Inhibits pyruvate dehydrogenase activity by phosphorylation of the E1 subunit PDHA1, and thereby regulates glucose metabolism and aerobic respiration. Can also phosphorylate PDHA2. Decreases glucose utilization and increases fat metabolism in response to prolonged fasting, and as adaptation to a high-fat diet. Plays a role in glucose homeostasis and in maintaining normal blood glucose levels in function of nutrient levels and under starvation. Plays a role in the generation of reactive oxygen species.
The switch of cellular metabolism from mitochondrial respiration to glycolysis is the hallmark of cancer cells and associated with tumor malignancy. However, the mechanism of this metabolic switch remains largely unknown. Herein, we reported that hypoxia-inducible factor-1 (HIF-1) induced pyruvate dehydrogenase kinase-3 (PDK3) expression leading to inhibition of mitochondrial respiration. Promoter activity assay, small interference RNA knockdown assay, and chromatin immunoprecipitation assay demonstrated that hypoxia-induced PDK3 gene activity was regulated by HIF-1 at the transcriptional level. Forced expression of PDK3 in cancer cells resulted in increased lactic acid accumulation and drugs resistance, whereas knocking down PDK3 inhibited hypoxia-induced cytoplasmic glycolysis and cell survival. These data demonstrated that increased PDK3 expression due to elevated HIF-1alpha in cancer cells may play critical roles in metabolic switch during cancer progression and chemoresistance in cancer therapy.
Pyruvate dehydrogenase kinase (PDK) isoforms are molecular switches that downregulate the pyruvate dehydrogenase complex (PDC) by reversible phosphorylation in mitochondria. We have determined structures of human PDK1 or PDK3 bound to the inhibitors AZD7545, dichloroacetate (DCA), and radicicol. We show that the trifluoromethylpropanamide end of AZD7545 projects into the lipoyl-binding pocket of PDK1. This interaction results in inhibition of PDK1 and PDK3 activities by aborting kinase binding to the PDC scaffold. Paradoxically, AZD7545 at saturating concentrations robustly increases scaffold-free PDK3 activity, similar to the inner lipoyl domain. Good DCA density is present in the helix bundle in the N-terminal domain of PDK1. Bound DCA promotes local conformational changes that are communicated to both nucleotide-binding and lipoyl-binding pockets of PDK1, leading to the inactivation of kinase activity. Finally, radicicol inhibits kinase activity by binding directly to the ATP-binding pocket of PDK3, similar to Hsp90 and Topo VI from the same ATPase/kinase superfamily.
The human pyruvate dehydrogenase complex (PDC) is regulated by reversible phosphorylation by four isoforms of pyruvate dehydrogenase kinase (PDK). PDKs phosphorylate serine residues in the dehydrogenase (E1p) component of PDC, but their amino-acid sequences are unrelated to eukaryotic Ser/Thr/Tyr protein kinases. PDK3 binds to the inner lipoyl domains (L2) from the 60-meric transacetylase (E2p) core of PDC, with concomitant stimulated kinase activity. Here, we present crystal structures of the PDK3-L2 complex with and without bound ADP or ATP. These structures disclose that the C-terminal tail from one subunit of PDK3 dimer constitutes an integral part of the lipoyl-binding pocket in the N-terminal domain of the opposing subunit. The two swapped C-terminal tails promote conformational changes in active-site clefts of both PDK3 subunits, resulting in largely disordered ATP lids in the ADP-bound form. Our structural and biochemical data suggest that L2 binding stimulates PDK3 activity by disrupting the ATP lid, which otherwise traps ADP, to remove product inhibition exerted by this nucleotide. We hypothesize that this allosteric mechanism accounts, in part, for E2p-augmented PDK3 activity.
