Exploiting direct electron detection to resolve protein-protein interactions in clathrin-mediated endocytosis

利用直接电子检测来解决网格蛋白介导的内吞作用中的蛋白质-蛋白质相互作用

• 批准号: BB/N008391/1
• 负责人: Corinne Smith
• 金额: $ 54.52万
• 依托单位: University of Warwick
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FN008391%2F1
• 关键词: Exploiting; direct; electron; detection; resolve

Clathrin-mediated endocytosis plays a central role in multiple cellular functions including nutrient uptake, synaptic vesicle recycling, signaling, maintenance of cell polarity and development. In addition, the endocytic apparatus is used by some viruses (notably HIV and influenza) and bacteria to gain entry into cells and there is accumulating evidence that mutations or differences in expression levels in endocytic proteins are associated with a wide range of diseases including neurodegenerative disease and cancer. Clathrin-mediated endocytosis operates through formation of a vesicle from the cell's membrane trapping cargo molecules inside. This process is controlled by a network of proteins which include clathrin and a set of adaptor proteins which bind to clathrin. Clathrin assembles to form a polyhedral cage which, together with its adaptor proteins, forms a coat around the vesicle. The vesicles then detach from the membrane and move to a location inside the cell to deliver its contents. It remains a mystery how so many different adaptor proteins coordinate with a single protein so that this cellular postal system can achieve its function. This proposal aims to elucidate how one or more adaptor proteins interact with clathrin cage structures by using two main approaches. First, and most importantly, exciting new developments in electron microscopy have provided a unique opportunity to push forward the resolution with which we can image these cages. High resolution images will reveal the detailed structures of the assembled multi-protein complex and show where components interact with one another. Secondly, by using advanced biophysical techniques we will measure how a set of adaptor proteins bind to clathrin individually and in pairs, to find out whether different adaptors use the same or different binding sites, or a combination of sites and if there is competition between adaptor proteins. Thirdly, the biophysical data will be extended by imaging clathrin cages bound to multiple adaptor proteins and comparing these to structures bound to single adaptors. By analysing difference images we will obtain highly detailed 3D information on how these cages assemble and disassemble and on the roles of multiple adaptor proteins in these processes. As a result, we will obtain key information on how clathrin coats specify and are involved in so many critical functions in cells.

网格蛋白介导的内吞作用在多种细胞功能中起核心作用，包括营养摄取、突触囊泡再循环、信号传导、细胞极性维持和发育。此外，内吞装置被一些病毒（特别是HIV和流感病毒）和细菌用于进入细胞，并且有越来越多的证据表明内吞蛋白的表达水平的突变或差异与包括神经变性疾病和癌症在内的广泛疾病相关。网格蛋白介导的内吞作用通过从细胞膜形成囊泡将货物分子捕获在内部来进行。该过程由蛋白质网络控制，所述蛋白质网络包括网格蛋白和一组与网格蛋白结合的衔接蛋白。网格蛋白组装形成多面体笼，其与其衔接蛋白一起形成囊泡周围的外壳。然后囊泡从膜上分离，并移动到细胞内的一个位置以输送其内容物。如此多的不同衔接蛋白如何与单个蛋白质协调，从而使这种细胞邮政系统能够实现其功能，这仍然是一个谜。该建议旨在阐明一个或多个衔接蛋白如何通过使用两种主要方法与网格蛋白笼结构相互作用。首先，也是最重要的，电子显微镜令人兴奋的新发展提供了一个独特的机会来推动我们可以对这些笼子进行成像的分辨率。高分辨率图像将揭示组装的多蛋白质复合物的详细结构，并显示组件之间的相互作用。其次，通过使用先进的生物物理技术，我们将测量一组衔接蛋白如何单独和成对地与网格蛋白结合，以找出不同的衔接蛋白是否使用相同或不同的结合位点，或位点的组合，以及衔接蛋白之间是否存在竞争。第三，生物物理数据将通过对与多个衔接子蛋白结合的网格蛋白笼进行成像并将其与单个衔接子结合的结构进行比较来扩展。通过分析差异图像，我们将获得关于这些笼子如何组装和拆卸以及多个衔接蛋白在这些过程中的作用的高度详细的3D信息。因此，我们将获得关于网格蛋白外套如何指定并参与细胞中如此多的关键功能的关键信息。

项目成果

CHC22 and CHC17 clathrins have distinct biochemical properties and display differential regulation and function.

• DOI: 10.1074/jbc.m117.816256
• 发表时间: 2017-12-22
• 期刊: The Journal of biological chemistry
• 作者: Dannhauser PN;Camus SM;Sakamoto K;Sadacca LA;Torres JA;Camus MD;Briant K;Vassilopoulos S;Rothnie A;Smith CJ;Brodsky FM
• 通讯作者: Brodsky FM

Multi-modal adaptor-clathrin contacts drive coated vesicle assembly.

• DOI: 10.15252/embj.2021108795
• 发表时间: 2021-10-01
• 期刊: The EMBO journal
• 作者: Smith SM;Larocque G;Wood KM;Morris KL;Roseman AM;Sessions RB;Royle SJ;Smith CJ
• 通讯作者: Smith CJ

Weak Molecular Interactions in Clathrin-Mediated Endocytosis.

• DOI: 10.3389/fmolb.2017.00072
• 发表时间: 2017
• 期刊: Frontiers in molecular biosciences
• 影响因子: 5
• 作者: Smith SM;Baker M;Halebian M;Smith CJ
• 通讯作者: Smith CJ

Capturing the mechanics of clathrin-mediated endocytosis

• DOI: 10.1016/j.sbi.2022.102427
• 发表时间: 2022-07-21
• 期刊: CURRENT OPINION IN STRUCTURAL BIOLOGY
• 影响因子: 6.8
• 作者: Smith，Sarah M.;Smith，Corinne J.
• 通讯作者: Smith，Corinne J.