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

Abstract

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.

GlSir2.1 of Giardia lamblia is a NAD+-dependent cytoplasmic deacetylase

“…Cell lysis was performed following the previously described procedure. Purification of inclusion bodies using denaturing agents followed a previously reported method (Sánchez-Lancheros et al., 2016). In addition, purified samples were run in a preparative 16 × 19 cm 10% SDS-PAGE.…”

Localization and phosphorylation of Plasmodium falciparum nicotinamide/nicotinate mononucleotide adenylyltransferase (PfNMNAT) in intraerythrocytic stages

“…HsNMNAT-1 contains one serine residue (Ser136), which is a phosphorylation target of protein kinase C enzymes, and is involved in the regulation of the catalytic activity of the self-modified PARP1 enzyme [ 21 ]. It has recently been found that Trypanosoma cruzi NMNAT has one or more serine-phosphorylated residues [ 22 ], which are possibly necessary for its regulation. PfNMNAT may be regulated by phosphorylation in some of its residues.…”

Protein Expression-Yeast

“…En nuestro grupo de investigación se identificó que la síntesis del NAD + en T. cruzi ocurre en el citosol, catalizada por la única NMNAT del parásito 80,136 , y ello llevó a generar la hipótesis de que un eficiente sistema de distribución del dinucleótido existe en el parásito; por ende, en este trabajó se optó por estudiar a los candidatos a transportador del dinucleótido de T. cruzi TcNdt1, TcNdt2 y TcNdt3, identificados en un trabajo desarrollado anteriormente 142 . A continuación, se presenta el primer apartado correspondiente a la caracterización in silico de los 3 candidatos.…”

“…Estudiar proteínas candidato a ser transportadores del NAD + en T. cruzi requiere de una aproximación en que se integren las particularidades de la biología del parásito, y el conocimiento construido acerca de los elementos que participan en el transporte de moléculas. Teniendo en cuenta que la homeóstasis del NAD + es vital, y que T. cruzi posee una única NMNAT citosólica 80,81 , debe existir un sistema de transporte que movilice esta molécula a lo largo de sus diferentes compartimentos subcelulares, sin embargo, hasta el momento este no ha sido identificado. De manera interesante, en otros organismos eucariotas se ha determinado que proteínas pertenecientes a la MCF son pieza clave de dichos sistemas 33,[49][50][51] .…”