Pyruvate Kinase Enzyme Activity Research Articles

Biochemical responses of the giant clam (Tridacna squamosa Lamarck, 1819) and the burrowing sea urchin (Echinometra mathaei (de Blainville, 1825)) to spatial and seasonal variations on the Red Sea coast of Egypt

This study aims to investigate the spatial and seasonal impacts on the biochemical responses of two bioindicators: Tridacna squamosa and Echinometra mathaei which inhabit the Red Sea coast of Egypt. Samples were collected from three sites during the summer of 2019 and the winter of 2020. Total protein, MDA, and enzyme activity of pyruvate kinase (PK) and lactate dehydrogenase (LDH) were measured in the gonads of the collected samples. The results confirmed that environmental variables vary at both spatial and seasonal levels, where the two species exhibited different patterns of biochemical response to internal and external stresses. In addition, both studied species did not resort to anaerobic respiration as a defense mechanism. Therefore, this study confirms that the use of biomarkers in the field shall be regarded as a general warning of internal and external stress.

Association of PKLR gene copy number, expression levels and enzyme activity with 2,3,7,8-TCDD exposure in individuals exposed to Agent Orange/Dioxin in Vietnam

2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) is the most toxic congener of dioxin and has serious long-term effects on the environment and human health. Pyruvate Kinase L/R (PKLR) gene expression levels and gene variants are associated with pyruvate kinase enzyme deficiency, which has been identified as the cause of several diseases linked to dioxin exposure. In this study, we estimated PKLR gene copy number and gene expression levels using real-time quantitative PCR (RT-qPCR) assays, genotyped PKLR SNP rs3020781 by Sanger sequencing, and quantified plasma pyruvate kinase enzyme activity in 100 individuals exposed to Agent Orange/Dioxin near Bien Hoa and Da Nang airfields in Vietnam and 100 healthy controls. The means of PKLR copy numbers and PKLR gene expression levels were significantly higher, while pyruvate kinase enzyme activity was significantly decreased in Agent Orange/Dioxin-exposed individuals compared to healthy controls (P < 0.0001). Positive correlations of PKLR gene copy number and gene expression with 2,3,7,8-TCDD concentrations were observed (r = 0.2, P = 0.045 and r = 0.54, P < 0.0001, respectively). In contrast, pyruvate kinase enzyme activity was inversely correlated with 2,3,7,8-TCDD concentrations (r = −0.52, P < 0.0001). PKLR gene copy number and gene expression levels were also inversely correlated with pyruvate kinase enzyme activity. Additionally, PKLR SNP rs3020781 was found to be associated with 2,3,7,8-TCDD concentrations and PKLR gene expression. In conclusion, PKLR copy number, gene expression levels, and pyruvate kinase enzyme activity are associated with 2,3,7,8-TCDD exposure in individuals living in Agent Orange/Dioxin-contaminated areas.

Impairments in SHMT2 expression or cellular folate availability reduce oxidative phosphorylation and pyruvate kinase activity

BackgroundSerine hydroxymethyltransferase 2 (SHMT2) catalyzes the reversible conversion of tetrahydrofolate (THF) and serine-producing THF-conjugated one-carbon units and glycine in the mitochondria. Biallelic SHMT2 variants were identified in humans and suggested to alter the protein’s active site, potentially disrupting enzymatic function. SHMT2 expression has also been shown to decrease with aging in human fibroblasts. Immortalized cell models of total SHMT2 loss or folate deficiency exhibit decreased oxidative capacity and impaired mitochondrial complex I assembly and protein levels, suggesting folate-mediated one-carbon metabolism (FOCM) and the oxidative phosphorylation system are functionally coordinated. This study examined the role of SHMT2 and folate availability in regulating mitochondrial function, energy metabolism, and cellular proliferative capacity in both heterozygous and homozygous cell models of reduced SHMT2 expression. In this study, primary mouse embryonic fibroblasts (MEF) were isolated from a C57Bl/6J dam crossed with a heterozygous Shmt2+/− male to generate Shmt2+/+ (wild-type) or Shmt2+/− (HET) MEF cells. In addition, haploid chronic myeloid leukemia cells (HAP1, wild-type) or HAP1 cells lacking SHMT2 expression (ΔSHMT2) were cultured for 4 doublings in either low-folate or folate-sufficient culture media. Cells were examined for proliferation, total folate levels, mtDNA content, protein levels of pyruvate kinase and PGC1α, pyruvate kinase enzyme activity, mitochondrial membrane potential, and mitochondrial function.ResultsHomozygous loss of SHMT2 in HAP1 cells impaired cellular folate accumulation and altered mitochondrial DNA content, formate production, membrane potential, and basal respiration. Formate rescued proliferation in HAP1, but not ΔSHMT2, cells cultured in low-folate medium. Pyruvate kinase activity and protein levels were impaired in ΔSHMT2 cells and in MEF cells exposed to low-folate medium. Mitochondrial biogenesis protein levels were elevated in Shmt2+/− MEF cells, while mitochondrial mass was increased in both homozygous and heterozygous models of SHMT2 loss.ConclusionsThe results from this study indicate disrupted mitochondrial FOCM impairs mitochondrial folate accumulation and respiration, mitochondrial formate production, glycolytic activity, and cellular proliferation. These changes persist even after a potentially compensatory increase in mitochondrial biogenesis as a result of decreased SHMT2 levels.

Effects of Fasting on Intermediary Metabolism Enzymes in the Liver and Muscle of Rainbow Trout

Fish can go through periods of feed deprivation, either due to natural causes influenced by temperature or management techniques in animal production, affecting their energy metabolism differently, depending on the duration of fasting in days and water temperature. This study showed the effect of different days of fasting before slaughter in rainbow trout by analyzing the intermediary metabolic enzymes in the muscle and liver. For this purpose, a non-fasting group was compared with trout that fasted for 5, 10, and 20 days (55, 107, and 200 degree days (°C d), respectively). A first phase of increased activity of enzymes involved in glycolysis was observed, increasing hexokinase enzyme activity in muscle and pyruvate kinase enzyme activity in muscle and liver. As the fasting days progressed, enzymes involved in the gluconeogenic and glycogenolytic pathways in the liver such as lactate dehydrogenase and glycogen phosphorylase increased their activity regarding the no fasted group. After 20 days of fasting (200 °C d), lipogenic and protein related enzyme activity depletion was observed, leading to significant changes in energy metabolism. Therefore, prolonged fasting with high degree days duration may compromise the energy supply for the vital development of rainbow trout.

