Regulation of Autophagy and Tumorigenesis by Bif-1

描述（由申请人提供）：该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“自食”过程，响应环境和细胞应激而被激活，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至溶酶体进行降解，在维持正常细胞稳态中发挥着重要作用。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。迄今为止，通过对酵母中自噬缺陷突变体的独立遗传筛选，已经鉴定出许多自噬相关（Atg）基因。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，并且是自噬体生物发生所必需的。 Bif-1，也称为 SH3GLB1 或内亲素 B1，最初被发现是一种 Bax 结合蛋白，包含氨基末端 N-BAR 结构域和羧基末端 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，在自噬过程中，Bif-1 可能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 结构域诱导膜弯曲来调节囊泡成核。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 在自噬体形成中发挥的重要作用，但与 Atg 缺陷小鼠不同，大多数 Bif-1 敲除小鼠发育正常。这种表型差异表明特定组织中存在未知因子，可以在胚胎和新生儿发育过程中功能性补偿 Bif-1 的缺乏。一个可能的候选者是 Bif-1 同系物内亲蛋白 B2。我们的初步结果表明，内亲素 B2 还与 Beclin 1 以及 UVRAG 相互作用，并且可以恢复 Bif-1 缺陷细胞中自噬体的形成。具体目标 2 将检查内亲蛋白 B2 在控制自噬方面是否与 Bif-1 具有冗余功能。此外，我们发现 Bif-1 消融显着促进小鼠自发性肿瘤的发展。由于 Bif-1 的缺失也会抑制 Bax/Bak 激活和线粒体凋亡，因此 Specific Aim 3 将确定 Bif-1 是否会诱导自噬体形成并抑制肿瘤发生，而与其与 Bax 的相互作用无关。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获得新的见解，而且还将有助于建立通过操纵自噬来预防和治疗癌症的新策略。公共健康相关性：我们相信，这项研究的成功实施不仅将获得对隔离膜起源和自噬囊泡成核和扩张的分子机制的新见解，而且还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. 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.