Protein-protein interactions of the nicotinamide/nicotinate mononucleotide adenylyltransferase of Leishmania braziliensis

BACKGROUND Nicotinamide adenine dinucleotide (NAD) plays a central role in energy metabolism and integrates cellular metabolism with signalling and gene expression. NAD biosynthesis depends on the enzyme nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT; EC: 2.7.7.1/18), in which converge the de novo and salvage pathways. OBJECTIVE The purpose of this study was to analyse the protein-protein interactions (PPI) of NMNAT of Leishmania braziliensis (LbNMNAT) in promastigotes. METHODS Transgenic lines of L. braziliensis promastigotes were established by transfection with the pSP72αneoαLbNMNAT-GFP vector. Soluble protein extracts were prepared, co-immunoprecipitation assays were performed, and the co-immunoprecipitates were analysed by mass spectrometry. Furthermore, bioinformatics tools such as network analysis were applied to generate a PPI network. FINDINGS Proteins involved in protein folding, redox homeostasis, and translation were found to interact with the LbNMNAT protein. The PPI network indicated enzymes of the nicotinate and nicotinamide metabolic routes, as well as RNA-binding proteins, the latter being the point of convergence between our experimental and computational results. MAIN CONCLUSION We constructed a model of PPI of LbNMNAT and showed its association with proteins involved in various functions such as protein folding, redox homeostasis, translation, and NAD synthesis.

Aerobic Swim Training Restores Aortic Endothelial Function by Decreasing Superoxide Levels in Spontaneously Hypertensive Rats

OBJECTIVE: We aimed to determine whether aerobic training decreases superoxide levels, increases nitric oxide levels, and improves endothelium-dependent vasodilation in the aortas of spontaneously hypertensive rats. METHODS: Spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) were distributed into 2 groups: sedentary (SHRsd and WKYsd, n=10 each) and swimming-trained (SHRtr, n=10 and WKYtr, n=10, respectively). The trained group participated in training sessions 5 days/week for 1 h/day with an additional work load of 4% of the animal’s body weight. After a 10-week sedentary or aerobic training period, the rats were euthanized. The thoracic aortas were removed to evaluate the vasodilator response to acetylcholine (10-10 to 10-4 M) with or without preincubation with L-NG-nitro-L-arginine methyl ester hydrochloride (L-NAME; 10-4 M) in vitro. The aortic tissue was also used to assess the levels of the endothelial nitric oxide synthase and nicotinamide adenine dinucleotide oxidase subunit isoforms 1 and 4 proteins, as well as the superoxide and nitrite contents. Blood pressure was measured using a computerized tail-cuff system. RESULTS: Aerobic training significantly increased the acetylcholine-induced maximum vasodilation observed in the SHRtr group compared with the SHRsd group (85.9±4.3 vs. 71.6±5.2%). Additionally, in the SHRtr group, superoxide levels were significantly decreased, nitric oxide bioavailability was improved, and the levels of the nicotinamide adenine dinucleotide oxidase subunit isoform 4 protein were decreased compared to the SHRsd group. Moreover, after training, the blood pressure of the SHRtr group decreased compared to the SHRsd group. Exercise training had no effect on the blood pressure of the WKYtr group. CONCLUSIONS: In SHR, aerobic swim training decreased vascular superoxide generation by nicotinamide adenine dinucleotide oxidase subunit isoform 4 and increased nitric oxide bioavailability, thereby improving endothelial function.