Characterization of C-Terminal Hydrophilic Tail of Human Zinc Transport Protein ZnT8, Proteases, and Metalloproteins of Achromobacter xylosoxidans

Abstract

This study comprises of characterization of metalloproteins; human zinc transport protein hZnT8, FecR and FecI involved in the iron metabolism in Achromobacter xylosoxidans and two viral proteases (TEV and HRV 3C from Tobacco Etch Virus and Human Rhinovirus, respectively). In humans, zinc transport is mediated by Cation Diffusion Facilitator (CDF) and Zinc-Iron Permease families. A CDF family member, hZnT8, is known to be involved in insulin processing and as an auto antigen in diabetes. Moreover, an R325W mutation in hZnT8 is also linked with pathogenesis of Type 1 and Type 2 diabetes. Therefore, several expression constructs were generated to study the effect of R325W mutation on the structure and function of hZnT8. Native hZnT8 and hZnT8 (R325W) C-terminal domains were produced by recombinant means and were found to be in tetrameric state and binding to Zn2+ ions with almost similar affinity. It was also revealed by the Circular dichroism and Fourier-transform infrared spectroscopy techniques that R325W mutation does not produce any significant change in the global structure of hZnT8 domain and this mutation may be tolerated in diabetic patients, therefore, the mutated hZnT8 could provide enough amount of zinc for insulin storage/maturation and release. During recombinant proteins production it was observed that the TEV protease cleavage is not 100% successful. However, like other proteases, activity of Human Rhinovirus 3C (HRV 3C) protease could be affected by buffer components and additives used for purification and stabilization of proteins. Hence, it necessitates the comparative study of activities of TEV and HRV 3C proteases. The activity of Tobacco Etch Virus (TEV) and HRV 3C proteases were assessed at 4 °C as well as 25 °C. Furthermore, the effect of elution buffers used for affinity based purification, salt ions, stability/solubility and reducing agents, and detergents were analyzed on the activity of the HRV 3C protease. The results showed that the HRV 3C protease performs better than the widely used TEV protease. For production of recombinant FecI and FecR proteins from A. xylosoxidans, HRV 3C protease site was used. A. xylosoxidans is an opportunistic hospital pathogen that causes serious infections which could be fatal. The pathway of iron metabolism in such bacteria can be targeted to hinder their xxi growth. In the iron metabolism pathway of A. xylosoxidans, the FecR, at its cytoplasmic N-terminal, signals the interacting extra-cytoplasmic function sigma factors such as FecI to recruit RNA polymerase for gene expression. The FecR-FecI interaction in A. xylosoxidans can, therefore, be exploited to develop new antimicrobial drugs. Thus, using the E. coli FecI and FecR sequences, their homologues were identified in A. xylosoxidans’ genome and cloned in E. coli expression vectors. Proteins were produced by recombinant means, HRV 3C protease was exploited instead of TEV protease for tag removal and interaction study of FecI and FecR was performed. However, the preliminary study did not show interaction of FecR with FecI. The overall conclusion of this study is that R325W mutation doesn’t affect the structure and hence the zinc sensing capability of hZnT8, however it could be further confirmed by x-ray diffraction analysis of hZnT8281-369 and hZnT8R325W crystals. Furthermore the HRV 3C could be the choice of protease for tag removal. Finally though our study regarding FecI and FecR does not report any mutual interaction, yet, further studies are needed under different conditions to find if there is any interaction between FecR and FecI.