BRE attenuates apoptosis through maintaining the cellular level of an apoptotic inhibitor XIAP.

以前的研究表明，BRE是一種能在內源和外源凋亡路徑中均發揮作用的抗凋亡蛋白。然而，我們卻完全不知道它發揮抗凋亡功能的分子機制和生物化學機制是怎樣的。在本論文中，我們報導了BRE通過保護細胞內的XIAP水準來發揮抗凋亡功能。XIAP是一種強大的內源性的半胱氨酸天冬氨酸蛋白酶的抑制劑，與BRE一樣，XIAP也在內源和外源凋亡路徑中均發揮作用。我們使用鼠Lewis細胞系為母細胞系，產生了shRNA介導的BRE基因敲除穩定細胞系。我們發現這種BRE敲除細胞系使得細胞對於在即使沒有放線菌酮作用下的腫瘤壞死因數α介導的凋亡也異常敏感。放線菌酮通過抑制抗凋亡蛋白的合成以發揮重要的促凋亡作用，相反地，腫瘤壞死因數-α通過NF-κB通路可以上調上述抗凋亡蛋白。蛋白質印跡結果顯示，上述通過NF-κB通路被上調且對放線菌酮敏感的抗凋亡蛋白中，只有XIAP的水準，而不是cIAP-1, cIAP-2 或者cFLIP的水準, 在BRE敲除細胞中明顯被降低。用具有高度同源性的人BRE在上述鼠BRE敲除細胞中恢復BRE的水準，XIAP水準也隨之上升。該實驗也證實了BRE正向調節XIAP的作用。並且當在上述BRE敲除細胞中恢復人BRE或者XIAP時，這些細胞對腫瘤壞死因數-α介導的凋亡的敏感性也都得以降低。當具有使蛋白中半胱氨酸殘基改變作用的N-乙基馬來醯亞胺被加入細胞裂解液中時，抗BRE抗體Mab489-7 對於BRE的識別作用會因為BRE蛋白中半胱氨酸殘基的改變而受到影響。與之對應地，裂解液中的XIAP水準也同時降低。該結果提示在細胞裂解液中，BRE保護XIAP的基團可能與半胱氨酸殘基有關。我們發現在凋亡過程中XIAP不僅被半胱氨酸天冬氨酸蛋白酶分解，它也會被蛋白體所降解。這提示我們XIAP的泛素化對於保持其穩定性有重要作用。我們已經知道BRE具有泛素鏈結合域，也可以形成具有降泛素作用的複合物。因此我們想通過實驗，瞭解BRE是否能降解掉鏈結在XIAP上的泛素鏈。實驗表明XIAP至少可以鏈結三種類型的泛素鏈，分別為K48型，K63型和K0型。然而BRE卻只特異性地降解鏈結在XIAP上的K0型泛素鏈或抑制XIAP上的K0型泛素鏈的形成。綜上所述，BRE特異性地降解鏈結在XIAP上的線型泛素鏈或者抑制該種泛素鏈的形成，並通過它影響XIAP的穩定性，從而在外源和內源凋亡通路中發揮抗凋亡的作用。BRE和XIAP之間的相互作用是間接的還是直接的仍有待進一步證實。BRE is a broad-spectrum anti-apoptotic protein, which attenuates both extrinsic and intrinsic apoptosis. However, the molecular and biochemical mechanisms by which BRE inhibits apoptosis remain completely unknown. Here I provide evidence that BRE attenuates apoptosis through maintaining the cellular level of XIAP, which is a potent endogenous inhibitor of caspases functioning in intrinsic and extrinsic pathways.Using a mouse Lewis lung carcinoma cell line D122, we found that shRNA-mediated stable depletion of BRE rendered the cells susceptible to TNF-α-induced apoptosis even in the absence of cycloheximide (CHX). CHX plays a critical pro-apoptotic role by inhibiting synthesis of anti-apoptotic proteins, which TNF-α also up-regulates through activation of NF-κB pathway. Western blot analysis of the NF-κB-driven and CHX-sensitive anti-apoptotic proteins revealed only XIAP, but not cIAP-1, cIAP-2, or cFLIP, was down-regulated in BRE-depleted cells. Reconstitution of the BRE-depleted mouse cells with highly homologous human BRE restored the XIAP protein level, confirming a positive regulatory role of BRE on XIAP protein expression. Furthermore, reconstitution of the BRE-depleted cells with human BRE or XIAP rendered the cells less sensitive to TNF-α-induced apoptosis.Addition of NEM (N-Ethylmaleimide), which binds irreversibly to cysteine residues of proteins, to cell lysates, was found to abrogate, the recognition of BRE by our anti-BRE antibody (Mab489-7) in Western blot analysis. Correspondingly, XIAP level was also found reduced in cell lysates. This correlation provides in vitro evidence that BRE has a protective role for XIAP, and that this role is related to cysteine residues of BRE.I have shown that that XIAP is not only cleaved by caspases during apoptosis, but also subjected to proteaosomal degradation, indicating that ubiquitination of XIAP is an important regulatory mechanism for the stability of this protein. As BRE is known to contain polyubiquitin-binding domains and forms complexes with deubiquitination activity, the issue of whether BRE could remove the ubiquitination of XIAP was investigated. I found that over-expressed XIAP