In vertebrate cells, mitochondrial Ca2+ uptake by the mitochondrial calcium uniporter (MCU) leads to Ca2+-mediated stimulation of an intramitochondrial pyruvate dehydrogenase phosphatase (PDP). This ...enzyme dephosphorylates serine residues in the E1α subunit of pyruvate dehydrogenase (PDH), thereby activating PDH and resulting in increased ATP production. Although a phosphorylation/dephosphorylation cycle for the E1α subunit of PDH from nonvertebrate organisms has been described, the Ca2+-mediated PDP activation has not been studied. In this work, we investigated the Ca2+ sensitivity of two recombinant PDPs from the protozoan human parasites Trypanosoma cruzi (TcPDP) and T. brucei (TbPDP) and generated a TcPDP-KO cell line to establish TcPDP’s role in cell bioenergetics and survival. Moreover, the mitochondrial localization of the TcPDP was studied by CRISPR/Cas9-mediated endogenous tagging. Our results indicate that TcPDP and TbPDP both are Ca2+-sensitive phosphatases. Of note, TcPDP-KO epimastigotes exhibited increased levels of phosphorylated TcPDH, slower growth and lower oxygen consumption rates than control cells, an increased AMP/ATP ratio and autophagy under starvation conditions, and reduced differentiation into infective metacyclic forms. Furthermore, TcPDP-KO trypomastigotes were impaired in infecting cultured host cells. We conclude that TcPDP is a Ca2+-stimulated mitochondrial phosphatase that dephosphorylates TcPDH and is required for normal growth, differentiation, infectivity, and energy metabolism in T. cruzi. Our results support the view that one of the main roles of the MCU is linked to the regulation of intramitochondrial dehydrogenases.
The mechanisms by which a high fat diet (HFD) promotes non-alcoholic fatty liver disease (NAFLD) appear to involve liver mitochondrial dysfunctions and redox imbalance. We hypothesized that a HFD ...would increase mitochondrial reliance on NAD(P)-transhydrogenase (NNT) as the source of NADPH for antioxidant systems that counteract NAFLD development. Therefore, we studied HFD-induced liver mitochondrial dysfunctions and NAFLD in C57Unib.B6 congenic mice with (Nnt+/+) or without (Nnt-/-) NNT activity; the spontaneously mutated allele (Nnt-/-) was inherited from the C57BL/6J mouse substrain. After 20 weeks on a HFD, Nnt-/- mice exhibited a higher prevalence of steatohepatitis and content of liver triglycerides compared to Nnt+/+ mice on an identical diet. Under a HFD, the aggravated NAFLD phenotype in the Nnt-/- mice was accompanied by an increased H2O2 release rate from mitochondria, decreased aconitase activity (a redox-sensitive mitochondrial enzyme) and higher susceptibility to Ca2+-induced mitochondrial permeability transition. In addition, HFD led to the phosphorylation (inhibition) of pyruvate dehydrogenase (PDH) and markedly reduced the ability of liver mitochondria to remove peroxide in Nnt-/- mice. Bypass or pharmacological reactivation of PDH by dichloroacetate restored the peroxide removal capability of mitochondria from Nnt-/- mice on a HFD. Noteworthy, compared to mice that were chow-fed, the HFD did not impair peroxide removal nor elicit redox imbalance in mitochondria from Nnt+/+ mice. Therefore, HFD interacted with Nnt mutation to generate PDH inhibition and further suppression of peroxide removal. We conclude that NNT plays a critical role in counteracting mitochondrial redox imbalance, PDH inhibition and advancement of NAFLD in mice fed a HFD. The present study provide seminal experimental evidence that redox imbalance in liver mitochondria potentiates the progression from simple steatosis to steatohepatitis following a HFD.
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•The Nnt genotype modifies the effects of high fat diet (HFD) on mouse liver.•The Nnt mutation from the C57BL/6J mouse aggravates HFD-induced fatty liver disease.•Functional Nnt preserves pyruvate metabolism and mitochondrial oxidative balance.•On HFD, mutated Nnt leads to the progression from simple steatosis to steatohepatitis.•The use of mice models carrying mutated Nnt in research must not be overlooked.
