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）：异常的刺猬（HH）信号传导促进大脑，皮肤，前列腺，子宫内膜和胃道癌。 HH转录调节剂，GLI蛋白或SPOP中的突变的扩增是泛素连接酶CRLSPOP的底物受体，与癌症的发展有关。 Spop通过线性旋转结合（SB）基序将诸如GLI3之类的底物募集到CRLSPOP。令人惊讶的是，我们的初步数据表明，GLI3包含许多弱SB图案。 SPOP具有两个寡聚结构域，共同促进自我关联到高阶Spop Homo-Oligomers，其大小取决于SPOP浓度。因此，GLI3和SPOP彼此之间是越来越多的，但是它们高价值在调节GLI3泛素化中的作用尚不清楚。有趣的是，SPOP将T点状结构定位在细胞核中，我们将其指定为核溅射“身体”。这些物体可以通过光学显微镜检测到，并可能通过多价相互作用来促进。我们假设（a）多价GLI3和SPOP组装成高阶Gli3/spop复合物，这可能形成细胞中的核汤匙“身体”； （b）多价性产生了CRLSPOP募集GLI3募集和泛素化的超敏性。多价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质所固有的挑战而被理解不足。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：1。通过（a）确定gli3及其序列/亲和力关系的SB基序的位置和亲和力，检验GLI3和SPOP高度多价的假设； （b）通过阐明两种SPOP寡聚结构域如何协同促进高阶Spop Homo-Oligomers以及它们的价值如何依赖于SPOP浓度。 2。检验以下假设：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，GLI3和SPOP在控制泛素化方面的功能； （b）通过将对GLI3的泛素化效率绘制为浓度和价值的函数，并确定汤匙低聚和底物在核汤匙“身体”中定位的作用。提高我们对GLI3水平调节的理解将为健康和疾病中的刺猬信号传递提供重要的见解。拟议的工作将对我们对新近鉴定的SPOP和无处不在但已研究的高阶蛋白质复合物的癌症突变产生重大影响。