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APOPTOSIS

SUBJECT : MOLECULAR BIOLOGYPROFESSOR : DR.M.A.AZEEM SIRPRSENTED BY : ROOHITH(1st MPHARM)COLLEGE: AL-AMEEN COLLEGE OF PHARMACY

Cell suicide

2 CONTENTS

Definition of Apoptosis

Process of apoptosis HISTORY EVENTS OF

APOPTOSISRegulators

of apoptosisMECHANISM

OF APOPTOSIS

Differentiation from Necrosis Pathways

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APOPTOSIS Greek word : Falling leaves (like in Autumn)

In the human body about 100,000 cells are produced every second by mitosis and a similar number die by apoptosis !!!

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DEFINATIONApoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, and global mRNA decay.

5 WHEN WOULD APOPTOSIS OCCUR…..

An orderly disposal of

cells that need to die

DNA has sustained too many injuries

Cell is infected with

a virus

Cell needs to be removed

for body parts to be formed

Cell is just too old and ’ its

time has come’

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PROGRAMMED CELL DEATH

CELL SUICIDE

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8 Importance of Apoptosis

1) Crucial for embryonic development-Errors in Apoptosis can lead to Birth Defects

2) Important for maintaining homeostasis- Cell death is balanced with mitosis to regulate cell number.

3) Improper regulation contributes to human disease- Neurodegenerative diseases

Parkinson’s Alzheimer’s

- Cancer- Autoimmune diseases e.g. (diabetes type I)- Viral diseases

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The tail of a tadpole is absorbed via apoptosis.

Most of the embryo development involves programmed cell death

10 History

Apoptotic principle was first described by KARL VOGT in 1842 “APOPTOSIS” term was coined by JAMES CORMACK 2002 NOBEL PRIZE IN MEDICINE was awarded to SYDNEY

BRENNER,HORVITZ and JOHN.E.SULSTON for their work in identifying genes that control Apoptosis.

John E Sulton won Nobel Prize in 2002 for his pioneering research in Apoptosis

11 How Apoptosis Differs from Necrosis?

1. Apoptosis is intrinsically controlled, necrosis is not2. Apoptosis is more rapid (2-4 hours) than necrosis3. Apoptosis is induced by endogenous or exogenous stimuli, necrosis is always

induced by exogenous harms 4. Apoptosis is limited to single or few cells at a time, and occurs among

healthy cell population, necrosis is usually more extensive & occurs in tissue exposed to injuries

5. Cell cytoplasm shrinks in apoptosis and swells in necrosis.6. Nucleosomes of apoptotic cells are 180 bp fragments, contrary to the

irregular ones in necrosis7. Apoptosis has no inflammation, while necrosis leads to liberation of pro-

inflammatory mediators8. Apoptosis has no systemic manifestations contrary to most inflammations

12 DIFFERENCE OFAPOPTOSIS FROM OTHER SORTS OF CELL DEATHS. Apoptosis is NOT cell death after injury

Cell death after injury is called NECROSIS

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

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15 Apoptosis vs Necrosis

Apoptosis

• Chromatin condensation• Cell Shrinkage• Preservation of Organellesand cell membranes• Rapid engulfment byneighboring cellspreventing inflammation

Necrosis

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

1972 Kerr Wyllie Currie

16 REGULATORS OF APOPTOSIS

MITOCHONDRIA. NITRIC OXIDE. CYTOCHROME C. Mitochondrial proteins known as SMACs (second mitochondria-

derived activator of caspases) .

17 MITOCHONDRIA

The mitochondria are essential to multicellular life. Without them, a cell ceases to respire aerobically and quickly dies. This fact forms the basis for some apoptotic pathways.

Apoptotic proteins that target mitochondria affect them in different ways. They may cause mitochondrial swelling through the formation of membrane pores, or they may increase the permeability of the mitochondrial membrane and cause apoptotic effectors to leak out.

These are very closely related to intrinsic pathway, and tumors arise more frequently through intrinsic pathway than the extrinsic pathway because of sensitivity.

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NITRIC OXIDE

Nitric oxide is able to induce apoptosis by helping to dissipate the membrane potential of mitochondria and therefore make it more permeable.