Characterizing the Effect of the Lysine Deacetylation Modification on Enzyme Activity of Pyruvate Kinase I and Pathogenicity of Vibrio alginolyticus.

Pyruvate kinase I (PykF) is one of the key enzymes of glycolysis and plays a crucial role in bacterial metabolism. Several acetylation sites of Vibrio alginolyticus PykF were reported in previous studies and then 11 sites were first verified in this study, however, the specific roles of PykF acetylation remains unclear. Overlap-PCR and homologous recombination were implied to delete V. alginolyticus pykF gene and constructed complementary strains of site-directed mutagenesis for the further research focus on the deacetylation regulation on PykF. The results showed that the pyruvate kinase activity was sharply suppressed in the deacetylation status of K52, K68, and K317 of PykF, as well as the extracellular protease activity was significantly decreased in the deacetylation status of K52 and K68, but not induced with K317. Moreover, the growth rates of V. alginolyticus were not influenced with these three deacetylation sites. The ΔpykF mutant exhibited a 6-fold reduction in virulence to zebrafish. Site-directed mutations of K52R and K68R also showed reduced virulence while mutations of K317R didn't. The in vitro experiments showed that PykF was acetylated by acetyl phosphate (AcP), with the increase of incubation time by AcP, the acetylation level of PykF increased while the enzyme activity of PykF decreased correspondingly. Besides, PykF was deacetylated by CobB deacetylase and in result that the deacetylation was significantly down-regulated while the pyruvate kinase activity of PykF increased. Moreover, deletion of cobB gene had no significant difference in pyruvate kinase activity. These results confirm that CobB can regulate the acetylation level and pyruvate kinase activity of PykF. In summary, the results of this study provide a theoretical basis for further understanding of the deacetylation modification of PykF. It provides a new idea for the prevention and cure of vibriosis.

Identification of a Digenic Inheritance of Pklr and KLF1 Mutations Resulting in Hemolytic Anemia in a Neonate from a Study of Genotype-Phenotype Correlation of Globin, Pklr and KLF1 in Thai Patients with Pyruvate Kinase (PK) Deficiency

Introduction: Pyruvate kinase deficiency (PK def) is an inherited disorder that affects red blood cells metabolism and causes a wide range of clinical manifestation from fetal anemia, severe neonatal jaundice, and chronic transfusion dependent anemia to fully compensated hemolytic anemia. This condition is rarely reported in Southeast Asian population due to a lack of knowledge and recognition by clinicians and unavailability of laboratory for confirmation. The majority of patients with PK def result from mutations of the PKLR genes encoding this enzyme. Our group has described for the first time (Viprakasit V et al., Blood 2014) that mutations in trans of an erythroid specific transcription factor; KLF1 that regulate PKLR expression, could result in clinical presentation mimic that of classical PK def. Moreover, α and β globin mutations are highly prevalent in our region (up to 40% carrier rate) but their roles as a genetic modifier in PK def remain unknown. In addition, mutations in KLF1 seems to be highly prevalent in our population (14.6 in 1000, n= 2464 chr.), it is of interest to determine the epistatic role of α and β globin, PKLR and KLF1 mutations in patients with classical presentation of PK def.Objectives: To characterize α and β globin, PKLR and KLF 1 mutations and correlate these with clinical presentation and outcome(s) of them in patients with PK def and their family members.Methods: After obtaining informed consent, 9 index PK def patients with their available family members were evaluated for PKLR and KLF1 mutations by direct genomic sequencing as previously described. Hematological parameters and hemoglobin analyses were performed using standard techniques. DNA analysis for all common α and β globin mutations leading to thalassemia and hemoglobinopathies found in this region was performed using our standard protocol.Results: We successfully identified PKLR mutations in 7 out of 9 index patients as shown in Table 1. All patients were diagnosed with clinical PK def following several measurement of biochemical activity. We found 11 different mutations in 6 compound heterozygotes (5 of two nucleotide mutations and one with 1 missense and a large PKLR deletion) and 1 homozygote. One mutation (c.941T>C =p.I314T) seemed to be recurrent since it was found in two families; one homozygous and one compounded with N118S. We failed to identified PKLR and KLF1 in only one patient (PK-9) suggesting that this patient might have a novel molecular mechanism that remains to be found. We found only one family with globin abnormality (Hb E trait in PK-1) without any deleterious effect on patient's clinical severity. Moreover, most of PK patients (except one) have no mutation in KLF1 genes. Interestingly, the PK-3 patient (who is an offspring of the PK-2) was subsequently found to be double heterozygous of PKLR (p.V548fs) and KLF1 (G176RfsX179 (+7bp)) mutations. This patient was previously diagnosed with PK def at birth when he had serious hemolytic anemia (Hb 10 g/dL, haptoglobin < 10 IU/L) with hyperbilirubinemia requiring phototherapy, based on a very low PK enzyme activity (6.5 IU/g Hb, 20% of normal level) and the PK level from his mother was also low (11 IU/g Hb). Since then, he had an uneventful history for 6 years, except mild anemia from iron deficiency anemia during infant period. Physical examination showed no hepatosplenomegaly. Currently his baseline Hb levels were around 12-12.5 g/dL with normal reticulocyte count (1%) and hypochromic microcytosis. There is no globin abnormality in this family. The PK enzyme activity at 6-year-of age showed an improved PK activity (41% of normal activity) without further hemolysis. Our finding showed for the first time a possible interaction between PKLR and KLF1 mutations in man and this digenic inheritance seems to cause a more serious phenotype than each of these carriers alone. However, since the clinical presentation of our index case (PK-3) appeared to be apparent only at the early age, this might suggest the possible impact of KLF1 mutations that were more serious at the early age (similar to those of patients with two KLF1 mutations; KLF1 disease) and might be further compensated by other hitherto mechanisms during erythroid development.Conclusions: We found no effects of globin mutations on the clinical phenotypes of patients with classical PK def. However, a mutation in KLF1 could deteriorate clinical presentations even in a carrier of PKLR mutation at the early age. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genotype-Phenotype Correlation and Molecular Heterogeneity in Pyruvate Kinase Deficiency: Data from the PKD Natural History Study

