Architecture and function of the mitochondrial VDAC/TSPO super-complex

转运蛋白18 kDa（TSPO）是一种保守的线粒体外膜蛋白，其参与调节线粒体和细胞生理学的各个方面，包括生物能量学、脂质代谢、胆固醇转运和类固醇合成，以及氧化应激和Ca 2+稳态的调节。TSPO蛋白的表达水平在不同组织中和在各种疾病状态期间变化，所述疾病状态例如神经变性疾病、炎性过程和癌症，以及神经精神障碍例如抑郁症。这些发现表明TSPO提供与细胞增殖、存活和凋亡相关的一般和特异性功能，但也影响突触传递和高级脑功能。为了能够完成这些多功能的任务，TSPO已被提议为多蛋白超复合物的一部分，从而与驻留在细胞溶质、线粒体外膜（OMM）、膜间隙、线粒体内膜和基质中的蛋白质伴侣相互作用。在OMM中与TSPO相互作用的最佳表征的效应蛋白之一是电压依赖性阴离子通道（VDAC），其是OMM中的主要孔，构成离子、ADP/ATP和代谢物的运输和交换的主要通道。TSPO在线粒体功能中的机制作用及其与外膜蛋白如VDAC的功能相互作用在很大程度上是未知的，这也是由于缺少关于所提出的TSPO超复合物的组装和结构的结构数据。由于需要天然膜环境或人工膜模拟物中含有TSPO的超复合物的结构信息，并且基于我们自己关于TSPO配体对线粒体和细胞活性的多个方面的影响的工作，当前的项目旨在（i）通过Cryo-EM单颗粒分析、电子晶体学和断层摄影术鉴定线粒体VDAC/TSPO-含有的超复合物的结构，（ii）将超复合物与另外的蛋白质例如己糖激酶、GSK 3 β、ATAD 3A和ATP/ADP载体组装在一起，和（iii）通过VDAC的生物物理学和药理学表征来表征VDAC/TSPO复合物内的调节能力和功能性相互作用，我们的结构和功能研究将揭示TSPO和VDAC在分子上的复杂相互作用，水平，并将有助于更深入地了解TSPO/VDAC介导的信号转导。

The translocator protein 18 kDa (TSPO) is a conserved outer mitochondrial membrane protein which is involved in the regulation of various aspects of mitochondrial and cellular physiology, including bioenergetics, lipid metabolism, cholesterol transport and steroid synthesis, as well as regulation of oxidative stress and Ca2+ homeostasis. Expression levels of the TSPO protein vary in different tissues and during various disease states such as neurodegenerative diseases, inflammatory processes and cancer, as well as neuropsychiatric disorders such as depression. These findings indicate that TSPO provides both general and specific functions related to cellular proliferation, survival and apoptosis, but also influences synaptic transmission and higher brain functions. To be able to fulfill these multifunctional tasks, TSPO has been proposed to be part of a multiprotein super-complex, thereby interacting with protein partners residing in the cytosol, outer mitochondrial membrane (OMM), intermembrane space, inner mitochondrial membrane, and the matrix. One of the best characterized effector proteins interacting with TSPO in the OMM is the voltage-dependent anion channel (VDAC), the major pore in the OMM, constituting the principle gateway for the transport and exchange of ions, ADP/ATP and metabolites. The mechanistic role of TSPO in mitochondrial function and its functional interaction with outer membrane proteins such as VDAC is largely unknown which is also due to missing structural data on the assembly and architecture of the proposed TSPO super-complexes.Due to the need for structural information on TSPO-containing super-complexes in native membrane environment or artificial membrane mimics, and based on our own work on the impact of TSPO ligands on the multiple aspects of mitochondrial and cellular activity, the current project aims at (i) identifying the architecture of the mitochondrial VDAC/TSPO-containing super-complex by means of Cryo-EM single particle analysis, electron crystallography and tomography, (ii) the assembly of the super-complex together with additional proteins, e.g. hexokinase, GSK3beta, ATAD3A, and the ATP/ADP carrier, and (iii) at characterizing the modulatory capacity and the functional interaction within the VDAC/TSPO complex by means of biophysical and pharmacological characterization of VDAC-mediated currents in native and reconstituted protein complexes of defined composition and structure.Our structural and functional studies will shed light on the complex interaction of TSPO and VDAC on a molecular level and will contribute to a deeper understanding of TSPO/VDAC-mediated signaling.