Apoptosis, Cell Signaling, and Necrosis – Comprehensive Study Notes

Cell Cycle Overview

• Interphase

  • Period between cell divisions; nuclear membrane intact, chromatin dispersed

  • Sub-phases and their hallmarks

  • G_1 (Gap 1) – cells in diploid state, growth & preparation for DNA synthesis

  • S (Synthesis) – DNA replication

  • G_2 (Gap 2) – preparation for mitosis, proofreading of replicated DNA

• Mitosis (M-phase)

  • Chromatin condenses; nuclear envelope disintegrates

  • Mitotic spindle forms, replicated chromosomes segregate to opposite poles

  • Cytokinesis yields two daughter cells

Cell-Cycle Check-Points & Molecular Control

• G_1 Check-point (“restriction point”)

  • DNA integrity assessment; damaged DNA must be repaired before entry into S phase

• G_2 Check-point

  • Requires complete, error-free DNA replication before mitosis can start

• Molecular governors

  • Cyclins – oscillating proteins; their periodic accumulation drives cell-cycle transitions

  • Cyclin-dependent kinases (cdk) – catalytically active only when bound to cyclins ➔ phosphorylate targets

  • Inhibitory proteins – Retinoblastoma protein (Rb) & p53 enforce arrest or apoptosis upon damage

Apoptosis – Programmed Cell Death

• Functional importance

  • Sculpting structures in morphogenesis

  • Immune tolerance (deletion of autoreactive lymphocytes)

  • Elimination of damaged, infected, or excess cells

  • Failure ➔ cancer, auto-immunity; excess ➔ degenerative or ischemic disease

• General features

  • Energy-dependent (ATP consumed)

  • Cell shrinkage, membrane blebbing, nuclear fragmentation, chromatin condensation

  • Plasma membrane integrity preserved until final stages; contents not spilled ➔ no inflammation

  • DNA cleavage occurs at internucleosomal intervals of \approx 185\,\text{bp} (DNA ladder)

Necrosis vs. Apoptosis

Molecular Machinery of Apoptosis

Caspases – Cysteine ASPartate-proteASES

• Synthesised as inactive procaspases (prodomain + large & small enzymatic subunits)

• Proteolytic cascade: initiator caspases (e.g. \text{C}8, \text{C}9) activate effector caspases (e.g. \text{C}3, \text{C}6, \text{C}_7)

• Active sites contain conserved Asp-28 & Asp-175 residues (numbering from caspase-3)

Extrinsic (Death-Receptor) Pathway

• Ligands: FasL, TNF-\alpha bind to receptors (e.g. CD95/Fas, TNFR)

• Ligand binding ➔ receptor trimerisation ➔ exposure of intracellular Death Domains (DD)

• Adapter FADD (Fas-Associated Death Domain) recruited; its DED binds procaspase-8

• Death-Inducing Signalling Complex (DISC) formed ➔ autocatalytic activation of caspase-8 ➔ cascade ➔ caspase-3 activation ➔ demolition phase

• Inhibitors: c-FLIP/FLIPs compete with caspase-8 at DISC

Intrinsic (Mitochondrial) Pathway

• Triggers: DNA damage, toxins, ROS, Ca^{2+} overload, UV radiation

• Mitochondrial outer membrane permeabilisation (MOMP) ➔ release of cytochrome c (requires ATP)

• Cyt c + Apaf-1 + dATP form apoptosome ➔ recruits/activates procaspase-9 ➔ activates effector caspases

• Bcl-2 family proteins regulate MOMP

  • Anti-apoptotic: Bcl-2, Bcl-xL – sequester Apaf-1 & inhibit Bax/Bak

  • Pro-apoptotic: Bax, Bak (oligomerise to form pores); Bid (truncated by caspase-8 links extrinsic to intrinsic pathway)

• Inhibitors of Apoptosis Proteins (IAPs) – XIAP, c-IAP1/2, survivin bind & inhibit active caspases; countered by Smac/DIABLO

