reduced mitochondrial membrane permeability, promoting cell survival. Outcome: ○ Increased necrosis/apoptosis when cyclophilin D activity is increased.  ○ Increased survival when its activity is inhibited. 8. Mitochondrial Permeability Transition Pore (MPTP) Action: Pore in the mitochondrial membrane that, when opened, leads to loss of mitochondrial potential, swelling, and release of pro-apoptotic factors. Direct Consequence of Change: ○ Excessive Opening: Leads to necrotic cell death or apoptosis via cytochrome c release. ○ Blocked Opening: Prevents cell death, allowing survival under stressful conditions. Outcome: ○ Increased necrosis/apoptosis with more frequent MPTP opening. ○ Cell survival with reduced MPTP opening. 9. Cytochrome c Action: Released from mitochondria during apoptosis, binds Apaf-1 to form the apoptosome, which activates procaspase-9. Direct Consequence of Change: ○ Release: Promotes the formation of the apoptosome and apoptosis. ○ Sequestration: Prevents apoptosome formation, blocking apoptosis. Outcome: ○ Increased apoptosis with cytochrome c release. ○ Reduced apoptosis with inhibited release. 10. Apaf-1 (Apoptotic Protease-Activating Factor 1) Action: Forms part of the apoptosome by binding cytochrome c, which activates procaspase-9. Direct Consequence of Change: ○ Upregulation: Enhances apoptosome formation and apoptosis. ○ Downregulation/Inhibition: Prevents apoptosome formation, blocking apoptosis. Outcome: ○ Increased apoptosis with higher Apaf-1 activity. ○ Reduced apoptosis with Apaf-1 inhibition, leading to cell survival. 11. Bcl-2 Action: Anti-apoptotic protein that prevents cytochrome c release by inhibiting pro-apoptotic proteins like Bax. Direct Consequence of Change: ○ Upregulation: Inhibits cytochrome c release, blocking apoptosis and promoting survival. ○ Downregulation/Inhibition: Leads to increased cytochrome c release and apoptosis. Outcome: ○ Increased survival with Bcl-2 upregulation. ○ Increased apoptosis with Bcl-2 inhibition, promoting cell death. Consequence in Terms of Division or Death: Upregulation of apoptotic components like Bax, caspases, Apaf-1, and cytochrome c leads to increased apoptosis (cell death). Downregulation or mutation of these components often results in reduced apoptosis, allowing increased cell survival, which can contribute to uncontrolled cell division, potentially leading to cancer. Changes in the MPTP, Cyclophilin D, or Bcl-2 can shift the balance toward necrosis, apoptosis, or survival, impacting both cell death and division. Overall, changes in these molecular processes can either tilt the balance toward excessive cell death (causing tissue damage and degenerative diseases) or excessive survival and division (leading to conditions like cancer).