Development Of A High-Throughput Screening Model For Identification For Expended CAG Repeats Aggregation Inhibitors

Irreversible protein aggregation has been recognized as a by-product of protein folding since Christian Anfinsen's ribonuclease refolding experiments, which proposed that the primary sequence of a protein determines its three-dimensional structure. Understanding this process is not only crucial to study the structural and functional genetics, but also to facilitate it on the design of therapeutics for the growing list of amyloid diseases (e.g. Huntingdon's disease, Alzheimer's disease).For this reason, we have developed a method by which soluble or insoluble protein can be easily distinguished in Escherichia coli. This method is based on constructing a "folding reporter" vector, in which a target protein is expressed as an N-terminal fusion with the reporter protein - hloramphenicol acetyltransferase(CAT), and we should observe that cells expressing fusions of an insoluble protein to CAT exhibit decreased resistance to chloramphenicol compared to fusions with soluble proteins.To test the ability of CAT reporting target protein's solubility, the genes encoded model proteins were cloned into the N terminus of CAT-fusion vector. The chloramphenicol resistance produced by the fusions correlates well with the solubility of these proteins expressed without CAT. In this system, the practical folding reporter gives a signal directly proportional to the amount of correctly folded protein, and requires no functional assay for the protein of interest or a prior knowledge of the tested protein's structure or biological function. So this system can definitely be widely used to the design of novel protein structures, theoretical and empirical studies of protein folding, screening large numbers of proteins and protein fragments for solubility, finding and modifying efficient folding partners and so on.The second part of our data is the application of report system to proteins involved in protein-folding disease-Huntingdon's disease(HD). HD is an inherited neurodegenerative disorder caused by polyglutamine(polyQ) expansions in the huntingtin protein(Ht).A central event in HD is the production of an N-terminal fragment of Ht that aggregates in affected neurons . Therefore, inhibiting polyglutamine protein aggregation or deposition would be an important target for HD therapy. So we construct the HD cell model choosing the different CAG lengths which will form soluble or insoluble proteins .We also make use of the well-known generation of functional P-galactosidase (p-gal) in the E. coli cytoplasm by complementation of two fragments that the plasmid we used carry one of them. The generation of functional p-gal is made dependent on the solubility of the fusion protein. Enzyme activity can be conveniently monitored by a color change. A correlation between protein solubility and a -complementation was also demonstrated. To verify weather the CAT resistance or a -gal activity correctly reports target protein solubility, we expressed all kinds of reconstructed plasmids and assayed their solubility by SDS-PAGE, at the same time we measured the P-gal activity of Qn-pCAR. The results reveal an unambiguous correlation between productive folding of the downstream a -fragment and consequent formation of p-gal activity and the solubility/folding of the upstream protein-Qn.Thus this HD model provides an efficient^ high-throughput and quick screening tool that inhibit aggregation in vitro,which make it practical to search for small molecule aggregation inhibitors and endogenous proteins that inhibit aggregation by screening cDNA expression libraryand to identify small peptides that interfere with pathogenic polyglutamine interactions(e.g. polyglutamine-binding peptide l,QBPl).Generally speaking, it is a good strategy to promote the folding or inhibit the aggregation of disease-related proteins, thereby shedding new light on the therapy of HD.