Bcl-2 protein regulation by conformational flexibility

多细胞生物通过细胞凋亡清除受损或多余的细胞，将对周围细胞和组织的危害降到最低。凋亡信号通路受Bcl-2家族蛋白活性控制，Bcl-2家族蛋白活性受相互作用和构象变化的复杂相互作用调控。Bcl-2蛋白构象的改变决定了Bcl-2家族蛋白的亚细胞定位、对结合伙伴的亲和力和凋亡活性。尽管广泛的研究只知道Bcl-2活性的细胞后果和非活性状态的结构，但活性Bcl-2蛋白构象的真实性质及其潜在的治疗靶点仍然是谜。该项目使用限制Bcl-2蛋白的策略，不仅研究构象变化的细胞后果，而且剖析变化本身。在先前的尝试中，产生了一系列促凋亡Bcl-2家族成员Bax的变体，其中包含不同的分子内链。虽然Bax的活性及其在激活后从细胞质到线粒体的定位主要被分子内系链降低，但一种系链的Bax变体即使在失活时也会在线粒体上形成定位。对这种受限Bax变异的分析显示对其调控不敏感，并揭示了一个以前未知的过程，该过程解释了促生存Bcl-2蛋白对Bax的抑制。因此，使用受限Bax变异体提供了对Bax调控的新见解，并将用于分析Bcl-2蛋白的构象变化。该项目还将研究BH3-only蛋白的折叠动力学，BH3-only蛋白调节Bcl-2蛋白。仅BH3蛋白含有BH3结构域，但大部分是非结构化的。该项目将试图研究细胞中这些结构的稳定性以及与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，该项目将研究辅助折叠的蛋白质，如伴侣蛋白和肽基脯氨酸顺/反式异构酶（PPIases），在多大程度上以及如何加速和促进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.