Explore the vast range of titles available.

In this study, approximately half the patients with red-cell pyruvate kinase deficiency who were treated with mitapivat had an improvement in their hemoglobin level and decreased hemolysis that was sustained for nearly 3 years. Patients who had missense mutations that allowed for synthesis of a hypofunctioning enzyme were most likely to have a response.

Pyruvate kinase deficiency, the most common genetic lesion in the glycolytic pathway, leads to chronic hemolytic anemia. Mitapivat, an oral agent, can activate some mutant enzymes and restore red-cell ATP generation. In this trial, hemoglobin levels increased from baseline by 1.5 g per deciliter or more at 24 weeks in 40% of the patients with pyruvate kinase deficiency who received mitapivat.

Endothelial nitric oxide synthase (eNOS) is protective against kidney injury, but the molecular mechanisms of this protection are poorly understood 1 , 2 . Nitric oxide-based cellular signalling is generally mediated by protein S -nitrosylation, the oxidative modification of Cys residues to form S -nitrosothiols (SNOs). S -nitrosylation regulates proteins in all functional classes, and is controlled by enzymatic machinery that includes S -nitrosylases and denitrosylases, which add and remove SNO from proteins, respectively 3 , 4 . In Saccharomyces cerevisiae , the classic metabolic intermediate co-enzyme A (CoA) serves as an endogenous source of SNOs through its conjugation with nitric oxide to form S -nitroso-CoA (SNO-CoA), and S -nitrosylation of proteins by SNO-CoA is governed by its cognate denitrosylase, SNO-CoA reductase (SCoR) 5 . Mammals possess a functional homologue of yeast SCoR, an aldo-keto reductase family member (AKR1A1) 5 with an unknown physiological role. Here we report that the SNO-CoA–AKR1A1 system is highly expressed in renal proximal tubules, where it transduces the activity of eNOS in reprogramming intermediary metabolism, thereby protecting kidneys against acute kidney injury. Specifically, deletion of Akr1a1 in mice to reduce SCoR activity increased protein S -nitrosylation, protected against acute kidney injury and improved survival, whereas this protection was lost when Enos (also known as Nos3 ) was also deleted. Metabolic profiling coupled with unbiased mass spectrometry-based SNO-protein identification revealed that protection by the SNO-CoA–SCoR system is mediated by inhibitory S -nitrosylation of pyruvate kinase M2 (PKM2) through a novel locus of regulation, thereby balancing fuel utilization (through glycolysis) with redox protection (through the pentose phosphate shunt). Targeted deletion of PKM2 from mouse proximal tubules recapitulated precisely the protective and mechanistic effects of S -nitrosylation in Akr1a1 −/− mice, whereas Cys-mutant PKM2, which is refractory to S -nitrosylation, negated SNO-CoA bioactivity. Our results identify a physiological function of the SNO-CoA–SCoR system in mammals, describe new regulation of renal metabolism and of PKM2 in differentiated tissues, and offer a novel perspective on kidney injury with therapeutic implications. AKR1A1-regulated protein S- nitrosylation protects against kidney injury through PKM2-mediated metabolic reprogramming.

Adaptation to the environment and extraction of energy are essential for survival. Some species have found niches and specialized in using a particular source of energy, whereas others—including humans and several other mammals—have developed a high degree of flexibility 1 . A lot is known about the general metabolic fates of different substrates but we still lack a detailed mechanistic understanding of how cells adapt in their use of basic nutrients 2 . Here we show that the closely related fasting/starvation-induced forkhead transcription factors FOXK1 and FOXK2 induce aerobic glycolysis by upregulating the enzymatic machinery required for this (for example, hexokinase-2, phosphofructokinase, pyruvate kinase, and lactate dehydrogenase), while at the same time suppressing further oxidation of pyruvate in the mitochondria by increasing the activity of pyruvate dehydrogenase kinases 1 and 4. Together with suppression of the catalytic subunit of pyruvate dehydrogenase phosphatase 1 this leads to increased phosphorylation of the E1α regulatory subunit of the pyruvate dehydrogenase complex, which in turn inhibits further oxidation of pyruvate in the mitochondria—instead, pyruvate is reduced to lactate. Suppression of FOXK1 and FOXK2 induce the opposite phenotype. Both in vitro and in vivo experiments, including studies of primary human cells, show how FOXK1 and/or FOXK2 are likely to act as important regulators that reprogram cellular metabolism to induce aerobic glycolysis. The Forkhead transcription factors FOXK1 and FOXK2, which are induced by starvation, reprogram cellular metabolism to induce aerobic glycolysis.

