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British Journal of Haematology Mar 2021Diagnosis of pyruvate kinase deficiency (PKD), the most common cause of hereditary non-spherocytic haemolytic anaemia, remains challenging in routine practice and no... (Clinical Trial)
Clinical Trial
Diagnosis of pyruvate kinase deficiency (PKD), the most common cause of hereditary non-spherocytic haemolytic anaemia, remains challenging in routine practice and no biomarkers for clinical severity have been characterised. This prospective study enrolled 41 patients with molecularly confirmed PKD from nine North American centres to evaluate the diagnostic sensitivity of pyruvate kinase (PK) enzyme activity and PK:hexokinase (HK) enzyme activity ratio, and evaluate the erythrocyte PK (PK-R) protein level and erythrocyte metabolites as biomarkers for clinical severity. In this population not transfused for ≥90 days before sampling, the diagnostic sensitivity of the PK enzyme assay was 90% [95% confidence interval (CI) 77-97%], whereas the PK:HK ratio sensitivity was 98% (95% CI 87-100%). There was no correlation between PK enzyme activity and clinical severity. Transfusion requirements correlated with normalised erythrocyte ATP levels (r = 0·527, P = 0·0016) and PK-R protein levels (r = -0·527, P = 0·0028). PK-R protein levels were significantly higher in the never transfused [median (range) 40·1 (9·8-73·9)%] versus ever transfused [median (range) 7·7 (0·4-15·1)%] patients (P = 0·0014). The PK:HK ratio had excellent sensitivity for PK diagnosis, superior to PKLR exon sequencing. Given that the number of PKLR variants and genotype combinations limits prognostication based on molecular findings, PK-R protein level may be a useful prognostic biomarker of disease severity and merits further study.
Topics: Adolescent; Adult; Anemia, Hemolytic, Congenital Nonspherocytic; Biomarkers; Child; Child, Preschool; Erythrocytes; Female; Hexokinase; Humans; Infant; Male; Middle Aged; Pyruvate Kinase; Pyruvate Metabolism, Inborn Errors; Severity of Illness Index
PubMed: 32463523
DOI: 10.1111/bjh.16724 -
Nucleic Acids Research Jul 2023In light of the numerous studies identifying post-transcriptional regulators on the surface of the endoplasmic reticulum (ER), we asked whether there are factors that...
In light of the numerous studies identifying post-transcriptional regulators on the surface of the endoplasmic reticulum (ER), we asked whether there are factors that regulate compartment specific mRNA translation in human cells. Using a proteomic survey of spatially regulated polysome interacting proteins, we identified the glycolytic enzyme Pyruvate Kinase M (PKM) as a cytosolic (i.e. ER-excluded) polysome interactor and investigated how it influences mRNA translation. We discovered that the PKM-polysome interaction is directly regulated by ADP levels-providing a link between carbohydrate metabolism and mRNA translation. By performing enhanced crosslinking immunoprecipitation-sequencing (eCLIP-seq), we found that PKM crosslinks to mRNA sequences that are immediately downstream of regions that encode lysine- and glutamate-enriched tracts. Using ribosome footprint protection sequencing, we found that PKM binding to ribosomes causes translational stalling near lysine and glutamate encoding sequences. Lastly, we observed that PKM recruitment to polysomes is dependent on poly-ADP ribosylation activity (PARylation)-and may depend on co-translational PARylation of lysine and glutamate residues of nascent polypeptide chains. Overall, our study uncovers a novel role for PKM in post-transcriptional gene regulation, linking cellular metabolism and mRNA translation.
Topics: Humans; Glutamates; Lysine; Poly ADP Ribosylation; Protein Biosynthesis; Proteomics; Pyruvate Kinase; Ribosomes
PubMed: 37224531
DOI: 10.1093/nar/gkad440 -
RNA (New York, N.Y.) Nov 2022About three decades ago, researchers suggested that metabolic enzymes participate in cellular processes that are unrelated to their catalytic activity, and the term... (Review)
Review
About three decades ago, researchers suggested that metabolic enzymes participate in cellular processes that are unrelated to their catalytic activity, and the term "moonlighting functions" was proposed. Recently developed advanced technologies in the field of RNA interactome capture now unveil the unexpected RNA binding activity of many metabolic enzymes, as exemplified here for the enzymes of glycolysis. Although for most of these proteins a precise binding mechanism, binding conditions, and physiological relevance of the binding events still await in-depth clarification, several well explored examples demonstrate that metabolic enzymes hold crucial functions in post-transcriptional regulation of protein synthesis. This widely conserved RNA-binding function of glycolytic enzymes plays major roles in controlling cell activities. The best explored examples are glyceraldehyde 3-phosphate dehydrogenase, enolase, phosphoglycerate kinase, and pyruvate kinase. This review summarizes current knowledge about the RNA-binding activity of the ten core enzymes of glycolysis in plant, yeast, and animal cells, its regulation and physiological relevance. Apparently, a tight bidirectional regulation connects core metabolism and RNA biology, forcing us to rethink long established functional singularities.
