Phosphoglycerate Kinase Activity Research Articles

Physiological Mechanisms of BvCPD Regulation in Sugar Beet Growth

Sugar beet is an important sugar crop, and its roots are mainly used for processing raw materials to produce products such as sugar, molasses, and saccharin, as well as being used as fodder for livestock. BvCPD, a key enzyme gene for brassinosteroid (BR) synthesis, regulates the development of parenchyma cells and vascular bundles by promoting BR synthesis, which promotes the expansion of the sugar beet taproot and influences the growth, development, and yield of sugar beets. This study investigated the impact of BvCPD on the physiological metabolism of sugar beet utilizing BvCPD overexpression, silent, and wild-type (WT) lines. BvCPD increased the chlorophyll content and maximum photochemical efficiency and improved the photosynthetic characteristics of sugar beet leaves. Simultaneously, BvCPD increased the rate of sugar beet taproot respiration and ATP content by enhancing the activities of phosphoglycerate kinase, alcohol dehydrogenase, sucrose synthase, and sucrose synthase catabolism. Moreover, BvCPD induced changes in the sugar fraction content, which increased the sugar yield of a single plant. In addition, BvCPD promoted water absorption, nitrogen accumulation, and lignin/cellulose synthesis activities, facilitated by increased activities of phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, cellulose synthase, and protein serine/threonine phosphatases, providing the requisite energy and materials for sugar beet growth. These findings not only provide a new perspective for understanding the physiological mechanisms regulating the growth of sugar beets but also provide a theoretical basis for the future improvement of sugar beet varieties through molecular breeding techniques.

A model for stimulation of enzyme activity by a competitive inhibitor based on the interaction of terazosin and phosphoglycerate kinase 1

The drug terazosin (TZ) binds to and can enhance the activity of the glycolytic enzyme phosphoglycerate kinase 1 (PGK1) and can increase ATP levels. That finding prompted studies of TZ in Parkinson's disease (PD) in which decreased neuronal energy metabolism is a hallmark feature. TZ was neuroprotective in cell-based and animal PD models and in large epidemiological studies of humans. However, how TZ might increase PGK1 activity has remained a perplexing question because structural data revealed that the site of TZ binding to PGK1 overlaps with the site of substrate binding, predicting that TZ would competitively inhibit activity. Functional data also indicate that TZ is a competitive inhibitor. To explore the paradoxical observation of a competitive inhibitor increasing enzyme activity under some conditions, we developed a mass action model of TZ and PGK1 interactions using published data on PGK1 kinetics and the effect of varying TZ concentrations. The model indicated that TZ-binding introduces a bypass pathway that accelerates product release. At low concentrations, TZ binding circumvents slow product release and increases the rate of enzymatic phosphotransfer. However, at high concentrations, TZ inhibits PGK1 activity. The model explains stimulation of enzyme activity by a competitive inhibitor and the biphasic dose-response relationship for TZ and PGK1 activity. By providing a plausible mechanism for interactions between TZ and PGK1, these findings may aid development of TZ or other agents as potential therapeutics for neurodegenerative diseases. The results may also have implications for agents that interact with the active site of other enzymes.

Calcium signal regulated carbohydrate metabolism in wheat seedlings under salinity stress.

This study aimed to explore the mechanism by which calcium (Ca) signal regulated carbohydrate metabolism and exogenous Ca alleviated salinity toxicity. Wheat seedlings were treated with sodium chloride (NaCl, 150mM) alone or combined with 500μM calcium chloride (CaCl2), lanthanum chloride (LaCl3) and/or ethylene glycol tetraacetic acid (EGTA) to primarily analyse carbohydrate starch and sucrose metabolism, as well as Ca signaling components. Treatment with NaCl, EGTA, or LaCl3 alone retarded wheat-seedling growth and decreased starch content accompanied by weakened ribulose-1,5-bisphosphate carboxylation/oxygenase (Rubisco) and Rubisco activase activities, as well as enhanced glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, alpha-amylase, and beta-amylase activities. However, it increased the sucrose level, up-regulated the sucrose phosphate synthase (SPS) and sucrose synthase (SuSy) activities and TaSPS and TaSuSy expression together, but down-regulated the acid invertase (SA-Inv) and alkaline/neutral invertase (A/N-Inv) activities and TaSA-Inv and TaA/N-Inv expression. Except for unchanged A/N-Inv activities and TaA/N-Inv expression, adding CaCl2 effectively blocked the sodium salt-induced changes of these parameters, which was partially eliminated by EGTA or LaCl3 presence. Furthermore, NaCl treatment also significantly inhibited Ca-dependent protein kinases and Ca2+-ATPase activities and their gene expression in wheat leaves, which was effectively relieved by adding CaCl2. Taken together, CaCl2 application effectively alleviated the sodium salt-induced retardation of wheat-seedling growth by enhancing starch anabolism and sucrose catabolism, and intracellular Ca signal regulated the enzyme activities and gene expression of starch and sucrose metabolism in the leaves of sodium salt-stressed wheat seedlings.

