【作者】 吴迪；

【导师】 李大为；

【作者基本信息】 苏州大学 ， 生物学， 2023， 硕士

【摘要】 目的:TRIM37是TRIM家族的E3泛素化连接酶,通过促进底物蛋白的泛素化修饰,诱导底物蛋白在蛋白酶体的降解,对底物蛋白的功能发挥调控作用。TRIM37在乳腺癌、肺癌等多种肿瘤组织中高表达,通过促进组蛋白泛素化修饰调控基因表达,促进肿瘤的生长。但也有研究发现TRIM37的缺失导致细胞有丝分裂过程中多极纺锤体的形成,影响基因组稳定性进而促进肿瘤的发生。TRIM37基因在人体内突变会导致一种罕见病,肌肝脑眼侏儒症(Mulibrey nanism),患者自发多种肿瘤;TRIM37基因敲除的小鼠也具有相似的表型,表明TRIM37在维持基因组稳定性及抑制肿瘤发生中发挥重要的作用。尽管TRIM37在调控肿瘤的发生及进展过程中发挥重要的作用,但是TRIM37蛋白的表达及功能在细胞内的调控机制还不明确。本研究旨在鉴定TRIM37的去泛素化酶,明确其在调控TRIM37蛋白稳定性及功能方面的重要作用,加深对肿瘤的发病机制理解,为治疗提供潜在的药物靶点。方法:(1)通过免疫共沉淀及Ni-NTA拉下实验建立了在哺乳动物细胞中纯化泛素化蛋白底物的方法。(2)采用去泛素化酶基因文库,通过与TRIM37共转染细胞,开展免疫共沉淀及免疫印迹技术高通量筛选并明确与TRIM37相互作用的去泛素化酶。(3)通过体外GST拉下实验,明确TRIM37能够与OTUD1在体外直接结合。(4)采用邻位连接技术(PLA)明确TRIM37能够与OTUD1在细胞内相互作用。(5)构建TRIM37和OTUD1结构域缺失突变体,通过免疫共沉淀及免疫印迹实验确定两个蛋白相互结合的结构域。(6)细胞内过表达或消减OTUD1,开展免疫印迹检测TRIM37蛋白的水平;采用放线菌酮(CHX)处理细胞,检测TRIM37蛋白水平,分析OTUD1对TRIM37蛋白稳定性的影响。(7)开展体外的去泛素化实验,将细胞内纯化的泛素化TRIM37底物蛋白与OTUD1蛋白及其酶活性缺失的突变体蛋白(OTUD1 C320S和H431R)共同孵育,免疫印迹检测TRIM37的泛素化水平。(8)开展PLA实验,比较对照及OTUD1消减的MCF7细胞系中TRIM37内源泛素化水平的差异。结果:(1)在哺乳动物细胞中成功纯化了泛素化的p53和TRIM37蛋白,为后续TRIM37的去泛素化酶功能研究提供了基础。(2)通过高通量筛选去泛素化酶文库,鉴定OTUD1可以与TRIM37蛋白结合。(3)反向的免疫共沉淀实验结果揭示TRIM37能够共沉淀OTUD1蛋白。(4)交互的GST拉下实验结果发现,TRIM37和OTUD1蛋白可以在体外直接结合。(5)PLA实验结果表明OTUD1与TRIM37在细胞内生理水平下存在相互作用。(6)OTUD1通过其OTU结构域与TRIM37蛋白的NLS结构域结合。(7)过表达OTUD1能够显著增加TRIM37蛋白的水平及稳定性,过表达OTUD1 C320S和H431R不能显著增加TRIM37蛋白的水平及稳定性。消减OTUD1的表达,TRIM37蛋白的水平及稳定性下降。(8)OTUD1可以在体外去除TRIM37蛋白的泛素化修饰,但是OTUD1 C320S和H431R不能去除TRIM37蛋白的泛素化修饰。(9)细胞内消减OTUD1后,TRIM37的泛素化水平增加。结论:OTUD1是TRIM37的去泛素化酶,通过与TRIM37蛋白直接结合,去除TRIM37蛋白的泛素化修饰,调控TRIM37蛋白的稳定性。OTUD1可能通过调控TRIM37的水平和功能参与细胞的有丝分裂及肿瘤的发生。更多还原

