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

项目摘要。 人阿利克斯（也称为PDCD 6 IP）在内-溶酶体途径、细胞凋亡、包膜病毒出芽和细胞凋亡中起作用。 其他重要的细胞信号传导和膜断裂过程。这些不同的功能是由其翻译后调节 修饰（PTM），特别是酪氨酸磷酸化和泛素化。我们最近发现阿利克斯通过 其富含脯氨酸的结构域（PRD）形成液体样凝聚物和淀粉样纤维，并且这两种组装体溶解 对酪氨酸残基的磷酸化和去磷酸化的改革。在这个ESI-MIRA提案中的项目扩展 基于这些令人兴奋的发现，并将揭示相分离，纤维化， 和阿利克斯的PTM。具体来说，我们将：确定阿利克斯的组件的结构特征，它们的调节， 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），阐明了功能性 阿利克斯聚合的相关性，以及阿利克斯缩合物向原纤维的时间依赖性转变的机制 （方向2），并表征阿利克斯和泛蛋白之间的相互作用，并确定阿利克斯之间的串扰 泛素化及其磷酸化介导的聚合（方向3）。阿利克斯的高级结构表征- 方向1.1的有序组装将揭示控制其相分离的相互作用热点和新的原子- PRD如何形成富含β折叠的原纤维的解析细节。方向1.2中的机制研究将阐明调节 以及通过脂质膜和酪氨酸去/磷酸化调节阿利克斯缩合物和原纤维，揭示了 激酶进入这些组件中的位点，以及酪氨酸残基的身份，其去磷酸化触发 阿利克斯聚合。方向1.3中的细胞研究将检查哺乳动物细胞中的阿利克斯聚合。沿方向 2.1，我们将确定聚合如何影响阿利克斯的功能。方向2.2的机制研究将阐明 时间依赖性硬化阿利克斯冷凝成原纤维，产生新的见解的作用相分离， 固定化方向3.1的结构和动力学研究将阐明阿利克斯和泛素之间的相互作用。 最后，在方向3.2中，我们将确定阿利克斯泛素化对其磷酸化介导的 聚合法上述研究建立在我们发现独特的阿利克斯组装体，它们通过PTM的调节， 阿利克斯的缓慢成熟浓缩成刚性原纤维，阿利克斯晚期内体膜的残基特异性细节 相互作用，以及阿利克斯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 阿利克斯组件的同质样品使其结构表征，选择性募集的发现， 阿利克斯的信号合作伙伴在其缩合物，和阿利克斯-泛素相互作用的解决方案，确保高可行性， 成功完成我们的研究。我们新开发的方法，包括一种新的标签策略， 核磁共振研究的阿利克斯组装，并生产毫克量的纯泛素化蛋白，承诺 突破性的见解阿利克斯聚合和泛素在阿利克斯生物学中的作用。总的来说，这些研究 将定义阿利克斯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.