Nitric oxide has been implicated in initiating and inhibiting apoptosis through its possible action as a signal molecule of subsequent pathways that activate apoptosis.

19 CYTOCHROME C

Cytochrome c is also released from mitochondria due to formation of a channel, the mitochondrial apoptosis-induced channel (MAC), in the outer mitochondrial membrane,and serves a regulatory function as it precedes morphological change associated with apoptosis.

Once cytochrome c is released it binds with Apoptotic protease activating factor – 1 (Apaf-1) and ATP, which then bind to pro-caspase-9 to create a protein complex known as an apoptosome.

The apoptosome cleaves the pro-caspase to its active form of caspase-9, which in turn activates the effector caspase-3.

20 Mitochondrial proteins known as SMACs (second mitochondria-derived activator of caspases)

Mitochondrial proteins known as SMACs (second mitochondria-derived activator of caspases) are released into the cell's cytosol following the increase in permeability of the mitochondia membranes.

SMAC binds to proteins that inhibit apoptosis (IAPs) thereby deactivating them, and preventing the IAPs from arresting the process and therefore allowing apoptosis to proceed.

IAP also normally suppresses the activity of a group of cysteine proteases called caspases,[19] which carry out the degradation of the cell, therefore the actual degradation enzymes can be seen to be indirectly regulated by mitochondrial permeability.

21What happens during Apoptosis Or events of apoptosis

A programmed series of events occur

Cell shrinkage (condensation of cytoplasm)

Breakdown of mitochondria; release of cytochrome C

Nuclear condensation

Nuclear fragmentation

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Cellular changes associated with

apoptosis

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• By activation of caspases• Caspases activate DNAses

Protein Cleavage

• Cleavage into oligonucleosomes• By Ca2+-and Mg2+-dependent

endonucleasesDNA

Breakdown

• Phosphatidylserine • Thrombospondin

Phagocytic Recognition

Biochemical features of Apoptosis

24 MECHANISM OF APOPTOSIS

A cell initiates intracellular apoptotic signaling in response to a stress, which may bring about cell suicide.

The binding of nuclear receptors by glucocorticoids,[13] heat,[13] radiation,nutrient deprivation,[13] viral infection,[13] hypoxia[13] and increased intracellular calcium concentration,[14] for example, by damage to the membrane, can all trigger the release of intracellular apoptotic signals by a damaged cell.

A number of cellular components, such as poly ADP ribose polymerase, may also help regulate apoptosis.[15]

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Before the actual process of cell death is precipitated by enzymes, apoptotic signals must cause regulatory proteins to initiate the apoptosis pathway.

This step allows those signals to cause cell death, or the process to be stopped, should the cell no longer need to die.

Several proteins are involved, but two main methods of regulation have been identified: the targeting of mitochondria functionality, or directly transducing the signal via adaptor proteins to the apoptotic mechanisms.

26Mechanisms of Apoptosis

Caspases are central initiators and executioners of apoptosis

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CASPASES

Caspases or cysteine-aspartic proteases or cysteine-dependent aspartate-directed proteases are a family of cysteine proteases that play essential roles in apoptosis (programmed cell death), necrosis, and inflammation.

Single chain of pro-enzymes. Contains an N-terminal domain, a small subunit

and a large subunit (similar to a ribosome). Apoptotic stimulus Activation Substrate

Cleavage Enzyme.

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3 TYPES OF CASPASES

Inflammatory Caspases: 1, 4, and 5 Initiator Caspases: 2, 8, 9, and 10

Long N-terminal domain Interact with effector caspases

Effector Caspases: 3, 6, and 7 Little to no N-terminal domain Initiate cell death

29 Mechanisms of Apoptosis

CASPASES14 different members of the caspases-family have been described in mammals

Active cysteine residue in the catalytic site

Specificity in cleavage after an Asp residue

Synthesized as inactive

zymogens (PROCASPASES)

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32DNA FRAGMENTATION - BIOCHEMICALHALLMARK OF APOPTOSIS

DNA cleaved into non-random fragments 180-200 bp fragments & multiples of this unit

