The role of weak multivalent interactions and phase separation in SPOP tumor suppressor function

弱多价相互作用和相分离在SPOP肿瘤抑制功能中的作用

基本信息

批准号：
10316227
负责人：
Tanja Mittag
金额：
$ 35.9万
依托单位：
ST. JUDE CHILDREN'S RESEARCH HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-01-01 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10316227
关键词：
Address Affect Affinity Androgen Receptor Behavior Binding Biochemical Biological Biological Process Biology Biophysics Cell physiology Cells Clinical Complex DAXX gene Data Death Domain Disease Endometrial Endometrial Carcinoma Epigenetic Process Functional disorder Future Genes In Vitro Individual Knowledge Lead Learning Liquid substance Malignant Neoplasms Malignant neoplasm of prostate Measures Mediating Membrane Modeling Mutate Mutation Nuclear Oils Organelles Outcome Phase Play Positioning Attribute Process Property Proteins Proto-Oncogenes Reagent Research Role Solid Specificity Substrate Interaction Substrate Specificity System Testing Therapeutic Therapeutic Intervention Tumor Suppressor Proteins Ubiquitination Validation Variant Vinegar Work biophysical properties c-myc Genes cellular imaging design driving force experience falls fluidity improved inhibitor insight macromolecule multidisciplinary mutant preservation protein function rare cancer rational design recruit response ubiquitin ligase

项目摘要

SUMMARY Liquid-liquid phase separation (LLPS), i.e. the ability of molecules to condense into liquid-like assemblies, compartmentalizes cells extensively and impacts many fundamental biological processes. Whether LLPS is required for function in cells remains largely unclear. One challenge in answering this question arises from the difficulty in modulating the ability to form condensates without affecting the proteins' function, because assembly and function are often mediated by the same interactions. It is possible that smaller, discrete complexes are able to facilitate the function. We will address this question in enzymatically active condensates of the tumor suppressor speckle-type POZ protein (SPOP). SPOP recruits substrates to a ubiquitin ligase for ubiquitination. We have recently shown that SPOP and substrates undergo LLPS via weak, multivalent interactions, which result in their colocalization in active, membraneless organelles. Prostate cancer mutations blunt the ability of SPOP to phase separate with substrates, leading to their separate localization in cells, to increased substrate levels, and transformation of susceptible cells. We have experience in characterizing multivalent, disordered and phase-separating systems, and have built the necessary in vitro biophysical, biochemical and cell biological approaches and reagents to tackle the above question. In the proposed work, we will first modulate the material properties of condensates to test the requirement of fluidity for effective enzymatic activity. Second, we will test whether designed monovalent substrates, which bind at similar affinities as their multivalent counterparts, can be ubiquitinated effectively in the absence of phase separation. Third, we will make use of cancer mutations that modulate the formation of condensates and discrete complexes in opposite directions to test which of the two are the major players in SPOP function. Forth, we will address the critical question whether the weak interactions that typically mediate LLPS are able to compartmentalize cells specifically. We will use SPOP endometrial cancer mutations, which alter substrate specificity, to identify the strongest motifs responsible for the specificity alteration. The results will provide a conservative measure of specificity-mediating affinities in phase-separating systems. Our rigorous, multidisciplinary studies will significantly advance the knowledge of the structural determinants of specificity in weak SPOP/substrate interactions that drive phase separation, of the necessity of phase separation for SPOP-mediated substrate ubiquitination, and of the biophysical basis for the dysfunction of several SPOP cancer mutations that are distinct from the well-characterized prostate cancer mutations. The expected results will therefore provide conceptual insights into the role of phase separation in biological function. While we use rare cancer-associated mutations mainly as guides towards understanding of normal SPOP function, our work may ultimately help guide target validation for developing therapeutics against SPOP- related cancers.

概括液-液相分离（LLPS），即分子凝结成液体状组装体的能力，广泛地划分细胞并影响许多基本的生物过程。是否 LLPS 细胞功能所需的物质在很大程度上仍不清楚。回答这个问题的一个挑战来自于很难在不影响蛋白质功能的情况下调节形成冷凝物的能力，因为组装和功能通常由相同的相互作用介导。更小的、离散的复合物能够促进该功能。我们将在酶活性缩合物中解决这个问题抑癌斑点型 POZ 蛋白 (SPOP)。 SPOP 将底物招募到泛素连接酶中泛素化。我们最近表明，SPOP 和底物通过弱、多价进行 LLPS 相互作用，导致它们共定位于活性的无膜细胞器中。前列腺癌突变削弱 SPOP 与底物相分离的能力，导致它们在细胞中单独定位，增加底物水平和易感细胞的转化。我们在表征多价、无序和相分离系统方面拥有丰富的经验，并建立了解决上述问题所需的体外生物物理、生物化学和细胞生物学方法和试剂问题。在拟议的工作中，我们将首先调制冷凝物的材料特性来测试有效酶活性的流动性要求。其次，我们将测试是否设计单价底物与其多价对应物以相似的亲和力结合，可以有效地泛素化不存在相分离。第三，我们将利用癌症突变来调节相反方向的凝聚态和离散复合物，以测试两者中哪一个是主要参与者 SPOP 功能。第四，我们将解决一个关键问题：通常调解的弱相互作用是否 LLPS 能够专门划分细胞。我们将使用 SPOP 子宫内膜癌突变，改变底物特异性，以确定导致特异性改变的最强基序。结果将提供相分离系统中特异性介导亲和力的保守测量。我们严谨的多学科研究将显着增进对结构决定因素的了解驱动相分离的弱 SPOP/底物相互作用的特异性，相的必要性 SPOP介导的底物泛素化的分离，以及功能障碍的生物物理基础几种与明确表征的前列腺癌突变不同的 SPOP 癌症突变。这因此，预期结果将为相分离在生物中的作用提供概念性见解。功能。虽然我们主要使用罕见的癌症相关突变作为理解正常突变的指南 SPOP 功能，我们的工作最终可能有助于指导目标验证，以开发针对 SPOP 的疗法相关癌症。