Lec13a Apoptosis

7/29/2019 Lec13a Apoptosis

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MOLECULAR BIOLOGY OF

APOPTOSIS

Definition

Apo: apart

Ptosis: fallen

Shedding of leaves from tress

During embriogenesis ------ occurs as

PCD Post-embrional life------- as apoptosis


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apoptosis

Apoptosis is used as a synonymous forPCD but PCD is physiological death, occursonly during embriogenesis.

It is a functional death and it is a good

mechanism to eliminate wasted, useless,unwanted, or crippled cells!


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Thymus

Prostate

Endometrium

Adrenal cortex

Lymphoid cells

Neurons are all subject to apoptosis


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Why have we developed such a self-

destructive system?

A. PCD allows a constant selection for thefittest cell in a colony

Every cell carries the molecular machinery

to do PCD! Cells that are sensitive to extracellular

signals will survive, cell that cannotcompete with their more vital sisters willundergo apoptosis.


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Ischemia

XRT

Toxins

Chemicals


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PCD machinery is silent until signals arriveto start PCD:

Signals:

damage to DNA

Activation of membrane receptors.Ligands are: peptides, cytokines, ATP,

ROS etc Deprivation of specific signals of GFs,

hormones or survival signals for apoptosis


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PHYSIOLOGICAL VS

PATHOLOGICAL CELL DEATH

Necrosis

Apoptosis

MOLECULAR PATHWAY OF APOPTOSIS

The initiating phase by signals

External that trigger receptors on the plasmamembrane.

Intracellular alterations


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Fas receptor?

Receptors for growth factors, cytokinesand hormones

Membrane alterations cause apoptosis.

Q. What kind of membrane alterations ??

A. Phospholipid redistributions, changes in

membrane charge, carbohydrate andsurface markers.


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Decision phase

Final decision " to live or die"

The final decision depends on expression ofseveral proto-oncogenes, called as Bcll-2 genefamily (B-Cell Leukemia Lymphoma)

Bcl-2 gene product protects B lymphocytes, Tcells against apoptosis induced by: Drugs

XRT

Heat shock

Oxidative stress

What are nuclear changes?


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MEASUREMENT OF APOPTOSIS:

TECHNIQUES BASED ONMORPHOLOGICAL CHANGES

Light microscopy

Electron microscopy

TECHNIQUES BASED ON DNAFRAGMENTATION

Measurement of endonuclease activity


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MEASUREMENT OF APOPTOSIS

TECHNIQUES BASED ON MEMBRANEALTERATIONS

Measurement of dye exclusion

TECHNIQUES BASED ON CYTOPLASMICCHANGES

Changes in intracellular enzyme activity

Measurement of calcium influx


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APOPTOSIS AND ILLNESS

APOPTOSIS AND OXIDATIVE STRESS

Background and introduction

Promotion of apoptosis by oxidative

stress Modulation of apoptosis by oxidative

stress


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Background and Intro:

Ox-stress can cause apoptotic or necrotic celldeath.

This section well talk about ways in which Ox-Stress can intersect apoptotic pathways.

ROS may accumulate due to toxic insults or

normal metabolic processes


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Cell shrinkage, chromatin condensation,internucleosomal DNA fragmentation andformation of apoptotic bodies are all

characteristic features of apoptosis. Several protease families have been

implicated in apoptosis the mostprominent being CASPASES

Caspase is aspartic acid-specific cysteineprotease which is found in zymogens almost inall cells!


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3 models of caspase action is proposed.

TNF receptor mediated

No receptor involvement

cytotoxic cell activation of caspase

Regardless of the mechanism, uponactivation caspases cleave many proteins

and finally DNA.


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A role for Ox-Stress in apoptosis has beenclarified by many scientists.

Promotion of apoptosis by OX-Stress:

Proposed mechanisms:

Fig

Modulation of apoptosis by oxidative

stress


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Apoptotic cell death can be switched tonecrosis during oxidative stress by 2mechanisms:

Inactivation of caspases due to oxidationof their active site thiol group by oxidantsor S-nitrosylation.

Decrease in ATP due to failure ofmitochondrial energy production byoxidants (Table 30.1).


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NO can also have dual effects onapoptosis

NO is reactive, unstable free radical gas

that can easily cross cell membranes. L-Arg------ NO

Low NO: Neurotransmitter, regulator in

vasodilation and platelet aggregation. High NO: Cytotoxicity


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NO may also mediate apoptosis:

Macrophages

Beta cell line

Thymocytes


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How???

Formation of iron-nitrosyl complexes withFeS-containing enzymes: This leads to

impairment of mitochondrial function -ATP depletion.

NO may directly damage DNA-mutagenesis

Generation of OONO- Apoptosis NO may inactivate several antioxidant

enzymes (CAT, GPx, SOD etc)


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NO exposure or iNOS activation mayinhibit apoptosis in

Lymphocytes

Endothelial cells

Neurons

Hepatocytes

Kidney cels


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How?? Direct inhibition of caspase (S-nitrosylation of the

active site Cys) R-S-NO is important component of signal

transduction cascades. S-nitrosylation can regulate many proteins: Enzymes Ion channels G-proteins Transcription factors NO may act as a modular switch to control

protein function via SH groups.


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For example, S-nitrosylation was shown to occurin:

Papain protease Calpain

NF-KB AP-1 These are all implicated in the regulation of

apoptosis. NO inhibition of caspase is reversible. Pro-caspase-3 was recently shown to be S-

nitrosylated on its catalytic site Cys (Cys 163). Nitrosylation/denitrosylation- may serve asa

regulatory mechanism just like.?

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Lec13a Apoptosis