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.

Structural insights into Plasmodium falciparum nicotinamide mononucleotide adenylyltransferase: oligomeric assembly

The biochemical pathways involved in nicotinamide adenine dinucleotide (NAD) biosynthesis converge at the enzymatic step catalysed by nicotinamide mononucleotide adenylyltransferase (NMNAT, EC: 2.7.7.1). The majority of NMNATs are assembled into homo-oligomeric states that comprise 2-6 subunits. Recently, the NMNAT of Plasmodium falciparum (PfNMNAT) has been identified as a pharmacological target. The enzymatic characterisation, cellular location, and tertiary structure of the PfNMNAT protein have been reported. Nonetheless, its quaternary structure remains to be explored. The present study describes the oligomeric assembly of the 6 x His-PfNMNAT recombinant protein using immobilised metal affinity chromatography coupled with size exclusion chromatography (SEC) and native protein electrophoresis combined with Ferguson plot graphing. These chromatographic approaches resulted in the elution of an active monomer from the SEC column, whereas the Ferguson plot indicated a dimeric assembly of the 6 x His-PfNMNAT protein.

Nicotinamide mononucleotide adenylyltransferase of Trypanosoma cruzi (TcNMNAT): a cytosol protein target for serine kinases

Nicotinamide/nicotinate adenine dinucleotide (NAD+/NaAD) performs essential functions in cell metabolism and energy production due to its redox properties. The nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1/18) enzyme catalyses the key step in the biosynthesis of NAD+. Previously, the enzyme NMNAT was identified in Trypanosoma cruzi (TcNMNAT), a pathogenic agent with epidemiological importance in Latin America. To continue with the functional characterisation of this enzyme, its subcellular location and its possible post-translational modifications were examined in this study. For this, polyclonal antibodies were generated in mice, with soluble and denatured recombinant protein being used to detect the parasite’s NMNAT. Immunodetection assays were performed on whole extracts of T. cruzi, and an approximation of its intracellular location was determined using confocal microscopy on wild and transgenic parasites, which revealed the cytosol distribution patterns. This localisation occurs according to the needs of the dinucleotides that exist in this compartment. Additionally, a bioinformatics study was performed as a first approach to establish the post-translational modifications of the enzyme. Possible phosphorylation events were experimentally analysed by western blot, highlighting TcNMNAT as a potential target for serine kinases.

Identification of the nicotinamide mononucleotide adenylyltransferase of Trypanosoma cruzi

The intracellular parasite Trypanosomacruzi is the aetiological agent of Chagas disease, a public health concern with an increasing incidence rate. This increase is due, among other reasons, to the parasite’s drug resistance mechanisms, which require nicotinamide adenine dinucleotide (NAD+). Furthermore, this molecule is involved in metabolic and intracellular signalling processes necessary for the survival of T. cruzithroughout its life cycle. NAD+biosynthesis is performed by de novo and salvage pathways, which converge on the step that is catalysed by the enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) (enzyme commission number: 2.7.7.1). The identification of the NMNAT of T. cruziis important for the development of future therapeutic strategies to treat Chagas disease. In this study, a hypothetical open reading frame (ORF) for NMNAT was identified in the genome of T. cruzi.The corresponding putative protein was analysed by simulating structural models. The ORF was amplified from genomic DNA by polymerase chain reaction and was further used for the construction of a corresponding recombinant expression vector. The expressed recombinant protein was partially purified and its activity was evaluated using enzymatic assays. These results comprise the first identification of an NMNAT in T. cruziusing bioinformatics and experimental tools and hence represent the first step to understanding NAD+ metabolism in these parasites.

Effect of overexpression of nicotinic acid mononucleotide adenylyltransferase on succinic acid production in Escherichia coli NZN111 / 生物工程学报

