Multivalent higher-order complexes regulate ubiquitination in Hedgehog signaling

描述（由申请人提供）：异常Hedgehog（Hh）信号传导促进脑、皮肤、前列腺、子宫内膜和胃肠道癌症。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）中的突变与癌症的发展相关。SPOP通过线性SPOP结合（SB）基序将底物如Gli 3募集到CRLSPOP。令人惊讶的是，我们的初步数据显示，Gli 3包含许多弱SB基序。SPOP具有两个寡聚化结构域，它们一起促进自缔合成更高阶的SPOP同源寡聚物，其大小取决于SPOP浓度。因此，Gli 3和SPOP彼此是多价的，但它们的高价在调节Gli 3泛素化中的作用尚不清楚。有趣的是，SPOP定位于细胞核中的T点状结构，我们将其命名为核SPOP体。“这些机构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Gli 3和SPOP组装成高阶Gli 3/SPOP复合物，其可在细胞中形成核SPOP“体”;和（B）多价产生Gli 3募集和CRLSPOP对蛋白质浓度的泛素化的超敏感性。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而知之甚少。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：1。通过（a）确定Gli 3中SB基序的位置和亲和力以及它们的序列/亲和力关系;和（B）通过阐明两个SPOP寡聚化结构域如何协同作用以促进更高级的SPOP同源寡聚体以及它们的价如何取决于SPOP浓度来检验Gli 3和SPOP是高度多价的假设。2.通过（a）确定高阶Gli 3/SPOP复合物的大小和亲和力的浓度依赖性;和（B）通过绘制Gli 3的泛素化效率作为浓度和化合价的函数，并通过确定SPOP寡聚化和底物结合对其在核SPOP“体”中定位的作用，检验Gli 3和SPOP多价性在控制泛素化中起作用的假设。提高我们对Gli 3水平调节的理解将为Hedgehog信号在健康和疾病中的作用提供重要的见解。这项工作将对我们理解SPOP中新发现的癌症突变以及普遍存在但研究不足的高阶蛋白质复合物产生重大影响。

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.