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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：
10543538
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 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10543538
关键词：
Affect Affinity Androgen Receptor Back 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 Learning Liquid substance Malignant Neoplasms Malignant neoplasm of prostate Measures Mediating Membrane Modeling Mutate Mutation Nuclear Oils Organelles Outcome Phase Physical condensation Play Positioning Attribute Predisposition 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 mimetics multidisciplinary mutant preservation protein function rare cancer rational design recruit response structural determinants 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.

摘要液-液相分离(LLP)，即分子凝聚成类液体组件的能力，广泛的细胞区隔，并影响许多基本的生物过程。有限责任合伙是否细胞功能所需的蛋白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在不影响蛋白质功能的情况下调节形成凝聚体的能力是困难的，因为组装和功能通常由相同的相互作用来调节。有可能是更小的、离散的复合体能够促进这一功能。我们将在酶活性凝析油中解决这个问题肿瘤抑制因子斑点型POZ蛋白(SPOP)。SPOP将底物招募到泛素连接酶上泛素化。我们最近已经证明，SPOP和底物通过弱的、多价的LLP 相互作用，导致它们在活性的、无膜的细胞器中共存。前列腺癌基因突变钝化SPOP与底物相分离的能力，导致它们在细胞中单独定位，以增加底物水平，并转化敏感细胞。我们在表征多价、无序和相分离系统方面有经验，并建立了解决上述问题所需的体外生物物理、生化和细胞生物学方法和试剂问题。在拟议的工作中，我们将首先调制凝析油的材料性质，以测试有效酶活性所需的流动性。第二，我们将测试设计的单价底物与其多价底物具有相似的亲和力，可以有效地泛素化。没有相分离。第三，我们将利用癌症突变来调节凝析油和离散复合体在相反的方向上，以测试这两者中的哪一个是主要参与者 SPOP函数。第四，我们将解决一个关键问题，即通常调解有限合伙人能够对细胞进行特定的分隔。我们将使用SPOP子宫内膜癌突变，改变底物特异性，以确定与特异性改变有关的最强基序。结果是将提供在相分离系统中调节亲和力的特异性的保守测量。我们严谨的多学科研究将极大地促进对结构决定因素的了解在驱动相分离的弱SPOP/底物相互作用中，相的必要性的专一性 SPOP介导的底物泛素化的分离及其功能障碍的生物物理基础几个SPOP癌症突变，与特征明确的前列腺癌突变不同。这个因此，预期的结果将提供对相分离在生物学中的作用的概念性见解功能。虽然我们主要使用罕见的癌症相关突变作为理解正常的指南 SPOP功能，我们的工作最终可能有助于指导针对SPOP的治疗开发的靶点验证- 相关癌症。