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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.

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.

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.

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.

Protein aggregation is mostly viewed as deleterious and irreversible causing several pathologies. However, reversible protein aggregation has recently emerged as a novel concept for cellular regulation. Here, we characterize stress-induced, reversible aggregation of yeast pyruvate kinase, Cdc19. Aggregation of Cdc19 is regulated by oligomerization and binding to allosteric regulators. We identify a region of low compositional complexity (LCR) within Cdc19 as necessary and sufficient for reversible aggregation. During exponential growth, shielding the LCR within tetrameric Cdc19 or phosphorylation of the LCR prevents unscheduled aggregation, while its dephosphorylation is necessary for reversible aggregation during stress. Cdc19 aggregation triggers its localization to stress granules and modulates their formation and dissolution. Reversible aggregation protects Cdc19 from stress-induced degradation, thereby allowing cell cycle restart after stress. Several other enzymes necessary for G1 progression also contain LCRs and aggregate reversibly during stress, implying that reversible aggregation represents a conserved mechanism regulating cell growth and survival. Saad et al.  identify stress-induced reversible protein aggregation as a protective mechanism to ensure cell cycle resumption and cell survival after stress in yeast.

Despite the fact that most cancer cells display high glycolytic activity, cancer cells selectively express the less active M2 isoform of pyruvate kinase (PKM2). Here we demonstrate that PKM2 expression makes a critical regulatory contribution to the serine synthetic pathway. In the absence of serine, an allosteric activator of PKM2, glycolytic efflux to lactate is significantly reduced in PKM2-expressing cells. This inhibition of PKM2 results in the accumulation of glycolytic intermediates that feed into serine synthesis. As a consequence, PKM2-expressing cells can maintain mammalian target of rapamycin complex 1 activity and proliferate in serine-depleted medium, but PKM1-expressing cells cannot. Cellular detection of serine depletion depends on general control nonderepressible 2 kinase-activating transcription factor 4 (GCN2-ATF4) pathway activation and results in increased expression of enzymes required for serine synthesis from the accumulating glycolytic precursors. These findings suggest that tumor cells use serine-dependent regulation of PKM2 and GCN2 to modulate the flux of glycolytic intermediates in support of cell proliferation.

Most cancer cells reprogram their glucose metabolic pathway from oxidative phosphorylation to aerobic glycolysis for energy production. By reducing enzyme activity of pyruvate kinase M2 (PKM2), cancer cells attain a greater fraction of glycolytic metabolites for macromolecule synthesis needed for rapid proliferation. Here we demonstrate that hydrogen sulfide (H 2 S) destabilizes the PKM2 tetramer into monomer/dimer through sulfhydration at cysteines, notably at C326, leading to reduced PKM2 enzyme activity and increased PKM2-mediated transcriptional activation. Blocking PKM2 sulfhydration at C326 through amino acid mutation stabilizes the PKM2 tetramer and crystal structure further revealing the tetramer organization of PKM2-C326S. The PKM2-C326S mutant in cancer cells rewires glucose metabolism to mitochondrial respiration, significantly inhibiting tumor growth. In this work, we demonstrate that PKM2 sulfhydration by H 2 S inactivates PKM2 activity to promote tumorigenesis and inhibiting this process could be a potential therapeutic approach for targeting cancer metabolism. Low level of pyruvate kinase M2 (PKM2) activity in cancer cells is essential for the dependence on aerobic glycolysis. Here the authors show that PKM2 sulfhydration by hydrogen sulfide destabilizes the PKM2 tetramer, leading to reduced PKM2 enzyme activity and enhanced proliferation of breast cancer cells.

