Apoptosis: Cell Death Chapter 5 PDF

**Chapter 5** - **Cell death** plays a crucial role in the development of animals and plants and continues into adulthood. - In a **healthy adult human**, billions of cells die every hour in the **bone marrow** and **intestine**. - **Tissues** do not shrink because **cell division**...

**Chapter 5** - **Cell death** plays a crucial role in the development of animals and plants and continues into adulthood. - In a **healthy adult human**, billions of cells die every hour in the **bone marrow** and **intestine**. - **Tissues** do not shrink because **cell division** balances **cell death** precisely. - These normal cell deaths are **suicides**. - **Cells** activate an **intracellular death program** and kill themselves in a controlled manner, a process called **programmed cell death**. **Definition** - **Apoptosis** is the most common form of **programmed cell death**. - The term originates from the Greek word meaning \"falling off.\" - The **cell surface** of apoptotic cells becomes chemically altered to allow **macrophages** to engulf them rapidly before their contents spill, avoiding a **damaging inflammatory response**. **Morphology** - Apoptotic cells exhibit distinct **morphological changes**: - Shrink and **condense**. - Collapse of the **cytoskeleton**. - Disassembly of the **nuclear envelope**. - Condensation and fragmentation of **nuclear chromatin**. - **Cell surface** often blebs and, if the cell is large, it breaks into **membrane-enclosed fragments** called **apoptotic bodies**. - The **surface** of the cell or apoptotic bodies becomes chemically altered. **Classical Examples of Apoptosis** - ***Animal development**:* - Sculpting **hands** and **feet**. - Regulating **cell numbers** in the developing **nervous system**. - Serving as a **quality control process** in the adaptive immune system development. - ***Metamorphosis**:* - Elimination of **unwanted cells**. - **Adult tissues**: - Removal of **abnormal**, **nonfunctional**, or **potentially dangerous cells**. - Elimination of **no longer needed cells**, such as **T** and **B lymphocytes** after resolving an infection. - Maintaining a ready supply of **short-lived neutrophils**. **Biochemistry** - **Biochemical changes** help identify apoptotic cells. - Cells undergoing apoptosis exhibit both **characteristic morphology** and **biochemical changes** that can serve as markers. 1. ***DNA Fragmentation:*** - During apoptosis, an **endonuclease** cleaves **chromosomal DNA** into fragments of distinctive sizes. - These cleavages occur in the **linker regions** between nucleosomes. - When analysed by **gel electrophoresis**, the fragments form a **ladder pattern**, characteristic of apoptotic cells. 2. ***Phosphatidylserine Flip**:* - The **negatively charged phospholipid phosphatidylserine**, normally found in the **inner leaflet** of the lipid bilayer, flips to the **outer leaflet** in apoptotic cells. - This serves as a **marker** for apoptotic cells and: - Signals **neighbouring cells** and **macrophages** to phagocytose the dying cell. - Blocks **inflammation** by inhibiting cytokine production in the **phagocytic cell**. 3. ***Loss of Mitochondrial Potential**:* - Apoptotic cells lose the **electric potential** across the inner mitochondrial membrane. - **Cytochrome C** is released from the **intermembrane space**, triggering further apoptotic signalling. **Apoptosis Machinery: Caspases (Overview)** - The **apoptosis machinery** relies on a family of proteases called **caspases**. - Caspases have a **cysteine** at their active site. - They cleave target proteins at specific **aspartic acid residues**. - **Caspases** are produced as inactive precursors called **procaspases**, which are activated by **proteolytic cleavage**. **Apoptosis: The Beginning** - **Healthy cells** continuously produce **procaspases**, ensuring the apoptosis machinery is always ready. - Apoptosis requires only a **trigger** to activate the machinery. - The activation of the first **procaspase** in the cascade involves: - **Initiator procaspases** that have a long prodomain containing a **caspase recruitment domain (CARD)**. - **CARD** facilitates the assembly with **adaptor proteins** into activation complexes when the cell receives an apoptotic signal. **Mechanism of Initiation** - Once incorporated into an **activation complex**: - **Initiator procaspases** are brought into proximity. - This proximity triggers their activation. - Activated initiator procaspases cleave one another, making the process **irreversible**.