Cell Death and Apoptosis

Cell Death

Introduction

• Multicellular organisms rely on both cell production and destruction for growth, development, and maintenance.

• Apoptosis is a programmed sequence of molecular events where a cell systematically destroys itself from within and is then eaten by other cells, leaving no trace.

• Tissue size is maintained by balancing cell production and cell death rates.

• Programmed cell death occurs through apoptosis, from the Greek word meaning “falling off”.

• Cells also undergo programmed cell death when damaged or infected to protect the organism.

Apoptosis vs. Necrosis

• Apoptosis:

• 10k to 10 million cells in our bodies die daily through apoptosis.

  • Cells shrink and condense.

  • The cytoskeleton collapses.

  • The nuclear envelope disassembles.

  • Nuclear chromatin condenses and breaks into fragments.

  • The cell surface bulges outward, forming membrane-enclosed apoptotic bodies.

  • The cell or apoptotic bodies are chemically altered to be engulfed by neighboring cells or macrophages.

  • Apoptosis is a neat process that is rapidly cleared away without causing inflammation.

• Necrosis:

  • Occurs in response to acute insults like trauma or lack of blood supply.

  • Cells swell and burst, spilling contents and causing inflammation.

  • Often caused by energy depletion, leading to metabolic defects and loss of ionic gradients.

• Necroptosis:

  • A form of programmed cell death triggered by specific regulatory signals.

Functions of Apoptosis

• Eliminates unwanted cells during development and in adult tissues.

• Sculpts structures like hands and feet during embryonic development.

• Eliminates cells when the structures they form are no longer needed, such as the tail of a tadpole during metamorphosis.

• Serves as a quality-control process, eliminating abnormal, misplaced, nonfunctional, or potentially dangerous cells.

• Eliminates developing T and B lymphocytes that are self-reactive or fail to produce useful antigen-specific receptors.

• Removes most lymphocytes activated by an infection after they have destroyed the responsible microbes.

• Maintains balance between cell death and cell division in adult tissues.

• Prevents cancer by causing cells with irreparable DNA damage to undergo apoptosis.

Caspases: Mediators of Apoptosis

• Apoptosis is mediated by a family of specialized intracellular proteases called caspases.

• Caspases cleave specific sequences in numerous proteins, leading to cell death and engulfment.

• Caspases have a cysteine at their active site and cleave target proteins at specific aspartic acids.

• Caspases are synthesized as inactive precursors and are activated only during apoptosis.

• Two major classes of apoptotic caspases:

  • Initiator caspases

  • Executioner caspases

Caspase Activation

• Initiator caspases begin the apoptotic process.

• Apoptotic signals trigger the assembly of large protein platforms that bring multiple initiator caspases together into large complexes.

• Within these complexes, caspases associate to form dimers, resulting in protease activation.

• Each caspase in the dimer cleaves its partner, stabilizing the active complex.

• The major function of initiator caspases is to activate executioner caspases.

• Executioner caspases exist as inactive dimers and are cleaved by initiator caspases to become active.

• Activated executioner caspases catalyze widespread protein cleavage events that kill the cell.

• The caspase cascade is destructive, self-amplifying, and irreversible.

• Proteins cleaved by caspases include:

  • Nuclear lamins

  • Proteins holding DNA-degrading endonuclease

  • Components of the cytoskeleton

  • Cell-cell adhesion proteins

Extrinsic Pathway

• Extracellular signal proteins binding to cell-surface death receptors trigger the extrinsic pathway of apoptosis.

• Death receptors are transmembrane proteins with an extracellular ligand-binding domain, a transmembrane domain, and an intracellular death domain.

• Death receptors are homotrimers and belong to the tumor necrosis factor (TNF) receptor family.

• Ligands that activate death receptors are also homotrimers and belong to the TNF family of signal proteins.

• Fas death receptor is activated by Fas ligand on killer lymphocytes.

• The death domains on the cytosolic tails of Fas death receptors bind intracellular adaptor proteins, which bind initiator caspases (caspase-8), forming a death-inducing signaling complex (DISC).

• Initiator caspases cleave their partners and then activate downstream executioner caspases to induce apoptosis.

• The extrinsic pathway can recruit the intrinsic apoptotic pathway to amplify the caspase cascade.

• Some cells produce the protein FLIP, which resembles an initiator caspase but lacks protease activity, blocking the apoptotic signal.

Intrinsic Pathway

• Cells activate the apoptosis program from inside the cell in response to stresses like DNA damage or developmental signals.

• In vertebrate cells, these responses are governed by the intrinsic, or mitochondrial, pathway of apoptosis.

• The intrinsic pathway depends on the release into the cytosol of mitochondrial proteins, such as cytochrome c, that normally reside in the intermembrane space.

• Released cytochrome c activates a caspase proteolytic cascade in the cytoplasm.

• When released into the cytosol, cytochrome c binds to an adaptor protein called Apaf1 (apoptotic protease activating factor-1), causing it to oligomerize into an apoptosome.

• The Apaf1 proteins in the apoptosome recruit initiator caspase-9 proteins, which are activated by proximity in the apoptosome.

