Characteristics of Mycobacterium tuberculosis PtpA interaction and activity on the alpha subunit of human mitochondrial trifunctional protein, a key enzyme of lipid metabolism.

During Mycobacterium tuberculosis (Mtb) infection, the virulence factor PtpA belonging to the protein tyrosine phosphatase family is delivered into the cytosol of the macrophage. PtpA interacts with numerous eukaryotic proteins modulating phagosome maturation, innate immune response, apoptosis, and potentially host-lipid metabolism, as previously reported by our group. In vitro, the human trifunctional protein enzyme (hTFP) is a bona fide PtpA substrate, a key enzyme of mitochondrial ß-oxidation of long-chain fatty acids, containing two alpha and two beta subunits arranged in a tetramer structure. Interestingly, it has been described that the alpha subunit of hTFP (ECHA, hTFPα) is no longer detected in mitochondria during macrophage infection with the virulent Mtb H37Rv. To better understand if PtpA could be the bacterial factor responsible for this effect, in the present work, we studied in-depth the PtpA activity and interaction with hTFPα. With this aim, we performed docking and in vitro dephosphorylation assays defining the P-Tyr-271 as the potential target of mycobacterial PtpA, a residue located in the helix-10 of hTFPα, previously described as relevant for its mitochondrial membrane localization and activity. Phylogenetic analysis showed that Tyr-271 is absent in TFPα of bacteria and is present in more complex eukaryotic organisms. These results suggest that this residue is a specific PtpA target, and its phosphorylation state is a way of regulating its subcellular localization. We also showed that phosphorylation of Tyr-271 can be catalyzed by Jak kinase. In addition, we found by molecular dynamics that PtpA and hTFPα form a stable protein complex through the PtpA active site, and we determined the dissociation equilibrium constant. Finally, a detailed study of PtpA interaction with ubiquitin, a reported PtpA activator, showed that additional factors are required to explain a ubiquitin-mediated activation of PtpA. Altogether, our results provide further evidence supporting that PtpA could be the bacterial factor that dephosphorylates hTFPα during infection, potentially affecting its mitochondrial localization or ß-oxidation activity.

Silencing of Mitochondrial Trifunctional Protein A Subunit (HADHA) Increases Lipid Stores, and Reduces Oviposition and Flight Capacity in the Vector Insect Rhodnius prolixus.

Rhodnius prolixus is an obligatory hematophagous insect, vector of Chagas disease. After blood meal, lipids are absorbed, metabolized, synthesized, and accumulated in the fat body. When necessary, stored lipids are mobilized, transported to other organs, or are oxidized to provide energy. Mitochondrial ß-oxidation is a cyclic conserved pathway, where degradation of long-chain fatty acids occurs to contribute to cellular energetic demands. Three of its reactions are catalyzed by the mitochondrial trifunctional protein (MTP), which is composed by hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunits alpha and beta (HADHA and HADHB, respectively). Here, we investigated the role of HADHA in lipid metabolism and reproduction of Rhodnius prolixus females. The expression of HADHA gene (RhoprHadha) was determined in the organs of starving adult insects. The flight muscle and ovary had higher expression levels when compared to the anterior and posterior midguts or the fat body. RhoprHadha gene expression was upregulated by blood meal in the flight muscle and fat body. We generated insects with RNAi-mediated knockdown of RhoprHadha to address the physiological role of this gene. RhoprHadha deficiency resulted in higher triacylglycerol content and larger lipid droplets in the fat body during starvation. After feeding, lifespan of the knockdown females was not affected, but they exhibited a decrease in oviposition, although hatching was the same in both groups. Silenced females showed lower forced flight capacity than the control ones, and their fat bodies had lower gene expression levels of Brummer lipase (RhoprBmm) and long-chain acyl-CoA synthetase 2 (RhoprAcsl2). Taken together, these findings indicate that HADHA is important to guarantee successful reproduction and efficient mobilization of lipid stores during starvation and flight.

Disturbance of mitochondrial functions provoked by the major long-chain 3-hydroxylated fatty acids accumulating in MTP and LCHAD deficiencies in skeletal muscle.

The pathogenesis of the muscular symptoms and recurrent rhabdomyolysis that are commonly manifested in patients with mitochondrial trifunctional protein (MTP) and long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) deficiencies is still unknown. In this study we investigated the effects of the major long-chain monocarboxylic 3-hydroxylated fatty acids (LCHFA) accumulating in these disorders, namely 3-hydroxytetradecanoic (3HTA) and 3-hydroxypalmitic (3HPA) acids, on important mitochondrial functions in rat skeletal muscle mitochondria. 3HTA and 3HPA markedly increased resting (state 4) and decreased ADP-stimulated (state 3) and CCCP-stimulated (uncoupled) respiration. 3HPA provoked similar effects in permeabilized skeletal muscle fibers, validating the results obtained in purified mitochondria. Furthermore, 3HTA and 3HPA markedly diminished mitochondrial membrane potential, NAD(P)H content and Ca(2+) retention capacity in Ca(2+)-loaded mitochondria. Mitochondrial permeability transition (mPT) induction probably underlie these effects since they were totally prevented by cyclosporin A and ADP. In contrast, the dicarboxylic analogue of 3HTA did not alter the tested parameters. Our data strongly indicate that 3HTA and 3HPA behave as metabolic inhibitors, uncouplers of oxidative phosphorylation and mPT inducers in skeletal muscle. It is proposed that these pathomechanisms disrupting mitochondrial homeostasis may be involved in the muscle alterations characteristic of MTP and LCHAD deficiencies.