ESCRT-III and MIT Protein Complexes in Cytokinesis

细胞分裂中的 ESCRT-III 和 MIT 蛋白质复合物

• 批准号: 8768131
• 负责人: WESLEY I. SUNDQUIST
• 金额: $ 29.8万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8768131
• 关键词: ATP phosphohydrolase; Adopted; Binding; Binding Sites; Biochemical; Biochemistry; Biological Assay; C-terminal; Cell Separation; Cell division; Cells; Complex; Cryoelectron Microscopy; Cytokinesis; Development; Disulfides; Electron Transport Complex III; Excision; Family; Filament; Genetic; Growth; Human; In Vitro; Ligand Binding; Liposomes; Malignant Neoplasms; Maps; Membrane; Molecular; Molecular Conformation; Molecular Structure; N-terminal; Pathway interactions; Peptide Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Polymers; Process; Proteins; Reaction; Recruitment Activity; Research; Resolution; Role; Series; Solutions; Sorting - Cell Movement; Staging; Structure; Tail; Testing; base; conformational conversion; constriction; crosslink; daughter cell; flexibility; polymerization; protein complex; protein function; public health relevance; reconstruction; research study; spastin; structural biology; structural genomics

DESCRIPTION (provided by applicant): The endosomal sorting complexes required for transport (ESCRT) play a conserved and essential role in cytokinetic abscission, the final step of cell division that physically separates daughter cells and can go awry during cancer development. The homologous family of ESCRT-III proteins plays key roles in this process by forming filaments that appear to constrict the intercellular bridge and promote abscission. ESCRT-III filaments also recruit MIT domain-containing proteins that we hypothesize provide a host of critical, but largely uncharacterized activities required for cytokinesis. To define these processes in molecular detail, we will analyze the structures, interactions and cytokinesis functions of the 12 human ESCRT-III and 21 MIT proteins. We have determined the crystal structure of the ESCRT-III protein, IST1, in its closed conformation and obtained a high resolution cryoEM structure of a helical co-polymer formed by IST1 and its ESCRT-III binding partner, CHMP1B. This is the first such reconstruction, and it reveals a remarkable assembly in which the two ESCRT-III subunits adopt radically different structures. The double-stranded helical filament comprises an outer strand of closed IST1 subunits and an inner strand of highly extended CHMP1B subunits. We will now test a series of structure-based mechanistic hypotheses for ESCRT-III filament formation and function, focusing on the importance of subunit conformational flexibility. IST1 can also form membrane-associated, double-stranded helical filaments that extrude membrane tubules from liposomes, and we will characterize this membrane remodeling reaction in biochemical detail. The MIT ATPases VPS4 and Spastin localize to intercellular bridges during cytokinesis by binding to MIT interaction motifs (MIM) within the exposed C-terminal tails of polymerized ESCRT-III subunits. Less well characterized MIT proteins have diverse effector domains that include kinases, proteases, and deubiquitinases, and we propose that a complex network of MIM-MIT interactions recruits these activities to the intercellular bridge to function in cytokinesis. In support of this idea, we have determined solution structures of three quite distinct ESCRT-III-MIT complexes and shown that an MIT kinase, ULK3, functions in cytokinesis, is recruited to intercellular bridges, and can phosphorylate ESCRT-III subunits. We have crystallized the ULK3 MIT-IST1 interaction complex and will now characterize its structure and associated biochemistry and then expand our studies to include global analyses of the structures and ESCRT-III interactions of all human MIT proteins. Finally, we will use cell-based assays to validate and extend our biochemical and structural studies and to screen the entire set of human MIT proteins for cytokinesis functions. Together, these studies will characterize the fundamental activities of ESCRT-III proteins; i.e., forming filaments, recruiting MIT proteins to function in cytokinesis, and facilitating membrane fission. Our studies will also identify the subset of MIT proteins that function in cytokinesis, elucidate their structural biology, and reveal how they are recruited.

描述（由申请人提供）：运输所需的内体分选复合物（ESCRT）在细胞分裂中发挥保守且重要的作用，细胞分裂是细胞分裂的最后一步，物理上分离子细胞，并且在癌症发展过程中可能会出错。 ESCRT-III 蛋白的同源家族在此过程中发挥着关键作用，通过形成似乎收缩细胞间桥并促进脱落的细丝。 ESCRT-III 丝还招募含有 MIT 结构域的蛋白质，我们假设这些蛋白质提供了胞质分裂所需的许多关键但基本上未表征的活性。为了从分子细节上定义这些过程，我们将分析 12 种人类 ESCRT-III 和 21 种 MIT 蛋白的结构、相互作用和胞质分裂功能。 我们确定了 ESCRT-III 蛋白 IST1 的闭合构象晶体结构，并获得了由 IST1 及其 ESCRT-III 结合伴侣 CHMP1B 形成的螺旋共聚物的高分辨率冷冻电镜结构。这是第一次此类重建，它揭示了一个非凡的组装，其中两个 ESCRT-III 亚基采用了截然不同的结构。双链螺旋丝由闭合 IST1 亚基的外链和高度延伸的 CHMP1B 亚基的内链组成。我们现在将测试一系列关于 ESCRT-III 丝形成和功能的基于结构的机制假设，重点关注亚基构象灵活性的重要性。 IST1 还可以形成膜相关的双链螺旋丝，从脂质体中挤出膜小管，我们将详细描述这种膜重塑反应的生化细节。 MIT ATP 酶 VPS4 和 Spastin 在胞质分裂过程中通过与聚合 ESCRT-III 亚基暴露的 C 末端尾部内的 MIT 相互作用基序 (MIM) 结合，定位于细胞间桥。不太明确的 MIT 蛋白具有多种效应域，包括激酶、蛋白酶和去泛素酶，我们提出 MIM-MIT 相互作用的复杂网络将这些活性招募到细胞间桥以在胞质分裂中发挥作用。为了支持这个想法，我们有 确定了三种截然不同的 ESCRT-III-MIT 复合物的溶液结构，并表明 MIT 激酶 ULK3 在胞质分裂中发挥作用，被募集至细胞间桥，并且可以磷酸化 ESCRT-III 亚基。我们已经结晶了 ULK3 MIT-IST1 相互作用复合物，现在将表征其结构和相关的生物化学，然后扩大我们的研究范围，包括所有人类 MIT 蛋白的结构和 ESCRT-III 相互作用的全局分析。 最后，我们将使用基于细胞的检测来验证和扩展我们的生化和结构研究，并筛选整套人类 MIT 蛋白的胞质分裂功能。这些研究将共同​​描述 ESCRT-III 蛋白的基本活性；即，形成丝，招募 MIT 蛋白在胞质分裂中发挥作用，并促进膜裂变。我们的研究还将鉴定在胞质分裂中起作用的 MIT 蛋白子集，阐明它们的结构生物学，并揭示它们是如何被招募的。