The Mechanism Of SIRT7 Protein Stability Regulated By TRIM25 And Its Functional Study

Human sirtuins are a class of histone deacetylase and require NAD+ for their catalytic activity,which are initially discovered in yeast Sir2 protein.Sirtuins all contain a conserved core catalytic domain:deacetylation domain.Seven sirtuins(Sirt1-7)have been identified in human genome,playing diverse roles in cellular activities such as calorie restriction,aging,metabolism,chromosome structure stability and transcription silence.So far,researches about Sirt7 gene are still insufficient and are not well characterized,the expression levers of Sirt7 mRNA were found to be increased in kinds of cancers,indicating that it may involves in the development and progression of cancer.Human Sirt7 gene locates in chromosome 17(17q25.3),Sirt7 mRNA is 1203 bp long,which encodes 10 exons and is composed of 400 amino acids,encoding a 44.9KDa protein.Sirt7 exists in nuclear,just like other sirtuins protein,the substracts of SIRT7 include histone proteins and non-histone proteins,but its deacetylase activity is not stronger than others.SIRT7 deacetylates PAF53,a subunit of RNA polymerase I complex,which is important for its interaction with rDNA promoter and promotes cell proliferation or inhibit cell apoptosis.A recent report has revealed that SIRT7 binds to prometors of target gene,and deacetylates H3K18 Ac and promotes transcriptional repression,which is required for its maintenance of oncogenic transformation.The ubiquitin-proteasome pathway is a significant mechanism in vivo and responsible for regulating protein level and its function.In this process,ubiquitin ligase E3 s play a vital role,which determine selective recognition and specificity degradation.TRIM(tripartite motif)family proteins contain a RING domain,which suggest potential ubiquitin E3 ligase activity.Here,we carry out a series of experiments around SIRT7 and its specific E3,we also establish Sirt7 knockout cell line in MCF-7 cell through CRISPR/Cas9 in order to study further in breast cancer.The main research contents and results show as follows:(1)Sirt7 gene knockout influences the p53 mRNA and protein expression levles in MCF-7 cells:Sirt7 gene was knockout through CRISPR/Cas9 in MCF-7 cell,however,it didn't affect the colony formation,but affect cell cycle which maybe through upregulating the mRNA and protein expression levles in MCF-7 cells.(2)TRIM25 protein specifically interacts with SIRT7 protein in 293 FT cells:According to the proteome data,we screened out proteins interacting with SIRT7 among of which,TRIM25 is our especially interest.To exame the idea further,we use co-immunoprecipitation and western blotting technology,and the results suggest that TRIM25 specifically interacts with SIRT7.(3)TRIM25 reduces SIRT7 protein stability and promotes its degradation:MCF7 cells or 293 FT cells were transfected with TRIM25 plasmids or empty vectors in the absence or presence of Sirt7 plasmids followed by treatment with CHX for different times.We examed the endogenous or exogeous SIRT7 protein expression levels respectly,and the WB results suggest that SIRT7 protein expression levels drecresed in the presence of TRIM25.Further more,SIRT7 ubiquitination was analyzed in the ub assay and the results indicating that TRIM25 leads SIRT7 to be polyubiquitinated.