Bcl-2 protein regulation by conformational flexibility

多细胞生物体通过细胞凋亡去除受损或多余的细胞，对周围细胞和组织的危险最小化。内源性凋亡信号传导途径受Bcl-2家族蛋白活性控制，Bcl-2家族蛋白活性通过相互作用和构象变化的复杂相互作用在其部分上调节。Bcl-2蛋白构象的变化决定了Bcl-2家族蛋白的亚细胞定位、结合伴侣亲和力和凋亡活性。尽管广泛的研究只有Bcl-2活性的细胞后果和非活性状态的结构是已知的，但活性Bcl-2蛋白构象的真实性质和潜在的治疗靶点仍然是谜。在先前的尝试中，产生了一组含有不同分子内系链的促凋亡Bcl-2家族成员Bax的变体。虽然Bax活性及其从细胞质到线粒体的定位在激活时主要是由分子内系链降低，但一个系链Bax变体即使在无活性时也组成性地定位在线粒体上。这种限制Bax变体的分析表明其调节不敏感，并揭示了一个以前未知的过程，占Bax抑制促生存Bcl-2蛋白。因此，限制Bax变体的使用提供了新的见解Bax调控，并将用于分析Bcl-2蛋白的构象变化。该项目还将研究调节Bcl-2蛋白的仅BH 3蛋白的折叠动力学。BH 3-only蛋白含有BH 3结构域，但大部分是非结构化的。该项目将试图研究这些结构在细胞中的稳定性以及与Bcl-2蛋白相互作用时可能发生的折叠动力学。在第三个核心领域，该项目将解决在何种程度上以及如何帮助折叠的蛋白质，如伴侣蛋白和肽基脯氨酰顺式/反式异构酶（PPIases），加速和促进Bcl-2家族成员的折叠事件，从而参与细胞凋亡的调节。

Multi-cellular organisms remove damaged or redundant cells with minimized danger to surrounding cells and tissues by apoptosis. The intrinsic apoptosis signaling pathway is controlled by Bcl-2 family protein activities, which are regulated on their part by a complex interplay of Interactions and conformational changes. The changes of Bcl-2 protein conformations determine the sub-cellular localization, affinity for binding partners and apoptotic activity of Bcl-2 family proteins. Despite extensive research only the cellular consequences of Bcl-2 activities and the structures of the inactive states are known, but the true nature of the active Bcl-2 protein conformations and thus potential therapeutic targets remain enigmatic.This project uses the strategy of constraining Bcl-2 proteins not only to study the cellular consequences of conformational changes but to dissect the changes itself. In a previous attempt a set of variants of the pro-apoptotic Bcl-2 family member Bax was generated containing different intramolecular tethers. While Bax activity and its localization from cytosol to mitochondria upon activation is mostly decreased by the intramolecular tethers, one tethered Bax variant localizes constitutively on mitochondria even when inactive. The analysis of this constrained Bax variant showed insensitivity to its regulation and revealed a previously unknown process that accounts for Bax inhibition by prosurvival Bcl-2 proteins. Thus, the use of constrained Bax variants provided novel insight into Bax regulation and will be used to analyze conformational changes in Bcl-2 proteins. This project will also examine the folding dynamics of BH3-only proteins, which regulate Bcl-2 proteins. BH3-only proteins contain a BH3 domain, but are largely unstructured. The project will attempt to investigate the stability of these structures in cells and folding dynamics that might occur upon interactions with Bcl-2 proteins. In a third core area the project will address to what extent and how proteins that assist folding, such as chaperones and peptidyl prolyl cis/trans isomerases (PPIases), accelerate and facilitate folding events in Bcl-2 family members and thus participate in the regulation of apoptosis.