E3泛素连接酶SPOP介导的HIPK2非降解型泛素化修饰调控前列腺癌细胞凋亡的机制研究

Speckle-type POZ Protein(SPOP) is an adaptor protein of the CUL3-RBX1 E3 ubiquitin ligase complex, and its gene is identified as one of the most frequently affected genes by somatic point mutations in prostate cancer, suggesting that SPOP is a potential key driver for prostate cancer occurrence and progression. However, how SPOP mutations contribute to prostate cancer remains to be elucidated. Our preliminary results showed that SPOP could interact with HIPK2 and trigger atypical polyubiquitination of HIPK2. This type of ubiquitination modification did not affect the instability of HIPK2, but rather actived HIPK2, dramatically improving the phosphorylation level of P53 at Ser 46. Strikingly, prostate cancer-associated mutants of SPOP were defective in interacting with HIPK2. So we hypothesize that the activation of HIPK2-P53 dependent apoptotic pathway is mediated by wild-type SPOP but not prostate cancer-associated mutants. To explore this hypothesis , at first, we will identify the interaction sites and ubiquitination sites using Tandem Affinity Purification (TAP) and Mass analysis. And then we will investigate the role of this atypical polyubiquitination of HIPK2 on apoptosis in a DNA damge model of prostate cancer cells. Finally, we will explore the SPOP mutations in prostate tissues and the relationship between SPOP mutations and apoptotic markers by immunohistochemical analysis. This study may provide a new theory for our understanding of the mechansim of prostate cancer progression.

锌指结构域蛋白（SPOP）是泛素E3连接酶复合体中的底物识别亚基，其基因在前列腺癌中高频突变，但这种突变对前列腺癌发生的作用机制尚不清楚。我们前期研究发现，SPOP与HIPK2相互作用，通过介导HIPK2的非降解型泛素化修饰提高了P53蛋白的磷酸化水平促进细胞凋亡；而前列腺癌来源的SPOP突变体不能与HIPK2相互作用。据此我们提出“突变的SPOP无法结合HIPK2而失去对细胞凋亡的调控从而诱发前列腺癌发生”的假说。本课题拟先利用串联亲和纯化和质谱分析鉴定SPOP-HIPK2的互作位点和泛素化修饰位点；然后在DNA损伤细胞模型中干预SPOP-HIPK2复合物形成，明确SPOP介导的HIPK2非降解型泛素化修饰在细胞凋亡中的作用。最后利用前列腺癌患者组织筛查SPOP基因突变情况，并通过免疫组化分析SPOP突变与凋亡标志物的相关性。本研究结果可望为揭示前列腺癌的发生提供新思路。

锌指结构域蛋白（SPOP）是泛素E3连接酶复合体中的底物识别亚基，其基因在前列腺癌中高频突变，但这种突变导致前列腺癌基因组不稳定的作用机制尚不清楚。我们通过研究证实，当细胞内的DNA损伤时，ATM通过促进SPOP的S119位磷酸化修饰促进其与HIPK2的相互作用，并促进其对HIPK2的非降解型泛素化修饰。HIPK2的非降解型泛素化修饰促进了HIPK2对HP1γ的磷酸化修饰能力，使HP1γ与H3K9me3解离开始DNA的损伤修复；而前列腺癌来源的SPOP突变体不能与HIPK2相互作用且对野生型SPOP存在显性负效应进而导致前列腺癌细胞的基因组不稳定促进前列腺癌的发生发展。本研究揭示了SPOP突变为何导致前列腺癌基因组不稳定的分子机制，有望为揭示前列腺癌的精准治疗提供新思路。