Importance of aspartate 4 in the Mg2+ dependent regulation of Leishmania major PAS domain-containing phosphoglycerate kinase

Magnesium is an important divalent cation for the regulation of catalytic activity. Recently, we have described that the Mg2+ binding through the PAS domain inhibits the phosphoglycerate kinase (PGK) activity in PAS domain-containing PGK from Leishmania major (LmPAS-PGK) at neutral pH 7.5, but PGK activity is derepressed at acidic pH 5.5. The acidic residue within the PAS domain of LmPAS-PGK is expected to bind the cofactor Mg2+ ion at neutral pH, but which specific acidic residue(s) is/are responsible for the Mg2+ binding is still unknown. To identify the residues, we exploited mutational studies of all acidic (twelve Asp/Glu) residues in the PAS domain for plausible Mg2+ binding. Mg2+ ion-dependent repression at pH 7.5 is withdrawn by substitution of Asp-4 with Ala, whereas other acidic residue mutants (D16A, D22A, D24A, D29A, D43A, D44A, D60A, D63A, D77A, D87A, and E107A) showed similar features compared to the wild-type protein. Fluorescence spectroscopic studies and isothermal titration calorimetry analysis showed that the Asp-4 is crucial for Mg2+ binding in the absence of both PGK's substrates. These results suggest that Asp-4 residue in the regulatory (PAS) domain of wild type enzymes is required for Mg2+ dependent repressed state of the catalytic PGK domain at neutral pH.

Hydrocortisone-Containing Animal-Derived Food Intake Affects Lipid Nutrients Utilization.

As the tremendous increases in consumption of animal-derived food, endogenous hydrocortisone migrating along the food chain to organism arouses extensive attention. This study aims to investigate the cumulative impacts of dietary hydrocortisone intake and mechanistic understanding on metabolism of lipid nutrients. A total of 120 porcine muscles samples with different concentrations of hydrocortisone are collected at three time points. An operational food chain simulation framework is constructed and 175 lipid molecules are identified by UHPLC-Q-Orbitrap HRMS. Compared to the control group, 66 lipid molecules are significantly different, including 17 triglycerides and 31 glycerophospholipids. Integrated analyses of lipidomics and proteomics indicate that hydrocortisone promotes adipose triglyceride lipase and hormone sensitive lipase activity to precondition for triglycerides hydrolysis. Quantitative lipidomics analysis shows the presence of hydrocortisone decreases the concentration of docosahexaenoic acid (3.66 ± 0.15-3.09 ± 0.12mgkg-1 ) and eicosapentanoic acid (0.54 ± 0.09-0.48 ± 0.06mgkg-1 ). A noteworthy increase of most saturated triglycerides concentration with the prolonging of time is observed. Hydrocortisone originating from animal-derived food induces glycerophospholipids degradation and triglycerides hydrolysis through promoting adipose triglyceride lipase, hormone sensitive lipase, and phosphoglycerate kinase activity and further intervenes lipid nutrients utilization.

Control of protein activity by photoinduced spin polarized charge reorganization

Considerable electric fields are present within living cells, and the role of bioelectricity has been well established at the organismal level. Yet much remains to be learnedabout electric-field effects on protein function. Here, we use phototriggered charge injection from a site-specifically attached ruthenium photosensitizer to directly demonstrate the effect of dynamic charge redistribution within a protein. We find that binding of an antibody to phosphoglycerate kinase (PGK) is increased twofold under illumination. Remarkably, illumination is found to suppress the enzymatic activity of PGK by a factor as large as three. These responses are sensitive to the photosensitizer position on the protein. Surprisingly, left (but not right) circularly polarized light elicits these responses, indicating that the electrons involved in the observed dynamics are spin polarized, due to spin filtration by protein chiral structures. Our results directly establish the contribution of electrical polarization as an allosteric signal within proteins. Future experiments with phototriggered charge injection will allow delineation of charge rearrangement pathways within proteins and will further depict their effects on protein function.