**Procaspase Activation** - ***Procaspases are split into two subunits:*** 1\. Large subunit 2. Small subunit - These subunits form a **heterodimer**, and two such dimers assemble into the **active tetramer**. **Executioner Caspase Activation** - The first caspases to be activated are the **initiator procaspases**. - These activate multiple **executioner procaspases**, creating an **amplifying chain reaction**. - **Executioner caspases** cleave various key proteins, including: 1\. Specific **cytosolic proteins**. 2. The **nuclear lamina**. - This leads to the **controlled death** of the cell. **Target Proteins** - **Executioner caspases** cleave a variety of target proteins, including: - **Nuclear lamina** - Proteins that keep **endonucleases** in their inactive form - **Cytoskeleton proteins** - **Cell-cell adhesion proteins** **Signalling Pathways** The two best understood signalling pathways that activate a caspase cascade leading to apoptosis in mammalian cells are: 1. **Extrinsic pathway** 2. **Intrinsic pathway** Each pathway uses its own initiator **procaspases** and activation complexes. **Extrinsic Pathway** - Triggered by **cell-surface Fas death receptors**. - ***Fas death receptors:*** - Belong to the **tumour necrosis factor (TNF) receptor family**. - ***Are homotrimeric transmembrane proteins with:*** - **Extracellular ligand-binding domain** - **Single transmembrane domain** - **Intracellular death domain**, required to activate the apoptotic program. **Fas Activation in the Extrinsic Pathway** - **Fas ligand** on the surface of a **killer lymphocyte** binds to **Fas death receptors** on the target cell. - The cytosolic tail of Fas recruits the adaptor protein **FADD** via their **death domains**. - Each **FADD protein** recruits an initiator procaspase (8, 10, or both) via their **death effector domains**, forming a **death-inducing signaling complex (DISC)**. - ***Within the DISC:*** - **Initiator procaspases** are brought into close proximity, activating them. - Activated procaspases cleave each other, stabilizing the active protease (now a caspase). - **Caspase-8** and **Caspase-10** then cleave and activate **executioner procaspases**, initiating a **caspase cascade** leading to apoptosis. **Inhibition of the Extrinsic Pathway** - Many cells produce **inhibitory proteins** to restrain the extrinsic pathway: **1. Extracellular Decoy Receptors**: - Have a **ligand-binding domain** but no **death domain**. - Bind death ligands but cannot activate apoptosis, competitively inhibiting death receptors. **2. Intracellular Blocking Proteins**: - Example: **FLIP**, which resembles an initiator procaspase but lacks a proteolytic domain. - FLIP competes with **procaspase-8** and **procaspase-10** for binding sites in the DISC, preventing their activation. These inhibitory mechanisms help prevent **inappropriate activation** of the extrinsic apoptosis pathway. **Intrinsic Pathway** - Triggered by **cellular stressors** such as: 1\. **Injury** 2. **DNA damage** 3. **Lack of oxygen** or nutrients **Release of Cytochrome C** - Cytochrome c, a component of the **electron transport chain**, is released from the **intermembrane space of mitochondria** during apoptosis. - **Fluorescence micrograph observations**: **1. *Control cells****:* - Transfected with a gene encoding **cytochrome-c-GFP** (green fluorescent protein). - Treated with a red dye that accumulates in mitochondria. - Overlapping green and red signals indicate **cytochrome-c-GFP** is in mitochondria. **2. *Apoptotic cells****:* - After ultraviolet light irradiation, **cytochrome-c-GFP** was released into the cytosol in apoptotic cells. - Cells at the bottom half of the micrograph released **cytochrome c**, while cells in the upper half retained it in mitochondria. **Mechanism of the Intrinsic Pathway** 1\. ***Cytochrome c release:*** - Once in the **cytosol**, cytochrome c binds to **Apaf1** (Apoptotic Protease Activating Factor-1). 