STRUCTURAL STUDIES OF HUMAN PROTEINS OF MEDICAL INTEREST

The aim of this thesis work was to determine the three-dimensional structure of three human proteins, Heat shock protein 60 (HSP60), Activation-induced cytidine deaminase (AICDA) and Cholesterol 7-alpha-monooxygenase (CYP7A1), by X-ray diffraction of single crystals. The human chaperonine HSP60 is a mitochondrial protein, expressed in a constitutive manner. The protein was expressed in E. coli and purified by immobilized metal ion affinity chromatography, using a histidine tag fused to the N-terminus of the protein, and by size exclusion chromatography. The crystallization trials do not give positive results, probably due to protein heterogeneity problems. Human AICDA is a cytidine deaminase, which is selectively expressed in B lymphocytes and plays a crucial role in antibody somatic hypermutation and class switch recombination. Protein expression was attempted in E. coli with different vectors and was successfully achieved with the pGEX-4T-1 plasmid, that allows to express GST at the N-terminus of the target protein; unfortunately, the purification protocol was not effective. CYP7A1 is a microsomal enzyme that catalyzes the conversion of cholesterol to 7α-hydroxycholesterol, the first reaction of bile acid synthesis and the rate-limiting step of the metabolic pathway; the protein is expressed only in the liver. The expression of human CYP7A1 in E. coli was troublesome and, although different vectors were tested, a good level of protein expression was not obtained. In order to overcome these problems, zebrafish (Danio rerio) CYP7A1 was expressed in E. coli, but without any substantial improvement. Another part of this thesis work concerned the determination of the three-dimensional structure of the human Methionine aminopeptidase 1 with two different inhibitors. The protein catalyzes the removal of the N-terminal methionine from nascent proteins. Inhibitors against this protein are of great medical interest because of their potential employment as anticancer drugs. This part of the thesis was performed at Dr. L. Mario Amzel's laboratory under the direct supervision of Dr. Sandra B. Gabelli - Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, School of Medicine (Baltimore, USA).