ROLE OF CALPAIN AND COFILIN IN APOPTOSIS REGULATION CHUA BOON TIN NATIONAL UNIVERSITY OF SINAGPORE 2004 ROLE OF CALPAIN AND COFILIN IN APOPTOSIS REGULATION CHUA BOON TIN B.Sc (Hons), IMPERIAL COLLEGE OF SCIENCE, TECHNOLOGY AND MEDICINE A THESIS SUBMITTED FOR THE DEGREE OF DOCTORAL OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINAGPORE 2004 Acknowledgements I would like to express my deepest gratitude to A/P Li Peng, my supervisor. Thank you for your guidance, patients and encouragement, throughout my candidature. My heartfelt thanks also go to Prof Porter A, A/P Yang XH and Prof Pallen C, for the time and critical discussion and most importantly encouragement during my yearly committee meeting. Many thanks to the fellow colleagues in AGP’s group and VY’s group for sharing reagents, information and providing technical support. Special thanks go to Dr Tan KO and A/P Yu V for the excellent collaboration. I would like to express my thanks to IMCB for giving me the opportunity to my PhD and providing the necessary resources to make my work possible. To the past and present colleagues in LP’s group, thank you for your support and company. Working with you all in the laboratory is an enjoyment. Special thank to Dr Volbracht for the patience and time to go through the writing of both the paper and the thesis. Thank you for your collaboration, sharing your technical experiences and interesting discussion. And most importantly, thanks for the trust in my ability. Also to ZhiHong and Darren, thank you for the encouragement and support these years. i To my special friend, Joy Tan. Thank you for being there for me these years, especially the last lag. My gratitude goes to my family and personal friends for the supports and understanding throughout these years. Most importantly, to Calvin, my husband and the pillar for my PhD journey, thank you for truly believing in my capability. Your love and encouragement have moved me in both the candidature and my life. ii Table of Contents Acknowledgements i Table of Contents iii Summary viii Abbreviations xii List of Figures xvi List of Schematic Diagrams and Tables xix List of Publications xx Chapter 1: Introduction I 1.1 Apoptosis – history 1.2 C.elegans, conservation of pathway 1.3 Extrinsic and Intrinsic apoptotic pathway 1.3.1. Role of caspases in apoptosis 1.3.2. Role of mitochondria in apoptosis 10 1.3.3. Role of Bcl-2 family proteins in apoptosis 14 1.3.4. Role of the death receptors in apoptosis 16 1.4 Apoptosis regulation 1.4.1. Regulation I: Caspase cascade 1.4.1.1 Activation of caspases 18 18 18 1.4.1.2 Interaction with inhibitors and non-functional caspase analog 1.4.1.3 Phosphorylation of caspases 19 20 1.4.1.4 Interaction with another proteolytic system – calcium activated cysteine proteases, calpains 21 1.4.2. Regulation II: Mitochondria regulation 22 1.4.2.1 Bcl-2 family member 23 iii 1.4.2.1.1. Anti-apoptotic Bcl-2 members 23 1.4.2.1.2. Pro-apoptotic Bcl-2 members 24 1.4.2.2. Mitochondrial translocation as initiation of apoptosis 26 1.4.2.2.1. Bcl-2 proteins 26 1.4.2.2.2. Non-Bcl-2 family proteins 27 1.4.2.2.3. Mitochondrial translocation of cofilin 29 Chapter 2: Introduction II 30 2.1 Cytoskeleton 30 2.1.1. Actin regulation by cofilin 30 2.1.1.1. Cofilin regulation 31 2.2 Cytoskeletal proteins and cell death 33 2.3 Cofilin and stress 35 Rational and Objectives 37 Chapter 3: Materials and Methods 38 3.1 Antibody list 38 3.2 Primer list 39 3.3 DNA methodology 40 3.3.1. Polymerase chain reaction (PCR) 40 3.3.2. Site-directed mutagensis (SDM) 41 3.3.3. Klneow/Kinase reaction 43 3.3.4. Agarose gel electrophoresis 43 3.3.5. Elution of DNA from agarose gel 44 3.3.6. Restriction enzymes (RE) digestion of plasmid DNA 44 3.3.7. Dephosphorylation of plasmid DNA 45 3.3.8. Ligation 45 3.3.9. Transformation by KCM method 45 3.3.10. Transformation by electroporation method 46 3.3.11. Mini-preparation of plasmid DNA 46 iv 3.3.12. Maxi-preparation of plasmid DNA 47 3.3.13. Preparation of KCM competent DH5α cells 48 3.3.14. Preparation of electroporation competent BL21(DE3) cells 48 3.3.15. Plasmid DNA sequencing 48 3.3.16. Ethanol precipitation of DNA 49 3.4 Mammalian cell culture, treatment and sub-cellular fractionation 50 3.4.1. Cell culture 50 3.4.2. Apoptotic stimulation 50 3.4.3. Apoptosis assay 50 3.4.4. Whole cell lysate preparation 51 3.4.5. Mitochondria and S100 preparation 51 3.4.6. Trypan blue exclusion assay 52 3.5 Protein methodology 52 3.5.1. Protein concentration determination by Bradford assay (Bio-Rad) 52 3.5.2. Modified Bradford assay for urea lysis sample 52 3.5.3. Western blot analysis 53 3.5.4. Two-dimensional gel electrophoresis (2-D) and mass spectrometry 53 3.5.5. Production and purification of 6-Histidine-tagged recombinant proteins 55 3.5.6. In vitro transcription and translation of