We report here that
Trypanosoma cruzi,
the etiologic agent of Chagas disease, possesses two unique paralogues of the mitochondrial calcium uniporter complex
TcMCU
subunit that we named
TcMCUc
and
...TcMCUd
. The predicted structure of the proteins indicates that, as predicted for the
TcMCU
and
TcMCUb
paralogues, they are composed of two helical membrane-spanning domains and contain a WDXXEPXXY motif. Overexpression of each gene led to a significant increase in mitochondrial Ca
2+
uptake, while knockout (KO) of either
TcMCUc
or
TcMCUd
led to a loss of mitochondrial Ca
2+
uptake, without affecting the mitochondrial membrane potential.
TcMCUc
-KO and
TcMCUd
-KO epimastigotes exhibited reduced growth rate in low-glucose medium and alterations in their respiratory rate, citrate synthase activity, and AMP/ATP ratio, while trypomastigotes had reduced ability to efficiently infect host cells and replicate intracellularly as amastigotes. By gene complementation of KO cell lines or by a newly developed CRISPR/Cas9-mediated knock-in approach, we also studied the importance of critical amino acid residues of the four paralogues on mitochondrial Ca
2+
uptake. In conclusion, the results predict a hetero-oligomeric structure for the
T. cruzi
MCU complex, with structural and functional differences, as compared with those in the mammalian complex.
Overall health relies on features of skeletal muscle that generally decline with age, partly due to mechanisms associated with mitochondrial redox imbalance and bioenergetic dysfunction. Previously, ...aged mice genetically devoid of the mitochondrial NAD(P)+ transhydrogenase (NNT, encoded by the nicotinamide nucleotide transhydrogenase gene), an enzyme involved in mitochondrial NADPH supply, were shown to exhibit deficits in locomotor behavior. Here, by using young, middle-aged, and older NNT-deficient (Nnt−/−) mice and age-matched controls (Nnt+/+), we aimed to investigate how muscle bioenergetic function and motor performance are affected by NNT expression and aging. Mice were subjected to the wire-hang test to assess locomotor performance, while mitochondrial bioenergetics was evaluated in fiber bundles from the soleus, vastus lateralis and plantaris muscles. An age-related decrease in the average wire-hang score was observed in middle-aged and older Nnt−/− mice compared to age-matched controls. Although respiratory rates in the soleus, vastus lateralis and plantaris muscles did not significantly differ between the genotypes in young mice, the rates of oxygen consumption did decrease in the soleus and vastus lateralis muscles of middle-aged and older Nnt−/− mice. Notably, the soleus, which exhibited the highest NNT expression level, was the muscle most affected by aging, and NNT loss. Additionally, histology of the soleus fibers revealed increased numbers of centralized nuclei in older Nnt−/− mice, indicating abnormal morphology. In summary, our findings suggest that NNT expression deficiency causes locomotor impairments and muscle dysfunction during aging in mice.
•Nnt−/− mice exhibit age-related muscle dysfunction and impaired motor performance.•Soleus had the highest NNT levels and was the most affected by aging and NNT loss.•Mitochondrial bioenergetics declined in soleus of middle-aged and old Nnt−/− mice.•Neuromotor assessment is advisable in elderly patients with NNT variants.
