Stoichiometry of homo- and hetero-complexes of Bcl-2 proteins at the single molecule level

Bcl2家族的蛋白形成了一个复杂的相互作用网络，决定了线粒体外膜(MOM)在细胞凋亡中的通透性。然而，Bcl2蛋白之间的多种相互作用如何整合以控制MOM通透性的分子机制仍然是该领域的关键问题之一。本项目的主要目的是揭示膜环境中Bcl2蛋白同质和异质复合体的化学计量比，并研究蛋白质浓度、磷酸化、膜曲率或其他家族成员如何调节这些复合体。我们的目标是通过使用定制的单分子技术来获得前所未有的细节，这将提供关于结合亲和力、结构组织、时间和种群异质性的定量信息，以及单个络合物的直接可视化。在我们以前对Bax的单分子研究的基础上，我们将解决以下具体问题：1)蛋白质浓度和膜曲率在Bax寡聚体物种分布中的作用。2)膜上CBID/Bax和BclxL/Bax复合物的化学计量学和寿命。3)Bax自激活与CBID激活在Bax寡聚物种最终分布中的相对贡献。4)膜上BOK寡聚体的化学计量学及CBID和BclxL的调节，以及Bfl2的抑制机制。5)Bcl2与PUMA、Bim或TBID的亲和力，以及磷酸化在这些蛋白相互作用中的作用和在细胞凋亡调控中的作用。6)膜环境中Bim的结构构象及其与DLC1、Bax或Bclxl的复合体。本研究的结果将对Bim的同源和异源复合体(包括它们的调控)提供详细的分子描述，从而改进现有的机制模型，使我们更接近于对线粒体凋亡是如何调控的功能的理解。鉴于Bcl2家族在多种疾病中的作用，我们的研究结果对于设计针对Bcl2家族的新的治疗干预方法和改进现有的治疗干预方法也将是重要的。

The proteins of the Bcl-2 family form a complex interaction network that determines the permeabilization of the mitochondrial outer membrane (MOM) in apoptosis. However, the molecular mechanism how the multiple interactions between Bcl-2 proteins are integrated to control MOM permeabilization remains one of the key questions in the field.The main objective of this project is to uncover the stoichiometry of homo- and hetero-complexes of Bcl-2 proteins in the membrane environment and to examine how protein concentration, phosphorylation, membrane curvature or other family members, regulate these complexes. We aim to achieve unprecedented detail thanks to the use of tailored single molecule techniques, which will provide quantitative information about binding affinity, structural organization, temporal and population heterogeneities, and direct visualization of individual complexes. Extending on our previous single molecule studies of Bax, we will address the following concrete questions:1) Role of protein concentration and membrane curvature in the distribution of Bax oligomeric species. 2) Stoichiometry and lifetime of cBid/Bax and Bcl-xL/Bax complexes in the membrane.3) Relative contribution of Bax auto-activation vs. activation by cBid in the final distribution of Bax oligomeric species. Effect of Bcl-xL on the process.4) Stoichiometry of Bok oligomers in the membrane and regulation by cBid and Bcl-xL, as well as the inhibitory mechanism of Bfl1.5) Affinity between Bcl-2 and PUMA, Bim or tBid, and role of phosphorylation on the interaction between these proteins and in apoptosis regulation. 6)Structural conformation of Bim alone and in complex with DLC1, Bax or Bcl-xL in the membrane environment.The outcome of this research will provide a detailed molecular description of the homo- and heterocomplexes of Bcl-2 proteins (including their regulation) that improves current mechanistic models and brings us closer to a functional understanding of how mitochondrial apoptosis is orchestrated. Given the implication of the Bcl-2 family in several diseases, the outcome of our research will also be important to design new ways of therapeutic intervention targeting the Bcl-2 family and improve the existing ones.