ABSTRACT
The human zinc transporter 8 (hZnT8) plays important roles in the storage of insulin in the secretory vesicles of pancreatic ß cells. hZnT8 consists of a transmembrane domain, with its N- and C-termini protruding into the cytoplasm. Interestingly, the exchange of arginine to tryptophan at position 325 in the C-terminal domain (CTD) increases the risk of developing type 2 diabetes mellitus (T2D). In the present study, the CTDs of hZnT8 (the wild-type (WT) and its disease risk variant (R325W)) were expressed, purified, and characterized in their native forms by biophysical techniques. The data reveal that the CTDs form tetramers which are stabilized by zinc binding, and exhibit negligible differences in their secondary structure content and zinc-binding affinities in solution. These findings provide the basis for conducting further structural studies aimed at unravelling the molecular mechanism underlying the increased susceptibility to develop T2D, which is modulated by the disease risk variant.
Subject(s)
Amino Acid Substitution , Diabetes Mellitus, Type 2/genetics , Zinc Transporter 8/chemistry , Zinc Transporter 8/metabolism , Zinc/metabolism , Arginine/metabolism , Crystallography, X-Ray , Humans , Models, Molecular , Protein Domains , Protein Multimerization , Protein Structure, Secondary , Scattering, Small Angle , Tryptophan/metabolism , X-Ray Diffraction , Zinc Transporter 8/geneticsABSTRACT
100K is an important scaffolding protein of adenoviruses including fowl adenovirus serotype 4 (FAdV-4) that causes inclusion body hepatitis-hydropericardium syndrome (IBH-HPS) in poultry. 100K carries out the trimerization of the major capsid hexon protein of the virus for the generation of new virions inside the target host cells. Despite its critical role for FAdV-4, no structural study, in particular, has been conducted so far. Here, the overexpression of soluble 100K protein was successfully carried out in E. coli using various expression constructs and purification yield of 3mg per litre culture volume was obtained. Gel filtration chromatography suggested that 100K protein exists in trimeric form. Circular dichroism and Fourier transform infrared spectroscopy clearly reveal that 100K protein folds with a high content of α-helices. The 3-dimentional homology model of the 100K protein, refined with molecular dynamics tools also depicts higher α-helical content within the protein model. Moreover, overexpressed recombinant 100K protein could be used to differentiate vaccinated and FAdV-4 infected chickens on the basis of higher serum anti 100K antibody titres. Our work provides preliminary structural and functional results to study biological role of the 100K protein and for further investigations to develop 100K inhibitors to control IBH-HPS in poultry.
