The Functional Interplay Between Phase Separation, Fibrillization, and Posttranslational Modifications of ALIX

项目摘要。 人Alix(也称为PDCD6IP)在内切溶酶体途径、细胞凋亡、包膜病毒萌发和 其他重要的细胞信号和膜断裂过程。这些不同的功能受其翻译后的调节 修饰(PTM)，特别是酪氨酸磷酸化和泛素化。我们最近发现Alix，通过 它的富含脯氨酸的结构域(PRD)形成液体状凝聚物和淀粉样原纤维，这两个组件都溶解 关于酪氨酸残基的磷酸化和去磷酸化的改革。ESI-MIRA提案中的项目扩展 在这些令人兴奋的发现的基础上，并将揭示相分离、玻璃化、 和Alix的PTMS。具体地说，我们将：确定Alix程序集的结构特征，通过 酪氨酸去磷酸化和膜，以及它们在哺乳动物细胞中的形成(方向1)，阐明功能 Alix聚合的相关性以及Alix凝聚物向原纤化转变的时间相关机制 (方向2)，并表征Alix和泛素之间的相互作用，并确定Alix之间的串扰 泛素化及其磷酸化介导的聚合(方向3)。Alix分子的结构表征 1.1方向的有序组装将揭示支配其相分离的相互作用热点和新颖的原子- 解析PRD如何形成富含β-Sheet的纤维的详细信息。方向1.2的机械论研究将阐明监管 以及脂膜和酪氨酸去磷酸化对Alix凝聚物和纤维的调节，揭示了 激酶进入其在这些组件中的位置，以及其去磷酸化触发的酪氨酸残基的身份 Alix聚合。方向1.3的细胞研究将检查哺乳动物细胞中的Alix聚合。在方向上 2.1，我们将确定聚合如何影响Alix的功能。方向2.2的机械论研究将阐明 Alix凝聚物随时间硬化成纤维，对相分离在 流光化。3.1方向的结构和动力学研究将阐明Alix和泛素之间的相互作用。 最后，在方向3.2中，我们将确定Alix泛素化对其磷酸化的影响 聚合反应。上述研究建立在我们发现独特的Alix组件、它们被PTMS调控、 Alix凝集物缓慢成熟为坚硬的纤维，Alix晚期内膜残基特有的细节 相互作用，以及Alix的磷酸化如何抑制这些相互作用。广泛的初步结果，包括高度 Alix组件的同质样本使其结构表征、选择性招募的发现 Alix在其缩合物中的信号合作伙伴，以及Alix-泛素在溶液中的相互作用，确保了高度的可行性 成功地完成了我们计划中的研究。我们新开发的方法，包括一种新的标签策略，以促进 Alix组件的核磁共振研究，以及生产毫克量的纯泛素化蛋白质，Promise 对Alix聚合和泛素在Alix生物学中的作用的开创性见解。总的来说，这些研究 将确定Alix多方面细胞和膜功能的分子机制。

Project Summary. Human ALIX (also known as PDCD6IP) functions in endo-lysosomal pathway, apoptosis, enveloped virus budding, and other essential cell signaling and membrane scission processes. These diverse functions are regulated by its posttranslational modifications (PTMs), specifically tyrosine phosphorylation and ubiquitination. We recently uncovered that ALIX, through its proline-rich domain (PRD), forms liquid-like condensates and amyloid fibrils, and that both these assemblies dissolve on phosphorylation and reform on dephosphorylation of its tyrosine residues. Projects in this ESI-MIRA proposal expand upon these exciting discoveries and will uncover the dynamic functional interplay between phase separation, fibrillization, and PTMs of ALIX. Specifically, we will: determine the structural characteristics of ALIX’s assemblies, their regulation by tyrosine de/phosphorylation and membranes, and their formation in mammalian cells (direction 1), elucidate the functional relevance of ALIX polymerization, and the mechanisms of the time-dependent transitions of ALIX condensates to fibrils (direction 2), and characterize the interactions between ALIX and ubiquitin, and determine the cross-talk between ALIX ubiquitination and its phosphorylation-mediated polymerization (direction 3). Structural characterization of ALIX’s higher- order assemblies in direction 1.1 will reveal the interactions hotspots that govern its phase separation and novel atomic- resolution details of how a PRD can form β-sheet rich fibrils. Mechanistic studies in direction 1.2 will elucidate regulation and modulation of ALIX condensates and fibrils by lipid membranes and tyrosine de/phosphorylation, revealing how a kinase accesses its sites within these assemblies, and the identity of tyrosine residues whose dephosphorylation triggers ALIX polymerization. Cellular studies in direction 1.3 will examine ALIX polymerization in mammalian cells. In direction 2.1, we will determine how polymerization affects ALIX’s functions. Mechanistic studies in direction 2.2 will elucidate time-dependent hardening of ALIX condensates into fibrils, yielding new insights into the role of phase separation in fibrillization. Structural and kinetic studies in direction 3.1 will elucidate the interactions between ALIX and ubiquitin. Finally, in direction 3.2, we will determine the impact of ALIX ubiquitination on its phosphorylation-mediated polymerization. The above studies build upon our discoveries of the unique ALIX assemblies, their modulation by PTMs, the slow maturation of ALIX condensates into rigid fibrils, residue-specific details of ALIX – late endosomal membrane interactions, and how ALIX’s phosphorylation inhibits these interactions. Extensive preliminary results, including highly homogenous samples of ALIX assemblies enabling their structural characterization, the discoveries of selective recruitment of ALIX’s signaling partners in its condensates, and of ALIX – ubiquitin interactions in solution, assure high feasibility of successfully completing our proposed studies. Our newly developed methods, including a new labeling strategy to facilitate NMR studies of ALIX assemblies, and the production of milligram quantities of pure ubiquitinated proteins, promise groundbreaking insights into ALIX polymerization and the role of ubiquitin in ALIX biology. Collectively, these studies will define molecular mechanisms that underlie ALIX’s multifaceted cellular and membrane functions.