A Comparative Study Of Strategies For Specific Substrate Screen Of Ubiquitin Ligase

Ubiquitination is an important post-translational modification that widely existed in eukaryotic cells.Most of the ubiquitinated protein substrates enter the proteasome to degrade to maintain the turnover cycle and homeostasis of various proteins in the cell.In addition to mediating degradation,ubiquitination can also induce changes in protein activity or localization,and thus participate in the regulation of other life activities.The entire ubiquitination machinery consists of four main components,namely ubiquitin-activating enzyme E1,ubiquitin-conjugating enzyme E2,ubiquitin ligase E3,and deubiquitinating enzyme DUB.These four components are coordinated with each other to form a complex and elaborate system.Among them,the main function of ubiquitin ligase E3 is to mediate the direct ubiquitination to specific substrates,and it is of great importance and specificity of the entire ubiquitination process.But the study on substrates screening techniques of E3 is progressing hardly,more intensive researches need to be launch.Proteomics is a new subject that can systematically identify and quantify all proteins in the sample and perform functional research.Its large-scale identification and precise quantitative advantages facilitate the screening of ubiquitin ligase substrates.Two commonly strategies including target gene knockout and overexpression have been used for ubiquitin ligase substrates screening,however,it is still unclear which one is more powerful and suitable for the globle screening of ubiquitinated substrates of ligases.In this study,we systematically compared these two strategies for screening ubiquitin ligase substrates,and tried to establish an efficient and specific screening technology for large-scale screening of a series of ubiquitin ligase substrates in Saccharomyces cerevisiae.We chose the pUC57 plasmid as expression vector and successfully constructed 40yeastubiquitinligaseE3overexpressingstrainsbasedonthetypical ubiquitination-studying strain,JMP024?Xu Lab?^[1].Only a few of the overexpressing strains showed slight growth retardation,and most other strains grew normally.In order to study oxygen related ubiquitin ligase substrates,we used oxidative stimulation sensitive gene HRT3 as an example and performed an in-depth comparison of substrate screening techniques.Studies showed that the introduction of the pUC57plasmid carrying the HRT3 high expression cassette seemed to affect the replication of the originally existing plasmid pUB221,which carring the His-Ub in the JMP024 strain,thereby influencing the effect of substrates screening.Thus,we further constructed a genomic integrated HRT3 overexpressing strain.Using the SILAC technique,we analyzed the ubiquitome of plasmid-overexpressing HRT3 strain?normal condition and treated with proteasome inhibitor MG132?,genomic integrated overexpressing HRT3strain,and HRT3 knockout strain.Compared with the control strain JMP024,the ubiquitome of the HRT3 knockout strain did not change significantly,while that of the genomic integrated HRT3 overexpressing strain showed a significantly globle up-regulated distribution.Data analysis showed that the HRT3 intergrated strain contributed 2.7 times?0.8:0.3?more ratio variety of the ubiquitination level of potential substrates than HRT3 knockout strain,and the standard deviation was twice that of the knockout strain?SD:0.16:0.08?.Moreover,the sequence coverage of the shared identified proteins in genomic integrated strain was increased 98%than that of knockout strain,which provided a basis for in-depth comparison of potential substrates'variation.In addition,the overexpressing strategy also showed clear advantage in the identification of reported substrates of HRT3,DLD3 and GRS1.This indicates that overexpressing strategy can achieve a more pronounced change in the level of ubiquitination of specific substrates and is therefore more suitable for the screening of E3 substrates.Analysis of the quantitative proteomics data found 54 potential substrates of HRT3and 10 potential modification sites,including a substrate likely to be associated with the HRT3 oxidation-stimulated phenotype,NCE103.This study provided effective technical support for the screening of ubiquitin ligase substrates.