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Identification of the nicotinamide mononucleotide adenylyltransferase of Trypanosoma cruzi.

Niño CH, Forero-Baena N, Contreras LE, Sánchez-Lancheros D, Figarella K, Ramírez MH - Mem. Inst. Oswaldo Cruz (2015)

Bottom Line: 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.These results comprise the first identification of an NMNAT in T. cruzi using bioinformatics and experimental tools and hence represent the first step to understanding NAD+ metabolism in these parasites.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Investigaciones Básicas en Bioquímica, Facultad de Ciencias, Universidad Nacional de Colombia, Bogotá, Colombia.

ABSTRACT
The intracellular parasite Trypanosoma cruzi 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. cruzi throughout 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. cruzi is 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. cruzi using bioinformatics and experimental tools and hence represent the first step to understanding NAD+ metabolism in these parasites.

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Related in: MedlinePlus

induction of the 6xHis-nicotinamide mononucleotide adenylyltransferase ofTrypanosoma cruzi (TcNMNAT) in total cellular extracts fromtransformed BL21 DE3 cells. A: sodium dodecyl sulfate polyacrylamide gelelectrophoresis on a 10% gel stained with Coomassie dye; B: western blot withalkaline phosphatase. The lanes include the molecular weight marker (kDa),induced negative control with a nontransformed strain (-) and differentinductions of transformed cells with the expression vector pET100D-TOPO-6xHis-TcNMNAT (BL21 clones I, II, III and IV).
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f04: induction of the 6xHis-nicotinamide mononucleotide adenylyltransferase ofTrypanosoma cruzi (TcNMNAT) in total cellular extracts fromtransformed BL21 DE3 cells. A: sodium dodecyl sulfate polyacrylamide gelelectrophoresis on a 10% gel stained with Coomassie dye; B: western blot withalkaline phosphatase. The lanes include the molecular weight marker (kDa),induced negative control with a nontransformed strain (-) and differentinductions of transformed cells with the expression vector pET100D-TOPO-6xHis-TcNMNAT (BL21 clones I, II, III and IV).

Mentions: Expression and purification of 6xHis-TcNMNAT - The cloned plasmids wereextracted and used to transform E. coli BL21 DE3 cells. The expressionof the recombinant protein, termed 6xHis-TcNMNAT, was induced at 37ºC overnight with 1mM IPTG (Fermentas). Expression was monitored using SDS-PAGE and WB against thehistidine tag that was added to the insert sequence during the cloning step in thepET-100 D-TOPO vector (Fig. 4). The SDS-PAGE inFig. 4A shows that only one of the TcNMNAT-BL21DE3 clones that were analysed overexpressed a 35-kDa band, which corresponded to theexpected molecular weight for the original aa sequence (32 kDa) with the histidine tagadded (3 kDa). The identity of this recombinant protein was confirmed using ananti-histidine antibody (Fig. 4B).


Identification of the nicotinamide mononucleotide adenylyltransferase of Trypanosoma cruzi.

Niño CH, Forero-Baena N, Contreras LE, Sánchez-Lancheros D, Figarella K, Ramírez MH - Mem. Inst. Oswaldo Cruz (2015)

induction of the 6xHis-nicotinamide mononucleotide adenylyltransferase ofTrypanosoma cruzi (TcNMNAT) in total cellular extracts fromtransformed BL21 DE3 cells. A: sodium dodecyl sulfate polyacrylamide gelelectrophoresis on a 10% gel stained with Coomassie dye; B: western blot withalkaline phosphatase. The lanes include the molecular weight marker (kDa),induced negative control with a nontransformed strain (-) and differentinductions of transformed cells with the expression vector pET100D-TOPO-6xHis-TcNMNAT (BL21 clones I, II, III and IV).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660618&req=5

f04: induction of the 6xHis-nicotinamide mononucleotide adenylyltransferase ofTrypanosoma cruzi (TcNMNAT) in total cellular extracts fromtransformed BL21 DE3 cells. A: sodium dodecyl sulfate polyacrylamide gelelectrophoresis on a 10% gel stained with Coomassie dye; B: western blot withalkaline phosphatase. The lanes include the molecular weight marker (kDa),induced negative control with a nontransformed strain (-) and differentinductions of transformed cells with the expression vector pET100D-TOPO-6xHis-TcNMNAT (BL21 clones I, II, III and IV).
Mentions: Expression and purification of 6xHis-TcNMNAT - The cloned plasmids wereextracted and used to transform E. coli BL21 DE3 cells. The expressionof the recombinant protein, termed 6xHis-TcNMNAT, was induced at 37ºC overnight with 1mM IPTG (Fermentas). Expression was monitored using SDS-PAGE and WB against thehistidine tag that was added to the insert sequence during the cloning step in thepET-100 D-TOPO vector (Fig. 4). The SDS-PAGE inFig. 4A shows that only one of the TcNMNAT-BL21DE3 clones that were analysed overexpressed a 35-kDa band, which corresponded to theexpected molecular weight for the original aa sequence (32 kDa) with the histidine tagadded (3 kDa). The identity of this recombinant protein was confirmed using ananti-histidine antibody (Fig. 4B).

Bottom Line: 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.These results comprise the first identification of an NMNAT in T. cruzi using bioinformatics and experimental tools and hence represent the first step to understanding NAD+ metabolism in these parasites.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Investigaciones Básicas en Bioquímica, Facultad de Ciencias, Universidad Nacional de Colombia, Bogotá, Colombia.

ABSTRACT
The intracellular parasite Trypanosoma cruzi 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. cruzi throughout 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. cruzi is 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. cruzi using bioinformatics and experimental tools and hence represent the first step to understanding NAD+ metabolism in these parasites.

Show MeSH
Related in: MedlinePlus