Genotype-Phenotype Correlation and Molecular Heterogeneity in Pyruvate Kinase Deficiency: Data from the PKD Natural History Study

Erythroid and Cardiovascular High Altitude-Selected Haplotypes in Andean Aymaras and Tibetans

Humans migrating out of Africa encountered new conditions, including living at high altitude. Tibetans and Andean Aymaras have inhabited regions of 4,000 meters or more for ~44,000 and 14,000 years respectively (Hu et al, PLoS Genet 2017, Rademaker et al, Science 2014). There is a distinct difference in erythroid phenotypes: Aymaras are polycythemic at high altitude, while most Tibetans are not. Mutations providing an advantage in highlanders will improve fitness under hypoxic conditions,including modulation of erythropoiesis through the hypoxia-inducible factor (HIF) pathway. There are few shared, naturally-selected gene regions in Aymaras and Tibetans and these have different phenotypic associations (Bigham et al, Am J Hum Biol 2013). Aymara high-altitude selected haplotypes have not been published. Tibetan EPAS 1 (encoding HIF2a protein) haplotypes in part originated from ancient Denisovans and entered the Tibetan genome through introgression (Huerta-Sánchez et al, Nature 2014). Tibetan variant prolyl hydroxylase 2 (PHD2), a negative regulator of HIFs encoded by EGLN1 gene, encodes in cis both PHD2D4E and PHD2C127S which together have increased activity in hypoxia (Lorenzo et al, Nature Genetics 2014) and, together with a Tibetan EPAS 1 haplotype, protects from high altitude polycythemia (Tashi et al, JMM 2017). We and others previously identified other Tibetans haplotypes that are not unique but are enriched in Tibetans, including a “Tibetan” haplotype of PKLR encoding liver- and red cell-specific pyruvate kinase (PK) (Simonson et al, Science 2010, and Yi et al, Science 2010).We studied Tibetan-enriched haplotypes of EGLN1, EPAS1 and PKLR in 72 Bolivian Andean Aymaras, all residing at 4,000 m, and compared them with 347 Tibetans living at altitudes of 200 m and 4,300 m (Table). We genotyped PHD2D4E and PHD2C127S variants of EGLN1 and 10 Tibetan specific single nucleotide polymorphisms (SNPs) of EPAS1, 5 Denisovan and 5 non-Denisovan, each under different linkage disequilibrium (Hu et all, PLoS Genet 2017), 7 Tibetan enriched PKLR SNPs, and searched for Aymara selected variants by whole genome sequencing.The prevalence of the Tibetan-selected EGLN1 and EPAS1 haplotypes increased with increasing altitude of residence in Tibetans, suggesting a continuous evolutionary advantage (Tashi et al, JMM 2017). Aymaras did not have the PHD2D4E haplotype, and PHD2C127S was found at lower prevalence in heterozygote form. Aymaras shared two of five non-Denisovan EPAS1 SNPs selected in Tibetans; one, rs130005507 G allele, had a similar prevalence to Tibetans, but another, rs142764723 C allele, had a lower prevalence. We report that >90% of Tibetans and ~50% of Aymaras, but only ~10% of Europeans, have the “Tibetan ” PKLR haplotype. Aymara females with homozygous PKLR haplotypes have lower hemoglobins than heterozygotes (p=0.022). Further, the PKLR transcript in reticulocytes decreases with increasing altitude and this progressive decrease is even more pronounced in the “Tibetan” haplotype. We found Aymaras' selected haplotypes encoding BRINP3, NOS2, TBX5 ; these genes are associated with cardiovascular development and function but not hypoxia sensing. They are not enriched in Tibetans.We conclude that Aymara highlanders do not have the Tibetan PHD2D4E mutation or Denisovan-like EPAS1 variants. Furthermore, they share only two of five Tibetan non- Denisovan EPAS1 variants. The absence of these variants in Aymaras supports that Tibetan and Aymara high altitude inhabitants do not have the same ancestry, that they developed different evolutionary adaptations (Bigham et al, Am J Hum Biol 2013), and that Tibetans' EGLN1 and EPAS1 mutations are unique to that part of the world. We hypothesize that decreased PK enzyme activity would be expected to increase 2,3-diphosphoglycerate (2,3-DPG) (as is shown in people with PK enzyme deficiency) and thus be beneficial to high altitude adaptation by progressively augmenting tissue oxygen delivery with increasing altitude. Based on its association with lower hemoglobin in Aymara females, the selected “Tibetan” PKLR haplotype, present in about half of Aymaras, may contribute to their hypoxic adaptation. Additional evaluation of evolutionary selected genes including PKLR, BRINP3, NOS 2, and TBX5 and their functional consequences are in progress with Aymaras living at El Alto (4,150 m), Cochabamba (2,500 m) and Santa Cruz, (416m), Bolivia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Effect of Methyl Parathion on The Pyruvate Kinase Enzyme Activity