Escherichia coli NZN111 is a promising strain with ldhA and pflB genes inactivated for the production of succinic acid. However, with these mutations, NAD+ could not be regenerated from NADH, and an unbalanced NADH/NAD+ ratio eliminated cell growth and glucose utilization under anaerobic conditions. Nicotinic acid mononucleotide adenylyltransferase (NAMNAT), encoded by the nadD gene, catalyzes the reaction from nicotinic acid mononucleotide (NaMN) to nicotinic acid adenine dinucleotide (NaAD) during the synthetic pathway of NAD(H). Overexpression of the nadD gene could enhance the concentration of NAD(H) and maintain a suitable NADH/NAD+ ratio. In this study, we constructed a recombinant strain E. coli NZN111/pTrc99a-nadD, and overexpressed NAMNAT with 1.0 mmol/L of IPTG under anaerobic conditions in sealed bottles. Compared to E. coli NZN111, the concentrations of NAD+ and NADH in the recombinant strain increased by 3.21-fold and 1.67-fold, respectively. The total concentration of NAD(H) was increased by 2.63-fold, and the ratio of NADH/NAD+ decreased from 0.64 to 0.42. The recombinant strain restored the cell growth and glucose utilization under anaerobic conditions. After 72 h, the recombinant strain could consume 14.0 g/L of glucose to produce 6.23 g/L of succinic acid, and the concentration of succinic acid was 19-fold higher than in E. coli NZN111.

Nicotinamide mononucleotide adenylyltransferase 1 gene NMNAT1 regulates neuronal dendrite and axon morphogenesis in vitro / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Wallerian degeneration is a self-destructive process of axonal degeneration that occurs after an axonal injury or during neurodegenerative disorders such as Parkinson's or Alzheimer's disease. Recent studies have found that the activity of the nicotinamide adenine dinucleotide (NAD) synthase enzyme, nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) can affect the rate of Wallerian degeneration in mice and drosophila. NMNAT1 protects neurons and axons from degeneration. However, the role of NMNAT1 in neurons of central nervous system is still not well understood.</p><p><b>METHODS</b>We set up the culture of primary mouse neurons in vitro and manipulated the expression level of NMNAT1 by RNA interference and gene overexpression methods. Using electroporation transfection we can up-regulate or down-regulate NMNAT1 in cultured mouse dendrites and axons and study the neuronal morphogenesis by immunocytochemistry. In all functional assays, FK-866 (CAS 658084-64-1), a highly specific non-competitive inhibitor of nicotinamide phosphoribosyltransferase was used as a pharmacological and positive control.</p><p><b>RESULTS</b>Our results showed that knocking down NMNAT1 by RNA interference led to a marked decrease in dendrite outgrowth and branching and a significant decrease in axon growth and branching in developing cortical neurons in vitro.</p><p><b>CONCLUSIONS</b>These findings reveal a novel role for NMNAT1 in the morphogenesis of developing cortical neurons, which indicate that the loss of function of NMNAT1 may contribute to different neurodegenerative disorders in central nervous system.</p>

Construction of recombinant lentiviral vectors for mouse NMNAT1 gene expression and its interference RNA / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct two recombinant lentiviral vectors carrying mouse NMNAT1 gene and RNAi targeting NMNAT1.</p><p><b>METHODS</b>According to GenBank, the full-length cDNA sequence of mouse NMNAT1, an interfering sequence targeting NMNAT1 and a negative sequence were designed, synthesized and inserted into plasmid pLenti6 lentiviral vector. The viral stock was prepared by cotransfection of plasmids and the packaging plasmid mix to 293T cells. The virus titer was tested by qPCR methods. After infection of Hela cells with these lentiviruses, the expression of NMNAT1 was detected by qPCR and Western blot.</p><p><b>RESULTS</b>All the recombinant plasmids were confirmed by sequencing. The titer of virus was over 2 X10(8) TU/mL. Hela cells infected with lentiviral vector carrying full length NMNAT1 gene successfully expressed high-level NMNAT1. The expression of NMNAT1 reduced to less than 30% after delivery of lentiviral vector carrying RNAi sequence.</p><p><b>CONCLUSION</b>The lentiviral vectors carrying full length NMNAT1 gene and RNAi sequence targeting NMNAT1 have been successfully constructed.</p>

Wld(S), Nmnats and axon degeneration--progress in the past two decades

A chimeric protein called Wallerian degeneration slow (Wld(S)) was first discovered in a spontaneous mutant strain of mice that exhibited delayed Wallerian degeneration. This provides a useful tool in elucidating the mechanisms of axon degeneration. Over-expression of Wld(S) attenuates the axon degeneration that is associated with several neurodegenerative disease models, suggesting a new logic for developing a potential protective strategy. At molecular level, although Wld(S) is a fusion protein, the nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1) is required and sufficient for the protective effects of Wld(S), indicating a critical role of NAD biosynthesis and perhaps energy metabolism in axon degeneration. These findings challenge the proposed model in which axon degeneration is operated by an active programmed process and thus may have important implication in understanding the mechanisms of neurodegeneration. In this review, we will summarize these recent findings and discuss their relevance to the mechanisms of axon degeneration.