Multivalent higher-order complexes regulate ubiquitination in Hedgehog signaling

描述(由申请人提供)：异常的Hedgehog(HH)信号促进脑癌、皮肤癌、前列腺癌、子宫内膜癌和胃道癌。HH转录调节因子Gli蛋白的扩增或泛素连接酶CRLSPOP底物受体SPOP的突变与癌症的发生有关。SPOP通过线性SPOP结合(SB)基序招募Gli3至CRLSPOP等底物。令人惊讶的是，我们的初步数据显示，Gli3包含许多弱的SB基序。SPOP有两个低聚结构域，这两个结构域共同促进了自缔合形成更高顺序的SPOP均低聚物，其大小取决于SPOP的浓度。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定位于核中的点状结构，我们称之为核SPOP“小体”。这些小体可以用光学显微镜检测到，并且很可能是通过多价相互作用来促进的。我们假设(A)多价Gli3和SPOP组装成高阶Gli3/SPOP复合体，可能在细胞内形成核SPOP“体”；(B)多价作用通过CRLSPOP对蛋白质浓度产生Gli3募集和泛素化的高度敏感性。多价性可能是一种调节信号的一般机制，但由于高阶复合体的异质性固有的挑战，人们对此知之甚少。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：1.通过(A)确定Sb基序在Gli3中的位置和亲和力以及它们的序列/亲和力关系来验证Gli3和SPOP高度多价的假设；以及(B)通过阐明两个SPOP低聚域如何协同作用来促进更高顺序的SPOP同源低聚物，以及它们的价态如何依赖于SPOP浓度。2.检验Gli3和SPOP的多价性在控制泛素化方面的作用，方法是：(A)确定高阶Gli3/SPOP复合体的大小和亲和力的浓度依赖性；(B)绘制Gli3的泛素化效率与浓度和价态的函数图，并确定SPOP寡聚和底物结合对其在核SPOP“小体”中定位的作用。提高我们对Gli3水平调节的理解将为了解Hedgehog信号在健康和疾病中的作用提供重要的见解。这项拟议的工作将对我们理解新发现的SPOP中的癌症突变以及普遍存在但未得到充分研究的高阶蛋白质复合体产生重大影响。

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.