2\. ***Apoptosome formation**:* - Cytochrome c binding causes **Apaf1** to oligomerize into a **wheel-like heptamer**, called the **apoptosome**. 3\. ***Recruitment of Initiator Procaspase-9**:* - **Apaf1** in the apoptosome recruit's **initiator procaspase-9**. 4\. ***Caspase cascade activation**:* - **Activated caspase-9** activates downstream **executioner procaspases**, leading to apoptosis. **Regulation Intrinsic pathway** **Bcl2** proteins **regulate** the **intrinsic pathway** of apoptosis. They **control** the release of **cytochrome c.** **Regulation of the Intrinsic Pathway** **Role of Bcl2 Proteins** 1. ***BH123 Proteins**:* - **Activated by apoptotic stimuli**. - Aggregate on the **outer mitochondrial membrane**. - Facilitate the release of **cytochrome c** and other proteins from the intermembrane space into the cytosol through an **unknown mechanism**. 2. ***Bcl2 Proteins (Anti-apoptotic)**:* - In the **absence of an apoptotic stimulus**, they bind to and inhibit **BH123 proteins** on the mitochondrial outer membrane. 3. ***BH3-only Proteins**:* - In the **presence of an apoptotic stimulus**, **BH3-only proteins** are activated. - Bind to anti-apoptotic **Bcl2 proteins**, preventing them from inhibiting **BH123 proteins**. - This allows **BH123 proteins** to aggregate on the mitochondrial outer membrane and promote the release of mitochondrial proteins into the cytosol. **Intrinsic pathway regulation** - **Bcl2** proteins are **not** the only ***intracellular regulators*** of apoptosis. - The ***IAPs (inhibitors of apoptosis)*** proteins also play an important part in ***suppressing apoptosis.*** - ***Intrinsic pathway regulation; inhibition: IAPs*** - ***IAPs*** are inhibitors of apoptosis **First discovered in viruses.** - Now it is known that it is **produced** in **many animals' cells.** **Proposed Model for the Roles of IAPs and Anti-IAPs in the Control of Apoptosis in Mammalian Cells** **In the Absence of an Apoptotic Stimulus** **1. *IAPs (Inhibitors of Apoptosis Proteins):*** - Prevent accidental apoptosis caused by the **spontaneous activation of procaspases**. - Located in the **cytosol**. - Bind to and inhibit any **spontaneously activated caspases**. **2. *Some IAPs function as ubiquitin ligases:*** - **Ubiquitylate** the caspases they bind to. - Mark caspases for **degradation in proteasomes** (not shown in the diagram). **Intrinsic pathway regulation; overcoming IAPs: anti-IAPs** **Role of Anti-IAP Proteins in the Intrinsic Pathway** 1\. ***Activation by an Apoptotic Stimulus**:* - When the intrinsic pathway is activated, proteins are released from the **mitochondrial intermembrane space**. 2\. ***Anti-IAP Proteins**:* - Bind to and block the **inhibitory activity** of IAPs. 3\. ***Cytochrome c Release**:* - Triggers the assembly of **apoptosomes**. 4\. ***Caspase Cascade Activation**:* - The apoptosome activates a **caspase cascade**, ultimately leading to **apoptosis**. **Extracellular Survival Factors Inhibit Apoptosis in various ways** **Three Ways Extracellular Survival Factors Can Inhibit Apoptosis** **(A) *Stimulation of Anti-apoptotic Gene Transcription*** 1. **Survival factors** suppress apoptosis by stimulating the transcription of genes encoding **anti-apoptotic Bcl2 proteins**, such as: 1. **Bcl2** itself. 2. **Bcl-XL**. **(B) *Activation of Serine/Threonine Protein Kinase Akt*** 1. ***Akt activation**:* - Many survival factors activate the **serine/threonine protein kinase Akt**. - Akt phosphorylates and inactivates **Bad**, a BH3-only pro-apoptotic protein. ***Effect of Bad phosphorylation**:* - When **Bad is not phosphorylated**, it promotes apoptosis by: - Binding to and inhibiting **Bcl2**. - When **Bad is phosphorylated**, it dissociates, freeing **Bcl2** to suppress apoptosis. **Other Akt actions**: - Akt suppresses apoptosis by phosphorylating and inactivating **gene regulatory proteins** of the **Fork head family**, which stimulate transcription of pro-apoptotic genes (details not shown). **(C) *Regulation in Drosophila*** - Some survival factors in **Drosophila** inhibit apoptosis by stimulating the **phosphorylation