Mechanisms of Assembly and Functional Regulation in Non-canonical Biomolecular Condensates

项目摘要 生物分子凝聚物是在活细胞中自组装的微米级无膜结构。 它们普遍存在于不同的生物体中，并存在于仅含蛋白质的集合体或与 核酸。生物分子冷凝物的形成对正常的细胞过程和错误的调节至关重要。 凝析油的组装或分解会导致各种病理现象。我们最近发现，病理性的 融合蛋白EML4-ALK在细胞质中自发形成微米级凝聚物，而缺乏 传统的凝析油形成结构域或序列基序。这种冷凝物引发了一种新的细胞模式 通过充当丰富信号蛋白的物理平台来进行信号传递。客户蛋白的募集 提高局部蛋白质浓度意味着一种通用策略，通过这种策略，多蛋白质缩合物可以实现 生物功能。该领域的一个主要差距是对凝析油以外的促进凝析油的基元知之甚少。 一些常规的基序，或关于蛋白质如何在凝聚体中协同工作以实现其生物功能。 我们提出了一个研究计划，系统地阐明蛋白质中的新基序和生物物理原理- 只有凝析油。这将通过利用CRISPR成像等创新方法来实现， 光遗传学操作，以及定制编写的分析和量化算法，追求两个相互关联的 研究主题。第一个主题是确定使蛋白质缩合成为可能的替代机制 形成方式：通过询问已知或已知的融合蛋白发现新的促进凝析油生成的基序 怀疑形成缩合物；确定这些主题的重要性和模块化；以及绘制主题图- 凝析油调节过程中的函数关系。第二个主题是揭示物理原理 调节多蛋白质缩合物的组成和动态。尽管分子细节可能有所不同， 这里展示的序列空间和原理广泛适用于各种蛋白质和 细胞过程。我们研究计划的长期目标是建立一个扩展的生物物理 了解生物分子凝聚物组装和跨细胞动态平衡和功能的基础 病理学。这种知识带来了新的机会，通过独立的 物理方法取代了干扰生化反应的传统方法，并提供了一种 生物物理框架，用于预防、诊断和治疗凝析油驱动的疾病。

Project Summary Biomolecular condensates are micrometer-scale membraneless structures that self-assemble in living cells. They are ubiquitous across diverse organisms and exist in protein-only conglomerates or protein associated with nucleic acids. Biomolecular condensate formation is critical to normal cellular processes and the mis-regulation of condensate assembly or disassembly drives various pathologies. We recently discovered that the pathological fusion protein EML4-ALK spontaneously forms micrometer-scale condensates in the cytoplasm while lacking conventional condensate-forming domains or sequence motifs. This condensate elicits a novel mode of cell signaling by acting as a physical platform that enriches signaling proteins. The recruitment of client proteins to elevate local protein concentrations implies a general strategy through which multi-protein condensates achieve biological function. A major gap in the field is that very little is known about condensate-promoting motifs beyond a few conventional motifs, or about how proteins work together in condensates to achieve their biological function. We propose a research program that systematically elucidates novel motifs and biophysical principles in protein- only condensates. This will be accomplished by leveraging innovative approaches such as CRISPR imaging, optogenetics manipulation, and custom-written analysis and quantification algorithms to pursue two interrelated research themes. The first theme is identification of alternative mechanisms that enable protein condensate formation by: discovering novel condensate-promoting motifs by interrogating fusion proteins known or suspected to form condensates; determining the essentiality and modularity of such motifs; and mapping motif- function relationships in condensate-mediated processes. The second theme is to uncover physical principles that regulate composition and dynamics in multi-protein condensates. Although the molecular details may differ, the sequence space and principles demonstrated here are broadly applicable to a diverse range of proteins and cellular processes. The long-term objective of our research program is to build an expanded biophysical foundation to understand biomolecular condensate assembly and functions across cellular homeostasis and pathology. Such knowledge brings new opportunities to modulate cellular processes through independent physical approaches instead of traditional ways of interfering with biochemical reactions, and provide a biophysical framework to prevent, diagnose, and treat condensate-driven diseases.