Exploration Of The Localization Of USP42 And Its Function And Mechanism In Cancer

The ubiquitin-proteasome proteolytic pathway is one of the general pathways of protein degradation in cells.And in the process,the ubiquitination and deubiquitination of protein is a dynamic process.Ubiquitin is a protein that contains 76 amino acids,and the molecular weight of it is about 8.5 KD.Nowadays the research of deubiquitinating enzymes(DUBs)is becoming a hotter and hotter.DUBs contain five families among which USP family is the largest one.The localization of the members of USP family has many types.Some of them located in the nucleus,some located in the cell nucleolus,some located in cytoplasm,and some located in both cytoplasm and nucleus.The different localization of the DUBs means that they have different function.There are some kinds of DUBs that located in nucleus,among which the localization of Ubiquitin Specific Peptidase 42 is the most special.SC35 is the marker of nuclear speckle.Most of those proteins who have the co-localization with nuclear speckle always regulated with RNA splicing.SC35 is one of splicing factors,which is needed to form spliceosome that also has numerous auxiliary proteins.Our research started with the exploration of the localization of Ubiquitin Specific Peptidase 42,and finding the most important domain that influenced the localization of it.And then we eager to find the mechanism of Ubiquitin Specific Peptidase 42 in cancer,through which we expected Ubiquitin Specific Peptidase 42 can be the new targets for cancer.In our research we found that it was C terminal which can influence the localization of it.Most importantly,the Lys rich domain and Arg rich domain can lead the special localization of Ubiquitin Specific Peptidase 42.At the same time,we also made it clear that Ubiquitin Specific Peptidase 42 and SC35 has co-localization.Furthermore we found the domain that can influenced the co-localization of Ubiquitin Specific Peptidase 42 and SC35,and this co-localization is enzyme activation dependent.In this study we also confirmed that the relationship between Ubiquitin Specific Peptidase and SC35 is indirect interaction.Next,we will explore to the protein that has direct interaction with Ubiquitin Specific Peptidase 42,through which we can find the function of Ubiquitin Specific Peptidase 42 in cancer.Objective: Make it clear that the most important domain of Ubiquitin Specific Peptidase 42 that influenced the localization of it.Make it clear that the co-localization of Ubiquitin Specific Peptidase 42 and SC35 is true,and find out the domain of Ubiquitin Specific Peptidase 42 that affected the co-localization of it and SC35.Explore the interaction between Ubiquitin Specific Peptidase 42 and SC35.Methods: Built up the different constructs of Ubiquitin Specific Peptidase 42 by the method of molecular cloning.These constructs,which were tagged with GFP,include deletion of different domains and mutation.To test the expression of these constructs we used Western blotting assay.At the same time,the localization of them were observed by immunofluorescence.We also used immunofluorescence to explore the co-localization of USP42 and SC35 in different cell lines.After transfection with GFP tagged constructs,the co-localization of exogenous USP42 and SC35 were observed by immunofluorescence.The interaction of USP42 and SC35 was detected by co-immunoprecipitation in 293 T which were transfected with different tagged USP42 and SC35.Results: All the constructs of USP42 have been confirmed by Western Blotting.And the results of immunofluorescence showed that only the constructs with C terminal had the same localization as wild type.Most importantly,the Lys rich domain and Arg rich domain could lead the special localization of Ubiquitin Specific Peptidase 42.The co-localization of endogenous USP42 and SC35 were observed in H1299 H1650,SKBR3,HCC827 cell lines,of which the co-localization in H1650 was remarkable,as well as the co-localization of exogenous USP42 and endogenous SC35,exogenous USP42 and exogenous SC35 in U2 OS.In addition,the colocalization of USP42 and SC35 was RNA independent.We found the domains that can influence the co-localization of Ubiquitin Specific Peptidase 42 and SC35 were Lys rich domain and Arg rich domain,and the enzyme active site of USP42 could influence the co-localization.The constructs of USP42 tagged with GFP were transfected in 293 T to determine the affection on SC35.The results of western blotting showed that the expression of these constructs could affect the expression of SC35.However,neither USP42 norSC35 could be co-immunoprecipitated in 293 T.Conclusions: 1.The most important domains of Ubiquitin Specific Peptidase 42 were Lys rich domain and Arg rich domain in C terminal that influenced its localization.2.USP42 co-localized with SC35,and the pattern of co-localization was RNA independent.3.It was Lys rich domain and Arg rich domain of USP42 that affected the colocalization with SC35.And the co-localization was enzyme active site dependent.4.The expression of USP42 could affect the expression of SC35,but the interaction between them was not direct.