33DNA FRAGMENTATION AND GELELECTROPHORESIS

Digestion of DNA starts after 2 hrs 3&4 hrs after initiation of apoptosis DNA is

almost all degraded DNA is fragmented with restriction

endonucleases Apoptosis induces 180 bp Laddering of DNA

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CELL DEATH CYCLEDAMAGE Physiological death signals

DEATH SIGNAL

PROAPOPTOTICPROTEINS

ANTIAPOPTOTICPROTEINS

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Physiological Intrinsicreceptor pathway damage pathway

MITOCHONDRIAL SIGNALS

Caspase cleavage cascade

Orderly cleavage of proteins and DNA

CROSSLINKING OF CELL CORPSES; ENGULFMENT(no inflammation)

APOPTOSIS

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STIMULI FOR APOPTIC CELL DEATH IN MAMALS

i. Growth factor deficiencies ii. Ionizing radiation/ viral

infection iii. Free radical toxicity iv. Death receptor activation

(such as Fas or CD95 triggering)

v. Metabolic or cell cycle disturbance

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Stimuli for Apoptotic Cell Death

38 APOPTOSIS PATHWAY

Apoptosis

Extrinsic pathway

Intrinsic pathway

Granzyme pathway

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EXTRINSIC- PATHWAY

Extrinsic pathway is also known as DEATH RECEPTOR PATHWAY.

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

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

Ligand binding causes the receptors to cluster and ultimately form a DEATH INDUCING SIGNALLING COMPLEX.

Upon DEATH INDUCING SIGNALLING COMPLEX formation , the extrinsic pathway has been to adapt the same effector caspase machinery as the intrinsic pathway.

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INTRINSICC PATHWAY OF APOPTOSIS.

The intrinsic signaling pathway for programmed cell death involves non-receptor–mediated intracellular signals, inducing activities in the mitochondria that initiate apoptosis.

Stimuli for the intrinsic pathway include viral infections or damage to the cell by toxins, free radicals, or radiation. Damage to the cellular DNA can also induce the activation of the intrinsic pathway for programmed cell death. These stimuli induce changes in the inner mitochondrial membrane that result in the loss of transmembrane potential, causing the release of pro-apoptotic proteins into the cytosol.

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Pro-apoptotic proteins activate caspases that mediate the destruction of the cell through many pathways. These proteins also translocate into the cellular nucleus, inducing DNA fragmentation, a hallmark of apoptosis7

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Apoptosis and Cancer

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•Apoptosis does not occur in Cancer•Cancerous cells trick and skip Apoptosis in number of waysInactivation of p53 [shooting the guard]Produce Bcl-2 or a protein which mimics Bcl-2Inhibits expression of Apaf-1

47 Bcl-2

Bcl2 was the first apoptosis-related gene ,recognized to play a role tumor genesis

BCL-2 is a human proto-oncogene located on chromosome 18.

Its product is an integral membrane protein (called Bcl-2) located in the membranes of the endoplasmic reticulum , nuclear envelope, and in the outer membrane of mitochondria.

The gene was discovered as the translocated locus in a B-cell leukemia (hence the name). This translocation is also found in some B-cell lymphomas

48Apoptosis and Autoimmune Disease

Autoimmune Lymph

Proliferative Syndrome[ALPS]

Apoptosis doesnot occur in self

reactive T & B cells

RBCHemolytic Anemia

NeutrophilNeutropenia

PlateletsThrombocyt

openia

49Apoptosis and HIV

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Infected CD4 cell induces Apoptosis in surrounding cells

• Deactivated Bcl-2

• Decreases CD4 Glycoprotein markers on innocent T cells ,getting them killed

Infected CD4 cell avoids Apoptosis in itselfDecreases Phosphatdylserine marker for itself allowing longer survival.

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Overview

Apoptosis is a good thingToo little of a good thing is

bad [Cancer]Too much of a good thing

is also bad [HIV]

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ANY QUEIRIES OR DOUBTS………..

52 References

Internet sources https://en.wikipedia.org/wiki/apptosis. Molecular biology by David P. Clark. Biotechnology by U. Satyanarayana; 1st edition, Reprinted(2011); Pg.

No.447-449 & 477-479.

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THANK YOU