Execution Phase

• Effector caspases cleave >100 substrates ➔ characteristic changes

  • Poly(ADP-ribose) polymerase (PARP) inactivation (DNA repair off)

  • Lamins, actin, fodrin cleavage (nuclear & cytoskeletal breakdown)

  • Phosphatidyl-serine externalisation (“eat-me” signal)

Summary Flowchart

( \text{Death receptor (Fas/TNF)} \;\xrightarrow{C8}\; C3,6,7 ) \quad \Longleftrightarrow \quad ( \text{Mitochondrion} \;\xrightarrow{Cyto\,c + Apaf\,1 + C9}\; C3,6,7 )

Clearance of Apoptotic Cells

• Rapid, programmed removal prevents leakage & auto-immunity

• “Eat-me” signals

  • Externalised phosphatidyl-serine (PS) recognised via annexin I bridges

  • Altered carbohydrates detected by lectin-like receptors

• Phagocyte receptors: Class A scavenger receptor (SRA), CD36 (class B), integrins \alphav\beta3 & \alphav\beta5

• Clearance functions beyond garbage disposal

  • Supplies pro-survival cytokines for tissue repair

  • Secretes anti-inflammatory mediators limiting collateral damage

  • Complements weak apoptotic signals (engulfment itself finalises death)

Pathological & Clinical Implications

• Cancer: apoptosis resistance (Bcl-2 over-expression, p53 loss, survivin up-regulation)

• Auto-immune disease: defective clearance ➔ auto-antigen exposure & auto-antibody production

• Neurodegeneration

  • Alzheimer: caspase cleavage of amyloid precursor protein (APP)

  • Huntington: poly-glutamine expanded huntingtin recruits caspase-8

  • Spinal muscular atrophy: loss of neuronal apoptosis inhibitory protein

• Cardiovascular

  • Ischemia/reperfusion injury; myocardial failure; Arrhythmogenic Right Ventricular Dysplasia (ARVD)

• Immune tolerance & chronic inflammation (e.g. arthritis) modulated by balanced apoptosis

Cell Signalling Fundamentals (Extended)

Principles

• Signal-producing cell secretes signalling molecule ➔ binds receptor on target cell

• One cell interprets limited signals owing to selective receptor & relay complement

Signalling Ranges

• Autocrine – self-targeting (e.g. IL-2 in T-cells)

• Paracrine – local diffusion (e.g. TGF-\beta)

• Endocrine – hormones via bloodstream (e.g. testosterone)

• Neuronal – synaptic neurotransmission (acetylcholine)

• Contact-dependent – membrane-bound ligand (e.g. Delta-Notch)

Receptors Classes

1. Ion-Channel-Linked – convert chemical ➔ electrical (nicotinic ACh receptor)

2. G-Protein-Coupled Receptors (GPCRs) – 7-pass transmembrane proteins

3. Enzyme-Linked Receptors – intrinsic or associated kinase activity (e.g. RTKs)

4. Intracellular (nuclear) receptors – for hydrophobic ligands (steroids, NO)

Signal Cascades – 4 Key Functions

1. Transduce – transform the stimulus form

2. Relay – propagate from membrane to effectors

3. Amplify – one receptor activates many downstream molecules (rhodopsin: 1 photon ➔ 10^6–10^7 Na^+ ions blocked)

4. Distribute/Modulate – branch to multiple targets, integrate other signals

GPCR Pathways

• Activation cycle: ligand → receptor → conformational change → GDP→GTP exchange on G\alpha → G\alpha ± G{\beta\gamma} act on effectors → intrinsic GTPase off-switch (cholera toxin locks G\alpha on)

• Ion channel regulation: ACh → G_{\beta\gamma} opens cardiac K^+ channel

• Second messengers

  • cAMP – adenylyl cyclase converts ATP → cAMP; degraded by phosphodiesterase; activates PKA (rapid cytosolic effects & slow transcriptional responses via CREB)

