Regulation of Autophagy and Tumorigenesis by Bif-1

描述(申请人提供)：这个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，在响应环境和细胞压力时被激活，并通过将未折叠的蛋白质、受损的细胞器和微生物病原体运送到溶酶体进行降解，在维持正常的细胞动态平衡方面发挥重要作用。这种细胞自我消化过程的失调可能会产生深远的后果，很可能在许多类型的疾病中发挥重要作用，包括癌症、自身免疫性疾病和神经退行性疾病。到目前为止，许多自噬相关(ATG)基因已经在酵母中通过独立的自噬缺陷突变体的遗传筛查得到了鉴定；然而，哺乳动物系统中自噬小体的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，是自噬小体生物发生所必需的。BIF-1，也被称为SH3GLB1或EndophinB1，最初被发现是一种Bax结合蛋白，含有一个氨基末端N-bar结构域和一个羧基末端SH3结构域，并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诱导自噬小体的形成是必不可少的。因此，在自噬过程中，Bif-1可能通过UVRAG在隔离膜或噬菌体上与Beclin 1相互作用，通过其N-bar结构域诱导膜弯曲来调节囊泡的成核。《特定目标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在自噬小体形成中扮演的重要角色，但大多数Bif-1基因敲除小鼠不同于ATG缺陷小鼠，发育正常。这种表型差异表明，在特定组织中存在一种未知因子(S)，该因子在功能上弥补了胚胎和新生儿发育过程中BIF-1的缺失。一个可能的候选者是Bif-1同系物内亲和素B2。我们的初步结果显示，亲内素B2还可以与Beclin 1和UVRAG相互作用，并能恢复Bif-1缺陷细胞的自噬小体形成。具体目标2将检查内嗜血素B2是否与Bif-1在控制自噬方面具有多余的功能。此外，我们发现，Bif-1消融显著增强了小鼠自发肿瘤的发展。由于Bif-1的缺失也抑制了Bax/Bak的激活和线粒体的凋亡，因此特异性靶点3将决定Bif-1是否诱导自噬小体的形成和抑制肿瘤的发生，而不是依赖于它与Bax的相互作用。我们相信，这项研究的成功实施不仅将对隔离膜的起源和自噬囊泡成核和扩张的分子机制有新的见解，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。

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.