underwent K48-linked, K63-linked, and K0-linked polyubiquitination, respectively. Overexpression of BRE led to the removal of or inhibited K0- but not K48- or K63-linked ubiquitination of XIAP.Taken together, I have provided evidence that BRE exerts its anti-apoptotic function through maintaining the cellular level of XIAP, a potent endogenous inhibitor of apoptosis. Promoting removal of linear ubiquitin chain of XIAP, or inhibition of the chain formation to prevent linear polyubiquitin-mediated proteasomal degradation of XIAP may be the mechanism by which BRE stabilizes this protein. Whether such interaction between BRE and XIAP is direct or indirect needs further investigation.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Li, Wei.Thesis (M.Phil.)--Chinese University of Hong Kong, 2012.Includes bibliographical references (leaves 114-126).Abstracts also in Chinese.Abstract --- p.iAcknowledgements --- p.ivList of Figures --- p.vList of Tables --- p.viiAbbreviations --- p.viiiChapter CHAPTER 1 --- : Introduction --- p.1Chapter 1.1 --- Apoptosis --- p.1Chapter 1.1.1 --- Overview of apoptosis --- p.1Chapter 1.1.2 --- Caspases --- p.1Chapter 1.1.3 --- Extrinsic and intrinsic apoptotic pathways --- p.2Chapter 1.2 --- NF-κB --- p.3Chapter 1.2.1 --- Overview of NF-κB --- p.3Chapter 1.2.2 --- NF-κB and two signaling complexes --- p.5Chapter 1.3 --- IAP --- p.7Chapter 1.3.1 --- Structure --- p.7Chapter 1.3.2 --- Function --- p.8Chapter 1.4 --- XIAP --- p.9Chapter 1.4.1 --- Discovery and function --- p.9Chapter 1.4.2 --- BIR domain of XIAP and its function as direct caspase inhibitor --- p.9Chapter 1.4.3 --- RING domain of XIAP and its function as E3 in ubiquitination --- p.10Chapter 1.4.4 --- XIAP associates with apoptosome --- p.12Chapter 1.4.5 --- Antagonists of XIAP --- p.13Chapter 1.4.6 --- Clinical significance of XIAP --- p.13Chapter 1.5 --- Ubiquitin and ubiquitination --- p.13Chapter 1.5.1 --- Overview of ubiquitin --- p.13Chapter 1.5.2 --- Ubiquitination process --- p.14Chapter 1.5.3 --- Ubiquitin-activating enzymes, E1s --- p.15Chapter 1.5.4 --- Ubiquitin-conjugating enzymes, E2s --- p.17Chapter 1.5.5 --- Ubiquitin-protein ligases, E3s --- p.18Chapter 1.5.6 --- Proteasomes --- p.21Chapter 1.5.7 --- Deubiquitinating enzymes --- p.23Chapter 1.6 --- Background of BRE --- p.27Chapter 1.6.1 --- DNA and RNA of BRE --- p.27Chapter 1.6.2 --- Protein --- p.28Chapter 1.6.3 --- BRE in two complexes --- p.29Chapter 1.6.4 --- The anti-apoptotic function of BRE --- p.31Chapter 1.6.5 --- BRE and ubiquitin --- p.33Chapter 1.7 --- RNA interference --- p.33Chapter 1.7.1 --- Mechanism of RNA interference --- p.33Chapter 1.7.2 --- Small hairpin RNA --- p.34Chapter CHAPTER 2 --- : Materials and methods --- p.36Chapter 2.1 --- Materials --- p.36Chapter 2.1.1 --- Primers used for cloning --- p.36Chapter 2.1.2 --- DNA clones used in the studies --- p.36Chapter 2.1.3 --- Materials for DNA manipulation --- p.44Chapter 2.1.4 --- Materials for protein manipulation --- p.45Chapter 2.1.5 --- Materials for virus manipulation --- p.45Chapter 2.1.6 --- Antibodies --- p.46Chapter 2.1.7 --- Chemicals --- p.46Chapter 2.1.8 --- Kits --- p.46Chapter 2.1.9 --- Culture media and reagents --- p.47Chapter 2.1.10 --- Bacterial strains used for transformation and cloning --- p.47Chapter 