Pyruvate dehydrogenase kinase (PDK) isoforms 2 and 3 were produced via co-expression with the chaperonins GroEL and GroES and purified with high specific activities in affinity tag-free forms. By using human components, we have evaluated how binding to the lipoyl domains of the dihydrolipoyl acetyltransferase (E2) produces the predominant changes in the rates of phosphorylation of the pyruvate dehydrogenase (E1) component by PDK2 and PDK3. E2 assembles as a 60-mer via its C-terminal domain and has mobile connections to an E1-binding domain and then two lipoyl domains, L2 and L1 at the N terminus. PDK3 was activated 17-fold by E2; the majority of this activation was facilitated by the free L2 domain (half-maximal activation at 3.3 microm L2). The direct activation of PDK3 by the L2 domain resulted in a 12.8-fold increase in k(cat) along with about a 2-fold decrease in the K(m) of PDK3 for E1. PDK3 was poorly inhibited by pyruvate or dichloroacetate (DCA). PDK3 activity was stimulated upon reductive acetylation of L1 and L2 when full activation of PDK3 by E2 was avoided (e.g. using free lipoyl domains or ADP-inhibited E2-activated PDK3). In marked contrast, PDK2 was not responsive to free lipoyl domains, but the E2-60-mer enhanced PDK2 activity by 10-fold. E2 activation of PDK2 resulted in a greatly enhanced sensitivity to inhibition by pyruvate or DCA; pyruvate was effective at significantly lower levels than DCA. E2-activated PDK2 activity was stimulated >/=3-fold by reductive acetylation of E2; stimulated PDK2 retained high sensitivity to inhibition by ADP and DCA. Thus, PDK3 is directly activated by the L2 domain, and fully activated PDK3 is relatively insensitive to feed-forward (pyruvate) and feed-back (acetylating) effectors. PDK2 was activated only by assembled E2, and this activated state beget high responsiveness to those effectors.
Activity of the mammalian pyruvate dehydrogenase complex is regulated by phosphorylation-dephosphorylation of three specific serine residues (site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) of the alpha subunit of the pyruvate dehydrogenase (E1) component. Phosphorylation is carried out by four pyruvate dehydrogenase kinase (PDK) isoenzymes. Specificity of the four mammalian PDKs toward the three phosphorylation sites of E1 was investigated using the recombinant E1 mutant proteins with only one functional phosphorylation site present. All four PDKs phosphorylated site 1 and site 2, however, with different rates in phosphate buffer (for site 1, PDK2 > PDK4 approximately PDK1 > PDK3; for site 2, PDK3 > PDK4 > PDK2 > PDK1). Site 3 was phosphorylated by PDK1 only. The maximum activation by dihydrolipoamide acetyltransferase was demonstrated by PDK3. In the free form, all PDKs phosphorylated site 1, and PDK4 had the highest activity toward site 2. The activity of the four PDKs was stimulated to a different extent by the reduction and acetylation state of the lipoyl moieties of dihydrolipoamide acetyltransferase with the maximum stimulation of PDK2. Substitution of the site 1 serine with glutamate, which mimics phosphorylation-dependent inactivation of E1, did not affect phosphorylation of site 2 by four PDKs and of site 3 by PDK1. Site specificity for phosphorylation of four PDKs with unique tissue distribution could contribute to the tissue-specific regulation of the pyruvate dehydrogenase complex in normal and pathophysiological states.
Cancer cells can undergo a metabolic reprogramming from oxidative phosphorylation to glycolysis that allows them to adapt to nutrient-poor microenvironments, thereby imposing a selection for aggressive variants. However, the mechanisms underlying this reprogramming are not fully understood. Using complementary approaches in validated cell lines and freshly obtained human specimens, we report here that mitochondrial respiration and oxidative phosphorylation are slowed in metastatic melanomas, even under normoxic conditions due to the persistence of a high nuclear expression of hypoxia-inducible factor-1α (HIF-1α). Pharmacologic or genetic blockades of the HIF-1α pathway decreased glycolysis and promoted mitochondrial respiration via specific reduction in the expression of pyruvate dehydrogenase kinase-3 (PDK3). Inhibiting PDK3 activity by dichloroacetate (DCA) or siRNA-mediated attenuation was sufficient to increase pyruvate dehydrogenase activity, oxidative phosphorylation, and mitochondrial reactive oxygen species generation. Notably, DCA potentiated the antitumor effects of elesclomol, a pro-oxidative drug currently in clinical development, both by limiting cell proliferation and promoting cell death. Interestingly, this combination was also effective against BRAF V600E-mutant melanoma cells that were resistant to the BRAF inhibitor vemurafenib. Cotreatment of melanomas with DCA and elesclomol in vivo achieved a more durable response than single agent alone. Our findings offer a preclinical validation of the HIF-1/PDK3 bioenergetic pathway as a new target for therapeutic intervention in metastatic melanoma, opening the door to innovative combinations that might eradicate this disease.
