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学情况中调节性细胞死亡的常见形式。线粒体凋亡通过促凋亡蛋白和抗凋亡蛋白的复杂网络来调节，这些蛋白主要是Bcl-2家族的成员。在Bcl-2家族中，两个促凋亡组（仅BH 3启动子和Bax/巴克效应子）和一个抗凋亡组（五种“Bcl-2样”蛋白）调节细胞色素c的释放，细胞凋亡的决定性步骤。关于Bcl-2家族蛋白的许多细节是已知的，但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Bcl-2家族内的多种潜在相互作用有关，产生了一个非常难以控制和理解的网络。我们已经观察到，线粒体细胞凋亡的主要触发因素，BH 3-only蛋白Bim，在线粒体外膜上发现不是作为单体，而是在一个大的复合物中，由小蛋白动力蛋白轻链1（DLC 1）协调，并特异性结合抗凋亡Bcl-2样蛋白Mcl-1。第二种仅含BH 3的蛋白质Bmf也被招募到这些复合物中。我们在体外重建了含有Bim的复合物，我们发现Bim与DLC 1和脂质膜的相互作用是复合物组装所必需的。我们已经获得了数据的结构变化引起的结合的DLC 1的Bim，并已开始的Bim-DLC 1复合物的结构分析。该项目的目标是达到Bim和含Bim复合物的结构理解，并推进我们对Bim在细胞凋亡过程中激活的理解，重点是早期淋巴细胞。我们将使用NMR和Bim结合伙伴Bmf，DLC 1和Mcl-1在体外表征的空间结构，诱导成内在无序的Bim蛋白，由他们的结合。我们将继续在体外纯化重组Bim-DLC 1-复合物，目的是通过冷冻EM获得高分辨率结构。这些研究将为突变方法提供信息，以研究已鉴定的结构和相互作用位点的功能相关性。在淋巴细胞中Bim依赖性凋亡的情况下，我们将研究小分子Bcl-2拮抗剂或通过上游促凋亡刺激启动Bim依赖性凋亡对Bim的激活，观察复合物组成的变化。在这些实验中，我们将使用小鼠淋巴细胞祖细胞和早期B细胞的实验系统。所有已建立的线粒体凋亡模型都假设Bcl-2家族成员之间存在一对一的相互作用，以启动或阻断凋亡。我们的数据表明，在Bcl-2家族的信号转导是更加复杂的，我们认为，确定的Bim复合物是一个重要的功能，在线粒体凋亡的调节。我们希望，这里提出的实验将澄清一些目前尚未解决的问题。

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.