Regulation of Autophagy and Tumorigenesis by Bif-1

项目描述（由申请人提供）：本项目的总体目标是确定Bif-1激活自噬和抑制肿瘤发生的分子机制。自噬是一种进化上保守的“自噬”过程，在环境和细胞应激下被激活，并通过将未折叠的蛋白质、受损的细胞器和微生物病原体传递给溶酶体进行降解，在维持正常的细胞稳态中起着重要作用。这种细胞自我消化过程的失调可能会产生深远的后果，并且很可能在许多类型的疾病中起主要作用，包括癌症、自身免疫性疾病和神经退行性疾病。迄今为止，许多自噬相关（Atg）基因已经通过酵母自噬缺陷突变体的独立遗传筛选鉴定出来；然而，哺乳动物系统中自噬体生物发生所需的分子机制尚未确定。我们最近发现Bif-1通过UVRAG与Beclin -1相互作用，是自噬体生物发生所必需的。Bif-1，也被称为SH3GLB1或亲内蛋白B1，最初被发现是一种bax结合蛋白，含有氨基端N-BAR结构域和羧基端SH3结构域，具有膜结合和弯曲活性。虽然Bif-1的SH3结构域足以与UVRAG结合，但N-BAR结构域对于Bif-1诱导自噬体形成是必不可少的。因此，Bif-1可能在自噬过程中通过隔离膜或吞噬细胞上的UVRAG与Beclin -1相互作用，通过其N- BAR结构域诱导膜曲率来调节囊泡成核。具体目标1将测试这种可能性。虽然我们的体外研究清楚地证明了Bif-1在自噬体形成中的重要作用，但与atg缺陷小鼠不同，大多数Bif-1敲除小鼠发育正常。这种表型差异表明，在胚胎和新生儿发育过程中，特定组织中存在一种未知因素，在功能上弥补了Bif-1的缺失。一个可能的候选者是Bif-1的同系物亲内蛋白B2。我们的初步结果表明，嗜内肽B2还与Beclin 1和UVRAG相互作用，并能恢复Bif-1缺陷细胞的自噬体形成。特异性目的2将研究嗜内蛋白B2是否与Bif-1在控制自噬方面共享冗余功能。此外，我们发现Bif-1消融显著促进小鼠自发性肿瘤的发展。由于Bif-1的缺失也抑制Bax/Bak激活和线粒体凋亡，特异性Aim 3将确定Bif-1是否诱导自噬体形成并抑制肿瘤发生，而不依赖于其与Bax的相互作用。我们相信，本研究的成功实施不仅将对自噬囊泡成核和扩张的分离膜的起源和分子机制有新的认识，而且将有助于建立通过操纵自噬来预防和治疗癌症的新策略。公共卫生相关性：我们相信，本研究的成功实施不仅将对自噬囊泡成核和扩张的分离膜的起源和分子机制有新的认识，而且将有助于建立通过控制自噬来预防和治疗癌症的新策略。

DESCRIPTION (provided by applicant): The overall objective of this project is to determine the molecular mechanisms by which Bif-1 activates autophagy and suppresses tumorigenesis. Autophagy, an evolutionarily conserved "self- eating" process, is activated in response to environmental and cellular stresses and plays a fundamental role in maintaining normal cellular homeostasis by delivering unfolded proteins, damaged organelles, and microbial pathogens to lysosomes for degradation. Dysregulation of this cellular self-digestion process can have profound consequences and most likely plays a major role in many types of diseases, including cancer, autoimmune diseases, and neurodegenerative disorders. To date, many autophagy-related (Atg) genes have been identified by independent genetic screens for autophagy-defective mutants in yeast; however, the molecular machinery required for the biogenesis of autophagosomes in mammalian systems has yet to be determined. We have recently discovered that Bif-1 interacts with Beclin 1 through UVRAG and is required for the biogenesis of autophagosomes. Bif-1, also known as SH3GLB1 or endophilin B1, was originally discovered as a Bax-binding protein that contains an amino-terminal N-BAR domain and a carboxy-terminal SH3 domain and demonstrates membrane binding and bending activities. Although the SH3 domain of Bif-1 is sufficient for binding to UVRAG, the N-BAR domain is indispensable for Bif-1 to induce autophagosome formation. Therefore, it is possible that Bif-1 interacts with Beclin 1 through UVRAG at the isolation membrane, or phagophore, during autophagy to regulate vesicle nucleation by inducing membrane curvature through its N- BAR domain. Specific Aim 1 will test this possibility. While our in vitro studies clearly demonstrated the vital role Bif-1 plays in autophagosome formation, the majority of Bif-1 knockout mice, unlike Atg-deficient mice, developed normally. This phenotypic discrepancy suggests that an unknown factor(s) exists in specific tissues that functionally compensate for the lack of Bif-1 during embryonic and neonatal development. A possible candidate is the Bif-1 homologue endophilin B2. Our preliminary results revealed that endophilin B2 also interacts with Beclin 1 as well as UVRAG and can restore autophagosome formation in Bif-1 deficient cells. Specific Aim 2 will examine whether endophilin B2 shares a function redundant with Bif-1 in the control of autophagy. Moreover, we found that Bif-1 ablation significantly enhances the development of spontaneous tumors in mice. Since loss of Bif-1 also suppresses Bax/Bak activation and mitochondrial apoptosis, Specific Aim 3 will determine whether Bif-1 induces autophagosome formation and suppresses tumorigenesis independently of its interaction with Bax. We believe that successful implementation of this research will not only gain novel insight into the origin of isolation membranes and the molecular mechanism responsible for autophagic vesicle nucleation and expansion, but will also contribute to the establishment of new strategies for the prevention and treatment of cancer through manipulation of autophagy. PUBLIC HEALTH RELEVANCE: We believe that successful implementation of this research will not only gain novel insight into the origin of isolation membranes and the molecular mechanism responsible for autophagic vesicle nucleation and expansion, but will also contribute to the establishment of new strategies for the prevention and treatment of cancer through manipulation of autophagy.