Mechanism of activation of pyruvate dehydrogenase by mitogens in pig lymphocytes.
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ABSTRACT:
SUBMITTER: Baumgarten E
PROVIDER: S-EPMC1152512 | biostudies-other | 1983 Nov
REPOSITORIES: biostudies-other
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Project description:The pyruvate dehydrogenase (E1) and acetyltransferase (E2) components of pig heart and ox kidney pyruvate dehydrogenase (PDH) complex were separated and purified. The E1 component was phosphorylated (alpha-chain) and inactivated by MgATP. Phosphorylation was mainly confined to site 1. Addition of E2 accelerated phosphorylation of all three sites in E1 alpha and inactivation of E1. On the basis of histone H1 phosphorylation, E2 is presumed to contain PDH kinase, which was removed (greater than 98%) by treatment with p-hydroxymercuriphenylsulphonate. Stimulation of ATP-dependent inactivation of E1 by E2 was independent of histone H1 kinase activity of E2. The effect of E2 is attributed to conformational change(s) induced in E1 and/or E1-associated PDH kinase. PDH kinase activity associated with E1 could not be separated from it be gel filtration or DEAE-cellulose chromatography. Subunits of PDH kinase were not detected on sodium dodecyl sulphate/polyacrylamide gels of E1 or E2, presumably because of low concentration. The activity of pig heart PDH complex was increased by E2, but not by E1, indicating that E2 is rate-limiting in the holocomplex reaction. ATP-dependent inactivation of PDH complex was accelerated by E1 or by phosphorylated E1 plus associated PDH kinase, but not by E2 plus presumed PDH kinase. It is suggested that a substantial proportion of PDH kinase may accompany E1 when PDH complex is dissociated into its component enzymes. The possibility that E1 may possess intrinsic PDH kinase activity is considered unlikely, but may not have been fully excluded.
| S-EPMC1152782 | biostudies-other
Project description:1. Pig heart pyruvate dehydrogenase phosphate complex in which all three sites of phosphorylation were completely phosphorylated was re-activated at a slower rate by phosphatase than complex predominantly phosphorylated in site 1. The ratio of initial rates of re-activation was approx. 1:5 with a comparatively crude preparation of phosphatase and with phosphatase purified by gel filtration and ion-exchange chromatography. 2. The ratio of apparent first-order rate constants during dephosphorylation of fully phosphorylated complex averaged 1/3.8/1.3 for site 1/site 2/site 3. Only site-1 dephosphorylation was linearly correlated with re-activation of the complex throughout dephosphorylation. Dephosphorylation of site 3 was linearly correlated with re-activation after an initial burst of dephosphorylation. 3. Because dephosphorylation of site 1 was always associated with dephosphorylation of site 2, it is concluded that dephosphorylation cannot be purely random. 4. The ratio of apparent first-order rate constants for dephosphorylation of site 1 (partially/fully phosphorylated complexes) averaged 1.72. This ratio is smaller than the ratio of approx. 5 for the initial rates of re-activation. Possible mechanisms involved in the diminished rate of re-activation of fully phosphorylated complex are discussed.
| S-EPMC1162854 | biostudies-other
Project description:Eukaryotic cells constantly have to fight against internal acidification. In mammals, this task is mainly performed by the ubiquitously expressed electroneutral Na(+)/H(+) exchanger NHE-1, which activates in a cooperative manner when cells become acidic. Despite its biological importance, the mechanism of this activation is still poorly understood, the most commonly accepted hypothesis being the existence of a proton-sensor site on the internal face of the transporter. This work uncovers mutations that lead to a nonallosteric form of the exchanger and demonstrates that NHE-1 activation is best described by a Monod-Wyman-Changeux concerted mechanism for a dimeric transporter. During intracellular acidification, a low-affinity form of NHE-1 is converted into a form possessing a higher affinity for intracellular protons, with no requirement for an additional proton-sensor site on the protein. This new mechanism also explains the activation of the exchanger by growth signals, which shift the equilibrium towards the high-affinity form.
| S-EPMC1298952 | biostudies-literature
Project description:Activating the macrophage NLRP3 inflammasome can promote excessive inflammation with severe cell and tissue damage and organ dysfunction. Here, we show that pharmacological or genetic inhibition of pyruvate dehydrogenase kinase (PDHK) significantly attenuates NLRP3 inflammasome activation in murine and human macrophages and septic mice by lowering caspase-1 cleavage and interleukin-1β (IL-1β) secretion. Inhibiting PDHK reverses NLRP3 inflammasome-induced metabolic reprogramming, enhances autophagy, promotes mitochondrial fusion over fission, preserves crista ultrastructure, and attenuates mitochondrial reactive oxygen species (ROS) production. The suppressive effect of PDHK inhibition on the NLRP3 inflammasome is independent of its canonical role as a pyruvate dehydrogenase regulator. Our study suggests a non-canonical role of mitochondrial PDHK in promoting mitochondrial stress and supporting NLRP3 inflammasome activation during acute inflammation.
