The Biochemistry of apoptosis

Transcription

1 The Biochemistry of apoptosis 1 1

2 The apoptosis is composed of multiple biochemical events 2 2

3 Biochemical, cellular, and molecular events in Apoptosis 1. Membrane blebbing; phosphatidyl serine exposure 2. Shrinkage of cytoplasm 3. Loss of membrane potential of mitochondria 4. Cytochrome C release 5. Caspases activation; cellular protein cleavage 6. Chromatin condensation 7. Internucleosomal cleavage of DNA; DNA fragmentation 3 8. Formation of apoptotic bodies 3

4 Membrane change 4 4

5 The asymmetry of the lipid bilayer Phosphatidylcholine, glycolipids and glycoproteins in outer monolayer Phosphatidylethanolamine and Phosphatidylserine in inner monolayer 5 5

6 Disruption of asymmetry of the plasma membrane in apoptosis Phosphatidylserine translocates to the extracellular monolayer Possible mechanism: inactivation of phospholipid translocator that is specific for transport phospholipids from non-cytosolic side to cytosolic side or activation of scramblase which transfers phospholipids non-specifically on either sides Detected by annexin V binding on the cellular surface 6 6

7 Annexin Annexin V is a member of a calcium and negatively charged phospholipid binding family of proteins with vascular anticoagulant activity. Various synonyms for annexin V exist: placental protein 4 (PP4), placental anticoagulant protein I (PAP I), calphobindin I (CPB-I), calcium dependent phospholipid binding protein 33 (CaBP33), vascular anticoagulant protein alpha (VACa), anchorin CII, lipocortin-v, endonexin II, and thromboplastin inhibitor. Largely found on the cytosolic face of plasma membranes, this molecule has high affinity for phospholipids in the presence of physiological concentrations of calcium. 7 7

8 Measurement of phosphatidyl serine exposure Fluorescence-labeled annexin V: FITC-annexin V, PE-annexin V Calcium containing buffer Measure by flow cytometry 8 or Detect the apoptotic cells on culture dishes 8

9 The mitochondrial change 9 9

10 The mitochondrial membrane potential The ph gradient (ΔpH) + The membrane potential (ΔV) Electrochemical proton gradient Exerts a proton-motive force 10 10

11 Measure changes in mitochondrial membrane potential Flow cytometry analysis Loss of membrane potential Measured by Rhodamine123 incorporation Rhodamine123 is a lipophilic, membrane permeable cationic fluorescent dye Amounts of oligomeric Bax Or Detection on cell culture and on loss of fluorescence by fluorescence microscopy 11 EMBO J. 2003, 22:

12 Establishment of detection of mitochondrial apoptosis by fluorescence microscopy and fluorometer H 2 O 2 Bright Field JC-1 aggregated (Cy3, red) Normal mitochondrial function JC-1 monomer (FITC, green) mitochondrial dysfunction 0 mm 0.3 mm 0.6 mm JC-1 aggregated (Cy3, red), Normal mitochondrial function JC-1 monomer (FITC, green), mitochondrial dysfunction When mitochondria possess highly positive charge inside which attracts JC-1 penetrates and aggregates 12 12

13 Release of cytochrome C Fluorescence-labeled punctate vs. diffuse Cyt c-gfp TMRE Merge Anti-cyt c TMRE, Tetramethylrh odamine ethyl ester, red UV stimulation Fractionation cell lysates Cytosol VS. mitochondrial 13 Nature Cell Biol. 2000, 2:156 13

14 The activation of caspases during apoptosis 14 14

15 Multiple apoptosis pathways activate caspases 15 Annu Rev Cell Dev Biol 2005, 21:35 15

16 Proteolytic cleavage mediates activativation of caspases pro Asp Small Asp Large 16 Science 1998, 281:

17 Measurement for caspases activation : Colorimetric assay for activity Caspase specific tetrapeptide conjugated with a chromophore After cleavage, free chromophore can be measured by reading in spectrophotometer Caspase 1 YVAD-pNA Caspase 2---VDVAD-pNA Caspase 3---DEVD-pNA Fluorogenic assay Caspase ---YVAD-AFC P-nitroaniline pna Reading optical density at 405 nm Caspase ---FITC-DEVD-FMK (cell permeable, non toxic and bind to irreversibly to the activated caspase) for detection in living cells 17 17

18 Indexes for caspases activation : Cleavage of procaspases Cleavage of caspase substrates, e.g., : PARP: poly (ADP-ribose) polymerase, a chromatinassociated enzyme and a role in DNA repair 18 Oncogene 2004, 23:

19 Nuclear changes during apoptosis 19 19

20 Major nuclear events during apoptosis Chromatin condensation DNA fragmentation 20 J. Exp. Med. 192:571 J Virol. 2004, 78:

21 Apoptotic effectors dismantle nucleus EndoG AIF: apoptosis-inducing factor, causing chromatin condensation (stage I) and 50 kb fragmentation Endo G: endonuclease G, causing 200-bp nucleosomal fragmentation Acinus: chromatin condensation factor CAD: caspase activated DNase 21 Nature 1999, 401:127 21

22 Measure DNA contents An index for cell death but not necessary for apoptosis Propidium iodide staining DNA Flow cytometry analysis SubG0/G1 fraction 22 Oncogene 2002, 21:

23 Dynamics of changes in nucleus during apoptosis EM 4,6-diamidino-2-phenylindole (DAPI) for nucleus staining Stage I: peripheral condensation 23 23

24 Nuclear apoptosis induced by AIF and CAD 24 J. Exp. Med. 192:571 24

25 DNA fragmentation Nucleosomal fragmentation DNA ladder assay 25 J virol. 2004, 78:

26 TdT: terminal deoxynucleotidyl transferase 26 TUNEL Assay TdT-mediated dutp nicked end labeling Template-independent addition of deoxyribonucleoside triphosphates to the 3'-hydroxyl ends of double- or singlestranded DNA with either blunt, recessed or overhanging ends. Detection: Biotinylated-dUTP Addition of enzymeconjugated streptavidin Detected by turning colorless substrates to colored substrates 3 -OH TdT Or by fluorescence detection using fluorescein-conjugated dutp 26

27 References Thornberry, N. A. and Lazebnik, Y.:Caspases: Enemies within. Science 1998, 281:1312 Boehning, D. et al.:cytochrome c binds to inositol (1,4,5) trisphosphate receptors, amplifying calcium-dependent apoptosis. Nature Cell Biol. 2003, 5:1051 Scorrano, L. et al.: BAX and BAK regulation of endoplasmic reticulum Ca2+:A control point for apoptosis. Science 2003, 300:135 Zamzami, N. and Kroemer, G.:Condensed matter in cell death. Nature 1999, 401:127 Goldstein, J. C. et al.:the coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant. Nature Cell Biol. 2000, 2:156 Arnoult, D. et al. Mitochondrial release of AIF and EndoG requires caspase activation downstream of Bax/Bak-mediated permeabilization. EMBO J. 2003, 22:

28 Problem and discussion Please speculate the possible advantages of chromatin condensation and DNA fragmentation for cells undergoing apoptosis