The Protein Interaction Analysis And Preliminary Functional Identification Of Human Heart Development Candidate Gene TRIM45and BZW2

Heart development is an extremely complicated and precise procedure including a series of morphogenesis. The regulatory network is mainly composed by cardiac transcription and differentiation factors. Abnormal expression of these genes will lead to early embryo lethal or cardiac diseases. Cardiac hypertrophy is one prevalent cardiac disease. Even until now, the researches about the mechanism of cardiac hypertrophy are still very hot.TRIM family proteins are very important in cell proliferation, differentiation, development, growth and cell apoptosis. In previous work, our lab cloned the human TRIM45gene. The full length of TRIM45is3584bp. Northern blot result shows that TRIM45is expressed in muscle, brain, pancreatic and heart. The yeast two-hybride result implies TRIM45protein might be interacting with SLC25A3and DNAJB6. In this thesis, the interactions between TRIM45and DNAJB6or SLC25A3have been verified in vivo by using Co-immunoprecipitation assay. DNAJB6gene belongs to the heat shock protein family (Hsp family). Published data indicates that DNAJB6blocked calcineurin-induced cardiomyocyte hypertrophic growth by interacting with NFATc3, and DNAJB6can inhibit the transcriptional activity of NFAT-Luc. In this thesis, this inhibition was repeated. To further understand the relationship between TRIM45and cardiac hypertrophy, the luciferase reporter assays of cardiac marker genes were investigated. Those luciferase reporters include ANF-Luc、NFAT-Luc、 SRF-Luc and MEF2C-Luc. The results show that all of them can be strongly suppressed by TRIM45. Interestingly, SLC25A3also can repress the NFAT-Luc. Taken together, those genes, TRIM45、SLC25A3and DNAJB6, are repressors of cardiac hypertrophy. And they may realize their functions by forming compounds with each other. Human BZW2gene is a novel gene which is functional unknown. It contains bzip domain and eIF5C domain. The BZW2gene is conserved in human, mouse, rat, zebrafish, chimpanzee, dog and chicken. BZW2gene is located at human chromosome7p21.1. And in mouse, it is at12;12B20The human BZW2gene and mouse BZW2gene have high identity which is around99%. The full length of human BZW2gene is1883bp. It encodes419amino acids. In this thesis, the coding region of the human BZW2gene was amplified by PCR and then was cloned into pGEX-4T-1vector. After confirmed by sequencing, the recombinant expression plasmid was transformed into BL21cells and the expressions of fusion proteins were induced by IPTG. After purification, the protein was used to immune the New Zealand white rabbits to prepare antibodies. The titer of BZW2antibody is1:50. Western blot was investigated by using mouse tissues. The result shows that BZW2is highly expressed in mouse heart, muscle and brain. By bioinformatics analysis, BZW2protein can physically interact with BZW1and PSTPIP1. The luciferase reporter assay result shows that BZW2can suppress the transcriptional activity of NF-kB and NFAT. This implies BZW2may be a potential cardiac developmental candidate.