| S-EPMC10117036 | biostudies-literature
Project description:1. The rate of adduct formation between NAD+ and enol-pyruvate at the active site of lactate dehydrogenase is determined by the rate of enolization of pyruvate in solution. 2. The proportion of enol-pyruvate solutions is less than 0.01%. 3. The overall dissociation constant of adduct formation is less than 5 X 10(-8) M for pig heart lactate dehydrogenase at pH 7.0. 4. The unusual kinetics for adduct formation previously observed in the case of rabbit muscle lactate dehydrogenase [Griffin & Criddle (1970) Biochemistry 9, 1195--1205] may be attributed to the concentration of enol-pyruvate in solution being considerably less than the concentration of enzyme.
| S-EPMC1186462 | biostudies-other
Project description:1. Monochloroacetate, dichloroacetate, trichloroacetate, difluoroacetate, 2-chloropropionate, 2,2'-dichloropropionate and 3-chloropropionate were inhibitors of pig heart pyruvate dehydrogenase kinase. Dichloroacetate was also shown to inhibit rat heart pyruvate dehydrogenase kinase. The inhibition was mainly non-competitive with respect to ATP. The concentration required for 50% inhibition was approx. 100mum for the three chloroacetates, difluoroacetate and 2-chloropropionate and 2,2'-dichloropropionate. Dichloroacetamide was not inhibitory. 2. Dichloroacetate had no significant effect on the activity of pyruvate dehydrogenase phosphate phosphatase when this was maximally activated by Ca(2+) and Mg(2+). 3. Dichloroacetate did not increase the catalytic activity of purified pig heart pyruvate dehydrogenase. 4. Dichloroacetate, difluoroacetate, 2-chloropropionate and 2,2'-dichloropropionate increased the proportion of the active (dephosphorylated) form of pyruvate dehydrogenase in rat heart mitochondria with 2-oxoglutarate and malate as respiratory substrates. Similar effects of dichloroacetate were shown with kidney and fat-cell mitochondria. Glyoxylate, monochloroacetate and dichloroacetamide were inactive. 5. Dichloroacetate increased the proportion of active pyruvate dehydrogenase in the perfused rat heart, isolated rat diaphragm and rat epididymal fat-pads. Difluoroacetate and dichloroacetamide were also active in the perfused heart, but glyoxylate, monochloroacetate and trichloroacetate were inactive. 6. Injection of dichloroacetate into rats starved overnight led within 60 min to activation of pyruvate dehydrogenase in extracts from heart, psoas muscle, adipose tissue, kidney and liver. The blood concentration of lactate fell within 15 min to reach a minimum after 60 min. The blood concentration of glucose fell after 90 min and reached a minimum after 120 min. There was no significant change in plasma glycerol concentration. 7. In epididymal fatpads dichloroacetate inhibited incorporation of (14)C from [U-(14)C]glucose, [U-(14)C]fructose and from [U-(14)C]lactate into CO(2) and glyceride fatty acid. 8. It is concluded that the inhibition of pyruvate dehydrogenase kinase by dichloroacetate may account for the activation of pyruvate dehydrogenase and pyruvate oxidation which it induces in isolated rat heart and diaphragm muscles, subject to certain assumptions as to the distribution of dichloroacetate across the plasma membrane and the mitochondrial membrane. 9. It is suggested that activation of pyruvate dehydrogenase by dichloroacetate could contribute to its hypoglycaemic effect by interruption of the Cori and alanine cycles. 10. It is suggested that the inhibitory effect of dichloroacetate on fatty acid synthesis in adipose tissue may involve an additional effect or effects of the compound.
