Molecular activation and activity of the BH3-only protein Bim

BH 3-only蛋白是Bcl-2-家族中线粒体凋亡的初始触发因子。然而，在许多情况下，仅BH 3蛋白自身是如何被激活的还不清楚。Bim是最突出的仅含BH 3的蛋白质之一; Bim在调节造血系统中的存活方面起着重要作用，并在许多细胞类型中作为肿瘤抑制因子发挥作用。Bim能够直接激活Bax/巴克，以及能够抑制所有抗凋亡Bcl-2样蛋白。Bim可能在所有细胞类型中组成型表达。尽管已知某些情况下Bim蛋白水平的增加与Bim诱导的细胞凋亡相关，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，这表明了翻译后调节。在这个项目中，我们已经确定了一个机制，我们认为这是重要的监管。我们发现，在线粒体外膜上，Bim通过与动力蛋白轻链1（DLC 1）结合而二聚化，从而形成大的蛋白质复合物。用完整细胞、分离的线粒体和脂质体进行的进一步实验表明，DLC 1与Bim的结合足以形成大的复合物，并且令人惊讶的是，DLC 1与抗凋亡Bcl-2蛋白的有效结合和抑制是必需的。大的复合物具有很少或没有促凋亡活性，在所有研究的细胞中发现，并且似乎还掺入抗凋亡Bcl-2蛋白。在这个项目中，我们将追求两个具体的目标，以了解Bim的监管。首先，我们将使用一些生物物理，生物化学和显微技术来阐明所观察到的复合物的分子性质。实验将针对理解化学计量，活性和动力学的大Bim含有复合物，以及结构的Bim内的复合物。其次，我们将努力了解这些复合物中Bim的抑制和激活的分子情况。将跟踪完整细胞中和响应于凋亡刺激的复合物的状态和分解。将进一步研究Bcl-2样蛋白在复合物形成和溶解中的招募和作用。在细胞凋亡诱导和淋巴细胞分化的模型中，我们将评估复合物形成对人类和小鼠细胞存活的重要性。我们相信，这些研究将阐明一种新的机制，调节的BH 3-唯一的蛋白质Bim，这可能作为一个模型，以了解其他BH 3-唯一的蛋白质，并欣赏激活的Bcl-2家族系统在线粒体。

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.