Impaired age-dependent increases in phosphoglycerate kinase activity in red blood cells of Parkinson's disease patients

BackgroundImpaired bioenergetics are partially involved in the pathogenesis of Parkinson's disease (PD). Phosphoglycerate kinase (PGK), an essential enzyme for glycolysis, has recently attracted attention due to its pathogenic role in PD and as a target for disease-modifying therapies. This study is aimed to evaluate the profiles of PGK activity in red blood cells (RBCs) of PD patients and controls. MethodsSixty-eight PD patients and thirty-four age-matched unrelated controls were enrolled. PGK activities of RBCs were measured by the established colorimetric assay and standardized by the same RBC samples. ResultsPGK activity of the PD group was significantly higher than that of the control group in participants aged sixty-five years or younger, whereas it was not significantly different between the two groups at any age. PGK activity was positively correlated with aging in the control group, but this was not noted in the PD group. On multivariable analysis by partial correlation in the PD group, PGK activity was negatively correlated with the specific binding ratio of dopamine transporter scintigraphy in the striatum. The levodopa-equivalent daily dose was not significantly correlated with the enzyme activity. ConclusionThe results support the following: 1) elevation of PGK activities in RBCs can be detected in relatively young PD patients and with normal aging; 2) the degree of striatonigral degeneration is associated with elevated PGK activities. These are important considerations when the PGK assay is applied as a diagnostic biomarker of PD and to therapeutically monitor PGK-enhancing treatments.

A nitrogen stress-inducible small RNA regulates CO2 fixation in Nostoc.

In the absence of fixed nitrogen, some filamentous cyanobacteria differentiate heterocysts, specialized cells devoted to fixing atmospheric nitrogen (N2). This differentiation process is controlled by the global nitrogen regulator NtcA and involves extensive metabolic reprogramming, including shutdown of photosynthetic CO2 fixation in heterocysts, to provide a microaerobic environment suitable for N2 fixation. Small regulatory RNAs (sRNAs) are major post-transcriptional regulators of gene expression in bacteria. In cyanobacteria, responding to nitrogen deficiency involves transcribing several nitrogen-regulated sRNAs. Here, we describe the participation of nitrogen stress-inducible RNA 4 (NsiR4) in post-transcriptionally regulating the expression of two genes involved in CO2 fixation via the Calvin cycle: glpX, which encodes bifunctional sedoheptulose-1,7-bisphosphatase/fructose-1,6-bisphosphatase (SBPase), and pgk, which encodes phosphoglycerate kinase (PGK). Using a heterologous reporter assay in Escherichia coli, we show that NsiR4 interacts with the 5′-untranslated region (5′-UTR) of glpX and pgk mRNAs. Overexpressing NsiR4 in Nostoc sp. PCC 7120 resulted in a reduced amount of SBPase protein and reduced PGK activity, as well as reduced levels of both glpX and pgk mRNAs, further supporting that NsiR4 negatively regulates these two enzymes. In addition, using a gfp fusion to the nsiR4 promoter, we show stronger expression of NsiR4 in heterocysts than in vegetative cells, which could contribute to the heterocyst-specific shutdown of Calvin cycle flux. Post-transcriptional regulation of two Calvin cycle enzymes by NsiR4, a nitrogen-regulated sRNA, represents an additional link between nitrogen control and CO2 assimilation.

Metabolic protein phosphoglycerate kinase 1 confers lung cancer migration by directly binding HIV Tat specific factor 1