Metil paration, tarımda ve genel alanlarda zararlılara karşı kullanılan organofosfat grubu bir insektisittir. İnsektisitlerin çevre ve canlı organizmalar üzerinde önemli derecede toksik etkileri bulunmaktadır. İnsanlarda farklı toksisiteye neden olan Metil paration’un metabolizmayı kontrol eden önemli enzimleri etkilediği bilinmektedir. Allosterik özellik gösteren Piruvat kinaz (EC.2.7.1.40), glikolizin son enzimidir ve glikoliz ile devamındaki moleküler yolaklarda kontrolü sağlamaktadır. Çalışmamızda Metil paration’nun Piruvat kinaz enzim aktivitesi üzerine etkisi araştırıldı. Metil paration’un LD50 dozu (7mg.kg-1) intraperitoneal olarak farklı periyotlarla (2, 4, 8, 16, 32, 64 ve 72 saat) erkek ve dişi sıçanlara uygulandı. Sıçanların karaciğer, böbrek, beyin ve ince bağırsak dokularında Piruvat kinaz enzim aktivitesi analiz edildi. Metil paration maruziyeti sonucunda sıçan dokularında genel olarak Piruvat kinaz enzim aktivitesinde artışlar belirlendi. Bu aktivasyonların özellikle beyin dokusunda daha erken saatlerde gerçekleşmiş olması dikkat çekicidir. Karaciğer, böbrek ve ince bağırsak dokularında aktivasyonların dişilerde erkeklerden daha erken saatlerde olduğu gözlendi. Sonuç olarak bir insektisit olan Metil paration uygulamasının sıçan dokularında piruvat kinaz enzim aktivitesinde artışlara neden olduğu bulundu. Metil parationun glikoz metabolizmasını ve buna bağlı olarak diğer metabolik yolakları etkilediği tartışıldı. Bu nedenle Metil paration insektisitinin metabolizmayı etkilediği, farklı sağlık sorunlarına neden olabileceği düşünülmeli ve Dünya Sağlık örgütünün önerdiği uygulama dozuna dikkat edilerek güvenli kullanım için önlemler alınmalıdır.

Loss of SHMT2 and Folate Deficiency Impair Energy Metabolism in Mouse Embryonic Fibroblasts Cells

ObjectivesSerine hydroxymethyltransferase 2 (SHMT2) catalyzes the reversible conversion of tetrahydrofolate (THF) and serine producing THF-conjugated one-carbon units and glycine in the mitochondria. Recently biallelic SHMT2 variants were identified in humans and suggested to alter the protein’s active site, potentially disrupting enzymatic function. Indeed, patient fibroblasts exhibited impaired oxidative capacity, however, with no changes in oxidative phosphorylation protein levels. Furthermore, immortalized cells models of total SHMT2 loss or folate deficiency exhibit decreased oxidative capacity and impaired mitochondrial complex I assembly and protein levels suggesting folate-mediated one-carbon metabolism (FOCM) and the oxidative phosphorylation system are functionally coordinated. We have developed a mouse model of reduced Shmt2 expression that provides an appropriate reference model to further study the deleterious human SHMT2 variants and their connection to folate metabolism. MethodsPrimary mouse embryonic fibroblasts (MEF) were isolated from a C57Bl/6 dam crossed with a heterozygous Shmt2+/– (HET) male. MEF embryos were genotyped, and Shmt2+/+ (wild type) or Shmt2+/– MEF cells were cultured for 4 doubling in 2 nM (deficient) or 25 nM folate (sufficient) medium. Cells were examined for proliferation, total folate levels, mtDNA content, protein levels of pyruvate kinase and PGC1α, pyruvate kinase enzyme activity, and mitochondrial membrane potential and function. ResultsLoss of Shmt2 and folate deficiency significantly reduced cell proliferation and oxygen consumption rate. Total cellular folate levels and pyruvate kinase enzyme activity and protein levels were reduced with folate deficiency. While there were no changes in mitochondrial DNA content or complex 1 proteins NDUFA9 or ND3, mitochondrial membrane potential was impaired and PGC1α protein levels were elevated in Shmt2+/– MEF cells. ConclusionsOur results demonstrate that loss of SHMT2 impairs cellular proliferation and that SHMT2 expression and folate status impact cellular energy metabolism and mitochondrial health. Funding SourcesThis study was financially supported by a President’s Council of Cornell Women Award and Cornell University Division of Nutritional Sciences funds.

Addition of tea tree oil (Melaleuca alternifolia) in diet minimize biochemical disturbances in silver catfish Rhamdia quelen exposed to the antiparasitic amitraz

In the present study, we evaluated diets supplemented with Melaleuca alternifolia essential oil (tea tree oil - TTO) as a growth promoter and its possible hepatoprotective effects in Rhamdia quelen exposed to the insecticide amitraz. Six groups (8 per group, in triplicate) were tested: control A (basal diet), B (basal diet + TTO 1.0 mL/kg), C (basal diet +0.84 mL/kg amitraz), D (basal diet +1.19 mL/kg amitraz), E (basal diet +1.0 mL/kg TTO + 0.84 mL/kg amitraz) and F (basal diet + TTO 1.0 mL/kg + 1.19 mL/kg amitraz). Initially R. quelen received diets according to each group. After 14 days, four groups were exposed to two levels of amitraz for a period of 7 days, and were later transferred to amitraz-free water for recovery. We measured levels of reactive oxygen species (ROS) and lipoperoxidation (LOOH) in liver tissue, and activities of enzymes involved in energy metabolism, adenylate kinase (AK) and pyruvate kinase (PK), in addition to zootechnical indices such as length, weight body and fish weight gain. ROS and LOOH levels increased significantly (p < 0.05) in fish exposed to amitraz concentrations, especially in groups of fish not supplemented with TTO. By contrast, AK and PK enzyme activities were significantly lower (p < 0.05) for the groups exposed to amitraz compared to the unexposed group. Finally, fish supplemented with TTO showed better (p < 0.05) zootechnical performance. Addition of TTO minimizes biochemical disturbances in fish exposed to amitraz and serves as a natural additive to improve fish production.

Improving the laboratory diagnosis of pyruvate kinase deficiency.

Pyruvate kinase (PK) deficiency is an autosomal recessive disease caused by mutations in the PKLR gene, which reduce erythrocyte PK enzyme activity and result in decreased energy synthesis in red cells, causing haemolytic anaemia. Historically, the investigation into pyruvate kinase deficiency (PKD) has been led by a red cell enzyme assay determining PK enzyme activity per unit of haemoglobin. For our laboratory, the reference range was set by Beutler et al. in 1977 when the test was first established. The introduction of genetic testing permitted the creation of reference sample datasets, with positive controls having two pathogenic variants causing disease. This permitted re-assessment of the enzyme assay's sensitivity and specificity, and was used to reassess the reference range of the enzyme assay. Using sequenced samples, we have devised an enzyme assay, DNA testing workflow, which minimises false negative/positive results and improves the diagnostic efficiency. This combined enzyme-DNA testing strategy should improve the diagnostic accuracy whilst limiting the number of expensive DNA tests. During this evaluation, 10 novel genetic variants were identified and are described.