Pyruvate dehydrogenase (PDH), the first component of the human pyruvate dehydrogenase complex, has two isoenzymes, somatic cell-specific PDH1 and testis-specific PDH2 with 87% sequence identity in the alpha subunit of alpha(2) beta(2) PDH. The presence of functional testis-specific PDH2 is important for sperm cells generating nearly all their energy from carbohydrates via pyruvate oxidation. Kinetic and regulatory properties of recombinant human PDH2 and PDH1 were compared in this study. Site-specific phosphorylation/dephosphorylation of the three phosphorylation sites by four PDH kinases (PDK1-4) and two PDH phosphatases (PDP1-2) were investigated by substituting serines with alanine or glutamate in PDHs. PDH2 was found to be very similar to PDH1 as follows: (i) in specific activities and kinetic parameters as determined by the pyruvate dehydrogenase complex assay; (ii) in thermostability at 37 degrees C; (iii) in the mechanism of inactivation by phosphorylation of three sites; and (iv) in the phosphorylation of sites 1 and 2 by PDK3. In contrast, the differences for PDH2 were indicated as follows: (i) by a 2.4-fold increase in binding affinity for the PDH-binding domain of dihydrolipoamide acetyltransferase as measured by surface plasmon resonance; (ii) by possible involvement of Ser-264 (site 1) of PDH2 in catalysis as evident by its kinetic behavior; and (iii) by the lower activities of PDK1, PDK2, and PDK4 as well as PDP1 and PDP2 toward PDH2. These differences between PDH2 and PDH1 are less than expected from substitution of 47 amino acids in each PDH2 alpha subunit. The multiple substitutions may have compensated for any drastic alterations in PDH2 structure thereby preserving its kinetic and regulatory characteristics largely similar to that of PDH1.
The human pyruvate dehydrogenase complex (PDC) is regulated by reversible phosphorylation by four isoforms of pyruvate dehydrogenase kinase (PDK). PDKs phosphorylate serine residues in the dehydrogenase (E1p) component of PDC, but their amino-acid sequences are unrelated to eukaryotic Ser/Thr/Tyr protein kinases. PDK3 binds to the inner lipoyl domains (L2) from the 60-meric transacetylase (E2p) core of PDC, with concomitant stimulated kinase activity. Here, we present crystal structures of the PDK3-L2 complex with and without bound ADP or ATP. These structures disclose that the C-terminal tail from one subunit of PDK3 dimer constitutes an integral part of the lipoyl-binding pocket in the N-terminal domain of the opposing subunit. The two swapped C-terminal tails promote conformational changes in active-site clefts of both PDK3 subunits, resulting in largely disordered ATP lids in the ADP-bound form. Our structural and biochemical data suggest that L2 binding stimulates PDK3 activity by disrupting the ATP lid, which otherwise traps ADP, to remove product inhibition exerted by this nucleotide. We hypothesize that this allosteric mechanism accounts, in part, for E2p-augmented PDK3 activity.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionUniProtKB
The human pyruvate dehydrogenase complex (PDC) is regulated by reversible phosphorylation by four isoforms of pyruvate dehydrogenase kinase (PDK). PDKs phosphorylate serine residues in the dehydrogenase (E1p) component of PDC, but their amino-acid sequences are unrelated to eukaryotic Ser/Thr/Tyr protein kinases. PDK3 binds to the inner lipoyl domains (L2) from the 60-meric transacetylase (E2p) core of PDC, with concomitant stimulated kinase activity. Here, we present crystal structures of the PDK3-L2 complex with and without bound ADP or ATP. These structures disclose that the C-terminal tail from one subunit of PDK3 dimer constitutes an integral part of the lipoyl-binding pocket in the N-terminal domain of the opposing subunit. The two swapped C-terminal tails promote conformational changes in active-site clefts of both PDK3 subunits, resulting in largely disordered ATP lids in the ADP-bound form. Our structural and biochemical data suggest that L2 binding stimulates PDK3 activity by disrupting the ATP lid, which otherwise traps ADP, to remove product inhibition exerted by this nucleotide. We hypothesize that this allosteric mechanism accounts, in part, for E2p-augmented PDK3 activity.