Phosphoglycerate kinase (PGK) is involved in glycolytic and various metabolic events. Dysfunction of PGK may induce metabolic reprogramming and the Warburg effect. In this study, we demonstrated that PGK1, but not PGK2, may play a key role in tumorigenesis and is associated with metastasis. We observed an inverse correlation between PGK1 and the survival rate in several clinical cohorts through bioinformatics statistical and immunohistochemical staining analyses. Surprisingly, we found that PGK1 was significantly increased in adenocarcinoma compared with other subtypes. Thus, we established a PGK1-based proteomics dataset by a pull-down assay. We further investigated HIV-1 Tat Specific Factor 1 (HTATSF1), a potential binding partner, through protein–protein interactions. Then, we confirmed that PGK1 indeed bound to HTATSF1 by two-way immunoprecipitation experiments. In addition, we generated several mutant clones of PGK1 through site-directed mutagenesis, including mutagenesis of the N-terminal region, the enzyme catalytic domain, and the C-terminal region. We observed that even though the phosphoglycerate kinase activity had been inhibited, the migration ability induced by PGK1 was maintained. Moreover, our immunofluorescence staining also indicated the translocation of PGK1 from the cytoplasm to the nucleus and its colocalization with HTATSF1. From the results presented in this study, we propose a novel model in which the PGK1 binds to HTATSF1 and exerts functional control of cancer metastasis. In addition, we also showed a nonenzymatic function of PGK1.

Global mapping of protein\u2013metabolite interactions in Saccharomyces cerevisiae reveals that Ser-Leu dipeptide regulates phosphoglycerate kinase activity

Protein–metabolite interactions are of crucial importance for all cellular processes but remain understudied. Here, we applied a biochemical approach named PROMIS, to address the complexity of the protein–small molecule interactome in the model yeast Saccharomyces cerevisiae. By doing so, we provide a unique dataset, which can be queried for interactions between 74 small molecules and 3982 proteins using a user-friendly interface available at https://promis.mpimp-golm.mpg.de/yeastpmi/. By interpolating PROMIS with the list of predicted protein–metabolite interactions, we provided experimental validation for 225 binding events. Remarkably, of the 74 small molecules co-eluting with proteins, 36 were proteogenic dipeptides. Targeted analysis of a representative dipeptide, Ser-Leu, revealed numerous protein interactors comprising chaperones, proteasomal subunits, and metabolic enzymes. We could further demonstrate that Ser-Leu binding increases activity of a glycolytic enzyme phosphoglycerate kinase (Pgk1). Consistent with the binding analysis, Ser-Leu supplementation leads to the acute metabolic changes and delays timing of a diauxic shift. Supported by the dipeptide accumulation analysis our work attests to the role of Ser-Leu as a metabolic regulator at the interface of protein degradation and central metabolism.

Dietary cinnamaldehyde supplementation improves the growth performance, oxidative stability, immune function, and meat quality in finishing pigs

The objective of this study was to evaluate the effects of different concentrations of cinnamaldehyde in feed on growth performance, oxidative stability, immune function, and meat quality in finishing pigs. Sixty pigs (Duroc × Landrace × Yorkshire; body weight = 70.10 ± 3.01 kg) were used in this study. The pigs were randomly allotted to three treatments (four replicates (pens) with five pigs per pen), control diet (basal diet), basal diet + 40 mg cinnamaldehyde and basal diet + 80 mg cinnamaldehyde per kg of feed. The results showed that dietary cinnamaldehyde increased pigs’ average daily gain(ADG), M. longissimus dorsi area(LA), pH24h, water maintain rate and serum interleukin 2 (IL-2)content (P <0.05), and decreased average backfat thickness, shear force, intramuscular fat, marbling Score, glycolytic potential (P <0.05). Cinnamaldehyde significantly increased glutathione peroxidase (GSH-Px) (P <0.05), while malondialdehyde (MDA) content and superoxide dismutase (SOD) enzyme activity decreased (P <0.05). In addition, cinnamaldehyde decreased the activities of glycolytic enzymes phosphoglycerate kinase (PGK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (P <0.05).These results demonstrated that dietary cinnamaldehyde could improve growth performance and ameliorate meat quality of finishing pigs by improving the living immunity, enhancing antioxidant capacity, and reducing glycolytic capacity of meat.

Regulation of Leishmaniamajor PAS domain-containing phosphoglycerate kinase by cofactor Mg2+ ion at neutral pH.

