Apoptosis

Xu Zhaoyang Department of cell biology Basicl medicine colledge Zhengzhou university

Apoptosis: Death comes for the Cell From Ingmar Bergman’s The Seventh Seal

“Death is a punishment to some, to others a gift and to many a favor.” – Seneca ■

Roman playwright Seneca’s keen assessment of death resonates with modern concepts of apoptosis, or programmed cell death. Although suicide by a living unit may seem contradictory to survival, apoptosis allows a cell to sacrifice itself for the benefit of a greater living unit.

The Nobel Prize in Physiology or Medicine 2002 ■

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The Nobel Assembly at Karolinska Institutet has awarded the Nobel Prize in Physiology or Medicine jointly to Sydney Brenner, Robert Horvitz and John Sulston for their discoveries concerning "genetic regulation of organ development and programmed cell death". By using the nematode Caenorhabditis elegans as a model system, the Laureates have identified key genes regulating these processes. They have also shown that corresponding genes exist in higher species, including man.

The Nobel Prize in Physiology or Medicine 2002

Sydney Brenner, born 1927, Robert Horvitz, born 1947, La Jolla, CA, USA. Cambridge, MA, USA.

John Sulston, born 1942, Cambridge, England.

Types of Cell death ■ ■ ■ ■ ■ ■

Cells die by one of two mechanisms – necrosis or apoptosis • Two physiologically different processes – Necrosis – death by injury – Apoptosis – death by suicide • Apoptosis and necrosis have different characteristics

Features of Apoptosis Vs Necrosis Apoptosis • Chromatin condensation • Cell Shrinkage • Preservation of Organelles and cell membranes • Rapid engulfment by neighboring cells preventing inflammation • Biochemical Hallmark: DNA FRAGMENTATION

Necrosis • Nuclear swelling • Cell Swelling • Disruption of Organelles • Rupture of cell and release of cellular contents • Inflammatory response

Detection of apoptotic cells • Microscopy – Cells have classic features (eg. small darkly stained nuclei) – Detection of free 3’ ends of DNA by TUNEL assay (terminal deoxytransferase-mediated dUTP-biotin nick end labeling) • Gel electrophoresis – Detect DNA ladder of 180 bp intervals caused by inter-nucleosomal DNA cleavage

Morphologic changes during apoptosis ■ ■

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Membranes become irregular Chromatin becomes condensed and segregated Condensation of cytoplasm DNA is fragmented Cell is fragmented and phagocytose

Morphological Features of Apoptosis

Transmission electron Micrograph

Scanning electron Micrograph

Cell undergoing apoptosis The membrane of the cell blebs as DNA is condensed and destroyed. The cell then shrinks and packages itself into an easilymovable material for macrophages (the body’s cleanup crew, indicated by the black arrow).

Removal of apoptotic cell by phagocytosis

Phagocytosis tags and receptors

Removal of cell corpses

The molecular mechanism of apoptosis ■

Triggers of apoptosis

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Genes implicated in apoptosis

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Molecular mechanism

Triggers of apoptosis • Programmed cell death in which many more cells are produced than survive (e.g. development of lymphocytes) • Toxic stimuli (viruses, chemicals, ionizing radiation) • Extracellular signals (Fas, p75 NGF-R, TNF) • DNA damage (p53)

Genes implicated in apoptosis ■

Ced

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Bcl-2

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Caspases(Cystein aspartate-specific protease)

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Fas/APO-1

Why study Caenorhabditis Elegans ? ■

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Reproduce very rapidly (3 week life span). Easy to induce mutations. Capable of reproducing as Hermaphrodites. Simple organism with only 1090 somatic cells, with 131 of 1090 somatic cells normally undergo PCD. Death of these cells is not required for viability. Development is invariant and has been mapped such that the fates of all cells are known. Special optics can be used to observe abnormal deaths in living organisms.

Bcl-2 Background ■

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Juxtaposed next to the Immunoglobulin heavy chain locus. > 50 Kb intron may facilitate recombination At the time was a unique sequence with limited sequence homology.

Bcl-2 family members • A very large family with 19 members identified • All have the BH3 domain (Bcl-2 Homology) – BH-3 is the pro-apoptotic domain exposed on activation

The function of Bcl-2 Act as dimers=either hetero or homodimers – Pro-apoptotic dimers (Bax) increase mitochondrial permeability – Anti-apoptotic members (Bcl-2, Bcl-XL) form dimers with pro-apoptotic members to inactivate them

The Bcl-2 Family

Bcl-2: Proposed Mechanisms of Action Binds and inhibits Proapoptotic Family Members • Regulates ion flux across the Mitochondria and stabilizes the membrane potential (PTP) • Regulates cytochrome C release. • Binds and inactivates APAF1 • ROS inhibition • Many others: Ca Homeostasis, RAF1 interaction • Regulates VDAC and thus ATP/ADP ratio

General features of Caspases ■

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Human ICE (interleukin-1βprocessing enzyme, 1992) and C. elegans ced-3 gene (1993) were the first identified caspases; So far, 11 members from human, 4 from C. elegans and 7 from Drosophila have been identified with obvious conservation in evolution. Apoptosis substrates are numerous (~40 and rising) and include PARP, DFF(ICAD), BID. Synthesized as inactive zymogens and activated by proteolysis (after Asp).

The function of Caspase Active cystein residue in the catalytic site. Specificity in cleavage after an Asp residue of the substrate proteins to carry out cell death program; – Caspases –8 and –9 are “initiator” caspases – Caspases –3 is the “effector” caspase – Caspase activation requires a stimulus ■

Proteolytic targets of effector caspases • Cytoskeletal regulatory proteins– Actin • Nuclear Lamins • Poly ADP-ribose polymerase (PARP)– PARP activity depletes ATP, thus cleavage of PARP may maintain store of ATP to drive apoptosis • DNA-fragmentation factor (DFF)

The regulation of capases:IAPs

The molecular mechanism of apoptosis • Intrinsic/ Mitochondrial Apoptosis – Regulated by Mitochondria – Cytochrome c release • Extrinsic/ Death Receptor Apoptosis – Activated by ligation of Death Receptors – Fas, TNF alpha • These pathways intersect at the effector caspases.

Intrinsic/Mitochondrial Pathway Cyto C+pro-caspase9+Apaf 1 =apoptosome

Extrinsic/ Death Receptor apoptosis FasL+Fas +FADD+pro-caspase8 death-inducing signaling complex （ DISC ） Sufficient caspase8

insufficient caspase8

activate caspase 3,6,7 Bid proteolyses apoptosis

tBid enter mitchondria CtyC release

Extrinsic/Death Receptor pathway

Example Question Compare the formation of the Death Initiation Signaling Complex (DISC) of the extrinsic pathway to the formation of the apoptosome of the intrinsic pathway. – What signal initiates the formation of each (an aggregation step)? – Where are the complexes formed in the cell? – What adaptor proteins mediate the formation of eachcomplex? – What are the initiator and effector caspases for each? – How are the caspases activated? What do they do?

The relation between apoptosis and medicine Development

Diseases

Death and the mouse’s paw

Dark fluorescence indicates apoptotic cells.