Effects of lycopene on skeletal muscle-fiber type and high-fat diet-induced oxidative stress

Increasing studies report that many natural products can participate in formation of muscle fibers. This study aimed to investigate the effect of lycopene on skeletal muscle-fiber type in vivo and in vitro. C2C12 myoblasts were used in vitro study, and the concentration of lycopene was 10 µM. In vivo, 8-week-old male C57/BL6 mice were used and divided into four groups (n=8): (1) ND: normal-fat diet; (2) ND+Lyc: normal-fat diet mixed with 0.33% w/w lycopene; (3) HFD: high-fat diet; and (4) HFD+Lyc: high-fat diet mixed with 0.33% w/w lycopene. The mice tissue samples were collected after 8 weeks feeding.We found that lycopene supplementation enhanced the protein expression of slow-twitch fiber, succinate dehydrogenase, and malic dehydrogenase enzyme activities, whereas lycopene reduced the protein expression of fast-twitch fibers, lactate dehydrogenase, pyruvate kinase enzyme activities. Moreover, lycopene can promote skeletal muscle triglyceride deposition, enhanced the mRNA expression of genes related to lipid synthesis, reduced the mRNA expression of genes related to lipolysis. And high-fat diet-induced dyslipidemia and oxidative stress were attenuated after lycopene supplementation. Additionally, lycopene supplementation reduced the glycolytic reserve but enhanced mitochondrial ATP production in C2C12 cells.These results demonstrated that lycopene affects the activities of metabolic enzymes in muscle fibers, promotes the expression of slow-twitch fibers, and enhanced mitochondrial respiratory capacity. We speculated that lycopene affects the muscle-fiber type through aerobic oxidation, suggesting that lycopene exerts potential beneficial effects on skeletal muscle metabolism.

Pyruvate Kinase (PK) Protein and Enzyme Levels in the Diagnosis and Clinical Phenotype of PK Deficiency

Background: Pyruvate Kinase (PK) deficiency is caused by mutations in the PKLR gene leading to chronic hereditary non-spherocytic hemolytic anemia. Diagnostic evaluation can be challenging with falsely normal PK enzyme activity levels in chronically transfused patients, in those with profound reticulocytosis, or in patients with mutations with unusual biochemical properties. Genetic testing can also be complex, as up to 20% of affected patients have new PKLR variants and up to 10% of patients have variants not routinely detected through exon sequencing. The phenotypic spectrum is broad, and biomarkers of clinical severity would be helpful for both prognosis and monitoring. Aims: To describe the correlation of PK enzyme activity, red cell PK (PK-R) protein level, and red cell metabolites [adenosine triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG)] with clinical phenotype. To estimate the sensitivity of PK enzyme activity to diagnose PK deficiency in a cohort of patients with genetically confirmed PK deficiency. Methods: Blood samples were collected from a subset of 41 patients enrolled in the PK Deficiency Natural History Study, all with two confirmed PKLR mutations and no red cell transfusions for ≥3 months prior to the blood sample. PK and hexokinase (HK) enzyme activity testing were performed using standard biochemical assays (Beutler 1985) at the Stanford Red Blood Cell Special Studies Laboratory (normal ranges: PK activity: 3.2-6.5 EU/gm Hb, HK activity: 0.14-0.37 EU/gm Hb). Baseline PK-R protein was quantitated by antibody-based capture and detection using a Meso Scale assay and the signal normalized to a control sample without PK deficiency. ATP and 2,3-DPG concentrations (μg/ml) in blood were analyzed using qualified tandem mass spectrometry methods and then normalized for individual hemoglobin (Hb) values (ATP and 2,3-DPG concentrations were converted to g/dL and divided by Hb (g/dL)). Spearman correlation coefficients were calculated to estimate the correlation between continuous variables, and Wilcoxon rank-sum testing was used to assess the association of continuous and binary variables. Results: The median age was 25.3 years (range 1.4-60.4) and 80% of patients were splenectomized (Table). The mean PK enzyme activity level was 1.1 EU/gm Hb with 90% (37/41) of patients with low PK activity. Normal PK activity was observed in 4 patients, 3 of whom had high HK activity and a low PK/HK ratio (normal mean PK/HK ratio 15.6, range: 8.7-22.5), with a sensitivity of 98% (40/41) when both PK activity and PK/HK ratio were used together. There were no correlations between PK enzyme activity and clinical severity indicators, including transfusion status (p=0.3), splenectomy status (p=0.4), or post-splenectomy Hb (r=0.109). Normalized ATP levels were significantly correlated with normalized 2,3-DPG levels (r=0.93) and higher in ever transfused (median=0.0022, range=0.0011-0.0029) compared with never transfused patients (median=0.0012 , range=0.0008-0.0020), (p=0.004). PK-R protein level was inversely correlated with the total number of transfusions prior to enrollment (r= -0.53) and was higher in never transfused (median=40.1%, range=9.8-73.9%) compared with ever transfused patients (median=7.7%, range=0.4%-15.1%), (p=0.0014). Conclusions: In this cohort with a molecularly confirmed diagnosis, PK deficiency was detected by enzyme testing with 98% sensitivity by utilizing both the PK activity and the PK/HK ratio. Although these assays cannot distinguish between heterozygotes and affected patients, the combination of PK activity and the PK/HK ratio is useful as a screening test to determine which patients require genetic testing. There was no correlation between PK enzyme activity and clinical phenotype. PK-R protein and ATP levels were inversely correlated with clinical severity and may be a helpful marker of disease phenotype. Disclosures Al-Samkari: Agios: Consultancy, Research Funding; Moderna: Consultancy; Dova: Consultancy, Research Funding. Glader:Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Chonat:Alexion: Other: advisory board; Agios Pharmaceuticals, Inc.: Other: advisory board. Thompson:bluebird bio, Inc.: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Baxalta: Research Funding. Kuo:Agios: Consultancy; Alexion: Consultancy, Honoraria; Apellis: Consultancy; Bioverativ: Other: Data Safety Monitoring Board; Bluebird Bio: Consultancy; Celgene: Consultancy; Novartis: Consultancy, Honoraria; Pfizer: Consultancy. Ravindranath:Agios Pharmaceuticals, Inc.: Other: I am site PI on several Agios-sponsored studies, Research Funding. Rothman:Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Agios: Honoraria, Research Funding. Kwiatkowski:Celgene: Consultancy; Terumo: Research Funding; Imara: Consultancy; Apopharma: Research Funding; Novartis: Research Funding; bluebird bio, Inc.: Consultancy, Research Funding; Agios: Consultancy. Kung:Agios Pharmaceuticals, Inc: Employment, Other: Stakeholder. Kosinski:Agios Pharmaceuticals, Inc: Employment, Other: Stakeholder. London:United Therapeutics: Consultancy; ArQule, Inc: Consultancy. Grace:Novartis: Research Funding; Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Decreased Activity and Stability of Pyruvate Kinase in Hereditary Hemolytic Anemia: A Potential Target for Therapy By AG-348 (Mitapivat), an Allosteric Activator of Red Blood Cell Pyruvate Kinase