• Activated caspase-9 molecules then activate downstream executioner caspases to induce apoptosis.

Bcl2 Proteins: Regulators of the Intrinsic Pathway

• The intrinsic pathway of apoptosis is tightly regulated by Bcl2 family proteins.

• Some Bcl2 family proteins are pro-apoptotic, while others are anti-apoptotic.

• The balance between these two classes of proteins determines whether a cell lives or dies by the intrinsic pathway of apoptosis.

• Anti-apoptotic Bcl2 family proteins inhibit apoptosis by blocking the release of cytochrome c and other intermembrane mitochondrial proteins into the cytosol.

• Pro-apoptotic proteins enhance the release.

• The anti-apoptotic Bcl2 family proteins, including Bcl2 and BclXL, share four Bcl2 homology (BH) domains (BH1–4).

• The pro-apoptotic Bcl2 family proteins consist of the effector proteins Bax and Bak, and the BH3-only proteins.

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Role of Pro-apoptotic Proteins

• When an apoptotic stimulus triggers the intrinsic pathway, pro-apoptotic effector Bcl2 family proteins become activated and aggregate to form oligomers in the mitochondrial outer membrane.

• This aggregation induces the release of cytochrome c and other intermembrane proteins.

• In mammalian cells, Bax and Bak are the main effector Bcl2 family proteins.

• Activated pro-apoptotic BH3-only proteins usually depend on the activation of Bax and Bak.

Role of Anti-apoptotic Proteins

• Anti-apoptotic Bcl2 family proteins, such as Bcl2 and BclXL, are located on the cytosolic surface of the outer mitochondrial membrane.

• They inhibit apoptosis by binding to and inhibiting pro-apoptotic Bcl2 family proteins.

• BH3-only proteins mediate the inhibition of anti-apoptotic proteins.

• BH3-only proteins bind to a hydrophobic groove on anti-apoptotic Bcl2 family proteins, neutralizing their activity, which enables the aggregation of Bax and Bak.

• BH3-only proteins provide the crucial link between apoptotic stimuli and the intrinsic pathway of apoptosis.

• In some cells, the extrinsic apoptotic pathway recruits the intrinsic pathway by activating the BH3-only protein Bid.

IAPs: Inhibitors of Apoptosis

• Cells employ multiple mechanisms to ensure that caspases are activated only when appropriate, one of which is the family of proteins call inhibitors of apoptosis (IAPs).

• IAPs bind to and inhibit activated caspases and can also polyubiquitylate caspases, marking them for destruction.

• The inhibitory barrier provided by IAPs can be neutralized by anti-IAP proteins.

• Anti-IAPs bind to the BIR domain of IAPs, preventing them from binding to a caspase.

• When the intrinsic pathway of apoptosis is activated, anti-IAPs are released from the mitochondrial intermembrane space, blocking IAPs in the cytosol.

Extracellular Survival Factors

• Some extracellular signal molecules stimulate apoptosis, while others inhibit it.

• Extracellular signal molecules that inhibit apoptosis are called survival factors.

• Most animal cells require continuous signaling from other cells to avoid apoptosis.

• Nerve cells, for example, compete for limited amounts of survival factors secreted by target cells.

• Survival factors usually bind to cell-surface receptors, activating intracellular signaling pathways that suppress the apoptotic program.

• Some survival factors stimulate the synthesis of anti-apoptotic Bcl2 family proteins.

• Others act by inhibiting the function of pro-apoptotic BH3-only proteins.

• In Drosophila, some survival factors act by phosphorylating and inactivating anti-IAP proteins.

Phagocytes and Apoptotic Cells

• Apoptotic cells and their fragments do not break open and release their contents, but instead, they are efficiently phagocytosed by neighboring cells, triggering no inflammatory response.

• This engulfment depends on chemical changes on the surface of the apoptotic cell, which displays signals that recruit phagocytic cells.

• Phosphatidylserine flips to the outer leaflet in apoptotic cells.

• A variety of soluble “bridging” proteins interact with the exposed phosphatidylserine on the apoptotic cell and with specific receptors on the surface of a neighboring cell or macrophage, initiating the engulfment process.

• Healthy cells express signal proteins on their surface that interact with inhibitory receptors on macrophages that block phagocytosis.

Consequences of Dysregulation of Apoptosis

• Excessive apoptosis contributes to tissue damage in conditions like heart attacks and strokes.

• Insufficient apoptosis can lead to autoimmune disease and tumor development.

• Mutations that inactivate the genes encoding the Fas death receptor or the Fas ligand prevent the normal death of some lymphocytes, causing these cells to accumulate in excessive numbers.

• Cancer cells often regulate their apoptotic program abnormally.

• High levels of Bcl2 protein promote cancer development by inhibiting apoptosis.

• Mutations in the gene encoding the tumor suppressor protein p53 enable cancer cells to survive and proliferate even when their DNA is damaged.

• Drugs that stimulate apoptosis can be used to treat cancers.

• Small chemicals that interfere with the function of anti-apoptotic Bcl2 family proteins are being developed.