【Abstract】 Objective:TRIM37 acts as an E3 ubiquitin ligase to regulate the stabilities of substrate proteins by promoting their ubiquitination and proteasomal degradation.Although TRIM37 promotes tumor growth by regulating histone ubiquitination and gene transcription,the controversial studies indicate that TRIM37 deficiency contributes to the formation of multipolar spindle in mitosis,which affects genome stability and leads to multiple tumors.TRIM37 gene mutation in human or loss in mouse can lead to a rare disease,Mulibrey nanism,in which a variety of tumors occur automatically.The intracellular molecular mechanism that maintains the stabilities and activities of TRIM37 remains unclear.This study aims to identify the deubiquitinating enzyme of TRIM37 and to further clarify its role in regulating genome stability and tumorigenesis.Methods:(1)The ubiquitinated substrate proteins were successfully purified from mammalian cells by Ni-NTA pull-down assay or co-immunoprecipitation.(2)The deubiquitinating enzyme expression library was used to screen the deubiquitinating enzyme that binds to TRIM37 by immunoprecipitation followed by western blotting.(3)The reciprocal GST pull-down assay was used to confirm the direct interaction between OTUD1 and TRIM37.(4)Proximity ligation assay(PLA)was used to confirm the interaction between TRIM37 and OTUD1 in cells.(5)TRIM37 and OTUD1 domain deletion mutants were constructed.The binding domains of TRIM37 and OTUD1 were identified by co-immunoprecipitation followed by western blotting.(6)TRIM37 protein level was detected by western blotting after OTUD1 was overexpressed or knocked down in cells.A pulse-chase experiment by cycloheximide(CHX)treatment was used to clarify the effects of OTUD1 on the stability of TRIM37 protein.(7)The in vitro deubiquitination assay was performed to determine the effects of OTUD1 and its deletion mutants(C320S and H431R)on the ubiquitination of TRIM37.(8)A PLA experiment was conducted to determine the endogenous ubiquitination level of TRIM37 in MCF7 control cells and OTUD1 knockdown cells.Results:(1)The ubiquitinated p53 and TRIM37 substrate proteins were successfully purified in mammalian cells.(2)High-throughput screening of a deubiquitinating enzyme library identified that OTUD1 binds to TRIM37.(3)Reciprocal immunoprecipitation assay showed that TRIM37 interacts with OTUD1.(4)The reciprocal GST pull-down assay indicated that TRIM37 and OTUD1 directly interacts in vitro.(5)The PLA assay showed that OTUD1 interacts with TRIM37 in cells.(6)OTUD1 binds to TRIM37 through its OTU domain and TRIM37 binds to OTUD1 through its NLS domain.(7)Overexpression of OTUD1 increased the level and stablity of TRIM37 protein,but overexpression of OTUD1 enzymatic activity deletion mutants C320S and H431R cannot increase the level and stability of TRIM37 protein.Conversely,knockdown of OTUD1 expression leads a reduction of TRIM37 protein and its stability.(8)The OTUD1 protein but not its mutants can effectively remove the ubiquitination of TRIM37 in vitro.(9)The ubiquitination level of TRIM37 was increased after OTUD1 was knocked down from MCF7 cells.Conclusion:OTUD1 is a deubiquitinating enzyme of TRIM37,which regulates the stability of TRIM37 by directly binding to TRIM37 protein and removing its ubiquitination modification.OTUD1 could play a critical role in maintaining genome stability and preventing tumorigenesis by regulating the function of TRIM37.These observations may deepen the current understanding of the tumorigenesis and provide potential drug targets for treatment.更多还原