MOLECULAR BIOLOGY OF APOPTOSIS

1. MOLECULAR BIOLOGY OF APOPTOSIS • Definition • Apo: apart • Ptosis: fallen • Shedding of leaves from tress • During embriogenesis ------ occurs as PCD • Post-embrional life------- as apoptosis

2. apoptosis • Apoptosis is used as a synonymous for PCD but PCD is physiological death, occurs only during embriogenesis. • It is a functional death and it is a good mechanism to eliminate wasted, useless, unwanted, or crippled cells!

3. Thymus • Prostate • Endometrium • Adrenal cortex • Lymphoid cells • Neurons are all subject to apoptosis

4. Why have we developed such a self-destructive system? • A. PCD allows a constant selection for the fittest cell in a colony • Every cell carries the molecular machinery to do PCD! • Cells that are sensitive to extracellular signals will survive, cell that cannot compete with their more vital sisters will undergo apoptosis.

5. Ischemia • XRT • Toxins • Chemicals

6. PCD machinery is silent until signals arrive to 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

7. PHYSIOLOGICAL VS PATHOLOGICAL CELL DEATH • Necrosis • Apoptosis • MOLECULAR PATHWAY OF APOPTOSIS • The initiating phase by signals • External that trigger receptors on the plasma membrane. • Intracellular alterations

8. Fas receptor? • Receptors for growth factors, cytokines and hormones • Membrane alterations cause apoptosis. • Q. What kind of membrane alterations ?? • A. Phospholipid redistributions, changes in membrane charge, carbohydrate and surface markers.

9. Decision phase • Final decision " to live or die" • The final decision depends on expression of several proto-oncogenes, called as Bcll-2 gene family (B-Cell Leukemia Lymphoma) • Bcl-2 gene product protects B lymphocytes, T cells against apoptosis induced by: • Drugs • XRT • Heat shock • Oxidative stress • What are nuclear changes?

10. MEASUREMENT OF APOPTOSIS: • TECHNIQUES BASED ON MORPHOLOGICAL CHANGES • Light microscopy • Electron microscopy • TECHNIQUES BASED ON DNA FRAGMENTATION • Measurement of endonuclease activity

11. MEASUREMENT OF APOPTOSIS • TECHNIQUES BASED ON MEMBRANE ALTERATIONS • Measurement of dye exclusion • TECHNIQUES BASED ON CYTOPLASMIC CHANGES • Changes in intracellular enzyme activity • Measurement of calcium influx

12. APOPTOSIS AND ILLNESS • APOPTOSIS AND OXIDATIVE STRESS • Background and introduction • Promotion of apoptosis by oxidative stress • Modulation of apoptosis by oxidative stress

13. Background and Intro: • Ox-stress can cause apoptotic or necrotic cell death. • This section we’ll talk about ways in which Ox-Stress can intersect apoptotic pathways. • ROS may accumulate due to toxic insults or normal metabolic processes

14. Cell shrinkage, chromatin condensation, internucleosomal DNA fragmentation and formation of “apoptotic bodies” are all characteristic features of apoptosis. • Several protease families have been implicated in apoptosis the most prominent being “CASPASES” • Caspase is aspartic acid-specific cysteine protease which is found in zymogens almost in all cells!

15. 3 models of caspase action is proposed. • TNF receptor mediated • No receptor involvement • cytotoxic cell activation of caspase • Regardless of the mechanism, upon activation caspases cleave many proteins and finally DNA.

16. A role for Ox-Stress in apoptosis has been clarified by many scientists. • Promotion of apoptosis by OX-Stress: • Proposed mechanisms: • Fig • Modulation of apoptosis by oxidative stress

17. Apoptotic cell death can be switched to necrosis during oxidative stress by 2 mechanisms: • Inactivation of caspases due to oxidation of their active site thiol group by oxidants or S-nitrosylation. • Decrease in ATP due to failure of mitochondrial energy production by oxidants (Table 30.1).

18. NO can also have dual effects on apoptosis • 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

19. NO may also mediate apoptosis: • Macrophages • Beta cell line • Thymocytes

20. How??? • Formation of iron-nitrosyl complexes with FeS-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)

21. NO exposure or iNOS activation may inhibit apoptosis in • Lymphocytes • Endothelial cells • Neurons • Hepatocytes • Kidney cels

22. 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.

23. For example, S-nitrosylation was shown to occur in: • 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….?