Self-Association and Conformational Stability of NAMPT Protein

Abstract

Nicotinamide phosphoribosyltransferase (NAMPT), also known as Visfatin and Pre B-cell colony enhancing factor (PBEF), is a rate-limiting enzyme in the salvage pathway required for nicotinamide adenine dinucleotide (NAD) biosynthesis. It is a highly conserved 52-kDa protein, found in living species from bacteria to humans [1]. The protein serves as a cytokine and is involved in cellular regulation influencing cancer, ischemia, obesity, and type-II diabetes [2, 3]. Despite being involved in many regulatory pathways, there is a paucity of information concerning the function of NAMPT, due to the limitations of in vivo assays, and lack of expression systems for the protein. Here, we successfully expressed the NAMPT protein using the pET-SUMO expression vector in E.coli strain SHuffle containing a hexa-His tag for protein purification. Activity assays demonstrated functionality of the protein. Moreover, initial biophysical characterization of the protein using circular dichroism revealed secondary structural elements consistent with crystallographic data. Dynamic light scattering showed the protein exists as large oligomeric units potentially involved in the NAMPT signal amplification pathway. Hydropathy analysis indicated possible hydrophobic patches on the protein surface that explains the native oligomeric state. Most striking, we discovered that NAMPT can be solubilized in n-dodecyl-β-D-maltopyranoside detergent in monomeric form. These findings open opportunities for further structural and functional investigations. Presently we are optimizing conditions for NMR experiments on NAMPT protein. These methods [4] are complementary to X-ray crystallography, and provide valuable information on the structure and dynamics, offering an important tool for understanding biological functioning.[1] T. Wang et al. (2006) Nat. Struct. Mol. Biol 13, 661.[2] S. M. Camp et al. (2015) Sci. Rep. 5, 13135.[3] A. Fukuhara et al. (2005) Science 307, 426.[4] T. R. Molugu et al. (2016) Chem. Rev. (in press).