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

ALIX 的相分离、纤维化和翻译后修饰之间的功能相互作用

• 批准号: 10700110
• 负责人: Lalit Deshmukh
• 金额: $ 39.5万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-15 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10700110
• 关键词: Affect; Amyloid Fibrils; Apoptosis; Biology; Cell membrane; Characteristics; Human; Kinetics; Label; Liquid substance; Mammalian Cell; Mediating; Membrane; Membrane Lipids; Methods; Molecular; Pathway interactions; Phase; Phosphorylation; Phosphorylation Inhibition; Phosphotransferases; Physical condensation; Polymers; Post-Translational Protein Processing; Process; Production; Proline-Rich Domain; Protein Dephosphorylation; Proteins; Regulation; Resolution; Role; Sampling; Signal Transduction; Site; Time; Tyrosine; Tyrosine Phosphorylation; Ubiquitin; Ubiquitination; Virus; beta pleated sheet; endosome membrane; insight; late endosome; milligram; novel; polymerization; recruit

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.

项目摘要。 人类ALIX（也称为PDCD6IP）在内部溶酶体途径，凋亡，包络病毒萌芽和 其他必要的细胞信号传导和膜分裂过程。这些潜水功能受到翻译后的调节 修饰（PTMS），特别是酪氨酸磷酸化和泛素化。我们最近通过 其富含脯氨酸的结构域（PRD），形成液状冷凝物和淀粉样蛋白纤维，并且这两个组件都溶解 关于光谱和改革的酪氨酸残留物的去磷酸化。 ESI-MIRA提案中的项目扩展 在这些令人兴奋的发现中，将发现相位分离，纤维化，纤维化之间的动态功能相互作用 和Alix的PTM。具体来说，我们将：确定Alix组装的结构特征，它们的调节 酪氨酸DE/磷酸化和膜，它们在哺乳动物细胞中的形成（方向1），阐明了功能 ALIX聚合的相关性以及Alix缩合到原纤维的时间依赖性过渡的机制 （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较高的结构表征 沿方向1.1方向组件的订单组件将揭示控制其相分离和新型原子的相互作用热点 分辨率的细节PRD如何形成富含β-折叠的原纤维。方向1.2方向的机械研究将阐明调节 以及通过脂质膜和酪氨酸DE/磷酸化对ALIX冷凝物和原纤维的调节，揭示了如何 激酶在这些组件中访问其位置，以及酪氨酸保留的身份，其去磷酸化触发了 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。方向 2.1，我们将确定聚合如何影响ALIX的功能。方向2.2的机械研究将阐明 ALIX将凝结成纤维的时间依赖性硬化，从 纤维化。方向3.1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3.2方向上，我们将确定ALIX泛素化对其磷酸化介导的影响 聚合。上述研究基于我们对独特的ALIX组件的发现，即PTMS的调节， ALIX凝结成刚性原纤维的缓慢成熟，ALIX的居住细节 - 晚期内体膜 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组件的同质样品具有结构性表征，选择性募集的发现 Alix在其冷凝物中的信号伴侣以及溶液中的Alix - 泛素相互作用的相互作用，确保高可行性 成功完成我们建议的研究。我们新开发的方法，包括一种新的标签策略来促进 ALIX组件的NMR研究以及毫克量的纯泛素蛋白的生产有望 对ALIX聚合的开创性见解和泛素在ALIX生物学中的作用。总的来说，这些研究 将定义基于ALIX多面细胞和膜功能的分子机制。