| S-EPMC1168183 | biostudies-other
Project description:The protein pirin, which is involved in a variety of biological processes, is conserved from prokaryotic microorganisms, fungi, and plants to mammals. It acts as a transcriptional cofactor or an apoptosis-related protein in mammals and is involved in seed germination and seedling development in plants. In prokaryotes, while pirin is stress induced in cyanobacteria and may act as a quercetinase in Escherichia coli, the functions of pirin orthologs remain mostly uncharacterized. We show that the Serratia marcescens pirin (pirin(Sm)) gene encodes an ortholog of pirin protein. Protein pull-down and bacterial two-hybrid assays followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization-tandem mass spectrometry analyses showed the pyruvate dehydrogenase (PDH) E1 subunit as a component interacting with the pirin(Sm) gene. Functional analyses showed that both PDH E1 subunit activity and PDH enzyme complex activity are inhibited by the pirin(Sm) gene in S. marcescens CH-1. The S. marcescens CH-1 pirin(Sm) gene was subsequently mutated by insertion-deletion homologous recombination. Accordingly, the PDH E1 and PDH enzyme complex activities and cellular ATP concentration increased up to 250%, 140%, and 220%, respectively, in the S. marcescens CH-1 pirin(Sm) mutant. Concomitantly, the cellular NADH/NAD(+) ratio increased in the pirin(Sm) mutant, indicating increased tricarboxylic acid (TCA) cycle activity. Our results show that the pirin(Sm) gene plays a regulatory role in the process of pyruvate catabolism to acetyl coenzyme A through interaction with the PDH E1 subunit and inhibiting PDH enzyme complex activity in S. marcescens CH-1, and they suggest that pirin(Sm) is an important protein involved in determining the direction of pyruvate metabolism towards either the TCA cycle or the fermentation pathways.
| S-EPMC1797226 | biostudies-literature
Project description:Metabolism in cancer serves to provide energy and key biomolecules that sustain cell growth, a process that is frequently accompanied by decreased mitochondrial use of glucose. Importantly, metabolic intermediates including mitochondrial metabolites are central substrates for post-translational modifications at the core of cellular signalling and epigenetics. However, the molecular means that coordinate the use of mitochondrial metabolites for anabolism and nuclear protein modification are poorly understood. Here, we unexpectedly found that genetic and pharmacological inactivation of Pyruvate Dehydrogenase A1 (PDHA1), a subunit of pyruvate dehydrogenase complex (PDC) that regulates mitochondrial metabolism16 inhibits prostate cancer development in different mouse and human xenograft tumour models. Intriguingly, we found that lipid biosynthesis was strongly affected in prostate tumours upon PDC inactivation. Mechanistically, we found that nuclear PDC controls the expression of Sterol regulatory element-binding transcription factor (SREBF) target genes by mediating histone acetylation whereas mitochondrial PDC provides cytosolic citrate for lipid synthesis in a coordinated effort to sustain anabolism. In line with the oncogenic function of PDC in prostate cancer, we find that PDHA1 and the PDC activator, Pyruvate dehydrogenase phospatase 1 (PDP1), are frequently amplified and overexpressed at both gene and protein level in these tumours. Taken together, our findings demonstrate that both mitochondrial and nuclear PDC sustains prostate tumourigenesis by controlling lipid biosynthesis thereby pointing at this complex as a novel target for cancer therapy.
2018-02-21 | GSE74245 | GEO
Project description:The human pyruvate dehydrogenase complex (PDC) comprises four multidomain components, E1, E3, E2 and an E3-binding protein (E3BP), the latter two forming the core as E2·E3BP sub-complex. Pyruvate flux through PDC is regulated via phosphorylation (inactivation) at E1 by four PDC kinases (PDKs), and reactivation by two PDC phosphatases. Up-regulation of PDK isoform gene expression is reported in several forms of cancer, while PDKs may be further activated by PDC by binding to the E2·E3BP core. Hence, the PDK: E2·E3BP interaction provides new therapeutic targets. We carried out both functional kinetic and thermodynamic studies to demonstrate significant differences in the activation of PDK isoforms by binding to the E2·E3BP core: (i) PDK2 needs no activation by E2·E3BP for efficient functioning, while PDK4 was the least effective of the four isoforms, and could not be activated by E2·E3BP. Hence, development of inhibitors to the interaction of PDK2 and PDK4 with E2·E3BP is not promising; (ii) Design of inhibitors to interfere with interaction of E2·E3BP with PDK1 and PDK3 is promising. PDK3 needs E2·E3BP core for activation, an activation best achieved by synergistic combination of E2-derived catalytic domain and tridomain.
| S-EPMC5322387 | biostudies-literature
Project description:The pyruvate dehydrogenase complex (PDHc) links glycolysis to the citric acid cycle by converting pyruvate into acetyl-coenzyme A. PDHc encompasses three enzymatically active subunits, namely pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. Dihydrolipoyl transacetylase is a multidomain protein comprising a varying number of lipoyl domains, a peripheral subunit-binding domain, and a catalytic domain. It forms the structural core of the complex, provides binding sites for the other enzymes, and shuffles reaction intermediates between the active sites through covalently bound lipoyl domains. The molecular mechanism by which this shuttling occurs has remained elusive. Here, we report a cryo-EM reconstruction of the native E. coli dihydrolipoyl transacetylase core in a resting state. This structure provides molecular details of the assembly of the core and reveals how the lipoyl domains interact with the core at the active site.
| S-EPMC8421416 | biostudies-literature