Recently, we described the PAS domain-containing phosphoglycerate kinase (PGK) from Leishmaniamajor (LmPAS-PGK) that shows acidic pH (5.5)-dependent optimum catalytic activity. The PAS domain of LmPAS-PGK is expected to regulate PGK activity during catalysis, but the mechanism of regulation by PAS domain at the molecular level is uncharacterized. In this work, we have utilized the full-length, PAS domain-deleted, and mutant enzymes to measure the enzymatic activity in the presence of divalent cation at various pH values. Catalytic activity measurement indicates that Mg2+ binding through PAS domain inhibits the PGK activity at pH 7.5, and this inhibition is withdrawn at pH 5.5. To identify the Mg2+ binding residues of the PAS domain, we exploited a systematic mutational analysis of all (four) His residues in the PAS domain for potential divalent cation binding. Replacement of His-57 with alanine resulted in depression in the presence of Mg2+ at pH 7.5, but H71A, H89A, and H111A showed similar characteristics with respect to the wild-type protein. Fluorescence and isothermal titration calorimetry studies revealed that H57 is responsible for Mg2+ binding in the absence of substrates. Thus, the protonated form of His57 at acidic pH 5.5 destabilizes the Mg2+ binding in the PAS domain, which is an essential requirement in the wild-type LmPAS-PGK for a conformational alteration in the sensor domain that, sequentially, activates the PGK domain, resulting in the synthesis of higher amounts of ATP.

The isc gene cluster expression ethanol tolerance associated improves its ethanol production by organic acids flux redirection in the ethanologenic Escherichia coli KO11 strain.

Fossil fuels consumption impacts the greenhouse gas emissions. Biofuels are considered as alternative renewable energy sources to reduce the fossil fuels dependency. Bioethanol produced by recombinant microorganisms is a widely suggested alternative to increase the yield in fermentation processes. However, ethanol and acetate accumulation under the fermentation process had been described as important stressors for the metabolic capabilities of the microorganisms, stopping the fermentation process and affecting the ethanol yield. Ethanol tolerance is a determining factor in the improvement of fermentative properties of microorganisms; however understanding of ethanol tolerance is limited. The engineered Escherichia coli KO11 strain has been studied in detail and used as an ethanologenic bacteria model. The strain is capable of using glucose and xylose for an efficient ethanol yield. In the current work, the effect of the iron-sulfur cluster (ISC) over-expression in the KO11 strain, on its tolerance and ethanol yield, was evaluated. Fatty acids profiles of membrane phospholipids in the E. coli KO11 were modified under ethanol addition, but not due to the hscA mutation. The hscA mutation provoked a decrease in ethanol tolerance in the Kmp strain when was grown with 2% ethanol, in comparison to KO11 parent strain. Ethanol tolerance was improved in the mutant Kmp complemented with the recombinant isc gene cluster (pJC10 plasmid) fromLD50 2.16% to LD50 3.8% ethanol. In batch fermentation on 1 L bioreactor using mineral medium with glucose (120g/L), the KO11 strain showed ethanol production efficiencies of ~ 76.9%, while the hscA mutant (Kmp) ~ 75.4% and the transformed strain Kmp(pJC10) showed ~ 92.4% efficiency. Ethanol amount increase in the engineered Kmp(pJC10) strain was correlated with less organic acids (such as acetate and lactate) production in the fermentation medium (2.3g/L), compared to that in the KO11 (17.05g/L) and the Kmp (16.62g/L). Alcohol dehydrogenase (ADH) activity was increased ~ 350% in the transformed Kmp(pJC10) strain, whereas in the Kmp mutant, the phosphoglycerate kinase (PGK), pyruvate kinase (PYK), and ADH activities were diminished, comparing to KO11. The results suggest that the isc system over-expression in the ethanologenic E. coli KO11 strain, increases ethanol yield mainly by improving ethanol tolerance and ADH activity, and by redirecting the metabolic flux from acetate synthesis to ethanol.

The effect of dietary garcinol supplementation on oxidative stability, muscle postmortem glycolysis and meat quality in pigs

The objective of this study was to evaluate the effects of dietary garcinol (0, 200, 400 and 600 mg/kg) on the growth performance, meat quality, postmortem glycolysis and antioxidative capacity of finishing pigs. Dietary garcinol increased pigs' average daily gain, pH 24h, a* and myoglobin content of longissimus dorsi (LM) (P < 0.05), and decreased feed/gain ratio, the L*24h, glycolytic potential, drip loss, shear force, and backfat depth (P < 0.05). The glutathione peroxidase (GPx), catalase (CAT) and total antioxidative capacity (T-AOC) were significantly increased by garcinol (P < 0.05), while the activity of lactate dehydrogenase (LDH) and malonaldehyde (MDA) content were decreased (P < 0.05). Moreover, garcinol decreased the p300/CBP-associated factor (PCAF) activity, the acetylation level and activities of glycolysis enzymes phosphoglycerate kinase 1 (PGK1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase-3 (PFKFB3) (P < 0.05). The results of this study showed that garcinol decreased postmortem glycolysis, and this may be due to the mechanism of decreasing glycolytic enzyme acetylation induced by PCAF. The present study indicates that garcinol can facilitate the growth performance of pigs and improve pork quality by changing postmortem glycolysis and antioxidative capacity.