The mechanisms by which mitochondrial metabolism supports cancer anabolism remain unclear. Here, we found that genetic and pharmacological inactivation of pyruvate dehydrogenase A1 (PDHA1), a subunit of the pyruvate dehydrogenase complex (PDC), inhibits prostate cancer development in mouse and human xenograft tumor models by affecting lipid biosynthesis. Mechanistically, we show that in prostate cancer, PDC localizes in both the mitochondria and the nucleus. Whereas nuclear PDC controls the expression of sterol regulatory element-binding transcription factor (SREBF)-target genes by mediating histone acetylation, mitochondrial PDC provides cytosolic citrate for lipid synthesis in a coordinated manner, thereby sustaining anabolism. Additionally, we found that PDHA1 and the PDC activator pyruvate dehydrogenase phosphatase 1 (PDP1) are frequently amplified and overexpressed at both the gene and protein levels in prostate tumors. Together, these findings demonstrate that both mitochondrial and nuclear PDC sustain prostate tumorigenesis by controlling lipid biosynthesis, thus suggesting this complex as a potential target for cancer therapy. Inactivation of pyruvate dehydrogenase A1 (PDHA1), a subunit of the pyruvate dehydrogenase complex (PDC) regulating mitochondrial metabolism, inhibits lipid biosynthesis and prostate cancer development in mouse and human xenograft tumor models.

Pyruvate is a keystone molecule critical for numerous aspects of eukaryotic and human metabolism. Pyruvate is the end-product of glycolysis, is derived from additional sources in the cellular cytoplasm, and is ultimately destined for transport into mitochondria as a master fuel input undergirding citric acid cycle carbon flux. In mitochondria, pyruvate drives ATP production by oxidative phosphorylation and multiple biosynthetic pathways intersecting the citric acid cycle. Mitochondrial pyruvate metabolism is regulated by many enzymes, including the recently discovered mitochondria pyruvate carrier, pyruvate dehydrogenase, and pyruvate carboxylase, to modulate overall pyruvate carbon flux. Mutations in any of the genes encoding for proteins regulating pyruvate metabolism may lead to disease. Numerous cases have been described. Aberrant pyruvate metabolism plays an especially prominent role in cancer, heart failure, and neurodegeneration. Because most major diseases involve aberrant metabolism, understanding and exploiting pyruvate carbon flux may yield novel treatments that enhance human health.

Diagnosis of pyruvate kinase deficiency (PKD) remains challenging in clinical practice. The pyruvate kinase (PK) to hexokinase (HK) activity ratio (PK/HK) was proposed to reduce the confounding effect of reticulocytosis on PK activity measurement. However, decreased PK activity and PK/HK ratios have also been observed in other anemias, raising doubts about their diagnostic value. We assessed the diagnostic accuracy of PK/HK ratio versus PK activity in differentiating PKD from other hereditary anemias. This study included 41 patients with molecularly confirmed PKD and 62 patients with other anemias. We also evaluated the influence of reticulocytosis and transfusions on erythrocyte PK activity. The PK/HK ratio showed 73% specificity, while PK activity alone achieved 95%. In PKD patients, reticulocytosis did not affect PK activity because reticulocyte PK activity was already markedly reduced (23-fold) compared with controls. In other anemias, decreases in PK activity were present in both reticulocytes and erythrocytes, but to a lesser extent. Transfusions contribute more to the false-normal result of PK activity than reticulocytosis. Measuring reticulocyte-specific PK activity during regular transfusions provided reliable results, as only patient-derived reticulocytes are present in the blood. PK activity demonstrates higher specificity than PK/HK ratio in diagnosing PKD. Reticulocytosis is not a confounder, while transfusions remain the main limitation. Reticulocyte-specific PK activity measurement may improve diagnostic accuracy in transfused patients.