protein 55 3.5.7. Caspase cleavage assay 56 3.5.8. Proteinase K digestion 57 3.5.9. Alkaline phosphatase assay 57 3.5.10. Indirect Immunofluorescent labeling 57 3.5.11. Transient transfection 58 3.5.12. siRNA transfection 58 3.5.13. Silver staining 59 3.5.14. Viability assay 59 3.5.15. Clonogenicity assay 60 3.5.16. Isolation of mitochondria and the in vitro assay of cytochrome c release 60 v Chapter 4: Result I: Direct caspase regulation by calcium/calpain 62 4.1. Cleavage of caspase-7 by MCF-7 cytosolic factors in the presence of calcium 63 4.2. Cleavage of caspase-7 by rat recombinant calpain II 65 4.3. Cleavage of caspase-8 and –9 by calpain II 65 4.4. Calpain proteolysis renders caspase-9 inactive 69 4.5. Truncated caspase-9 inhibits dATP/cyt c induced caspase-9 and –3 activation 71 4.6. Activated calpain cleaves endogenous caspases 71 4.7. Pulsing of SH-SY5Y cells with calcium protects cells from H7-induced apoptosis 73 Discussion I 75 Conclusions and future perspective 82 Chapter 5: Result II: Mitochondrial translocation of cofilin induces apoptosis via the intrinsic pathway 83 5.1. Cofilin translocation into mitochondria in early stage of apoptosis 83 5.2. Silencing of cofilin by siRNA prevents cytochrome c release and apoptosis 90 5.3. Dephosphorylated cofilin localise on mitochondria of apoptotic cells 94 5.4. Expression of cofilin S3D mutant reduces endogenous cofilin translocation and inhibits STS-induced apoptosis 97 5.5. Identification of mitochondrial-targeting domains on cofilin 100 5.6. Mitochondrial-targeting of cofilin induces apoptosis 101 5.7. A functional actin-binding domain is required for cofilin-induced apoptosis but not mitochondrial localisation 106 vi Discussion II 109 Conclusions and future perspective 120 References 122 vii Summary Apoptosis, a form of programmed cell death, plays an important role in the development of multicellular organism and the maintenance of homeostasis in adults. Understanding and exploring the process of apoptosis will allow us to gain insights into the fundamentals of cell death and also provide an alternative therapeutic approach to pathological conditions such as cancer treatment and neurodegenerative disorders. Three decades of research have revealed the mechanism by which the death signal is transduced in the doom cell. Apoptosis can be activated generally via either the extrinsic or the intrinsic pathway. In the former, death signal is transduced from the ligation of death ligands onto the death receptors, which in turn recruit others cytoplasmic proteins to form the large death complex at the peripheral membrane. The intrinsic pathway is activated when the pro-apoptotic proteins, in response to intracellular stress, translocate to the mitochondria outer membrane. Both pathways result in the activation of caspases. Activation of caspases, the cysteine proteases, resulted in the distinctive morphological changes associated with apoptosis. The enzyme cascade forms the main disintegration force that halts the cellular repair system, the transcription and translation system, cell division and dismantles the cellular remains into apoptotic bodies. Therefore, regulation of the caspases’ activity became one of the foci in apoptosis regulation. viii Conclusions and future perspective In the process of screening for proteins translocating in and out of mitochondria, we identified cofilin, an actin binding and depolymerising factor. Cofilin is inactivated by the upstream Rho GTPases via phosphorylation by LIM kinase as well as via the binding of PIP2. Cofilin dephosphorylation or activation is observed in the event that requires actin reorganisation such as chemotaxis. Our studies identified a novel role of dephosphorylated cofilin. At the early stage of death signal transduction, the protein undergoes dephosphorylation which may result in its disengagement with 14-3-3ζ proteins and exposure of the mitochondrial localisation signal at 15-30 aa. This change may result in translocation of the protein to the mitochondrial outer membrane where it exerts its potent death effect. How cofilin translocates to the mitochondria as we proposed, would require further structural studies. Mitochondrial translocation of the cofilin is independent of its actin-binding domain, however, cofilin can only induce cytochrome c release from the mitochondria in the presence of functional actin-binding domain. Detailed mechanisms of how the cytoskeletal protein-induced cell death awaits further analysis. Based on the data, we speculate that cofilin either acts in a cytoskeletal independent manner, interacting with anti-apoptotic proteins and thus inducing mitochondrial release of cytochrome c, or it exerts its actin-depolymerising function reorganising the actin cytoskeleal network around the organelle causing dysfunction, subsequently resulting in the release of cytochrome c. 