The human pyruvate dehydrogenase complex (PDC) is regulated by reversible phosphorylation by four isoforms of pyruvate dehydrogenase kinase (PDK). PDKs phosphorylate serine residues in the dehydrogenase (E1p) component of PDC, but their amino-acid sequences are unrelated to eukaryotic Ser/Thr/Tyr protein kinases. PDK3 binds to the inner lipoyl domains (L2) from the 60-meric transacetylase (E2p) core of PDC, with concomitant stimulated kinase activity. Here, we present crystal structures of the PDK3-L2 complex with and without bound ADP or ATP. These structures disclose that the C-terminal tail from one subunit of PDK3 dimer constitutes an integral part of the lipoyl-binding pocket in the N-terminal domain of the opposing subunit. The two swapped C-terminal tails promote conformational changes in active-site clefts of both PDK3 subunits, resulting in largely disordered ATP lids in the ADP-bound form. Our structural and biochemical data suggest that L2 binding stimulates PDK3 activity by disrupting the ATP lid, which otherwise traps ADP, to remove product inhibition exerted by this nucleotide. We hypothesize that this allosteric mechanism accounts, in part, for E2p-augmented PDK3 activity.
Activity of the mammalian pyruvate dehydrogenase complex is regulated by phosphorylation-dephosphorylation of three specific serine residues (site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) of the alpha subunit of the pyruvate dehydrogenase (E1) component. Phosphorylation is carried out by four pyruvate dehydrogenase kinase (PDK) isoenzymes. Specificity of the four mammalian PDKs toward the three phosphorylation sites of E1 was investigated using the recombinant E1 mutant proteins with only one functional phosphorylation site present. All four PDKs phosphorylated site 1 and site 2, however, with different rates in phosphate buffer (for site 1, PDK2 > PDK4 approximately PDK1 > PDK3; for site 2, PDK3 > PDK4 > PDK2 > PDK1). Site 3 was phosphorylated by PDK1 only. The maximum activation by dihydrolipoamide acetyltransferase was demonstrated by PDK3. In the free form, all PDKs phosphorylated site 1, and PDK4 had the highest activity toward site 2. The activity of the four PDKs was stimulated to a different extent by the reduction and acetylation state of the lipoyl moieties of dihydrolipoamide acetyltransferase with the maximum stimulation of PDK2. Substitution of the site 1 serine with glutamate, which mimics phosphorylation-dependent inactivation of E1, did not affect phosphorylation of site 2 by four PDKs and of site 3 by PDK1. Site specificity for phosphorylation of four PDKs with unique tissue distribution could contribute to the tissue-specific regulation of the pyruvate dehydrogenase complex in normal and pathophysiological states.