Background: Reactive oxygen species (ROS) play an important role in the complex and multifactorial pathophysiology of hereditary hemolytic anemia like sickle cell disease (SCD), β-thalassemia and hereditary xerocytosis (HX). Increased intracellular levels of oxidative stress disrupt normal cell functioning and may contribute to premature red blood cell (RBC) clearance from the circulation. Pyruvate kinase (PK) is a key regulatory enzyme of glycolysis, the cell's main source of energy. Because PK is very sensitive to redox balance we hypothesized that increased levels of oxidative stress in SCD, β-thalassemia and HX impairs proper enzyme function, thereby compromizing RBC energy metabolism. This may contribute to disease pathophysiology. Aims: To investigate if secondary deficiency of PK is common in SCD, thalassemia, and HX, and to investigate if PK in these disorders is able to respond to treatment with the allosteric PK activator AG-348 (mitapivat). Methods: Enzymatic activities of red cell PK and hexokinase (HK) were measured together with PK-thermostability in order to assess relative PK activity and enzyme stability. Purified RBCs were incubated with AG-348 (3.33μM) for 24 hours after which PK activity and ATP response was measured. RBCs of SCD patients were also analyzed with the oxygenscan, a newly developed method that characterizes individual sickling behavior by oxygen gradient ektacytometry (Rab et al, Am J Hematol, 2019). Individual tendency to sickle is reflected by Point-of-Sickling (PoS) that indicates the specific pO2 at which RBCs start to sickle during deoxygenation under shear stress. Results: Thirty-eight patients and 21 healthy controls (HC) were included. The patient cohort consisted of patients homozygous for HbS (HbSS, n=26), patients compound heterozygous for HbS and HbC (HbSC, n=4), β-thalassemia major (regularly transfused, n=3), and hereditary xerocytosis (n=5). Patients showed reticulocytosis and, in line with this, a concomitant increase in HK activity. In contrast however, relative PK activity was decreased significantly compared to HK in HbSS, β-thalassemia and HX patients, but not in HbSC patients (Figure 1A). PK thermostability was significantly decreased compared to healthy controls in HbSS patients and patients with HX (Figure 1B). In HbSC and β-thalassemia patients, PK-thermostability was comparable to HC. PK thermostability strongly correlated with absolute reticulocyte count (ARC), indicating that patients displaying the highest degree of PK instability had the highest reticulocyte count (Figure 1C). This suggests that in general, a higher degree of PK instability is associated with more severe anemia due to a high hemolytic rate. In SCD patients, PK-thermostability inversely correlated with PoS, indicating that decreased PK stability is associated with sickling at higher pO2 (r=-0.646, p&amp;lt;0.001, Figure 1F). When purified RBCs were incubated with 3.33μM of the allosteric PK-activator AG-348, an increase in PK activity was seen in all patients and HCs, with a mean increase of 122% in HbSS (range 111-139%, n=6), 137% in β-thalassemia (n=1), 163% in HX (range 152-174%, n=2) and 143% in HC (range 113-173%, n=9, Figure 1E). Accordingly, ATP-levels increased in all patients and HCs, with a mean increase of 133% in HbSS (range 125-141%, n=5), 144% patient with β-thalassemia (n=1), 121% in HX (range 112-129, n=3), and 132% in HCs (range 101-149%, n=9, Figure 1E). Conclusion: PK enzyme activity and stability is compromised in patients with various forms of hereditary hemolytic anemia. This implies that PK stability and, hence, compromised red cell metabolism could contribute to the complex pathophysiology of these diseases. In SCD patients, reduced PK-thermostability is associated with higher PoS, which we previously have shown to be associated with more severe disease (Rab et al, Am J Hematol, 2019, ASH 2019 abstract ID128870). This is confirmed by the correlation of decreased PK-thermostability with increased reticulocyte count as presented in this study. Current studies are in progress to further substantiate the underlying mechanism(s) involved, and to investigate whether AG-348 may ameliorate clinical features such as hemolysis, sickling tendency and iron overload. Disclosures Rab: RR Mechatronics: Research Funding. Bos:RR Mechatronics: Research Funding. Kosinski:Agios Pharmaceuticals, Inc: Employment, Other: Stakeholder. Kung:Agios Pharmaceuticals, Inc: Employment, Other: Stakeholder. van Beers:Agios Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Research Funding; RR Mechatronics: Research Funding. van Wijk:Agios Pharmaceuticals: Consultancy, Research Funding; RR Mechatronics: Research Funding.