  • IP3 & DAG – PLC-β cleaves PIP2 → IP3 (opens ER Ca^{2+} channels) + DAG (with Ca^{2+} recruits & activates PKC)

  • Ca^{2+} – binds calmodulin → CaM-kinase cascade

Enzyme-Linked Receptors

• Receptor Tyrosine Kinases (RTKs)

  • Ligand-induced dimerisation → trans-autophosphorylation → docking sites for SH2/PTB-containing proteins

  • Major branches

  • PI3-K/AKT (cell survival)

  • PLC-γ/IP3/DAG (calcium & PKC)

  • Ras/MAPK (proliferation)

• Ras–MAPK cascade

  • Grb2-SOS act as GEF for Ras (monomeric G-protein)

  • Activated Ras → Raf (MAPKKK) → MEK (MAPKK) → ERK (MAPK) → cytoplasmic targets & nuclear gene regulators

  • Ras mutations (cannot hydrolyse GTP) cause constitutive signalling (≈30 % human cancers)

JAK-STAT Pathway

• Cytokine receptor lacks intrinsic kinase; bound JAK kinases phosphorylate each other & receptor

• STATs dock, get phosphorylated, dimerise, translocate to nucleus → immediate gene activation

SMAD/TGF-\beta Pathway

• TGF-\beta binds serine/threonine kinase receptor → receptor phosphorylates SMAD2/3 → binds SMAD4 → nuclear complex regulates development & ECM genes

Intracellular Hydrophobic Signal Molecules

• Steroid hormones (cortisol, estradiol, testosterone), thyroxine cross membrane → bind cytosolic or nuclear receptors → receptor-ligand complex acts as transcription factor

• Nitric Oxide (NO) diffuses, activates guanylyl cyclase → GTP \rightarrow cGMP (vasodilation; Viagra blocks cGMP breakdown)

Molecular Switches

• Phosphorylation: kinases vs phosphatases (Ser/Thr, Tyr)

• GTP-binding proteins: G-proteins (trimeric) & small GTPases (e.g. Ras, Rho, Rab)

Network Cross-Talk

• Signals interweave (GPCR-derived Ca^{2+} can modulate RTK pathways; PKC influences MAPK etc.)

• Figure 16-38 shows convergence on MAPK, PKA, CaM-kinase, PKC

Necrosis – Irreversible Cell Injury

Definition & Causes

• Necrosis: spectrum of morphological changes following rapid cell death within living tissue

• Common triggers: ischemia, physical trauma, chemical toxins, immunologic injury

• Mechanism: protein denaturation + enzymatic digestion by lysosomal hydrolases; membrane disruption ➔ inflammation

Major Morphologic Types

1. Coagulative necrosis

   • Architecture preserved (“tombstone” outlines), tissue firm

   • Typical of heart, kidney, spleen infarcts

2. Liquefactive necrosis

   • Tissue transforms to viscous liquid; creamy yellow pus in abscess; characteristic of brain infarcts

3. Gangrenous necrosis (clinical term)

   • Dry gangrene: arterial occlusion – limb black, dry, clear demarcation, no bacteria

   • Wet gangrene: venous/concurrent infection – bowel, diabetic foot; swollen, foul odour, poor prognosis

   • Gas gangrene: Clostridial infection produces gas bubbles

4. Caseous necrosis

   • Cheese-like, white, friable; typical of tuberculosis; combination of coagulative & liquefactive features

5. Fat necrosis

   • Pancreas & breast; lipase-released fatty acids + Ca^{2+} ➔ chalky white saponification

6. Fibrinoid necrosis

   • Immune-mediated vascular injury; deposition of fibrin-like eosinophilic material in vessel walls (e.g. malignant hypertension, vasculitis)

Cellular Response Continuum

\text{Normal} \;\rightarrow\; \text{Adaptation} \;\rightarrow\; \text{Reversible injury} \;\rightarrow\; \text{Irreversible injury} \;\begin{cases}\text{Necrosis} \ \text{Apoptosis}\end{cases}