ESCRT-III and MIT Protein Complexes in Cytokinesis

描述(由申请人提供)：运输所需的内体分选复合体(ESCRT)在细胞动力学脱落中发挥着保守和必要的作用，细胞分裂的最后一步是物理上分离子细胞，并可能在癌症发展过程中出错。ESCRT-III蛋白的同源家族在这一过程中起着关键作用，它形成的细丝似乎可以收缩细胞间的桥梁并促进脱落。ESCRT-III细丝也招募含有MIT结构域的蛋白，我们推测这些蛋白提供了细胞质分裂所需的一系列关键的、但基本上未确定的活动。为了在分子上详细定义这些过程，我们将分析12个人ESCRT-III和21个MIT蛋白的结构、相互作用和细胞质功能。我们测定了ESCRT-III蛋白IST1的闭合构象的晶体结构，并获得了IST1与其ESCRT-III结合伙伴CHMP1B形成的螺旋共聚物的高分辨低温EM结构。这是第一次这样的重建，它揭示了一个引人注目的组装，其中两个ESCRT-III亚基采用了完全不同的结构。双链螺旋细丝包括闭合的IST1亚基的外链和高度延伸的CHMP1B亚基的内链。我们现在将测试一系列基于结构的机制假说，用于ESCRT-III细丝的形成和功能，重点是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细丝，从脂质体中挤出膜小管，我们将从生化细节上表征这种膜重塑反应。在细胞质分裂过程中，MIT ATPase Vps4和Spastin通过与聚合ESCRT-III亚基暴露的C末端尾部的MIT相互作用基序(MIM)结合，定位于细胞间桥。特性不太好的MIT蛋白具有不同的效应结构域，包括激酶、蛋白酶和去泛素酶，我们认为MIM-MIT相互作用的复杂网络将这些活性招募到细胞间桥上，从而在细胞质分裂中发挥作用。为了支持这一想法，我们有 测定了三个相当不同的ESCRT-III-MIT复合体的溶液结构，并表明MIT激酶ULK3参与胞质分裂，被招募到细胞间桥，并可以磷酸化ESCRT-III亚单位。我们已经结晶了ULK3 MIT-IST1相互作用复合体，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，包括对所有人类MIT蛋白的结构和ESCRT-III相互作用的全球分析。最后，我们将使用基于细胞的分析来验证和扩展我们的生化和结构研究，并筛选全套人类MIT蛋白的胞质分裂功能。总之，这些研究将描述ESCRT-III蛋白的基本活性，即形成细丝，招募MIT蛋白发挥胞质分裂功能，以及促进膜分裂。我们的研究还将确定在细胞质分裂中发挥功能的MIT蛋白质子集，阐明它们的结构生物学，并揭示它们是如何被招募的。

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.