PS1519 DECREASED ACTIVITY AND STABILITY OF PYRUVATE KINASE IN SICKLE CELL DISEASE AND THALASSEMIA: A POTENTIAL TARGET FOR THERAPY

Background:Reactive oxygen species (ROS) play an important role in the complex and multifactorial pathophysiology of sickle cell disease (SCD) and thalassemia. In SCD, auto‐oxidation of red blood cell (RBC) hemoglobin produces high levels of ROS that exceed anti‐oxidant capacity. In β–thalassemia, auto‐oxidation of unpaired α‐globins produces high levels of ROS. Increased intracellular levels of oxidative stress disrupt normal cell functioning and may contribute to premature clearance from the circulation. Pyruvate kinase (PK) is a key regulatory enzyme of glycolysis, the cell's only source of energy. Because PK is very sensitive to redox balance we hypothesized that increased levels of oxidative stress in hemoglobinopathies impairs proper enzyme function and thereby compromizes RBC energy metabolism. This may contribute to disease pathophysiology.Aims:To investigate if secondary deficiency of PK is common in SCD and thalassemia, and to investigate if PK in these disorders is able to respond to treatment with the allosteric PK activator AG‐348.Methods:Enzymatic activities of red cell PK and hexokinase (HK) were measured together with PK thermal stability (residual activity of PK) in order to assess relative PK activity and enzyme stability. RBC lysates were incubated with AG‐348 (3.33 uM) for 2 hours after which PK thermal stability, PK activity and ATP response was measured. Red blood cells of SCD patients were also analyzed with the oxygenscan, a newly developed method that characterizes individual sickling behavior (Rab et al, Am J Hematol, 2019).Results:Twenty‐eight patients and 18 healthy controls (HC) were included. The patient cohort consisted of patients with HbSS (n = 16), HbS/β‐thalassemia (n = 1), β‐thalassemia major (n = 3, regularly transfused), and HbSC (n = 3). Patients showed reticulocytosis and a concomitant increase in HK activity. However, PK activity was relatively decreased significantly compared to HK in SCD and β‐thalassemia patients, but not in HbSC patients (Figure 1A). PK thermal stability also was significantly decreased in SCD patients compared to healthy controls. In HbSC and β‐thalassemia patients PK‐thermal stability was comparable to HC (Figure 1B). When purified RBCs were incubated with 3.33uM of the allosteric PK‐activator AG‐348 an increase in PK activity was seen in all patients and HCs, with a median of 122% in SCD (range 120–138%, n = 3), 127% in β‐thalassemia and 142% in HC (range 130–166%, n = 5, Figure 1D). Accordingly, ATP‐levels increased in all patients and HCs, with a median of 126% in SCD (range 121–129%, n = 3), 146% in 1 patient with β‐thalassemia, and 122% in HCs (range 100–148%). In SCD patients PK‐thermal stability inversely correlated significantly with oxygenscan parameters, in particular the point‐of‐sickling (PoS). This indicates that RBCs tend to sickle at a higher pO2 when PK is more unstable. Interestingly, the 3 patients with most stable PK are HbSC patients (2) and a HbS/dβ‐thalassemia patient with HbF levels of 35% (Figure 1C).Summary/Conclusion:PK enzyme activity and stability is compromised in patients with SCD and β‐thalassemia. In SCD, reduced PK thermal stability is associated with higher PoS, which is associated with more severe disease. Current studies are in progress to further substantiate the underlying mechanism(s), and to investigate whether AG‐348 may ameliorate pathophysiological features such as hemolysis and sickling tendency.image

Nutritional regulation of pyruvate kinase and phosphoenolpyruvate carboxykinase at the enzymatic and molecular levels in cobia Rachycentron canadum.

Despite being a carnivorous fish species, cobia (Rachycentron canadum) can utilize high levels of dietary carbohydrate (up to 360gkg-1). By contrast, rainbow trout (also carnivorous) cannot, due to the absence of molecular induction of glycolytic enzyme and inhibition of gluconeogenic enzyme gene expressions such as pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK). We hypothesized that this phenomenon is species-specific and will not be observed in cobia. Our results show that, at the molecular level, the mRNA abundance of the important glycolytic (PK) and gluconeogenic (PEPCK) enzymes in cobia liver are regulated by dietary carbohydrate-to-lipid (CHO:L) ratios and nutritional status (fed, unfed, and refed). Significantly upregulated hepatic PK and depressed PEPCK gene expressions were observed when the fish were fed with an increasing CHO/L-ratio diet or were refed. However, in contrast to gene expression, there was no significant effect of dietary CHO/L ratios on PK enzyme activity. The decrease in PEPCK activity was significantly found between low CHO/L ratio and high CHO/L ratio diets, whereas the moderate CHO/L ratio group showed intermediate values. But PEPCK activity appeared to be independent of nutritional status. These results suggest that nutritional regulation is obvious, at least at the molecular level, in the key hepatic enzymes (PK and PEPCK) of the glucose metabolism pathway, in response to different dietary CHO/L ratios and to the transition from being starved to fed. Determining whether other key enzymes involved in hepatic glucose metabolism contribute to glucose tolerance in cobia is necessary for further investigation of this phenomenon at the enzymatic and molecular levels.

The Spectrum of Genetic Defects in Indian Patients with Rare Congenital Anemias: Next Generation Sequencing Based Approach

