Study On The Expression Of Prmt5 Related Protein And Apoptosis In Medaka

Prmt5(Protein arginine N-methyltransferases 5)is a type Ⅱ protein arginine methyltransferase that catalyzes the methylation of a variety of histones and non-histones and participates in various biological processes such as transcriptional regulation,proliferation,apoptosis,metabolism,cell integrity,genome stability and immune response.Medaka is a model organism and a good material for developmental and immunobiological studies.Our group previously screened proteins that may interact with medaka Prmt5 by yeast two-hybrid technology,including Mep50(Methylosome protein 50),Prdm1a(PR domain zinc finger protein 1a),Prdm1 b,Tim-3(T-cell immunoglobulin and mucin domain containing-3),which are all related to apoptosis.Mep50 is a protein containing multiple WD40 repeat motifs.It is involved in cell proliferation,differentiation and apoptosis.It can also bind to Prmt5 to form a complex and participate in m RNA splicing.Prdm1 is a member of the PRDM family,which is involved in primordial germ cell generation and specialization,embryonic development,immune response,tumorigenesis,apoptosis and other functions.Prdm1 has a pair of homologous genes Prdm1 a and Prdm1 b.Tim-3,Tim-1 and Tim-4 belong to the Tim family.They play an important role in immune regulation,tumorigenesis and apoptosis.The specific research results are as follows:1.Expression,purification and interaction of Prmt5 related proteins in medaka.(1)The expression and interaction of Prmt5,Mep50,Prdm1 a and Prdm1 b in Medaka.In the prokaryotic(E.coli)expression system,the full-length and truncated Prmt5,Mep50,Prdm1 a and Prdm1 b expression plasmids were successfully constructed using p ET28-MHL as a vector.The full-length and truncated histones of Prmt5 and Mep50 were mainly expressed in the form of inclusion bodies,and some were expressed in soluble form.The Prmt5(290-462 aa),Prmt5(461-631 aa)and Mep50(1-105 aa),Mep50(1-150 aa)recombinant proteins expressed in soluble form were purified,and the protein with high purity and good stability was successfully obtained.The full-length recombinant protein inclusion bodies of Prmt5 and Mep50 were also successfully obtained with high purity and good stability after renaturation and affinity chromatography.In the eukaryotic(insectbaculovirus)expression system,the full-length genes of Prmt5,Mep50,Prdm1 a and Prdm1 b were cloned into the p Fast Bac1 plasmid to construct the recombinant transfer vector,and the recombinant shuttle plasmids Bacmid-Prmt5,Bacmid-Mep50,Bacmid-Prdm1 a and Bacmid-Prdm1 b were obtained.The recombinant shuttle plasmid was transfected into Sf9 insect cells,and the recombinant proteins of Mep50,Prmt5,Prdm1 a and Prdm1 b were successfully expressed.In HEK-293 T cells,FLAG-tagged medaka Prmt5 and HA-tagged medaka Mep50 were overexpressed alone or together.Coimmunoprecipitation experiments showed that medaka Prmt5 interacted with medaka Mep50.The binding sites of Prmt5 and Mep50 were predicted by Pymol software,and the binding sites may be ARG45 of Prmt5 and ASP97 of Mep50.Our group previously found that Prmt5 interacts with Prdm1 a and Prdm1 b by yeast two-hybrid technology,and Mep50 also interacts with Prdm1 a and Prdm1 b.Therefore,we used the ZDOCK molecular docking tool for protein-protein docking,and obtained the protein docking complexes of medaka Prmt5:Prdm1a,Prmt5:Prdm1b,Mep50:Prdm1a,Mep50:Prdm1b.The binding site prediction found that multiple amino acid residues were involved in the interaction.The binding sites of Prmt5 and Prdm1 a may include THR319[O] of Prmt5 and TYR808[OH] of Prdm1 a.The binding sites of Prmt5 and Prdm1 b may include GLN450[NE2] of Prmt5 and SER466[O] of Prdm1 b.The binding sites of Mep50 and Prdm1 a may include HIS305[O] of Mep50 and TYR396[N]of Prdm1 a.The binding sites of Mep50 and Prdm1 b may include ASN257[O] of Mep50 and ILE281[N]of Prdm1 b.(2)Analysis of Tim-1 and Tim-4 expression in medaka.We also studied the expression characteristics of Tim-1 and Tim-4,which belong to the same family as Tim-3.Both Tim-1 and Tim-4 are members of the Tim family.The Tim-1 protein contains an Ig-like domain and is a transmembrane protein,while the Tim-4 protein contains two Ig-like domains and is a secretory protein.Tim-1 and Tim-4 were mainly ectopic expressed in the cytoplasm of HEK-293 T cells.We also observed that Tim-1 signal also appeared on the cell membrane,while Tim-4 signal also appeared on the surface of the cell membrane.Tim-1 and Tim-4 are widely expressed in various tissues of adult fish,and are highly expressed in liver,kidney,spleen and intestine.2.Studies on apoptosis of medaka Prmt5 and xylitol selenite.(1)Study on anti-apoptosis of Medaka Prmt5.The anti-apoptotic effect of Prmt5 was explored.It was found that the optimal concentration of cisplatin for Hela cells was 25 mg/L,and the optimal concentration of cisplatin for CO cells was 35mg/L;cisplatin could significantly induce the apoptosis of Hela cells,but not CO cells.We found that overexpression of medaka Prmt5 could not significantly inhibit cisplatin-induced apoptosis of Hela cells.The reason may be that medaka Prmt5 could not function in human cells such as Hela,or medaka Prmt5 could not play a role in cisplatin-induced apoptosis signaling pathway in Hela cells.(2)The mechanism of apoptosis induced by xylitol selenite in SMMC-7221 cells.The optimal concentration of xylitol selenite to induce apoptosis of SMMC-7221 cells was determined to be 0.5 mg/L by MTT assay.SMMC-7221 cells were treated with 0.5 and 1 mg/L xylitol selenite for 12,24,36 and 48 h.The expression of apoptosis-related genes Caspase-3,Caspase-8 and Caspase-9 was detected by q PCR.It was found that the expression of Caspase-3 gene was up-regulated in all periods,while the expression of Caspase-8 and Caspase-9 was up-regulated only at 24 h,and there was no significant change at other times.SMMC-7221 cells were treated with 0.5 mg/L xylitol selenite for 12,24,36 and 48 h.The expression of Caspase-3,Caspase-8 and Caspase-9 proteins was detected by Western blot.It was found that only Caspase-3 protein was activated,and Caspase-8 and Caspase-9 proteins were not activated.After SMMC-7221 cells were treated with 0.25,0.5,1,2 mg/L xylitol selenite for 24 h,Western blot results showed that Caspase-3 protein was activated.The results showed that the apoptosis of SMMC-7221 cells induced by xylitol selenite was accompanied by the activation of Caspase-3.In summary,we expressed and purified medaka Prmt5 and its interacting proteins Mep50,Prdm1 a,and Prdm1 b,and studied their binding sites.The expression characteristics of Tim-1 and Tim-4belonging to the same family as Tim-3 were studied.The study on the role of Prmt5 and xylitol selenite in apoptosis will help us to understand how Prmt5 and its related proteins participate in the regulation of apoptosis,which will help us to understand the function of protein methylation in fish development and provide a theoretical basis for promoting the development of aquaculture.In addition,the study of how apoptosis-related compounds play a role in apoptosis can also help to study the treatment of human diseases.