Mitochondrial complexes containing the pro-apoptotic Bcl-2-family protein Bim: structure and regulatory function

线粒体凋亡是免疫系统和其他生物环境中常见的调节性细胞死亡形式。线粒体的凋亡是通过一个复杂的促凋亡和抗凋亡蛋白网络来调节的，这些蛋白大多是Bcl2家族的成员。在Bcl2家族中，两个促凋亡基团(仅BH3启动子和Bax/Bak效应基团)和一个抗凋亡基团(五个‘类Bcl2’蛋白)调节细胞色素c的释放，细胞色素c是细胞凋亡的决定性步骤。关于Bcl2家族蛋白的许多细节是已知的，但关于它们的分子相互作用和活性的实质性方面仍然不清楚。这种不确定性与Bcl2家族内大量潜在的相互作用有关，从而产生了一个在体内很难控制和理解的网络。我们观察到，线粒体凋亡的主要触发因素是BH3-Only蛋白Bim，它不是以单体形式存在于线粒体外膜上，而是在一个大的复合体中，与小蛋白动力蛋白轻链1(DLC1)配位，并与抗凋亡的Bcl-2样蛋白Mcl-1特异性结合。第二种仅与BH3结合的蛋白质BMF也被招募到这些复合体中。我们在体外重建了含有Bim的复合体，我们发现Bim-与DLC1和脂膜的相互作用是复杂组装所必需的。我们已经获得了DLC1结合引起的Bim结构变化的数据，并开始了Bim-DLC1-络合物的结构分析。本项目的目标是了解BIM及其含BIM的复合体的结构，并促进我们对BIM在细胞凋亡过程中的激活的理解，重点是早期淋巴细胞。我们将使用核磁共振和Bim结合伙伴BMF，DLC1和Mcl-1在体外表征空间结构，这种空间结构是由他们的结合诱导到内在无序的Bim蛋白中的。我们将继续对体外重组的Bim-DLC1-络合物进行纯化，以期通过冷冻-EM获得高分辨率结构。这些研究将为突变方法提供信息，以调查已确定的结构和相互作用位点的功能相关性。在Bim依赖的淋巴细胞凋亡的情况下，我们将研究Bim被小分子Bcl-2拮抗剂激活或通过上游促凋亡刺激启动Bim依赖的凋亡，观察复合体组成的变化。在这些实验中，我们将使用小鼠淋巴祖细胞和早期B细胞的实验系统。所有已建立的线粒体凋亡模型都假定Bcl2家族成员之间存在一对一的相互作用来启动或阻止细胞凋亡。我们的数据表明，在Bcl2-家族中的信号转导更加复杂，我们认为所鉴定的Bim-复合体是调控线粒体凋亡的一个重要特征。我们希望这里提出的实验将澄清一些目前悬而未决的问题。

Mitochondrial apoptosis is a common form of regulated cell death in the immune system and other situations of biology. Mitochondrial apoptosis is regulated through a complex network of pro- and anti-apoptotic proteins, mostly members of the Bcl-2-family. Within the Bcl-2-family, two pro-apoptotic groups (the BH3-only initiators and the Bax/Bak effectors) and one anti-apoptotic group (five ‘Bcl-2-like’ proteins) regulate the release of cytochrome c, a decisive step of apoptosis. Many details about Bcl-2-family proteins are known, but substantial aspects of their molecular interactions and activity are still unclear. This uncertainty is linked to the multitude of potential interactions within the Bcl-2-family, generating a network that is very difficult to control and to understand in vivo. We have observed that a main trigger of mitochondrial apoptosis, the BH3-only protein Bim, is found on the outer mitochondrial membrane not as monomers but in a large complex, coordinated by the small protein dynein light chain 1 (DLC1), and specifically binding the anti-apoptotic Bcl-2-like protein Mcl-1. A second BH3-only protein, Bmf, was also recruited into these complexes. We have re-built Bim-containing complexes in vitro, where we have found that Bim-interactions with both DLC1 and lipid membranes are required for complex assembly. We have obtained data on structural changes induced in Bim by the binding of DLC1 and have started a structural analysis of Bim-DLC1-complexes. The goal of this project is to arrive at a structural understanding of Bim and Bim-containing complexes and to advance our understanding of the activation of Bim during apoptosis, focussing on early lymphocytes. We will use NMR and the Bim-binding partners Bmf, DLC1 and Mcl-1 in vitro to characterize spatial structure that is induced into the intrinsically disordered Bim protein by their binding. We will continue purifying in vitro-reconstituted Bim-DLC1-complexes with the aim to obtain high-resolution structure by cryo-EM. These studies will inform mutation approaches to investigate the functional relevance of identified structural and interaction sites. In situations of Bim-dependent apoptosis in lymphocytes we will then study the activation of Bim by small-molecule Bcl-2-antagonists or by the initiation of Bim-dependent apoptosis through upstream pro-apoptotic stimuli, observing changes in the composition of the complexes. In these experiments we will use an experimental system of mouse lymphocyte progenitor cells and early B cells. All established models of mitochondrial apoptosis assume one-on-one interactions between Bcl-2-family members to initiate or to block apoptosis. Our data indicate that signal transduction in the Bcl-2-family is even more complex, and we believe that the identified Bim-complexes are an important feature in the regulation of mitochondrial apoptosis. We hope that the experiments proposed here will clarify some of the currently unresolved issues.