| Background and aims:Ubiquitin-Proteasome System(UPS)is mainly involved in more than half of protein degradation in eukaryotic cells.E3 ubiquitin ligases are considered to be the most important components in determining the specificity of the ubiquitin proteasome pathway.They can be classified into three subfamilies based on their working mechanisms and structures:HECT(Homologous to E6-AP Carboxyl Terminus)domain E3 ubiquitin ligases,RING(Really Interesting New Gene)domain E3 ubiquitin ligases,RBR(RING-Between-RING)domain E3 ubiquitin ligases.Among them,RING domain ubiquitin ligases are the most diverse E3 ubiquitin ligase family.TRIM 17(The tripartite motif 17)belongs to the TRIM(The tripartite motif)protein family,which is one of the largest RING domain E3 ubiquitin ligases.Currently known biological functions of TRIM 17 include regulation of gene transcription,protein homeostasis,autophagy,apoptosis,maintenance of innate immune function,and modification of tumor progression.Through systematic analysis of comparative genomics,our laboratory found that TRIM 17 was lost in long-lived underground species naked mole rat(Heterocephalus glaber,NMRs)and blind mole rat(Spalax galili,BMRs).TRIM 17 has also been reported to be lost in whales and pangolins.All of these species have long life-time.Therefore,we hypothesize that TRIM 17 may have biological functions that affect longevity.The stability of telomeres is the key factor determining cell lifespan.Telomere is a special cap-like structure at the end of chromosome.Whenever a cell divides,50 to 100 bases of the telomere repeat sequence are lost,and the telomere gradually shortens.When telomeres become too short to protect the ends of chromosomes,causing cells to stop dividing,leading to cell aging and death,resulting in a shortened cell lifespan.This thesis mainly explores whether TRIM 17 regulates animal longevity by identifying its interacting proteins and its associated functions.Methods:First,the TRIM 17 plasmid containing a FLAG tag at the N-terminus was constructed by standard molecular cloning techniques and transfected into HEK293T cells along with an empty vector for comparison.Then,cells were harvested to obtain cell lysates.FLAG-TRIM 17 and its interacting proteins were immunoprecipitated with FLAG M2 affinity gel.The purified samples were separated by SDS-PAGE and visualized by silver staining.The gels were excised and proteins were digested with trypsin to obtain peptides.Then the samples were desalted and analyzed by mass spectrometry.Through database search,quantitative analysis and bioinformatics analysis,the interacting proteins of TRIM 17 and their main functions were obtained and some of the interacting proteins of TRIM 17 were verified by immunoprecipitation and Western blotting experiments.We used proteasome inhibition and ubiquitination assay to verify the effect of TRIM 17 on the stability and ubiquitination of a key interacting protein.In addition,we used Q-FISH(Quantitative Fluorescent In Situ Hybridization)technique and TRAP(Telomeric Repeat Amplification Protocol)telomerase activity detection kit to measure telomeres and telomerase stabilityResults:Immunoprecipitation and label-free quantitative proteomics were used to successfully identify TRIM 17-interacting proteins in HEK293T cells.Three biological replicates resulted in the identification of 30 TRIM17-interacting proteins.Bioinformatics analysis showed that the TRIM17-interacting proteins were mainly involved in the regulation of telomere stability,telomere localization,protein folding,localization,transportation and other biological processes.Biochemical experiments demonstrated that TRIM 17 could reduce the CCT1 protein level.Proteasome inhibitor treatment and ubiquitination experiments revealed that TRIM 17 overexpression decreased the stability of CCT1 and enhanced the level of ubiquitin proteasome degradation of CCT1.Q-FISH real-time and telomerase activity analyses disclosed that TRIM17 could affect the stability of telomeres and telomerase.Conclusion:Through the combination of immunoprecipitation and quantitative proteomics,we identified 30 TRIM17-interacting proteins,and further found that TRIM 17 downregulated the level of its interacting protein CCT1 stability and upregulated the level of ubiquitin proteasome degradation of CCT1.TRIM17 also affects the stability of telomeres and telomerase.These results not only enrich our understanding of the biological functions of TRIM 17,but also provide an experimental foundation underlying for the subsequent exploration of the potential biological functions and mechanisms of TRIM17. |