The mechanisms by which a high-fat diet (HFD) promotes non-alcoholic fatty liver disease (NAFLD) appear to involve liver mitochondrial dysfunction and redox imbalance. The functional loss of the ...enzyme NAD(P)+ transhydrogenase, a main source of mitochondrial NADPH, results in impaired mitochondrial peroxide removal, pyruvate dehydrogenase inhibition by phosphorylation, and progression of NAFLD in HFD-fed mice. The present study aimed to investigate whether pharmacological reactivation of pyruvate dehydrogenase by dichloroacetate attenuates the mitochondrial redox dysfunction and the development of NAFLD in NAD(P)+ transhydrogenase-null (Nnt−/−) mice fed an HFD (60% of total calories from fat). For this purpose, Nnt−/− mice and their congenic controls (Nnt+/+) were fed chow or an HFD for 20 weeks and received sodium dichloroacetate or NaCl in the final 12 weeks via drinking water. The results showed that HFD reduced the ability of isolated liver mitochondria from Nnt−/− mice to remove peroxide, which was prevented by the dichloroacetate treatment. HFD-fed mice of both Nnt genotypes exhibited increased body and liver mass, as well as a higher content of hepatic triglycerides, but dichloroacetate treatment attenuated these abnormalities only in Nnt−/− mice. Notably, dichloroacetate treatment decreased liver pyruvate dehydrogenase phosphorylation levels and prevented the aggravation of NAFLD in HFD-fed Nnt−/− mice. Conversely, dichloroacetate treatment elicited moderate hepatocyte ballooning in chow-fed mice, suggesting potentially toxic effects. We conclude that the protection against HFD-induced NAFLD by dichloroacetate is associated with its role in reactivating pyruvate dehydrogenase and reestablishing the pyruvate-supported liver mitochondrial capacity to handle peroxide in Nnt−/− mice.
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•Dichloroacetate (DCA) prevented the aggravation of NAFLD in HFD-fed Nnt−/− mice.•DCA reduced body and liver mass, and hepatic triglycerides in HFD-fed Nnt−/− mice.•DCA decreased the levels of liver pyruvate dehydrogenase phosphorylation.•DCA reestablished the ability of Nnt−/− liver mitochondria to metabolize peroxide.•Chow-fed mice on DCA treatment presented moderate hepatocyte ballooning.
Mitochondrial calcium transport in trypanosomes Docampo, Roberto; Vercesi, Anibal E.; Huang, Guozhong
Molecular and biochemical parasitology,
09/2014, Volume:
196, Issue:
2
Journal Article
Peer reviewed
Open access
•Discovery of MCU in trypanosomes led to the finding of the MCU gene in mammals.•While MCU is essential in trypanosomes it is not essential in mice.•Other components of the trypanosome MCU complex ...are MCUb, MICU1 and MICU2.•Mitochondrial Ca2+ is essential to regulate trypanosome bioenergetics.
The biochemical peculiarities of trypanosomes were fundamental for the recent molecular identification of the long-sought channel involved in mitochondrial Ca2+ uptake, the mitochondrial Ca2+ uniporter or MCU. This discovery led to the finding of numerous regulators of the channel, which form a high molecular weight complex with MCU. Some of these regulators have been bioinformatically identified in trypanosomes, which are the first eukaryotic organisms described for which MCU is essential. In trypanosomes MCU is important for buffering cytosolic Ca2+ changes and for activation of the bioenergetics of the cells. Future work on this pathway in trypanosomes promises further insight into the biology of these fascinating eukaryotes, as well as the potential for novel target discovery.
is the agent of Chagas disease, and the finding that this parasite possesses a mitochondrial calcium uniporter (TcMCU) with characteristics similar to that of mammalian mitochondria was fundamental ...for the discovery of the molecular nature of MCU in eukaryotes. We report here that ablation of
, or its paralog
, by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 led to a marked decrease in mitochondrial Ca
uptake without affecting the membrane potential of these cells, whereas overexpression of each gene caused a significant increase in the ability of mitochondria to accumulate Ca
While
knockout (KO) epimastigotes were viable and able to differentiate into trypomastigotes, infect host cells, and replicate normally, ablation of
resulted in epimastigotes having an important growth defect, lower rates of respiration and metacyclogenesis, more pronounced autophagy changes under starvation, and significantly reduced infectivity. Overexpression of
, in contrast to what was proposed for its mammalian ortholog, did not result in a dominant negative effect on TcMCU.
The finding of a mitochondrial calcium uniporter (MCU) in
was essential for the discovery of the molecular nature of this transporter in mammals. In this work, we used the CRISPR/Cas9 technique that we recently developed for
to knock out two components of the uniporter: MCU, the pore subunit, and MCUb, which was proposed as a negative regulator of MCU in human cells. In contrast to what occurs in human cells, MCU is not essential, while MCUb is essential for growth, differentiation, and infectivity; has a bioenergetic role; and does not act as a dominant negative subunit of MCU.
