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: 9187013
• 负责人: Tanja Mittag
• 金额: $ 31.1万
• 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/9187013
• 关键词: Address; Affinity; BTB/POZ Domain; Binding; Binding Sites; Biochemical; Bioinformatics; Biological; Biological Assay; Biological Process; Biophysics; Brain; Cancer Etiology; Cell Nucleus; Cells; Cellular biology; Complex; Data; Dependence; Development; Disease; Dissociation; Endometrial; Endometrial Carcinoma; Erinaceidae; Future; GLI Family Protein; GLI gene; Genetic Transcription; Health; Heterogeneity; Homo; In Vitro; Ligase; Location; Malignant Neoplasms; Malignant neoplasm of prostate; Mediating; Memory; Metabolism; Mutate; Mutation; Nature; Nuclear; Outcome; Pattern; Property; Prostate; Protein Engineering; Proteins; RNA; Recruitment Activity; Regulation; Research; Role; Signal Transduction; Signaling Protein; Skin; Source; Stomach; Structure; System; Techniques; Testing; Therapeutic; Tissues; Ubiquitination; Up-Regulation; Validation; Work; biophysical model; biophysical techniques; experience; improved; in vivo; innovation; insight; light microscopy; novel; novel therapeutics; protein complex; public health relevance; 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）：异常的 Hedgehog (Hh) 信号传导会促进脑癌、皮肤癌、前列腺癌、子宫内膜癌和胃癌。 Hh 转录调节因子 Gli 蛋白的扩增或 SPOP（泛素连接酶 CRLSPOP 的底物受体）的突变与癌症的发生有关。 SPOP 通过线性 SPOP 结合 (SB) 基序将 Gli3 等底物招募到 CRLSPOP。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 具有两个寡聚结构域，它们共同促进自缔合成更高阶的 SPOP 同聚寡聚物，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 在细胞核中定位点状结构，我们将其称为核 SPOP“体”。这些物体可以通过光学显微镜检测到，并且可能是通过多价相互作用促进的。我们假设 (a) 多价 Gli3 和 SPOP 组装成更高阶的 Gli3/SPOP 复合物，可能在细胞中形成核 SPOP“体”； (b) 多价性使 Gli3 募集和 CRLSPOP 泛素化对蛋白质浓度超敏感。多价可能是调节信号传导的通用机制，但由于高阶复合物的异质性所固有的挑战，人们对其知之甚少。我们将使用生物物理、结构、生物化学和细胞生物学技术的创新组合来： 1. 通过 (a) 确定 Gli3 中 SB 基序的位置和亲和力及其序列/亲和力，检验 Gli3 和 SPOP 高度多价的假设关系; (b) 阐明两个 SPOP 寡聚结构域如何协同作用以促进更高阶的 SPOP 同源寡聚物以及它们的化合价如何取决于 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，检验 Gli3 和 SPOP 的多价性在控制泛素化中起作用的假设； (b) 将 Gli3 的泛素化效率绘制为浓度和化合价的函数，并确定 SPOP 寡聚化和底物结合对其在核 SPOP“体”中定位的作用。提高我们对 Gli3 水平调节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。拟议的工作将对我们对 SPOP 中新发现的癌症突变以及普遍存在但尚未充分研究的高阶蛋白质复合物的理解产生重大影响。