Multivalent higher-order complexes regulate ubiquitination in Hedgehog signaling

多价高阶复合物调节 Hedgehog 信号传导中的泛素化

• 批准号: 8986797
• 负责人: Tanja Mittag
• 金额: $ 30.97万
• 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 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8986797
• 关键词: Address; Affinity; BTB/POZ Domain; Binding; Binding Sites; Biochemical; Bioinformatics; Biological; Biological Assay; Biological Process; Brain; Cancer Etiology; Cell Nucleus; Cells; Cellular biology; Complex; Data; Dependence; Development; Disease; Dissociation; Endometrial; Erinaceidae; Future; GLI gene; Health; Heterogeneity; Homo; In Vitro; Ligase; Location; Malignant Neoplasms; Malignant neoplasm of prostate; Mediating; Memory; Metabolism; Modeling; Mutate; Mutation; Nature; Nuclear; Outcome; Pattern; Property; Prostate; Protein Engineering; Proteins; Qualifying; RNA; Recruitment Activity; Regulation; Research; Role; Signal Transduction; Signaling Protein; Skin; Source; Stomach; Structure; System; Techniques; Testing; Therapeutic; Tissues; Ubiquitination; Up-Regulation; Validation; Work; base; biophysical techniques; experience; improved; in vivo; innovation; insight; light microscopy; novel; novel therapeutics; protein complex; receptor; response; smoothened signaling pathway; tool; ubiquitin ligase

DESCRIPTION (provided by applicant): Aberrant Hedgehog (Hh) signaling promotes brain, skin, prostate, endometrial, and gastric tract cancers. Amplification of the Hh transcriptional regulators, the Gli proteins, or mutations in SPOP, the substrate receptor of the ubiquitin ligase CRLSPOP, are associated with the development of cancer. SPOP recruits substrates such as Gli3 to CRLSPOP through linear SPOP-binding (SB) motifs. Surprisingly, our preliminary data show that Gli3 contains many weak SB motifs. SPOP has two oligomerization domains that together facilitate self-association into higher-order SPOP homo-oligomers, whose size depends on the SPOP concentration. Hence, Gli3 and SPOP are multivalent for each other, but the role of their high valency in regulating Gli3 ubiquitination is unclear. Interestingly, SPOP localizes t punctate structures in the nuclei, which we designate as nuclear SPOP "bodies." These bodies can be detected by light microscopy and are likely facilitated by multivalent interactions. We hypothesize that (a) multivalent Gli3 and SPOP assemble into higher-order Gli3/SPOP complexes, which may form nuclear SPOP "bodies" in cells; and (b) multivalency generates ultrasensitivity of Gli3 recruitment and ubiquitination by CRLSPOP to protein concentration. Multivalency may be a general mechanism to regulate signaling but is poorly understood because of challenges inherent to the heterogeneous nature of higher-order complexes. We will use an innovative combination of biophysical, structural, biochemical, and cell biological techniques to: 1. Test the hypothesis that Gli3 and SPOP are highly multivalent by (a) determining the location and affinities of SB motifs in Gli3 and their sequence/affinity relationship; and (b) by elucidating how the two SPOP oligomerization domains synergize to promote higher-order SPOP homo-oligomers and how their valency depends on SPOP concentration. 2. Test the hypothesis that multivalency of Gli3 and SPOP functions in controlling ubiquitination by (a) determining the concentration-dependence of size and affinity of higher-order Gli3/SPOP complexes; and (b) by charting the ubiquitination efficiency towards Gli3 as a function of concentration and valency, and by determining the role of SPOP oligomerization and substrate binding for its localization in nuclear SPOP "bodies". Improving our understanding of the regulation of Gli3 levels will provide important insight into Hedgehog signaling in health and disease. The proposed work will have significant impact for our understanding of newly identified cancer mutations in SPOP and for ubiquitous but understudied higher-order protein complexes.

描述（由申请人提供）：异常刺猬（Hh）信号可促进脑癌、皮肤癌、前列腺癌、子宫内膜癌和胃癌。Hh转录调控因子、Gli蛋白或SPOP（泛素连接酶CRLSPOP的底物受体）突变的扩增与癌症的发生有关。SPOP通过线性SPOP结合（SB）基序将Gli3等底物招募到CRLSPOP中。令人惊讶的是，我们的初步数据显示Gli3包含许多弱SB基序。SPOP有两个寡聚结构域，它们共同促进自结合成高阶SPOP同质寡聚物，其大小取决于SPOP浓度。因此，Gli3和SPOP彼此是多价的，但它们的高价在调节Gli3泛素化中的作用尚不清楚。有趣的是，SPOP定位于细胞核中的点状结构，我们将其称为核SPOP“体”。这些体可以通过光学显微镜检测到，并可能通过多价相互作用促进。我们假设(a)多价Gli3和SPOP组装成高阶Gli3/SPOP复合物，这可能在细胞中形成核SPOP“体”；(b)多价性使CRLSPOP对Gli3的募集和泛素化对蛋白浓度具有超敏感性。多价性可能是调节信号传导的一般机制，但由于高阶复合物的异质性所固有的挑战，人们对多价性知之甚少。我们将使用生物物理、结构、生化和细胞生物学技术的创新组合来：通过(a)确定Gli3中SB基序的位置和亲和关系及其序列/亲和关系，验证Gli3和SPOP具有高多价性的假设；(b)阐明两个SPOP寡聚结构域如何协同促进高阶SPOP同质寡聚物，以及它们的价如何取决于SPOP浓度。2. 通过(a)确定高阶Gli3/SPOP复合物的大小和亲和力的浓度依赖性，验证Gli3和SPOP的多价性在控制泛素化中的作用；(b)通过绘制Gli3泛素化效率与浓度和价的函数图，并通过确定SPOP寡聚化和底物结合对其在核SPOP“体”中的定位的作用。提高我们对Gli3水平调控的理解将为了解健康和疾病中的Hedgehog信号传导提供重要的见解。这项工作将对我们理解SPOP中新发现的癌症突变和普遍存在但研究不足的高阶蛋白质复合物具有重大影响。