Activity of the mammalian pyruvate dehydrogenase complex is regulated by phosphorylation-dephosphorylation of three specific serine residues (site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) of the alpha subunit of the pyruvate dehydrogenase (E1) component. Phosphorylation is carried out by four pyruvate dehydrogenase kinase (PDK) isoenzymes. Specificity of the four mammalian PDKs toward the three phosphorylation sites of E1 was investigated using the recombinant E1 mutant proteins with only one functional phosphorylation site present. All four PDKs phosphorylated site 1 and site 2, however, with different rates in phosphate buffer (for site 1, PDK2 > PDK4 approximately PDK1 > PDK3; for site 2, PDK3 > PDK4 > PDK2 > PDK1). Site 3 was phosphorylated by PDK1 only. The maximum activation by dihydrolipoamide acetyltransferase was demonstrated by PDK3. In the free form, all PDKs phosphorylated site 1, and PDK4 had the highest activity toward site 2. The activity of the four PDKs was stimulated to a different extent by the reduction and acetylation state of the lipoyl moieties of dihydrolipoamide acetyltransferase with the maximum stimulation of PDK2. Substitution of the site 1 serine with glutamate, which mimics phosphorylation-dependent inactivation of E1, did not affect phosphorylation of site 2 by four PDKs and of site 3 by PDK1. Site specificity for phosphorylation of four PDKs with unique tissue distribution could contribute to the tissue-specific regulation of the pyruvate dehydrogenase complex in normal and pathophysiological states.
Any process that results in a change in state or activity of a cell (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a fatty acid stimulus.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
Butyrate, a short-chain fatty acid produced by the colonic bacterial fermentation is able to induce cell growth inhibition and differentiation in colon cancer cells at least partially through its capacity to inhibit histone deacetylases. Since butyrate is expected to impact cellular metabolic pathways in colon cancer cells, we hypothesize that it could exert its antiproliferative properties by altering cellular metabolism. We show that although Caco2 colon cancer cells oxidized both butyrate and glucose into CO(2) , they displayed a higher oxidation rate with butyrate as substrate than with glucose. Furthermore, butyrate pretreatment led to an increase cell capacity to oxidize butyrate and a decreased capacity to oxidize glucose, suggesting that colon cancer cells, which are initially highly glycolytic, can switch to a butyrate utilizing phenotype, and preferentially oxidize butyrate instead of glucose as energy source to produce acetyl coA. Butyrate pretreated cells displayed a modulation of glutamine metabolism characterized by an increased incorporation of carbons derived from glutamine into lipids and a reduced lactate production. The butyrate-stimulated glutamine utilization is linked to pyruvate dehydrogenase complex since dichloroacetate reverses this effect. Furthermore, butyrate positively regulates gene expression of pyruvate dehydrogenase kinases and this effect involves a hyperacetylation of histones at PDK4 gene promoter level. Our data suggest that butyrate exerts two distinct effects to ensure the regulation of glutamine metabolism: it provides acetyl coA needed for fatty acid synthesis, and it also plays a role in the control of the expression of genes involved in glucose utilization leading to the inactivation of PDC.
Any process that results in a change in state or activity of a cell (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a glucose stimulus.
The chemical reactions and pathways involving glucose, the aldohexose gluco-hexose. D-glucose is dextrorotatory and is sometimes known as dextrose; it is an important source of energy for living organisms and is found free as well as combined in homo- and hetero-oligosaccharides and polysaccharides.
The switch of cellular metabolism from mitochondrial respiration to glycolysis is the hallmark of cancer cells and associated with tumor malignancy. However, the mechanism of this metabolic switch remains largely unknown. Herein, we reported that hypoxia-inducible factor-1 (HIF-1) induced pyruvate dehydrogenase kinase-3 (PDK3) expression leading to inhibition of mitochondrial respiration. Promoter activity assay, small interference RNA knockdown assay, and chromatin immunoprecipitation assay demonstrated that hypoxia-induced PDK3 gene activity was regulated by HIF-1 at the transcriptional level. Forced expression of PDK3 in cancer cells resulted in increased lactic acid accumulation and drugs resistance, whereas knocking down PDK3 inhibited hypoxia-induced cytoplasmic glycolysis and cell survival. These data demonstrated that increased PDK3 expression due to elevated HIF-1alpha in cancer cells may play critical roles in metabolic switch during cancer progression and chemoresistance in cancer therapy.
