Mitochondrial NAD+-dependent malic enzyme from Anopheles stephensi: a possible novel target for malaria mosquito control

Abstract

BackgroundAnopheles stephensi mitochondrial malic enzyme (ME) emerged as having a relevant role in the provision of pyruvate for the Krebs' cycle because inhibition of this enzyme results in the complete abrogation of oxygen uptake by mitochondria. Therefore, the identification of ME in mitochondria from immortalized A. stephensi (ASE) cells and the investigation of the stereoselectivity of malate analogues are relevant in understanding the physiological role of ME in cells of this important malaria parasite vector and its potential as a possible novel target for insecticide development.MethodsTo characterize the mitochondrial ME from immortalized ASE cells (Mos. 43; ASE), mass spectrometry analyses of trypsin fragments of ME, genomic sequence analysis and biochemical assays were performed to identify the enzyme and evaluate its activity in terms of cofactor dependency and inhibitor preference.ResultsThe encoding gene sequence and primary sequences of several peptides from mitochondrial ME were found to be highly homologous to the mitochondrial ME from Anopheles gambiae (98%) and 59% homologous to the mitochondrial NADP+-dependent ME isoform from Homo sapiens. Measurements of ME activity in mosquito mitochondria isolated from ASE cells showed that (i) Vmax with NAD+ was 3-fold higher than that with NADP+, (ii) addition of Mg2+ or Mn2+ increased the Vmax by 9- to 21-fold, with Mn2+ 2.3-fold more effective than Mg2+, (iii) succinate and fumarate increased the activity by 2- and 5-fold, respectively, at sub-saturating concentrations of malate, (iv) among the analogs of L-malate tested as inhibitors of the NAD+-dependent ME catalyzed reaction, small (2- to 3-carbons) organic diacids carrying a 2-hydroxyl/keto group behaved as the most potent inhibitors of ME activity (e.g., oxaloacetate, tartronic acid and oxalate).ConclusionsThe biochemical characterization of Anopheles stephensi ME is of critical relevance given its important role in bioenergetics, suggesting that it is a suitable target for insecticide development.

Infection with Plasmodium berghei ookinetes alters protein expression in the brain of Anopheles albimanus mosquitoes

“…The malate dehydrogenase reflects a higher energetic demand. Interestingly, this enzyme has been studied as a potential target for species-specific insecticidal compounds [ 53 ].…”

Wdfy3 regulates glycophagy, mitophagy, and synaptic plasticity

“…Mitochondria were precipitated by centrifuging the supernatant for 10 min at 10,300 Â g. The supernatant was called the PM fraction. Untargeted proteomics was performed at the UCD core facility as described in detail before, 27 followed by functional analysis [27][28][29][30] to determine glucose metabolic pathways in cortex from each genotype allowing to construct a "functional fingerprinting" of intermediary metabolism. Integration of pathways and metabolic outcomes was performed by using bioinformatics approaches as described before.…”

Cellular Redox State Acts as Switch to Determine the Direction of NNT-Catalyzed Reaction in Cystic Fibrosis Cells

“…To test this, the inhibitors of NADP-dependent enzymes were added separately to cells and after 10 min NADPH level was assayed and compared to that of control cells treated with vehicle only. NADPH level did not change significantly in Wt cells treated with oxalate (OXA, 1 mM), a specific inhibitor of malic enzyme [ 26 ], or oxalomalate (OXM, 3 mM), a competitive inhibitor of NADP+-dependent isocitrate dehydrogenase [ 27 ], but it was markedly reduced by 6-aminonicotinamide (6AN, 5 mM, by approximately 50%), compound that inhibit G6PDH [ 24 ], and by GSNO (1 mM, by approximately 45%) (see inset to Figure 2 A’).…”

“…To test this, the inhibitors of NADP-dependent enzymes were added separately to cells and after 10 min NADPH level was assayed and compared to that of control cells treated with vehicle only. NADPH level did not change significantly in Wt cells treated with oxalate (OXA, 1 mM), a specific inhibitor of malic enzyme [26], or oxalomalate (OXM, 3 mM), a competitive inhibitor of NADP + -dependent isocitrate dehydrogenase [27], but it was markedly reduced by 6-aminonicotinamide (6AN, 5 mM, by approximately 50%), compound that inhibit G6PDH [24], and by GSNO (1 mM, by approximately 45%) (see inset to Figure 2A'). When GSNO was added to CF cells (No-NNT cells), we surprisingly observed much higher NADPH level (45%) than that of CF cells not treated with GSNO (Figure 2A); on When GSNO was added to CF cells (No-NNT cells), we surprisingly observed much higher NADPH level (45%) than that of CF cells not treated with GSNO (Figure 2A); on the contrary after the same treatment NADH level was lower (45%) in No-NNT cells than in untreated cells (Figure 2B).…”