Nicotinamide phosphoribosyltransferase interaction with polyphenolic modulator in the presence of ATP
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Nicotinamide adenine dinucleotide (NAD) is critical in human physiology, the molecule is part of redox reactions vital to metabolism and it is a substrate for multiple signaling proteins in the body. During times of stress in the body, which if chronic can lead to lifelong illness, NAD consumption by signaling proteins is increased and NAD concentration can be depleted. Age related illnesses are often the result of compounding stresses placed on the body over time. A novel way to approach understanding age-related illness, such as diabetes type II and neurodegenerative diseases, is through investigation of NAD modulation. The importance of NAD is exemplified by the redundancy of routes in which NAD is produced in vivo. The predominant pathway in humans is the salvage pathway. The rate-limiting enzyme of the salvage pathway and key component of this research is Nicotinamide phosphoribosyltransferase (NAMPT). NAMPT is a 55 kDa homodimer. It contains autophosphorylation and transferase activity which leads to the recycling of NAD in the body. The enzyme’s reaction happens sequentially with autophosphorylation increasing the enzyme’s affinity for the next reactant, phosphoribosyl pyrophosphate (PRPP). When PRPP binds the phosphorylated enzyme the affinity for NAM increases and promotes the conversion of NAM to NNM. Due to the physiological importance of NAD, NAMPT has become an enzyme of interest in multiple fields including metabolism, oncology, and neurology. Identification of NAMPT modulators would aid the understanding of NAMPT’s physiological role as well as NAD modulation in disease. Through years of research, the Almeida Lab at Rhode Island College has identified a small polyphenolic compound, Compound 20, which consistently enhances NAMPT’s activity. Compound 20 is part of the anthocyanin family. Its modulation of NAMPT activity has been studied through enzyme activity coupled fluorescence assays. To move forward with investigations of Compound 20 as an allosteric enhancer of NAMPT, the physical interaction between the enzyme and compound needed to be confirmed. This research aimed to confirm binding between Compound 20 and NAMPT via fluorescence polarization assays. Further, it examined the hypothesis that the autophosphorylation of NAMPT enhanced Compound 20’s binding with the enzyme. Analysis of fluorescence polarization values revealed Compound 20 and NAMPT bind with a high affinity, while there is a low binding affinity between phosphorylated enzyme, NAMPTPO4, and Compound 20. In conclusion, Compound 20’s binding to NAMPT was confirmed. Further the hypothesis that the phosphorylation of NAMPT increased Compound 20’s binding affinity for the enzyme was disproved. Alternative hypotheses were investigated, and it is more likely Compound 20 binds prior to the enzyme’s phosphorylation and reaction onset. Overall this research corroborates Compound 20 as an allosteric modulator of NAMPT. The identification of this enhancer may lead to novel insights on NAMPT’s physiological role, as well insights on the effects of enhanced NAD concentration in human diseases.