BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956

Apoptosis:Extrinsic Pathway

BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956

Extrinsic pathway also known as the “death receptor pathway”.

The extrinsic pathway begins outside the cell through activation of pro-apoptotic receptors on the cell surface.

The pro-apoptotic receptors are activated by molecules known as pro-apoptotic ligands.

Ligand binding causes receptors to cluster and ultimately form a death-inducing signaling complex (DISC).

Upon DISC activation, the extrinsic pathway has been seen to adopt the same effector caspase machinery as the intrinsic pathway

Introduction

BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956

Based on the triggering stimulus and nature of the components involved two Apoptotic pathways can be differentiated:

One involving Receptor Systems Second triggered by Cytotoxic Stress Receptor mediated pathways include those

activated by Death ligands. Stress effects that can induce Apoptosis are

Gamma- and UV radiation, treatment with cytotoxic drugs such as Actinomycin D.

Types of Extrinsic Pathways

BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956

• Death Receptors (DRs) are Cell surface Receptors.

• They transmit Apoptotic signals initiated by specific ligands.

• Play a central role in instructive Apoptosis.• Death receptors (DRs) activate Death

Caspases within seconds of ligand binding, causing an Apoptotic demise of the cell within hours.

Death Receptors

BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956

• DRs (Death Receptors) belong to the superfamily of TNFR (Tumor Necrosis Factor Receptor).

• TNFR (Tumor Necrosis Factor Receptor) are characterized by a Cys-rich Extracellular Domain.

• These have a homologous Intracellular Domain known as the Death Domain.

• The Fas receptor binds the Fas ligand (FasL), a transmembrane protein part of the TNF family. The interaction between Fas and FasL results in the formation of the death-inducing signaling complex (DISC), which contains the FADD, caspase-8 and caspase-10.

Death Receptors

BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956

• FADD (Fas-Associated via Death Domain)• TRADD (Tumor Necrosis Factor Receptor-1-

Associated Death Domain) or Daxx • The Adapter-molecules contain Death

Domains so that they can interact with the DRs and transmit the Apoptotic signal to the death-machinery. The best-characterized Death Receptors are Fas and TNFR1 (Tumor Necrosis Factor Receptor-1).

Adapter Molecules

BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956

Death Inducing Signaling Complex (DISC) formation requires following steps:

a. FasL (homotrimeric protein) acts as ligand for Fas causing oligomerization of its Receptor on binding.

b. Clustering of the Death Domains and binding of cofactor FADD.

c. Binding of FADD protein via its DED (Death Effector Domain) motif to a homologous motif in Procaspase8.

Complex of Fas, FADD and ProCaspase8 is called the DISC

Death Inducing Signaling Complex

BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956

• Caspase play the central role in the transduction of DR apoptotic signals.

• Caspases are proteins that are highly conserved, cysteine-dependent aspartate-specific proteases.

• There are two types of caspases: • initiator caspases, caspase 8,10,9,2, and • effector caspases, caspase 3,7,6.

• The activation of initiator caspases requires binding to specific oligomeric adaptor protein.

• Effector caspases are then activated by these active initiator caspases.

Caspases

BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956

• Disc formation is followed by blockage of FADD cofactor function by interaction with the regulator FLIP (FLICE Inhibitory Protein).

• Recruitment by FADD • Procaspase8 oligomerization • FADD activation through self-cleavage of

oligomerized Procaspase8.• Activation of downstream Caspases

(Caspase3 and 7) by activated Caspase8 committing the cell to Apoptosis.

Cell Apoptosis

BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956

Animation

BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956

After a cell receives stimulus, it undergoes organized degradation of cellular organelles by activated caspases. A cell undergoing apoptosis shows a characteristic morphology:

• Cell shrinkage and rounding are shown because of the breakdown of the proteinaceous cytoskeleton by caspases.

• The cytoplasm appears dense, and the organelles appear tightly packed.

• Chromatin undergoes condensation into compact patches against the nuclear envelope (also known as the perinuclear envelope) in a process known as pyknosis, a hallmark of apoptosis.

• The nuclear envelope becomes discontinuous and the DNA inside it is fragmented in a process referred to as karyorrhexis. The nucleus breaks into several discrete chromatin bodies ornucleosomal units due to the degradation of DNA.

• The cell membrane shows irregular buds known as blebs.• The cell breaks apart into several vesicles called apoptotic bodies,

which are then phagocytosed.

Execution

BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956

• The removal of dead cells by neighboring phagocytic cells has been termed efferocytosis.

• Dying cells that undergo the final stages of apoptosis display phagocytotic molecules, such as phosphatidylserine, on their cell surface.

•  Phosphatidylserine is normally found on the cytosolic surface of the plasma membrane, but is redistributed during apoptosis to the extracellular surface by a protein known as scramblase.

• These molecules mark the cell for phagocytosis by cells possessing the appropriate receptors, such as macrophages.

• Upon recognition, the phagocyte reorganizes its cytoskeleton for engulfment of the cell.

• The removal of dying cells by phagocytes occurs in an orderly manner without eliciting an inflammatory response.

Removal of dead cells