The Heterotetrameric Assembly And Catalytic Function Of 17β-hydroxysteroid Dehydrogenase Type 8 And Carbonyl Reductase Type 4

In eukaryotes cells, mitochondria play a very critical role, and provide energy for cells. Mitochondrial dysfunction can result mitochondrial diseases, so it is very important to research the proteins involved in mitochondrial metabolism. It has found that a new fatty acid synthesis pathway exists in mitochondria, the related enzymes has drawn the widespread attention. The 3-ketoacyl-acyl carrier protein(ACP) reductase(KAR) is one of the mitochondrial fatty acid synthases and composed by 17β-hydroxysteroid dehydrogenase 8(17β-HSD8) and carbonyl reductase 4(CBR4). KAR is the last enzyme in this pathway identified very recently, and its subunit assembly mechanism remains vague. In this thesis, we investigate the assembly mechanism of Hs17β-HSD8 and HsCBR4. The main contents are as follows.l) We express and purify the Hs17β-HSD8 and HsCBR4 using of Escherichia coli(E. coli) expression system. In recent studies, Hs17β-HSD8 and HsCBR4 are localized mainly in inclusion bodies when they are separately expressed in E. coli. In order to increase the content of the soluble proteins, we use two methods: reducing induction temperature and introducing chaperonins. We overexpress the HsCBR4 in different temperatures, and choose 18 degrees as the induction temperatures. On this basis, several chaperonins: dnaK, dnaJ, grpE, groEL, groES and trigger factor were co-expressed with the Hs CBR4. The soluble content analysis shows that groEL-groES are the best combination. We finally isolated the HsCBR4 from E. coli. By using the same condition, we also overexpressed and purified the Hs17β-HSD8.2) The self-assembly of Hs17β-HSD8 and HsCBR4 proteins was studied in both a physiological buffer solution and E. coli cell extract. We choose 4 degree as the assembly temperature of the Hs17β-HSD8 and HsCBR4 proteins, the assembly costs about 60 h with a slow rate. We then try to research the assembly of that two protein in the E. coli cell extract in order to study the factors which might effect the protein assembly. The results showed that Hs17β-HSD8 and HsCBR4 are almost completely assembled in a short time in the cell extract, hinting the existence unknown factors in the cells which can promote the assembly of these two proteins.3) The interaction of Hs17β-HSD8 and HsCBR4 are studied in E. coli by using bimolecular fluorescence complementation(BiFC). We construct the fluorescence complementation system and choose the red fluorescent protein mCherry which has the feature of long emission wavelength. First, the cDNA of mCherry protein is cut into two parts between amino acids 159/160, and then fused in frame with HsCBR4 and Hs17β-HSD8. The resulting constructs are transferred into E. coli. Finally, the fluorescent signals of the cells are characterized using fluorescence microscopy. The results confirmed that the two proteins interact with each other and exist as complex in E. coli cells.