Activity of the mammalian pyruvate dehydrogenase complex is regulated by phosphorylation-dephosphorylation of three specific serine residues (site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) of the alpha subunit of the pyruvate dehydrogenase (E1) component. Phosphorylation is carried out by four pyruvate dehydrogenase kinase (PDK) isoenzymes. Specificity of the four mammalian PDKs toward the three phosphorylation sites of E1 was investigated using the recombinant E1 mutant proteins with only one functional phosphorylation site present. All four PDKs phosphorylated site 1 and site 2, however, with different rates in phosphate buffer (for site 1, PDK2 > PDK4 approximately PDK1 > PDK3; for site 2, PDK3 > PDK4 > PDK2 > PDK1). Site 3 was phosphorylated by PDK1 only. The maximum activation by dihydrolipoamide acetyltransferase was demonstrated by PDK3. In the free form, all PDKs phosphorylated site 1, and PDK4 had the highest activity toward site 2. The activity of the four PDKs was stimulated to a different extent by the reduction and acetylation state of the lipoyl moieties of dihydrolipoamide acetyltransferase with the maximum stimulation of PDK2. Substitution of the site 1 serine with glutamate, which mimics phosphorylation-dependent inactivation of E1, did not affect phosphorylation of site 2 by four PDKs and of site 3 by PDK1. Site specificity for phosphorylation of four PDKs with unique tissue distribution could contribute to the tissue-specific regulation of the pyruvate dehydrogenase complex in normal and pathophysiological states.
The series of molecular signals initiated by binding of a ligand to any of the peroxisome proliferator activated receptors (alpha, beta or gamma) in the nuclear membrane, and ending with the initiation or termination of the transcription of target genes.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
The nuclear receptors peroxisome proliferator-activated receptors (PPARs) are known for their critical role in the metabolic syndrome. Here, we show that they are direct regulators of the family of pyruvate dehydrogenase kinase (PDK) genes, whose products act as metabolic homeostats in sensing hunger and satiety levels in key metabolic tissues by modulating the activity of the pyruvate dehydrogenase complex. Mis-regulation of this tightly controlled network may lead to hyperglycemia. In human embryonal kidney cells we found the mRNA expression of PDK2, PDK3 and PDK4 to be under direct primary control of PPAR ligands, and in normal mouse kidney tissue Pdk2 and Pdk4 are PPAR targets. Both, treatment of HEK cells with PPARbeta/delta-specific siRNA and the genetic disruption of the Pparbeta/delta gene in mouse fibroblasts resulted in reduced expression of Pdk genes and abolition of induction by PPARbeta/delta ligands. These findings suggest that PPARbeta/delta is a key regulator of PDK genes, in particular the PDK4/Pdk4 gene. In silico analysis of the human PDK genes revealed two candidate PPAR response elements in the PDK2 gene, five in the PDK3 gene and two in the PDK4 gene, but none in the PDK1 gene. For seven of these sites we could demonstrate both PPARbeta/delta ligand responsiveness in context of their chromatin region and simultaneous association of PPARbeta/delta with its functional partner proteins, such as retinoidXreceptor, co-activator and mediator proteins and phosphorylated RNA polymerase II. In conclusion, PDK2, PDK3 and PDK4 are primary PPARbeta/delta target genes in humans underlining the importance of the receptor in the control of metabolism.