of the anti-IAP protein Hid**. - ***Hid regulation**:* - When **not phosphorylated**, Hid promotes cell death by inhibiting **IAPs**. - When **phosphorylated**, hid no longer inhibits IAPs, which then become active and block apoptosis. **Either Excessive or insufficient apoptosis can contribute to disease** **Disorders Associated with Abnormal Apoptosis** **1. *Excessive Apoptosis*** **Examples**: - **Heart attacks and strokes**: - In acute conditions, many cells die due to necrosis caused by **ischemia** (inadequate blood supply). - However, some less-affected cells die by apoptosis. - ***Future hope**:* - Drugs such as **caspase inhibitors** that block apoptosis might save cells in these conditions. **2. *Insufficient Apoptosis*** - ***Causes**:* - **Mutations in genes** encoding the Fas death receptor or the Fas ligand prevent the normal death of some lymphocytes. - ***Effects**:* - Causes these cells to **accumulate in excessive numbers** in the spleen and lymph glands. - Leads to **autoimmune diseases**, where lymphocytes react against the individual's tissues. **3. *Role in Tumours and Cancer*** - **Decreased apoptosis** is a significant factor in tumor development as cancer cells often regulate the apoptotic program abnormally. - ***Role of Bcl2 gene**:* - First identified in a common form of **lymphocyte cancer** in humans. - A chromosomal translocation causes excessive production of the **Bcl2 protein**. - Increased Bcl2 levels protect lymphocytes from apoptosis, resulting in **B-cell lymphoma**. - ***Impact of high Bcl2 levels**:* - Prolongs cell survival and increases cell numbers by inhibiting apoptosis. - Decreases sensitivity to anticancer drugs that kill cancer cells by inducing apoptosis. **4. *p53 Mutation in Cancer*** - ***Role of p53**:* - A tumour suppressor protein that promotes apoptosis or cell-cycle arrest in response to DNA damage. - Its loss allows cells to survive and proliferate even with DNA damage. - This leads to the accumulation of more mutations, making cancer cells more malignant. - ***Impact on treatment**:* - Many anticancer drugs rely on a **p53-dependent mechanism** to induce apoptosis. - The **loss of p53 function** makes cancer cells less sensitive to these drugs. **Summary** **Overview of Programmed Cell Death (Apoptosis):** 1. ***Definition and Purpose:*** - Cells can activate a death program to kill themselves in a controlled way, termed **programmed cell death**. - This process allows elimination of irreversibly damaged or unnecessary cells, benefiting the organism. 2. ***Characteristics of Apoptosis:*** - Cells **shrink**, become **dense**, and **frequently fragment**. - Neighbouring cells or macrophages **rapidly phagocytose** the fragments, preventing cytoplasmic content leakage. 3. ***Role of Caspases:*** - Apoptosis relies on **proteolytic enzymes** called **caspases** to destroy intracellular components. - Caspases are present as inactive **procaspases** in animal cells. - **Initiator procaspases** are activated by bringing them into **activation complexes**. - Activated initiator procaspases cleave **executioner procaspases**, triggering a cascade of **irreversible proteolysis**. **Pathways for Caspase Activation:** 1. ***Extrinsic Pathway:*** - Activated by **extracellular ligands** binding to **cell-surface death receptors**. - Death receptors recruit **procaspases-8 and -10** via **adaptor proteins** to form the **DISC** (death-inducing signalling complex). 2. ***Intrinsic Pathway:*** - Triggered by **intracellular stress signals**. - **Cytochrome c**, released from the **mitochondrial membrane**, activates **Apaf1**, which forms an **apoptosome**. - The apoptosome recruits and activates **procaspase-9**. **Regulation of Apoptosis:** 1. ***Role of Bcl2 Proteins:*** - Extracellular signal proteins and intracellular **Bcl2 proteins** and **IAP proteins** tightly regulate apoptosis. - These proteins ensure cells only undergo apoptosis when it benefits the organism. 2. ***Pro-apoptotic vs Anti-apoptotic:*** - **Anti-apoptotic Bcl2 proteins** prevent mitochondrial intermembrane release. - **Pro-apoptotic Bcl2 proteins** promote cytochrome c release, activating caspases and facilitating apoptosis.