Neutrophils rely predominantly on glycolytic metabolism for their biological functions, including reactive oxygen species (ROS) production. Although pyruvate kinase M2 (PKM2) is a glycolytic enzyme known to be involved in metabolic reprogramming and gene transcription in many immune cell types, its role in neutrophils remains poorly understood. Here, we report that PKM2 regulates ROS production and microbial killing by neutrophils. Zymosan-activated neutrophils showed increased cytoplasmic expression of PKM2. Pharmacological inhibition or genetic deficiency of PKM2 in neutrophils reduced ROS production and Staphylococcus aureus killing in vitro. In addition, this also resulted in phosphoenolpyruvate (PEP) accumulation and decreased dihydroxyacetone phosphate (DHAP) production, which is required for de novo synthesis of diacylglycerol (DAG) from glycolysis. In vivo, PKM2 deficiency in myeloid cells impaired the control of infection with Staphylococcus aureus . Our results fill the gap in the current knowledge of the importance of lower glycolysis for ROS production in neutrophils, highlighting the role of PKM2 in regulating the DHAP and DAG synthesis to promote ROS production in neutrophils. Neutrophil activation has been shown to rely on the pentose phosphate pathway (PPP) for NADPH generation and reactive oxygen species production. In this study, the authors identify a mechanism of neutrophil activation that is independent of the PPP but relies on the glycolytic enzyme pyruvate kinase M2 instead.

Studying patients with long-term diabetes who lack diabetic nephropathy reveals that targeting pyruvate kinase M2 protects against renal disease. Diabetic nephropathy (DN) is a major cause of end-stage renal disease, and therapeutic options for preventing its progression are limited. To identify novel therapeutic strategies, we studied protective factors for DN using proteomics on glomeruli from individuals with extreme duration of diabetes (ł50 years) without DN and those with histologic signs of DN. Enzymes in the glycolytic, sorbitol, methylglyoxal and mitochondrial pathways were elevated in individuals without DN. In particular, pyruvate kinase M2 (PKM2) expression and activity were upregulated. Mechanistically, we showed that hyperglycemia and diabetes decreased PKM2 tetramer formation and activity by sulfenylation in mouse glomeruli and cultured podocytes. Pkm -knockdown immortalized mouse podocytes had higher levels of toxic glucose metabolites, mitochondrial dysfunction and apoptosis. Podocyte-specific Pkm2 -knockout (KO) mice with diabetes developed worse albuminuria and glomerular pathology. Conversely, we found that pharmacological activation of PKM2 by a small-molecule PKM2 activator, TEPP-46, reversed hyperglycemia-induced elevation in toxic glucose metabolites and mitochondrial dysfunction, partially by increasing glycolytic flux and PGC-1α mRNA in cultured podocytes. In intervention studies using DBA2/J and Nos3 ( eNos ) KO mouse models of diabetes, TEPP-46 treatment reversed metabolic abnormalities, mitochondrial dysfunction and kidney pathology. Thus, PKM2 activation may protect against DN by increasing glucose metabolic flux, inhibiting the production of toxic glucose metabolites and inducing mitochondrial biogenesis to restore mitochondrial function.

Eukarya pyruvate kinases possess glutamate at position 117 (numbering of rabbit muscle enzyme), whereas bacteria have either glutamate or lysine. Those with E117 are K+-dependent, whereas those with K117 are K+-independent. In a phylogenetic tree, 80% of the sequences with E117 are occupied by T113/K114/T120 and 77% of those with K117 possess L113/Q114/(L,I,V)120. This work aims to understand these residues’ contribution to the K+-independent pyruvate kinases using the K+-dependent rabbit muscle enzyme. Residues 117 and 120 are crucial in the differences between the K+-dependent and -independent mutants. K+-independent activity increased with L113 and Q114 to K117, but L120 induced structural differences that inactivated the enzyme. T120 appears to be key in folding the protein and closure of the lid of the active site to acquire its active conformation in the K+-dependent enzymes. E117K mutant was K+-independent and the enzyme acquired the active conformation by a different mechanism. In the K+-independent apoenzyme of Mycobacterium tuberculosis, K72 (K117) flips out of the active site; in the holoenzyme, K72 faces toward the active site bridging the substrates through water molecules. The results provide evidence that two different mechanisms have evolved for the catalysis of this reaction.