Control of intracellular reactive oxygen species (ROS) concentrations is critical for cancer cell survival. We show that, in human lung cancer cells, acute increases in intracellular concentrations of ROS caused inhibition of the glycolytic enzyme pyruvate kinase M2 (PKM2) through oxidation of Cys³⁵⁸. This inhibition of PKM2 is required to divert glucose flux into the pentose phosphate pathway and thereby generate sufficient reducing potential for detoxification of ROS. Lung cancer cells in which endogenous PKM2 was replaced with the Cys³⁵⁸ to Ser³⁵⁸ oxidation-resistant mutant exhibited increased sensitivity to oxidative stress and impaired tumor formation in a xenograft model. Besides promoting metabolic changes required for proliferation, the regulatory properties of PKM2 may confer an additional advantage to cancer cells by allowing them to withstand oxidative stress.

The pyruvate dehydrogenase complex (PDC) bridges glycolysis and the citric acid cycle. In human, PDC deficiency leads to severe neurodevelopmental delay and progressive neurodegeneration. The majority of cases are caused by variants in the gene encoding the PDC subunit E1α. The molecular effects of the variants, however, remain poorly understood. Using yeast as a eukaryotic model system, we have studied the substitutions A189V, M230V, and R322C in yeast E1α (corresponding to the pathogenic variants A169V, M210V, and R302C in human E1α) and evaluated how substitutions of single amino acid residues within different functional E1α regions affect PDC structure and activity. The E1α A189V substitution located in the heterodimer interface showed a more compact conformation with significant underrepresentation of E1 in PDC and impaired overall PDC activity. The E1α M230V substitution located in the tetramer and heterodimer interface showed a relatively more open conformation and was particularly affected by low thiamin pyrophosphate concentrations. The E1α R322C substitution located in the phosphorylation loop of E1α resulted in PDC lacking E3 subunits and abolished overall functional activity. Furthermore, we show for the E1α variant A189V that variant E1α accumulates in the Hsp60 chaperonin, but can be released upon ATP supplementation. Our studies suggest that pathogenic E1α variants may be associated with structural changes of PDC and impaired folding of E1α.

Background Pyruvate is a precursor for various compounds in the chemical, drug, and food industries and is therefore an attractive target molecule for microbial production processes. The fast-growing bacterium Vibrio natriegens excels with its specific substrate uptake rate as an unconventional chassis for industrial biotechnology. Here, we aim to exploit the traits of V. natriegens for pyruvate production in fermentations with low biomass concentrations. Results We inactivated the pyruvate dehydrogenase complex in V. natriegens Δ vnp12 , which harbors deletions of the prophage regions vnp12 . The resulting strain V. natriegens Δ vnp12 Δ aceE was unable to grow in minimal medium with glucose unless supplemented with acetate. In shaking flasks, the strain showed a growth rate of 1.16 ± 0.03 h − 1 and produced 4.0 ± 0.3 g Pyr L − 1 within 5 h. We optimized the parameters in an aerobic fermentation process and applied a constant maintenance feed of 0.24 g Ac h − 1 which resulted in a maximal biomass concentration of only 6.6 ± 0.4 g CDW L − 1 and yielded highly active resting cells with a glucose uptake rate (q S ) of 3.5 ± 0.2 g Glc g CDW −1 h − 1 . V. natriegens Δ vnp12 Δ aceE produced 41.0 ± 1.8 g Pyr L − 1 with a volumetric productivity of 4.1 ± 0.2 g Pyr L − 1 h − 1 . Carbon balancing disclosed a gap of 30%, which we identified partly as parapyruvate. Deletion of ligK encoding the HMG/CHA aldolase in V. natriegens Δ vnp12 Δ aceE did not impact biomass formation but plasmid-based overexpression of ligK negatively affected growth and led to a 3-fold higher parapyruvate concentration in the culture broth. Notably, we also identified parapyruvate in supernatants of a pyruvate-producing Corynebacterium glutamicum strain. Cell-free bioreactor experiments mimicking the biological process also resulted in parapyruvate formation, pointing to a chemical reaction contributing to its synthesis. Conclusions We engineered metabolically highly active resting cells of V. natriegens producing pyruvate with high productivity at a low biomass concentration. However, we also found that pyruvate production is accompanied by parapyruvate formation in V. natriegens as well as in a pyruvate producing C. glutamicum strain. Parapyruvate formation seems to be a result of chemical pyruvate conversion and might be supported biochemically by an aldolase reaction.