Topics: Animals; Glyceraldehyde-3-Phosphate Dehydrogenases; Glycolysis; Phosphoglycerate Kinase; Pyruvate Kinase; RNA; Saccharomyces cerevisiae; Transcription, Genetic
PubMed: 35973722
DOI: 10.1261/rna.079210.122 -
Nature Jan 2019Endothelial nitric oxide synthase (eNOS) is protective against kidney injury, but the molecular mechanisms of this protection are poorly understood. Nitric oxide-based...
Endothelial nitric oxide synthase (eNOS) is protective against kidney injury, but the molecular mechanisms of this protection are poorly understood. 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. 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). Mammals possess a functional homologue of yeast SCoR, an aldo-keto reductase family member (AKR1A1) 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.
Topics: Acute Kidney Injury; Aldehyde Reductase; Animals; Cell Line; Coenzyme A; Female; Glycolysis; HEK293 Cells; Humans; Kidney Tubules, Proximal; Male; Metabolic Engineering; Mice; Mutation; Nitric Oxide Synthase Type III; Oxidation-Reduction; Oxidoreductases; Pentose Phosphate Pathway; Protein Multimerization; Pyruvate Kinase
PubMed: 30487609
DOI: 10.1038/s41586-018-0749-z -
BioMed Research International 2015In the early twentieth century, Otto Heinrich Warburg described an elevated rate of glycolysis occurring in cancer cells, even in the presence of atmospheric oxygen (the... (Review)
Review
In the early twentieth century, Otto Heinrich Warburg described an elevated rate of glycolysis occurring in cancer cells, even in the presence of atmospheric oxygen (the Warburg effect). Recently it became a therapeutically interesting strategy and is considered as an emerging hallmark of cancer. Hypoxia inducible factor-1 (HIF-1) is one of the key transcription factors that play major roles in tumor glycolysis and could directly trigger Warburg effect. Thus, how to inhibit HIF-1-depended Warburg effect to assist the cancer therapy is becoming a hot issue in cancer research. In fact, HIF-1 upregulates the glucose transporters (GLUT) and induces the expression of glycolytic enzymes, such as hexokinase, pyruvate kinase, and lactate dehydrogenase. So small molecules of natural origin used as GLUT, hexokinase, or pyruvate kinase isoform M2 inhibitors could represent a major challenge in the field of cancer treatment. These compounds aim to suppress tumor hypoxia induced glycolysis process to suppress the cell energy metabolism or enhance the susceptibility of tumor cells to radio- and chemotherapy. In this review, we highlight the role of natural compounds in regulating tumor glycolysis, with a main focus on the glycolysis under hypoxic tumor microenvironment.
Topics: Animals; Cell Hypoxia; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Glucose Transport Proteins, Facilitative; Glycolysis; Hexokinase; Humans; Hypoxia-Inducible Factor 1; Neoplasm Proteins; Pyruvate Kinase; Tumor Microenvironment
PubMed: 25685782
DOI: 10.1155/2015/354143 -
Developmental Cell Oct 2017Cellular survival during periods of acute environmental stress is essential for single-celled organisms. Reporting in Nature Cell Biology, Saad et al. (2017) identify... (Review)
Review
Cellular survival during periods of acute environmental stress is essential for single-celled organisms. Reporting in Nature Cell Biology, Saad et al. (2017) identify reversible aggregation of the metabolic enzyme pyruvate kinase under environmental stress and propose a method to protect and store proteins for immediate use post-stress.
Topics: Animals; Cell Survival; Eating; Humans; Proteins; Pyruvate Kinase; Stress, Physiological; Yeasts
PubMed: 29017028
DOI: 10.1016/j.devcel.2017.09.015 -
The New England Journal of Medicine Apr 2008Malaria that is caused by Plasmodium falciparum is a significant global health problem. Genetic characteristics of the host influence the severity of disease and the...
