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

Abstract

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.

Fig. 1:. expression of the LbNMNAT-green fluorescent protein (GFP) in Leishmania braziliensis promastigotes. (A) Results from 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and protein visualisation using Coomassie R-250. M, molecular weight marker (kDa). (B) The samples were transferred onto polyvinylidene difluoride (PVDF) membranes and immunodetected using the anti-GFP (1:1000) antibody. Lanes 1-3: soluble protein fraction of non-transfected, empty vector-transfected, and recombinant vector-transfected (pSP72RαneoαLbNMNAT-GFP) promastigotes, respectively. (C) Direct fluorescence was observed in the different cellular lines.

Fig. 2:. Co-immunoprecipitation of proteins associated with the LbNMNAT protein. (A and B) Co-immunoprecipitates from promastigotes expressing only green fluorescent protein (GFP). (C and D) Co-immunoprecipitates from promastigotes expressing recombinant LbNMNAT-GFP. (A and C) Results from 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and proteins visualisation with colloidal Coomassie (G-250). The box samples, corresponding to the immunoprecipitates, were cut and analysed by Nano-LC-MS/MS. (B and D) Western blots obtained after using anti-GFP primary antibody (1:500) and anti-rabbit biotin secondary antibody (1:5000). 1. Clarified extract. 2. Post-immunoprecipitation soluble proteins. 3. Immunoprecipitates. The asterisks indicate possible products of processing or degradation of recombinant LbNMNAT-GFP.

Fig. 3:. the proteins identified by co-immunoprecipitation and mass spectrometry (Co-IP-MS/MS) in the biological replicates 1 and 2 belong to diverse functional categories.

Fig. 4:. protein-protein interaction network of the LbNMNAT protein (STRING V.10.5). Reliable type view. The strongest associations are shown with thicker lines. Parameters: Score (0.4), no additional nodes; sources of interaction used: experimental, databases, co-expression, co-occurrence, gene fusion, and neighbourhood. LbNMNAT protein: node XP_001563913.1 (A4H990). Red: nodes associated with the metabolism of nicotinamide and nicotinate. Blue: nodes with RNA-binding motif. Grey: proteins not associated with cellular routes.