摘要
目的验证RabGEF1(Rab guanine nucleotide exchange factor 1)为线性泛素化修饰的新底物。方法在pEF6/MycHis C载体中克隆人RabGEF1基因。通过免疫共沉淀(Co-IP)实验验证RabGEF1和HOIP的相互作用。利用GST-pulldown实验探索RabGEFl与HOIP相互作用结构域。通过免疫荧光实验验证RabGEF1和HOIP的相互作用与亚细胞定位。应用体内泛素化实验检测RabGEF1的线性泛素化修饰。通过NTA-His泛素化实验进一步明确RabGEF1能够发生线性泛素化修饰。将RabGEF1的泛素化蛋白质样品进行质谱分析,根据质谱结果提示的RabGEF1泛素化位点构建赖氨酸位点突变质粒,进一步在体内泛素化实验中验证RabGEF1的线性泛素化修饰位点。结果RabGEF1与HOIP存在相互作用,且HOIP通过ZF-NEF结构域与RabGEF1发生直接相互作用。RabGEF1与HOIP共同定位于细胞质。LUBAC介导RabGEF1发生线性泛素化修饰依赖于LUBAC酶活性,RabGEF1泛素化修饰位点为K158。结论RabGEFl为线性泛素化修饰的新底物,且K158是其发生线性泛素化修饰的位点。
Objective To verify that Rab guanine nucleotide exchange factor 1(RabGEFl)is a novel substrate of linear ubiquitin.Methods Human RabGEFl gene was cloned into pEF6/Myc-His C vector.The interaction between RabGEFl and HOIP was verified by Co-IP experiment.The interaction domain between RabGEFl and HOIP was analyzed by GST-pulldown.The interaction and subcellular localization were verified by immunofluorescence.The linear ubiquitination of RabGEFl was detected by in vivo ubiquitination experiment.The NTA-His ubiquitination assay further confirmed that RabGEF1 can undergo linear ubiquitination modification.The ubiquitinated RabGEF1 proteinsamples were subjected to mass spectrometry to analyze the specific site modified by ubiquitin.The mutant plasmid of potential RabGEFl ubiquitination site was constructed according to the mass spectrometry results,and the ubiquitination site of RabGEFl was further verified with RabGEFl KR mutant plasmids in vivo ubiquitination experiment.Results RabGEFl interacts with HOIP via ZF-NEF domain.RabGEFl co-locates with HOIP in the cytoplasm.LUBAC mediated linear ubiquitination modification of RabGEFl depends on LUBAC enzyme activity,and the ubiquitination modification site of RabGEFl is K158.Conclusion RabGEFl is a novel substrate for linear ubiquitination modification,and the site of linear ubiquitination modification is K158.
作者
黄彬
张令强
张学利
HUANG Bin;ZHANG Ling-Qiang;ZHANG Xue-Li(Shanghai Fengxian District Central Hospital Affiliated to Anhui University of Technology,Shanghai 201499,China;Institute of Biomics,Academy of Military Medicine,Academy of Military Sciences,National Center for Protein Science(Beijing),Beijing 102200,China)
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2023年第4期749-759,共11页
Progress In Biochemistry and Biophysics
基金
国家自然科学基金(81772959)
奉贤区科委社会类科技发展基金(20191101)资助项目。