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Cell 90, 405-413. 135 [...]... phosphorylated cofilin in F-actin depolymerisation function Figure 5.4.2 Cofilin S3D acts in a dominant negative manner blocking cofilin translocation Figure 5.4.3 Cofilin S3D blocks translocation of endogenous cofilin Figure 5.5 Mitochondrial-localised cofilin induces apoptosis Figure 5.6.1 Cofilin1 5-166, cofilin1 5-30/106-166 and M -cofilin trigger caspase-dependent apoptosis Figure 5.6.2 M -cofilin triggers... 5.6.3 Recombinant Cofilin1 5-166 does not induce cytochrome c release from isolated mitochondria Figure 5.7.1 Mitochondrial localisation of cofilin is independent on its actin-binding activity Figure 5.7.2 Mitochondrial-localised cofilin mutant-induced apoptosis is dependent on the actin-binding domain Figure 5.7.3 Cofilin KQ3 inhibits STS-induced apoptosis xviii List of Schematic Diagrams and Tables... accumulation of cofilin prior to cytochrome c release Figure 5.1.5 Sub-cellular localisation of cofilin Figure 5.1.6 Apoptosis and mitochondrial translocation of cofilin induced by STS is blocked by Bcl-xL overexpression Figure 5.1.7 Cofilin is localised to the mitochondrial outer membrane xvi Figure 5.2.1 Silencing of cofilin expression using cofilin siRNA Figure 5.2.2 Lack of cytochrome c release and apoptotic... morphology in cofilin knockdown cells Figure 5.2.3 Depletion of endogenous cofilin protein inhibits apoptosis Figure 5.2.4 Cofilin silencing confers cellular viability Figure 5.3.1 Phosphorylation status of cofilin Figure 5.3.2 Exclusively dephosphorylated cofilin is observed in neutrophil-like HL60 upon PMA activation Figure 5.4.1 Cofilin S3A and S3D mutants mimicked the dephosphorylated and the phosphorylated... Conservation of apoptotic pathway in C.elegans and mammalian system Diagram 2 The extrinsic and intrinsic apoptotic signal transduction pathways Diagram 3 The role of mitochondria in apoptosis Diagram 4 Schematic structure of calpain II Diagram 5 Cofilin is regulated by Rho family of small GTPase Diagram 6 Protein alignment of human destrin and cofilin Diagram 7 Rasmol model of human destrin protein (1AK6)... cysteine protease in the presence of calcium Figure 4.2 Direct cleavage of caspase-7 by rat recombinant calpain II Figure 4.3 Calpain cleaves both caspase-8 and –9 Figure 4.4 Cleavage of caspase-9 by calpain renders it incapable of caspase-3 activation Figure 4.5 Cleavage of caspase-9 by calpain blocked dATP and cytochrome c induced caspase-3 cleavage Figure 4.6 Time course of calpain activation and. .. independent of caspase activation as well as its actin-binding capacity Using over-expression system, cofilin localised on the organelle induced massive caspase-dependent cell death The proapoptotic effect required the actin-binding property Although, we had shown domains required for cofilin translocation and apoptosis induction, the direct mechanism of cell death induction is not clear As actin-binding ability... 9 (ced-9), 4 (ced-4) and 3 (ced-3) (Horvitz, 2001) Gain -of- function of ced-9 gene in C.elegans prevented the death of the 131 cells, suggesting a protective role of the protein (Hengartner et al., 1992) On the contrary, loss -of- function of ced-4 and ced-3 resulted in the living of the 131 doomed cells, indicating an opposing role of these two proteins with Ced-9 function (Ellis and Horvitz, 1986) More... depolymerising factor AIF Apoptosis- inducing factor AP Calf Intestinal Alkaline Phosphatase Apaf-1 Apoptotic protease-activating factor-1 ATP Adenosine triphosphate Bcl-2 B-cell lymphoma 2 proteins BH domain Bcl-2 homology domain BIR Baculoviral IAP repeat BSA Bovine serum albumin CaCl2 Calcium chloride CARD Caspase recuitment domain Calp In I Calpain inhibitor I Calp In II Calpain inhibitor II Ced Cell... that cofilin induced cell death is actin-dependent The change of cytoskeletal network around the organelle may serve to comprise the integrity of the mitochondria and aid in the release of apoptogenic factors from the organelle Alternatively, cofilin may play a novel role in death induction when translocated to the mitochondria x Previous studies have linked cytoskeletal proteins to apoptosis It is often . TIN NATIONAL UNIVERSITY OF SINAGPORE 2004 ROLE OF CALPAIN AND COFILIN IN APOPTOSIS REGULATION CHUA BOON TIN NATIONAL UNIVERSITY OF SINAGPORE 2004 ROLE OF CALPAIN AND. translocation of cofilin induces apoptosis via the intrinsic pathway 83 5.1. Cofilin translocation into mitochondria in early stage of apoptosis 83 5.2. Silencing of cofilin by siRNA prevents. Mitochondrial-localised cofilin induces apoptosis Figure 5.6.1. Cofilin 15-166 , cofilin 15-30/106-166 and M -cofilin trigger caspase-dependent apoptosis Figure 5.6.2. M -cofilin triggers cytochrome