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.

概括液态液相分离（LLP），即分子凝结成液体样组件的能力，广泛地划分细胞，并影响许多基本的生物学过程。 LLP是否是在细胞中功能所必需的很大程度上不清楚。回答这个问题的一个挑战是在不影响蛋白质功能的情况下调节形成冷凝水的能力的困难，因为组装和功能通常由相同的相互作用介导。可能会较小，离散复合物能够促进该功能。我们将以酶活性的冷凝水来解决这个问题肿瘤抑制型POZ蛋白（SPOP）的抑制作用。 Spop将基材招募到泛素连接酶泛素化。我们最近表明，SPOP和底物通过弱的多价经历LLP 相互作用，导致它们在活跃的无膜细胞器中进行共定位。前列腺癌突变钝锯与SPOP与底物分离的能力，导致其在细胞中的单独定位，以底物水平增加，易感细胞的转化。我们有表征多价，无序和分离系统的经验，并建立了必要的体外生物物理，生化和细胞生物学方法和试剂以应对上述问题。在拟议的工作中，我们将首先调节冷凝水的材料特性以测试对有效酶活性的流动性要求。其次，我们将测试是否设计单价以与多价对应物相似的亲和力结合的底物可以有效地泛滥没有相分离。第三，我们将利用调节形成的癌症突变凝结和离散复合物朝相反的方向测试，以测试两者中的哪一个是 SPOP功能。第四，我们将解决一个关键的问题 LLP能够专门将细胞分离。我们将使用Spop子宫内膜癌突变，改变底物特异性，以确定负责特异性改变的最强基序。结果将提供一个保守的衡量相分离系统中特异性的亲和力的衡量标准。我们严格的多学科研究将显着提高对结构决定因素的了解驱动相分离的弱铲/底物相互作用的特异性，相位的必要性分离蒸馏介导的底物泛素化，以及生物物理基础的功能障碍的生物物理基础几种与特征良好的前列腺癌突变不同的SPOP癌突变。这因此，预期的结果将提供概念性的见解，以了解相位分离在生物学中的作用功能。虽然我们将罕见的癌症相关突变主要用作理解正常的指南 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-的治疗剂相关的癌症。