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

• 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
• 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

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.

总结 液-液相分离（LLPS），即分子冷凝成液体状组件的能力， 广泛地分隔细胞并影响许多基本的生物过程。LLP是否 在细胞中的功能所需的基本上仍然不清楚。回答这个问题的一个挑战来自于 难以调节形成缩合物的能力而不影响蛋白质的功能，因为 组装和功能通常由相同的相互作用介导。有可能更小的离散的 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 肿瘤抑制斑点型POZ蛋白（SPOP）。SPOP将底物募集到泛素连接酶， 泛素化我们最近表明，SPOP和底物通过弱的多价 相互作用，这导致它们在活跃的无膜细胞器中共定位。前列腺癌突变 钝化SPOP与底物相分离的能力，导致它们在细胞中的单独定位， 底物水平增加和易感细胞的转化。 我们在表征多价、无序和相分离体系方面具有丰富的经验， 解决上述问题所必需的体外生物物理、生物化学和细胞生物学方法和试剂 问题在所提出的工作中，我们将首先调节冷凝物的材料性质，以测试 有效酶活性的流动性要求。第二，我们将测试是否设计单价 底物以与多价对应物相似的亲和力结合，可以有效地被泛素化， 不存在相分离。第三，我们将利用癌症突变来调节 凝聚物和离散复合物在相反的方向，以测试这两个是主要的球员， SPOP功能。第四，我们将解决的关键问题，是否弱相互作用，通常调解 LLPS能够特异性地划分细胞。我们将使用SPOP子宫内膜癌突变， 改变底物特异性，以鉴定负责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，多学科的研究将显着推进的结构决定因素的知识， 在驱动相分离的弱SPOP/底物相互作用中的特异性， 分离SPOP介导的底物泛素化，以及 几种SPOP癌症突变与特征明确的前列腺癌突变不同。的 因此，预期的结果将为相分离在生物学中的作用提供概念性的见解。 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癌症的指南， SPOP功能，我们的工作可能最终有助于指导靶点验证，以开发针对SPOP的治疗方法。 相关癌症