Pyruvate is the final metabolite of glycolysis and can be converted into acetyl coenzyme A (acetyl-CoA) in mitochondria, where it is used as the substrate for the tricarboxylic acid cycle. Pyruvate ...availability in mitochondria depends on its active transport through the heterocomplex formed by the mitochondrial pyruvate carriers 1 and 2 (MPC1/MPC2). We report here studies on MPC1/MPC2 of
, the etiologic agent of Chagas disease. Endogenous tagging of
(
) and
with 3×
showed that both encoded proteins colocalize with MitoTracker to the mitochondria of epimastigotes. Individual knockout (KO) of
and
genes using CRISPR/Cas9 was confirmed by PCR and Southern blot analyses. Digitonin-permeabilized
-KO and
-KO epimastigotes showed reduced O
consumption rates when pyruvate, but not succinate, was used as the mitochondrial substrate, while α-ketoglutarate increased their O
consumption rates due to an increase in α-ketoglutarate dehydrogenase activity. Defective mitochondrial pyruvate import resulted in decreased Ca
uptake. The inhibitors UK5099 and malonate impaired pyruvate-driven oxygen consumption in permeabilized control cells. Inhibition of succinate dehydrogenase by malonate indicated that pyruvate needs to be converted into succinate to increase respiration.
-KO and
-KO epimastigotes showed little growth differences in standard or low-glucose culture medium. However, the ability of trypomastigotes to infect tissue culture cells and replicate as intracellular amastigotes was decreased in
-KOs. Overall,
MPC1 and MPC2 are essential for cellular respiration in the presence of pyruvate, invasion of host cells, and replication of amastigotes.
is the causative agent of Chagas disease. Pyruvate is the end product of glycolysis, and its transport into the mitochondrion is mediated by the mitochondrial pyruvate carrier (MPC) subunits. Using the CRISPR/Cas9 technique, we generated individual
(
) and
knockouts and demonstrated that they are essential for pyruvate-driven respiration. Interestingly, although glycolysis was reported as not an important source of energy for the infective stages, MPC was essential for normal host cell invasion and intracellular replication.
The interaction between supraphysiological cytosolic Ca2+ levels and mitochondrial redox imbalance mediates the mitochondrial permeability transition (MPT). The MPT is involved in cell death, ...diseases and aging. This study compared the liver mitochondrial Ca2+ retention capacity and oxygen consumption in the long-lived red-footed tortoise (Chelonoidis carbonaria) with those in the rat as a reference standard. Mitochondrial Ca2+ retention capacity, a quantitative measure of MPT sensitivity, was remarkably higher in tortoises than in rats. This difference was minimized in the presence of the MPT inhibitors ADP and cyclosporine A. However, the Ca2+ retention capacities of tortoise and rat liver mitochondria were similar when both MPT inhibitors were present simultaneously. NADH-linked phosphorylating respiration rates of tortoise liver mitochondria represented only 30% of the maximal electron transport system capacity, indicating a limitation imposed by the phosphorylation system. These results suggested underlying differences in putative MPT structural components e.g. ATP synthase, adenine nucleotide translocase (ANT) and cyclophilin D between tortoises and rats. Indeed, in tortoise mitochondria, titrations of inhibitors of the oxidative phosphorylation components revealed a higher limitation of ANT. Furthermore, cyclophilin D activity was approximately 70% lower in tortoises than in rats. Investigation of critical properties of mitochondrial redox control that affect MPT demonstrated that tortoise and rat liver mitochondria exhibited similar rates of H2O2 release and glutathione redox status. Overall, our findings suggest that constraints imposed by ANT and cyclophilin D, putative components or regulators of the MPT pore, are associated with the enhanced resistance to Ca2+-induced MPT in tortoises.
Mitochondrial permeability transition (MPT) is a non-selective inner membrane permeabilization that may precede necrotic and apoptotic cell death. Although this process has a specific inhibitor, ...cyclosporin A, little is known about the nature of the proteinaceous pore that results in MPT. Here, we review data indicating that MPT is not a consequence of the opening of a pre-formed pore, but the consequence of oxidative damage to pre-existing membrane proteins.
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