The Screening Of Transcription Factors's Interaction And Prokaryotic Expression Of Restin

Restin is a novel gene of MAGE gene family which was cloned by our lab and got from all-trans retinoic acid-induced HL60 cells, the GenBank accession No. of this gene is NM₀14061. Restin, 1475bp full-length, locates on Xp11.22, has only one exon and encodes 219 amino acids. The protein molecular weight is 24.4 kD and the isoelectric point is 9.05. A untypical bipartite nuelear localization singal in the N-termina l8-25 aa and a MHD in the 88-176 aa of Restin. Bioinformatics analysis shows that the relationship between Restin and MAGE familyПtype proteins such as MAGED and Necdin is highly homologous.The tissue expression of Restin showed that it highly expressed in testis and final terminal differentiation cells, also highly expressed in brain, nerve and prostate tissue rather than in tumors. Analysis of the promoter region of Restin showed that it has a binding site of p53. p53 was able to upregulate Restin indirectly. Our evidence supported that STAT-1a plays an important role in the atRA-induced transcriptional up-regulation of restin.Previous study show that p75NTR,CDK5RAP2,KPNA2,MOAP1,PIAS4,TADA3L,NAP1L5 interacted with Restin. Our previous study identified that the transcription factor ATF3 interaction with Restin too. TeherPakov study identified that both Restin and Necdin interacted with intracellular domain of p75NTR directly including involving in p75 NTR pathway and accelerating nerve cell differentiation. Salehi found that MAGE D1, another homologous protein of Restin, also interacted with the p75 NTR and induced the apoptosis of nerve growth factor. The p75NTR signaling pathway regulated cell survival and differentiation, apoptosis, axonal growth and morphogenesis in different physiological state. Our previous work found that Restin over-expression in MCF-7 cells would lead to G1 arrest, which maybe relate to p75NTR signaling pathway, but the mechanism was limited.Restin is a nuclear protein, and transcription factor which is final effector molecule of transduction pathway regulated gene transcription in cell nucleus. Therefore, it is important to screen the transcription factors which interacte with Restin. Two-hybrid technology commonly uses for detecting the interaction between proteins and has the merits of low false positive rate and a high sensitivity. Our team constructed signal transducer library of eucaryotic cell by mammalian two-hybrid system. In this research, we identified 55 transcription factors interacted with Restin from the library, which included cell cycle regulation, stress reaction and protein modification and so on by using mammalian cell two-hybrid system.Experimental results showed that Restin could interact with ZNF238, ZNF239 and ZNF486 obviously in cold stimulation condition. They were zinc finger proteins whose family had extensive functions such as embryonic development, sex definition, hormone secretion, cell differentiation, senescence and apoptosis, and related to pathological processes of many diseases. Previous study demonstrated that ZNF238 played a crucial role in neuronal proliferation, migration, and differentiation in the developing cerebral cortex. ZNF239 interacted with lamin A/C and the nuclear matrix in transcription inhibition. ZNF486 biological function was unclear.The results in protein-protein interaction database showed that proteins CDK5RAP2,KPNA2,MOAP1,PIAS4,TADA3L,NAP1L5 which interacted with Restin were detected by yeast two-hybrid array. We cloned and constructed pACT-TADA3L and pACT-KPNA2 to further verification the interaction by mammalian two-hybrid system. Results showed that Restin interaction with TADA3L and KPNA2.Obtain active protein was crucial for studying the function of unknown gene. In order to obtain active protein, we have developed prokaryotic expression system for Restin protein. We constructed expression vectors of pET41a(+)-Restin and pET44a(+)-Restin, and transformed them into Rosetta-gamiTM2(DE3). And the recombinated vectors successfully expressed soluble fusion protein.In summary, we detected three transcription factors ZNF238, ZNF239 and ZNF486 which interacted with Restin and further confirmed the interaction between TADA3L, KPNA2 and Restin. We achieved soluble fusion protein expression of Restin by optimizing plasmid vector and host strain of prokaryotic expression system, which laid a solid foundation for further study about biological functions of Restin.