Malaria that is caused by Plasmodium falciparum is a significant global health problem. Genetic characteristics of the host influence the severity of disease and the ultimate outcome of infection, and there is evidence of coevolution of the plasmodium parasite with its host. In humans, pyruvate kinase deficiency is the second most common erythrocyte enzyme disorder. Here, we show that pyruvate kinase deficiency provides protection against infection and replication of P. falciparum in human erythrocytes, raising the possibility that mutant pyruvate kinase alleles may confer a protective advantage against malaria in human populations in areas where the disease is endemic.
Topics: Adult; Animals; Erythrocytes; Female; Genetic Predisposition to Disease; Humans; Malaria, Falciparum; Male; Mutation; Phagocytosis; Plasmodium falciparum; Polymorphism, Single Nucleotide; Pyruvate Kinase
PubMed: 18420493
DOI: 10.1056/NEJMoa072464 -
International Journal of Molecular... Jan 2022Eukarya pyruvate kinases possess glutamate at position 117 (numbering of rabbit muscle enzyme), whereas bacteria have either glutamate or lysine. Those with E117 are...
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 , 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.
Topics: Amino Acid Sequence; Animals; Apoenzymes; Binding Sites; Catalysis; Catalytic Domain; Glutamic Acid; Lysine; Models, Molecular; Mycobacterium tuberculosis; Phylogeny; Potassium; Protein Conformation; Pyruvate Kinase; Rabbits
PubMed: 35163274
DOI: 10.3390/ijms23031347 -
International Journal of Molecular... Jun 2014Cancer metabolism reprogramming or alterations in epigenetics are linked to an incidence of cancer. It is apparent that epigenetic changes have been found in tumors,... (Review)
Review
Cancer metabolism reprogramming or alterations in epigenetics are linked to an incidence of cancer. It is apparent that epigenetic changes have been found in tumors, therefore, the complete epigenome and entire pathways relevant to cell metabolism are subject to epigenetic dysregulation. Here, we review the pyruvate kinase M2 (PKM2) isoform, a glycolytic enzyme involved in ATP generation and pyruvate production, which plays an essential role in tumor metabolism and growth, and also functions as a protein kinase that phosphorylates histones during genes transcription and chromatin remodeling. We also discuss the potential role of PKM2 in the dynamic integration between metabolic reprogramming and alterations in epigenetics during carcinogenesis and cancer progression.
Topics: Animals; Carcinogenesis; Cellular Reprogramming; Chromatin Assembly and Disassembly; Energy Metabolism; Humans; Pyruvate Kinase
PubMed: 24972138
DOI: 10.3390/ijms150711435 -
The Biochemical Journal May 1999Pyruvate carboxylase (PC; EC 6.4.1.1), a member of the biotin-dependent enzyme family, catalyses the ATP-dependent carboxylation of pyruvate to oxaloacetate. PC has been... (Review)
Review
Pyruvate carboxylase (PC; EC 6.4.1.1), a member of the biotin-dependent enzyme family, catalyses the ATP-dependent carboxylation of pyruvate to oxaloacetate. PC has been found in a wide variety of prokaryotes and eukaryotes. In mammals, PC plays a crucial role in gluconeogenesis and lipogenesis, in the biosynthesis of neurotransmitter substances, and in glucose-induced insulin secretion by pancreatic islets. The reaction catalysed by PC and the physical properties of the enzyme have been studied extensively. Although no high-resolution three-dimensional structure has yet been determined by X-ray crystallography, structural studies of PC have been conducted by electron microscopy, by limited proteolysis, and by cloning and sequencing of genes and cDNA encoding the enzyme. Most well characterized forms of active PC consist of four identical subunits arranged in a tetrahedron-like structure. Each subunit contains three functional domains: the biotin carboxylation domain, the transcarboxylation domain and the biotin carboxyl carrier domain. Different physiological conditions, including diabetes, hyperthyroidism, genetic obesity and postnatal development, increase the level of PC expression through transcriptional and translational mechanisms, whereas insulin inhibits PC expression. Glucocorticoids, glucagon and catecholamines cause an increase in PC activity or in the rate of pyruvate carboxylation in the short term. Molecular defects of PC in humans have recently been associated with four point mutations within the structural region of the PC gene, namely Val145-->Ala, Arg451-->Cys, Ala610-->Thr and Met743-->Thr.
Topics: Animals; Evolution, Molecular; Gene Expression Regulation, Enzymologic; Gluconeogenesis; Humans; Lipids; Pyruvate Kinase; Signal Transduction
PubMed: 10229653
DOI: 10.1042/bj3400001