Requirements for cytosolic chaperones in the de novo folding of septin proteins

细胞功能需要由不同多肽组成的大分子复合物的组装 以精确方式相互作用的子单元。蛋白质序列通常不足以保证在拥挤的环境中 细胞环境中每个新生亚基仅产生正确的蛋白质-蛋白质相互作用。不正确 构象和相互作用导致细胞缺陷和人类疾病。细胞如何确保新- 合成的多肽组装成功能复合物的过程仍然知之甚少。脓毒蛋白 组装成高度保守的细胞骨架异源寡聚物，这些寡聚物已成为许多领域的核心参与者 细胞过程。所有septin亚基在结构上都是相关的，但异源寡聚物的组织是紧密的 由尚不清楚的机制控制。影响septin蛋白折叠的突变会导致男性不育， 野生型脓毒症的错误折叠可能导致其他疾病（例如阿尔茨海默病）。分子伴侣 促进许多蛋白质的正确折叠和组装，但尚不清楚脓毒症是否在其中。的 长期目标是了解 Septin 异源正确从头组装的分子要求 低聚物。该应用的目标是确定在脱除过程中与酵母败血症结合的伴侣。 novo biogenesis，定义介导这些相互作用的脓毒症的分子特征，并 确定它们对 de novo septin 折叠动力学的影响。酵母模型已被开发出来 测量 septin 折叠动力学，以及快速、灵敏地绘制 septin 伴侣图谱的新工具 相互作用并确定它们沿着隔膜折叠途径发生的顺序。中心假设 胞质伴侣网络与新生脓蛋白结合以促进天然折叠和功能 septin 寡聚化。该模型是根据在 申请人的实验室。该项目的基本原理是 Septin 错误折叠和 人类疾病表明 Septin 折叠在 septin 功能中的重要性，但细胞对 septin 折叠未知。对这个过程的更深入的理解可能会直接转化为关于以下方面的见解： 脓毒症相关人类疾病的分子基础。该模型将通过提出两个问题来测试 制定为具体目标：（1）伴侣如何识别新生败血症？； (2) 陪伴者有哪些 de novo septin 折叠需要吗？第一个目标是采用尖端的体内方法来识别 septin-伴侣相互作用并确定它们在 septin 生物发生过程中发生的顺序。 强大的遗传学用于剖析单个脓毒症特定区域在这些关联中的作用。 第二个目标利用四种平行、独立的隔膜折叠测定法来确定哪个伴侣蛋白- 脓毒症关联在功能上很重要。这种创新方法首次探索折叠角色 对于与脓毒蛋白相互作用的伴侣，使用以前从未应用于此问题的方法。该提案的 重要性在于它有可能解释脓毒蛋白突变或伴侣功能障碍如何导致疾病。

Cellular function requires the assembly of macromolecular complexes composed of distinct polypeptide subunits that interact in precise ways. Protein sequence is often insufficient to guarantee that in the crowded cellular environment each nascent subunit makes only the correct protein-protein interactions. Incorrect conformations and interactions lead to cellular defects, and human disease. How cells ensure that newly- synthesized polypeptides assemble into functional complexes remains poorly understood. Septin proteins assemble into highly conserved cytoskeletal hetero-oligomers that have emerged as central players in many cellular processes. All septin subunits are structurally related, yet the organization of hetero-oligomers is tightly controlled by mechanisms that remain unclear. Mutations that affect septin protein folding cause male infertility, and misfolding of wildtype septins may contribute to other diseases (e.g. Alzheimer's). Molecular chaperones promote the proper folding and assembly of many proteins, but it is not known if septins are among them. The long-term goal is to understand the molecular requirements for proper de novo assembly of septin hetero- oligomers. The goals of this application are to identify the chaperones that engage yeast septins during de novo biogenesis, to define the molecular features in the septins that mediate these interactions, and to determine their effects on the kinetics of de novo septin folding. A yeast model has been developed to measure the kinetics of septin folding, as well as new tools to rapidly and sensitively map septin-chaperone interactions and identify the order in which they occur along the septin folding pathway. The central hypothesis is that a network of cytosolic chaperones engages nascent septins to promote native folding and functional septin oligomerization. This model was generated from extensive preliminary observations made in the applicant's laboratory. The rationale for this project is that emerging links between septin misfolding and human disease point to the importance of septin folding in septin function, yet the cellular requirements for septin folding are unknown. Deeper understanding of this process may translate directly to insights regarding the molecular basis of septin-associated human diseases. This model will be tested by pursuing two questions formulated as specific aims: (1) How do chaperones recognize nascent septins?; (2) Which chaperones are required for de novo septin folding? In the first aim, cutting-edge in vivo methods are employed to identify septin-chaperone interactions and determine the sequential order in which they occur during septin biogenesis. Powerful genetics are used to dissect the roles of specific regions of individual septins in these associations. The second aim exploits four parallel, independent assays for septin folding to determine which chaperone- septin associations are functionally important. This innovative approach explores for the first time folding roles for septin-interacting chaperones, using methods never before applied to this question. The proposal's significance lies in its potential to explain how septin mutations or chaperone dysfunctions cause disease.