Lecture 24

Apoptosis

Apoptosis Overview

  • Apoptosis is a distinctive and important mode of "programmed" cell death.

Historical Context

  • Nobel Prize in Physiology or Medicine 2002 awarded to:

    • John E. Sulston

    • Sydney Brenner

    • Robert Horvitz

  • Recognized for discoveries concerning genetic regulation of organ development and programmed cell death, particularly in Caenorhabditis elegans (C. elegans).

Observational Studies in C. elegans

  • The lineage of all cells in C. elegans is known, allowing observation of cells in vivo.

    • Researchers were able to see cells "dying" during development.

  • Mutants in C. elegans were isolated, showing impaired or failed clearance of apoptotic cells.

    • Examples of types:

      • Wild-type

      • ced-1 mutant

  • Microscopy images noted with a measurement of 10 µm.

Apoptotic Pathway Construction

  • The apoptotic pathway includes stages:

    • Specification

    • Killing

    • Execution

  • Key components include:

    • Genes: crn-1, crn-4, eor-1, cps-6, crn-5, eor-2, HSNs, wah-1, cyp-13, tra-1, nuc-1, crn-6, crn-2, egl-1, ced-9, ced-4, ced-3, crn-3, hlh-2, hlh-3, ces-2, ces-1, ced-1, ced-6, ced-7, ced-2, ced-10, ced-12, psr-1, ced-5.

  • The apoptosis process includes:

    • DNA degradation

    • Corpse engulfment

Functional Roles of Apoptosis in Development

  • Apoptosis sculpting is crucial in developing organisms, exemplified by:

    • Sculpting digits in the developing mouse paw.

    • Clear connecting tissue to form joints in arthropods.

  • Normally functioning apoptosis results in organized cell death without distress to neighboring cells.

    • Contrast with necrosis:

      • Necrotic cells release contents into their surroundings, causing damage.

Mechanism of Apoptosis

  • Apoptosis depends on a proteolytic cascade mediated by Cysteine Aspartate Proteases (Caspases).

    • Caspase-9 is described as an initiator caspase.

    • Caspase-3 is identified as an effector caspase.

    • Caspases activate by cutting strategic proteins in the cell; involve cleavage next to aspartate amino acids.

    • They are cysteine proteases utilizing sulfur from cysteine for cleavage reactions.

Caspase Classification

  • Initiator caspases (Caspase 8 and 9) activate executioner caspases (Caspase 3, 6, and 7).

  • Executioner caspases are synthesized as procaspases and activated by cleavage at aspartic acids by other caspases.

    • Structure includes large and small subunits.

  • Initiator caspases are inactive monomers that activate upon dimerization and self-cleavage.

Activation and Amplification of Apoptosis

  • A cascade of caspase activations enhances the magnitude of apoptosis:

    • One molecule of active caspase can activate multiple downstream molecules.

    • Targets include cytosolic proteins and nuclear lamins.

Role of Caspase-Activated DNase (CAD)

  • CAD catalyzes the hydrolytic cleavage of DNA, leading to apoptotic cell death.

    • Inactive CAD (iCAD) is cleaved by active executioner caspases, allowing for DNA cleavage between nucleosomes, resulting in chromatin fragments.

Activation Pathways of Apoptosis

  • Cells employ two main activation pathways:

    • Extrinsic Pathway:

      • Initiated by cell-surface death receptors through external signals (e.g., Fas ligand).

      • Activation involves clustering of inactive Fas death receptors with FADD adaptor proteins and inactive caspase-8.

    • Intrinsic Pathway:

      • Triggered by internal signals such as DNA damage, leading to the release of cytochrome c from mitochondria.

      • Cytochrome c binds to Apaf1, leading to apoptosome formation.

Role of Apaf1

  • Apaf1 (apoptotic protease activating factor 1) assembles into a huge complex known as the apoptosome, which activates caspases.

  • Apaf1 activation is critical for assembly and oligomerization in response to cytochrome c.

Inhibitory Mechanisms

  • The activity of caspases can be regulated by inhibitors of apoptosis (IAPs), which bind and inhibit various procaspases.

  • Bcl-2 proteins play a significant role in the regulation of the intrinsic pathway:

    • Pro-apoptotic Bcl-2 proteins create pores in the mitochondrial membrane.

    • Anti-apoptotic Bcl-2 proteins prevent the activation of pro-apoptotic Bcl-2 proteins.

Recognition of Apoptotic Cells

  • Phosphatidylserine (PtdSer) exposure on apoptotic cells signals for removal by neighboring healthy cells; normally, PtdSer is contained within the cell.

Distinction with Other Cell Death Mechanisms

  • Ferroptosis:

    • A distinct, iron-dependent form of cell death characterized by lipid peroxidation in cells, differing from both apoptosis and necrosis.

Summary of Key Points

  • Apoptosis is mediated by the proteases known as caspases.

  • Caspases (ex: Caspase-9) initiate the apoptosis pathway, while Caspase-3 functions as an effector.

  • The apoptosis process can be initiated from either the intrinsic pathway (via mitochondria and cytochrome c) or the extrinsic pathway (via cell-surface receptors).

  • Key genes and components related to apoptosis include various caspases, Bcl-2 family proteins, and adaptors like Apaf-1.

  • Mechanisms to inhibit unwanted apoptosis involve IAPs and regulatory roles of Bcl-2 proteins.

  • Understanding these processes offers insights into cell regulation mechanisms, potential therapeutic approaches in diseases, especially cancer and developmental disorders.