Structural And Functional Study Of Mutant Proteins Of Human Phosphoribosylpyrophosphate Synthetase (hPRS) And Of Melanoma Differentiation-associated Antigen 5(MDA5)

1. Structural and Functional Study of Mutant Proteins of Human Phosphoribosylpyrophosphate Synthetase (hPRS1) and hPRS2Human phosphoribosylpyrophosphate synthetase (PRS, E.C. 2.7.6.1) is an important enzyme catalyzing the synthesis of PRPP from ATP and ribose 5-phosphate (R5P), which regulates the biosynthesis of purine nucleotides and pyrimidine nucleotides. Three highly homologous PRS isoforms (PRS1, PRS2 and PRS3) are encoded by the prs genes. To date, seven point-mutant PRS1s have been identified from primary gout patients. The kinetic mechanisms and structure of PRS1 have been reported; nevertheless, how these point mutations lead to excessive enzyme activity as well as alter the structure of PRS1 is still uncertain. What role does another isoform PRS2 play in causing primary gout is not certain. In this research, the recombinant wild-type PRS1, the point-mutant PRS1 and PRS2 have been respectively expressed in E. coli BL21 (DE3) and Rosetta cells, and purified by Ni–Affinity chromatography and gel filtration, and studied by spectroscopy methods such as synchrotron radiation circular dichroism spectroscopy, dynamic light scattering and sedimentation velocity experiments. The results were discussed based on the crystal structure analysis of wt-PRS1. Dynamic light scattering and sedimentation velocity experiments indicate that after adding the substrate ATP, the monomeric wt-PRS1 in solution assembles into hexamer. This indicates that the substrate ATP could induce monomeric wild-type PRS1 to form hexamer, while no such aggregation effect was observed on some mutant PRS1. I also show that the enzymatic activity of mutant PRS1 is higher than wild-type PRS1, while the mixing of PRS1 and PRS2 has lower activity than either. Further, based on the structure of hPRS1, I assumed two structural alteration could be the reason of superactivity of one mutant PRS1. The efforts to get the conditions of crystallization of mutant PRS1 and PRS2 are under way.2. Cloning, Expression, Purification and the structural research of several domains of human Melanoma Differentiation-associated Antigen 5(MDA5)Melanoma differentiation associated antigen 5(MDA5), an important pattern recognition receptor in innate immune system, senses some particular infectious virus dsRNA and triggers dangerous single. It can induce cells to produce type I interferon not relying on Toll like receptor 3. MDA5 is consisted of two repeats of the caspase recruitment domain (CARD) at its N-terminus, DExD/H box helicases domain and Repression domain at its C-terminus. The helicase domain could bind viruses'RNA, and this would change RIG-I conformation to release CARD, which is masked when the cell is uninfected. The CARD domain acts as a signaling domain, which interacts with a downstream molecule Virus-induced signaling adaptor (VISA). Ten selected sections of MDA5 protein were cloned, and the recombinant plasmid was constructed and transformed into E. coli strain BL21(DE3) or ROSETTA(DE3). Only two of them were expressed in the form of inclusion body. Refolding of them produced proteins with poor stability. One of the section ( Helicase ATP Binding Domain) was reprogrammed and reconstructed into plasmid. It was then expressed as soluble protein. Purification and crystallization of it have been performed. The efforts to get the conditions of crystallization are under way.