Molecular activation and activity of the BH3-only protein Bim

仅 BH3 蛋白 Bim 的分子激活和活性

• 批准号: 245716980
• 负责人: Professor Dr. Georg Häcker
• 金额: --
• 依托单位: Department für Medizinische Mikrobiologie, Virologie und Hygiene
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/245716980?language=en
• 关键词: Molecular; activation; activity; BH3; only

BH3-only proteins are the initial triggers of mitochondrial apoptosis within the Bcl-2-family. How BH3-only proteins are activated themselves however is in many instances unclear. Bim is one of the most prominent BH3-only proteins; Bim plays a substantial role in regulating survival in the haematopoietic system and functions as a tumour suppressor in many cell types. Bim is capable of direct activation of Bax/Bak, as well as able to inhibit all anti-apoptotic Bcl-2-like proteins. Bim is constitutively expressed in probably all cell types. Although some cases are known where an increase in Bim protein levels correlates with Bim-induced apoptosis, in other cases Bim becomes active without such protein induction, suggesting post-translational regulation. In this project we have identified a mechanism that we believe is important for this regulation. We found that, on the outer mitochondrial membrane, Bim dimerizes through binding to dynein light chain 1 (DLC1), leading to the formation of large protein complexes. Further experiments with intact cells, isolated mitochondria and liposomes suggest that DLC1-binding to Bim is sufficient for large complex formation and is, surprisingly, required for efficient binding to and inhibition by anti-apoptotic Bcl-2 proteins. Large complexes have little or no pro-apoptotic activity, are found in all cells investigated and appear also to incorporate anti-apoptotic Bcl-2 proteins. In this project we will pursue two specific aims to understand the regulation of Bim. Firstly, we will use a number of biophysical, biochemical and microscopic techniques to elucidate the molecular nature of the observed complexes. Experiments will be directed at understanding stoichiometry, activity and dynamics of large Bim containing complexes, as well as the structure of Bim within the complexes. Secondly, we will endeavour to understand the molecular circumstances of inhibition and activation of Bim in these complexes. Status and disassembly of complexes in intact cells and in response to apoptotic stimuli will be followed. Recruitment and role of Bcl-2-like proteins in formation and dissolution of the complexes will further be studied. In models of apoptosis induction and of lymphocyte differentiation we will assess the importance of complex formation for survival of human and mouse cells. We believe that these studies will elucidate a novel mechanism of regulation of the BH3-only protein Bim, which may serve as a model to understand other BH3-only proteins, and to appreciate the activation of the Bcl-2-family system at mitochondria.

BH3-Only蛋白是Bcl2家族中线粒体凋亡的初始触发物。然而，在许多情况下，只有BH3的蛋白质本身是如何被激活的还不清楚。Bim是最重要的BH3-Only蛋白之一；Bim在调节造血系统的存活方面发挥着重要作用，并在许多细胞类型中发挥肿瘤抑制作用。BIM不仅能直接激活Bax/Bak，还能抑制所有抗凋亡的Bcl2样蛋白。BIM可能在所有细胞类型中都有结构性表达。虽然已知一些情况下Bim蛋白水平的增加与Bim诱导的细胞凋亡有关，但在其他情况下，Bim在没有这种蛋白诱导的情况下变得活跃，这表明翻译后调节。在这个项目中，我们确定了一种我们认为对这一监管很重要的机制。我们发现，在线粒体膜外膜上，Bim通过与动力蛋白轻链1(DLC1)结合而二聚化，从而形成大的蛋白质复合体。对完整的细胞、分离的线粒体和脂质体的进一步实验表明，DLC1与Bim的结合足以形成大的复合体，令人惊讶的是，它是有效结合抗凋亡的Bcl-2蛋白并抑制其活性所必需的。大的复合体很少或没有促凋亡活性，在所有被研究的细胞中都发现了，而且似乎也包含了抗凋亡的Bcl-2蛋白。在这个项目中，我们将追求两个具体目标，以了解BIM的监管。首先，我们将使用一些生物物理、生化和显微技术来阐明所观察到的络合物的分子性质。实验将旨在了解含有BIM的大分子络合物的化学计量、活性和动力学，以及BIM在络合物中的结构。其次，我们将努力了解这些络合物中Bim抑制和激活的分子情况。将跟踪完整细胞中复合体的状态和分解，以及对凋亡刺激的反应。类Bcl2蛋白的募集及其在复合体形成和溶解中的作用将进一步研究。在诱导细胞凋亡和淋巴细胞分化的模型中，我们将评估复合体形成对人类和小鼠细胞存活的重要性。我们相信，这些研究将阐明BH3-Only蛋白Bim的一种新的调控机制，这可能为理解其他BH3-Only蛋白以及了解线粒体上Bcl2-家族系统的激活提供一个模型。