IntroductionDiagnosis of inherited anemias is based on automated red cell indices, morphology and reticulocyte count in an appropriate clinical presentation and a stepwise diagnostic algorithm needs to be followed. Major inherited causes of hemolysis include the hemoglobinopathies, membranopathies and enzymopathies. In contrast, ineffective erythropoiesis, a term that refers to a disturbance in proliferation, differentiation and maturation of erythroblasts, includes megaloblastic anemia, myelodysplastic syndromes, thalassemias, and congenital dyserythropoietic anemias (CDA). Phenotypes vary from severe transfusion-dependent hemolytic anemia (HA) to fully compensated hemolysis. Patients are encountered where the entire spectrum of diagnostic tests cannot identify the etiology. Causal genes implicated are numerous, making a gene-by-gene approach time consuming, expensive and laborious. Hence, the use of targeted resequencing can expedite molecular diagnosis, genetic counseling and prenatal diagnosis if indicated.ObjectivesThis study aimed at determining the spectrum of mutations in uncommon HA and CDA.MethodsSixty-one patients (HA=44, CDA=17) with clinical and laboratory evidence suggestive of HA/CDA were enrolled after excluding common causes of anemias. Various biochemical and molecular tests were used to exclude glucose-6-phosphate dehydrogenase (G6PD) deficiency, hemoglobinopathies, autoimmune HA, hereditary spherocytosis and pyruvate kinase (PK) deficiency. Peripheral blood genomic DNA was extracted using QIAamp DNA Blood Midi Kit, quantified on NanoDrop 2000 spectrophotometer and Qubit® 2.0 Fluorometer. Common G6PD, PKLR variants, SEC23B mutation were excluded by molecular tests. Since the genetic defects in Indian patients with unexplained HA/CDA are not available, we first tested 16 patients with a comprehensive commercially available sequencing panel that covers 4,813 clinically-relevant genes. We next sought to streamline testing and designed a customized panel of 55 genes (Illumina). Variants were annotated and classified and the most likely disease-causing variants were validated by Sanger sequencing in the patient and available affected and unaffected family members.ResultsOut of 61 patient's sequenced, unexpected pyruvate kinase (PK) deficiency were found in 11 patients. PK enzyme activity assay was within normal limits in all these cases. Three patients with G6PD deficiency, 2 patients with glucose-6-phosphate isomerase deficiency and 1 case with hexokinase deficiency were found. Of 5 patients with stomatocytes on peripheral blood film, 3 had Mediterranean stomatocytosis/ macrothrombocytopenia (ABCG5/ABCG8) and 2 were found to have overhydrated hereditary stomatocytosis (RHAG).We also found 6 cases of HA to have a mutation in erythrocyte membrane protein-coding genes. Heterozygous nonsense mutation in the ANK1 gene was found in 2 cases and compound heterozygous missense mutations in ANK1 were seen in 1. Missense mutations were also found in SPTB gene in other 3 cases. Xerocytosis or dehydrated hereditary stomatocytosis (PEIZO1) was found in 3 patients.Seventeen cases for suspected CDA were studied and SEC23B mutations were present in 7. In one patient only heterozygous mutation was found. There could be the possibility of intronic mutation or long deletion/insertion in her. Notably, 6 patients with CDA showed mutations in PKLR (n=3), 1 each had mutations in MTRR, SPTB and PIEZO1 genes. We also found a mutation in GATA1 gene in a patient of CDA with thrombocytopenia.Conclusion(s)HA/CDA showed highly varied etiology. Our experience demonstrates the high diagnostic yield (>75%) of targeted resequencing for molecular diagnosis of unexplained anemias. It is cost effective and can expedite the diagnosis where conventional testing is not helpful. Timely detection of the etiology was helpful in genetic counseling. It not only offered therapeutic benefits to our patients but may help us in future antenatal diagnosis if required. Therapeutic implications include performing splenectomy which can ameliorate the anemia in selected subgroups of patients (HS, PK deficiency, CDA type II). DisclosuresNo relevant conflicts of interest to declare.

Discovery of Pyruvate Kinase as a Novel Target of New Fungicide Candidate 3-(4-Methyl-1,2,3-thiadiazolyl)-6-trichloromethyl-[1,2,4]-triazolo-[3,4- b][1,3,4]-thiadizole.

Target identification is an essential basis for novel-pesticide development in new molecular design and lead optimization. 3-(4-Methyl-1,2,3-thiadiazolyl)-6-trichloromethyl[1,2,4]triazolo[3,4- b][1,3,4]thiadizole (YZK-C22) is a novel fungicide candidate with specific antifungal activity. We investigated its mode of action, and our studies indicated that YZK-C22 showed no cross resistance against Saccharomyces cerevisiae mutants with classic fungicide targets. Mec1 and Rad53 are two kinases that respond to DNA-replication damage, and the efficacy test showed that YZK-C22 could not perform its fungicidal activity by inhibiting DNA repair. Target screening by drug-affinity-responsive target stability (DARTS) showed that pyruvate kinase (PK), a key enzyme in the glycolytic pathway, was the potent new fungicidal target of YZK-C22. Fifty-eight differentially expressed proteins (DEPs) primarily involved in the metabolic process were identified by isobaric tags for relative and absolute quantification analysis (iTRAQ) in S. cerevisiae, and protein expression in the citrate cycle decreased with treatment of 5 mg/L YZK-C22, which was consistent with the results of DARTS. Molecular-docking analysis further validated that YZK-C22 could dock into the active center of PK instead of phosphoenolpyruvate. The enzyme activity of PK from S. cerevisiae was competitively inhibited with a Ki of 3.33 ± 0.28 μmol/L, and the cell-growth inhibition of S. cerevisiae was released by supplementation with pyruvic acid, whereas the growth of S. cerevisiae was not recovered by adding PK's substrate (phosphoenolpyruvate) or allosteric regulator (fructose-1,6-bisphosphate). The present studies uncovered and validated the primary target of the new, potent fungicidal candidate YZK-C22; our results provide a successful, valuable, and applicable case of target discovery and identification for novel-fungicide development.

Construction of thermo-tolerant yeast based on an artificial protein quality control system (APQC) to improve the production of bio-ethanol

A major challenge for the production of bio-ethanol is to develop yeast that can sustain growth under high temperature. Here, we show the application of an artificial protein quality control (APQC) system to an industrial Saccharomyces cerevisiae strain for improving thermo-tolerance, a key trait for a bio-ethanol fermentation program. All engineered strains exhibited a significantly higher growth rate and cell viability at 37 °C, which conferred increased thermo-tolerance and more efficient glucose conversion to ethanol. Furthermore, the analysis of ubiquitination and the enzyme activities of pyruvate kinase (PK) and malate dehydrogenase (MDH) indicated strengthened protein quality, implying that proteostasis had been effectively improved at higher temperature. Industrial material fermentation results from yeast-RSP5-UBC4 (combined expression of ubiquitin conjugated enzyme E2 and ubiquitin ligase E3) showed a 2.4% increase in ethanol production. All these findings prove that APQC is suitable for improving yeast resistance to high temperature during industrial bio-ethanol production.