Enhancing glycolysis attenuates Parkinson's disease progression in models and clinical databases.

Parkinson's disease (PD) is a common neurodegenerative disease that lacks therapies to prevent progressive neurodegeneration. Impaired energy metabolism and reduced ATP levels are common features of PD. Previous studies revealed that terazosin (TZ) enhances the activity of phosphoglycerate kinase 1 (PGK1), thereby stimulating glycolysis and increasing cellular ATP levels. Therefore, we asked whether enhancement of PGK1 activity would change the course of PD. In toxin-induced and genetic PD models in mice, rats, flies, and induced pluripotent stem cells, TZ increased brain ATP levels and slowed or prevented neuron loss. The drug increased dopamine levels and partially restored motor function. Because TZ is prescribed clinically, we also interrogated 2 distinct human databases. We found slower disease progression, decreased PD-related complications, and a reduced frequency of PD diagnoses in individuals taking TZ and related drugs. These findings suggest that enhancing PGK1 activity and increasing glycolysis may slow neurodegeneration in PD.

Characterization of filarial phosphoglycerate kinase

Phosphoglycerate kinase (PGK) is a key enzyme of glycolysis which also acts as a mediator of DNA replication and repair in the nucleus. We have cloned and expressed PGK in Brugia malayi. The rBmPGK was found to be 415 amino acid residues long having 45 kDa subunit molecular weight. This enzyme was also identified in different life stages of bovine filarial parasite Setaria cervi. The enzyme activity was highest in microfilarial stage followed by adult female and male as also shown by real time PCR in the present study. Further using BmPGK primers the cDNA prepared from S. cervi was amplified and sequenced which showed 100% homology with Brugia malayi PGK. B. malayi and S. cervi, PGK consists of conserved calmodulin binding domain (CaMBD) having 21 amino acids. In the present study we have shown the CaMBD binds to calcium-calmodulin and regulates its activity. The binding of calmodulin (CaM) with CaMBD was confirmed using calmodulin agarose binding pull down assay, which showed that the rBmPGK binds to CaM agarose-calcium dependent manner. The effect of CaM-Ca2+on the activity of rBmPGK was studied at different concentration of CaM (0.01–5.0 μM) and calcium chloride (0.01–100 μM). The rBmPGK was activated up to 85% in the presence of CaM at 1 μM and 10 μM concentration of CaCl2. Interestingly this activation was abrogated by metal chelator EDTA. Similar results were shown in case of Setaria cervi PGK. A significant increase (90 ± 10) % in ScPGK activity was observed in the presence of CaM and CaCl2 at 1.0 μM and 1.0 mM respectively, further increase in the conc. of CaCl2, the activity of ScPGK was found to be decreased like rBmPGK. Bioinformatics studies have also confirmed the interaction between CaMBD and CaM which showed CaM interacted to Phe 206, Gln 220, Arg 223 and Asn 224 of rBmPGK CaM binding domain.On the basis of these findings, it has been suggested that the activity of filarial PGK could be regulated in cells by Ca2+-CaM depending upon the concentration of calcium. To the best of our knowledge this is first report in filarial parasite.

Malonylome Analysis Reveals the Involvement of Lysine Malonylation in Metabolism and Photosynthesis in Cyanobacteria.