2.1.11 --- Instrumentation --- p.47Chapter 2.2 --- Methods --- p.48Chapter 2.2.1 --- Construction of plasmids --- p.48Chapter 2.2.2 --- Plasmids preparation --- p.53Chapter 2.2.3 --- Cell culture --- p.53Chapter 2.2.4 --- Cell transfection --- p.54Chapter 2.2.5 --- Generation of stable transfectants --- p.55Chapter 2.2.6 --- Western blot analysis --- p.55Chapter 2.2.7 --- Chemical treatment --- p.56Chapter 2.2.8 --- Apoptosis assays by the flow cytometry --- p.57Chapter 2.2.9 --- Immunoprecipitation --- p.58Chapter 2.2.10 --- BRE containing retrovirus generation and transduction --- p.58Chapter 2.2.11 --- BRE containing adenovirus generation and transduction --- p.61Chapter CHAPTER 3 --- : BRE attenuates apoptosis through maintaining the cellular level of an apoptotic inhibitor XIAP --- p.65Chapter 3.1 --- Establishment of cell lines with BRE expression stably knocked down by shRNA --- p.65Chapter 3.2 --- BRE-depleted cells are more sensitive to apoptosis --- p.67Chapter 3.3 --- BRE-depleted cells are susceptible to TNF-α induced apoptosis in the absence of cycloheximide --- p.69Chapter 3.4 --- Reduction of XIAP in BRE-depleted cells --- p.72Chapter 3.5 --- Recovery of BRE restores XIAP in BRE-depleted cells --- p.74Chapter 3.6 --- Recovery of XIAP or BRE to BRE-depleted cells renders the cellsless sensitive to TNF-α induced apoptosis --- p.76Chapter 3.7 --- N-Ethylmaleimide (NEM) affects BRE and XIAP --- p.79Chapter 3.7.1 --- NEM affects BRE staining by anti-BRE (Mab489-7) antibody --- p.79Chapter 3.7.2 --- NEM affects XIAP --- p.81Chapter 3.7.3 --- NEM reduces XIAP instead of antibody-binding failure --- p.83Chapter 3.8 --- Ubiquitination of XIAP is important for its stability --- p.85Chapter 3.8.1 --- VAD cannot preserve XIAP upon 16 hours of etoposide treatment --- p.85Chapter 3.8.2 --- Degradation of XIAP in response to etoposide in the presence of VAD is due to proteasomal degradation --- p.87Chapter 3.9 --- BRE leads to the removal of or inhibits ubiquitination of XIAP --- p.89Chapter 3.9.1 --- Immunoprecipitation of ubiquitinated XIAP --- p.89Chapter 3.9.2 --- BRE leads to the removal of or inhibits endogenous ubiquitination of XIAP --- p.92Chapter 3.9.3 --- BRE does not affect exogenous K48-linked ubiquitination of XIAP --- p.94Chapter 3.9.4 --- BRE does not affect exogenous K63-linked ubiquitination of XIAP --- p.96Chapter 3.9.5 --- BRE leads to the removal of or inhibits exogenous K0-linked ubiquitination of XIAP --- p.98Chapter CHAPTER 4 --- : Discussion --- p.100Chapter 4.1 --- Summary --- p.100Chapter 4.2 --- Shortcoming and improvement in future experiment --- p.101Chapter 4.3 --- Significance of the research findings --- p.102Chapter 4.4 --- Possibility of BRE to attenuate apoptosis through affecting other NF-κB-activated anti-apoptotic proteins --- p.103Chapter 4.5 --- BRE may function with other deubiquitinase to mediate deubiquitination of XIAP --- p.104Chapter 4.6 --- Comparison of methodologies --- p.109Chapter 4.7 --- Lysine-linkage specificity of the ubiquitination of XIAP --- p.110Chapter 4.8 --- Possibility of BRE to maintain XIAP level utilizing other mechanisms --- p.111Chapter 4.9 --- Interpretation of NEM-related data --- p.112Chapter 4.10 --- Conclusion --- p.112Reference --- p.114Supplementary result --- p.12