As a recently validated reversible post translational modification, lysine malonylation regulates diverse cellular processes from bacteria to mammals, but its existence and function in photosynthetic organisms remain unknown. Cyanobacteria are the most ancient group of photosynthetic prokaryotes and contribute about 50% of the total primary production on Earth. Previously, we reported the lysine acetylome in the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). Here we performed the first proteomic survey of lysine malonylation in Synechocystis using highly accurate tandem mass spectrometry in combination with affinity purification. We identified 598 lysine malonylation sites on 339 proteins with high confidence in total. A bioinformatic analysis suggested that these malonylated proteins may play various functions and were distributed in diverse subcellular compartments. Among them, many malonylated proteins were involved in cellular metabolism. The functional significance of lysine malonylation in the metabolic enzyme activity of phosphoglycerate kinase (PGK) was determined by site-specific mutagenesis and biochemical studies. Interestingly, 27 proteins involved in photosynthesis were found to be malonylated for the first time, suggesting that lysine malonylation may be involved in photosynthesis. Thus our results provide the first lysine malonylome in a photosynthetic organism and suggest a previously unexplored role of lysine malonylation in the regulation of metabolic processes and photosynthesis in Synechocystis as well as in other photosynthetic organisms.

Inhibition of Setaria cervi protein tyrosine phosphatases by Phenylarsine oxide: A proteomic and biochemical study

Phenylarsine oxide (PAO), a specific protein tyrosine phosphatase (PTP) inhibitor significantly decreased the motility and viability of Setaria cervi ultimately leading to its death. The PTP activity present in the cytosolic and detergent soluble fractions as well as on surface of these parasites was significantly inhibited by PAO. A marked alteration in protein spots abundance after proteomic analysis showed 14 down-regulated and 9 upregulated spots in the treated parasites as compared to the control. The PTP inhibition led to increase in the cytosolic and mitochondrial calpain activity in these parasites. PAO also blocked the ATP generation in the parasite depicted by reduced activity of phosphoglycerate kinase and expression of enolase. An increased ROS level, induced lipid peroxidation/protein carbonyl formation and decreased activity of different antioxidant enzymes like thioredoxin reductase, glutathione reductase and glutathione transferases was also observed in the PAO treated parasites. PAO, thus disturbs the overall homeostasis of the filarial parasite by inhibiting PTPs. Thereby suggesting that these molecules could be used as a good chemotherapeutic target for lymphatic filariasis.

The glycosomal-membrane associated phosphoglycerate kinase isoenzyme A plays a role in sustaining the glucose flux in Trypanosoma cruzi epimastigotes

In Trypanosoma cruzi three isoenzymes of phosphoglycerate kinase (PGK) are found which are simultaneously expressed: the cytosolic isoenzyme PGKB as well as two glycosomal enzymes, PGKA and PGKC. In this paper, we show that PGKA in T. cruzi epimastigotes is associated to the glycosomal membrane; it is responsible for about 23% of the glycosomal PGK activity, the fraction that remains in the pellet after osmotic shock treatment of purified organelles, in contrast to the 77% soluble activity that is mainly attributed to PGKC. Antibodies against the unique 80 amino-acid insertion of PGKA blocked almost completely the glucose consumption by epimastigotes that were partially permeabilized with digitonin. These results indicate that PGKA is the predominant isoenzyme for sustaining glycolysis through the glycosomes of these parasites.

T.P.30: Recurrent episodes of myoglobinuria, mental retardation and epilepsy but no haemolysis in two brothers with phosphoglycerate kinase deficiency

We report two brothers, aged 14 and 16, with a mild intellectual deficiency. Both brothers presented recurrent episodes of generalized weakness following effort since the age of 4. At the age of 8, after spending twenty minutes playing in a swimming pool, the youngest brother presented an episode of weakness, together with a painful swelling of the left thigh and myoglobinuria (max CK 540,000 UI/l). He was hospitalized in an intensive care unit and treated by intravenous fluids and loop diuretics. 4 months later, a muscle biopsy disclosed abnormal vacuolisation with slightly increased muscle lipid content. An extensive workup excluded a mitochondrial disease, a defect of fatty acid oxidation and Mac Ardle disease. At the age of 13, the youngest brother presented a refractory status epilepticus, complicated by rhabdomyolysis and acute renal failure (max CK 710,000 UI/l). Physical examination of both brothers, between acute episodes, was completely normal and CK level was approximately 500 UI/l ( N PGK1 gene sequencing revealed a splice site consensus sequence change that co-segregates with decreased PGK activity. Its putative functional consequences are currently under investigation. Conclusion: Phosphoglycerate kinase (PGK) deficiency, a rare defect in glycolysis pathway, has its place in the differential diagnosis of metabolic myopathies. The association of exercise intolerance and recurrent episodes of myoglobinuria with haemolytic anaemia, intellectual deficiency or epilepsy is highly suggestive of the disease.