Any process that modulates the frequency, rate or extent of the chemical reactions and pathways resulting in the formation of acetyl-CoA from pyruvate.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
Cancer cells can undergo a metabolic reprogramming from oxidative phosphorylation to glycolysis that allows them to adapt to nutrient-poor microenvironments, thereby imposing a selection for aggressive variants. However, the mechanisms underlying this reprogramming are not fully understood. Using complementary approaches in validated cell lines and freshly obtained human specimens, we report here that mitochondrial respiration and oxidative phosphorylation are slowed in metastatic melanomas, even under normoxic conditions due to the persistence of a high nuclear expression of hypoxia-inducible factor-1α (HIF-1α). Pharmacologic or genetic blockades of the HIF-1α pathway decreased glycolysis and promoted mitochondrial respiration via specific reduction in the expression of pyruvate dehydrogenase kinase-3 (PDK3). Inhibiting PDK3 activity by dichloroacetate (DCA) or siRNA-mediated attenuation was sufficient to increase pyruvate dehydrogenase activity, oxidative phosphorylation, and mitochondrial reactive oxygen species generation. Notably, DCA potentiated the antitumor effects of elesclomol, a pro-oxidative drug currently in clinical development, both by limiting cell proliferation and promoting cell death. Interestingly, this combination was also effective against BRAF V600E-mutant melanoma cells that were resistant to the BRAF inhibitor vemurafenib. Cotreatment of melanomas with DCA and elesclomol in vivo achieved a more durable response than single agent alone. Our findings offer a preclinical validation of the HIF-1/PDK3 bioenergetic pathway as a new target for therapeutic intervention in metastatic melanoma, opening the door to innovative combinations that might eradicate this disease.
Any process that modulates the rate, frequency or extent of glucose metabolism. Glucose metabolic processes are the chemical reactions and pathways involving glucose, the aldohexose gluco-hexose.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
Cancer cells can undergo a metabolic reprogramming from oxidative phosphorylation to glycolysis that allows them to adapt to nutrient-poor microenvironments, thereby imposing a selection for aggressive variants. However, the mechanisms underlying this reprogramming are not fully understood. Using complementary approaches in validated cell lines and freshly obtained human specimens, we report here that mitochondrial respiration and oxidative phosphorylation are slowed in metastatic melanomas, even under normoxic conditions due to the persistence of a high nuclear expression of hypoxia-inducible factor-1α (HIF-1α). Pharmacologic or genetic blockades of the HIF-1α pathway decreased glycolysis and promoted mitochondrial respiration via specific reduction in the expression of pyruvate dehydrogenase kinase-3 (PDK3). Inhibiting PDK3 activity by dichloroacetate (DCA) or siRNA-mediated attenuation was sufficient to increase pyruvate dehydrogenase activity, oxidative phosphorylation, and mitochondrial reactive oxygen species generation. Notably, DCA potentiated the antitumor effects of elesclomol, a pro-oxidative drug currently in clinical development, both by limiting cell proliferation and promoting cell death. Interestingly, this combination was also effective against BRAF V600E-mutant melanoma cells that were resistant to the BRAF inhibitor vemurafenib. Cotreatment of melanomas with DCA and elesclomol in vivo achieved a more durable response than single agent alone. Our findings offer a preclinical validation of the HIF-1/PDK3 bioenergetic pathway as a new target for therapeutic intervention in metastatic melanoma, opening the door to innovative combinations that might eradicate this disease.
Cancer cells can undergo a metabolic reprogramming from oxidative phosphorylation to glycolysis that allows them to adapt to nutrient-poor microenvironments, thereby imposing a selection for aggressive variants. However, the mechanisms underlying this reprogramming are not fully understood. Using complementary approaches in validated cell lines and freshly obtained human specimens, we report here that mitochondrial respiration and oxidative phosphorylation are slowed in metastatic melanomas, even under normoxic conditions due to the persistence of a high nuclear expression of hypoxia-inducible factor-1α (HIF-1α). Pharmacologic or genetic blockades of the HIF-1α pathway decreased glycolysis and promoted mitochondrial respiration via specific reduction in the expression of pyruvate dehydrogenase kinase-3 (PDK3). Inhibiting PDK3 activity by dichloroacetate (DCA) or siRNA-mediated attenuation was sufficient to increase pyruvate dehydrogenase activity, oxidative phosphorylation, and mitochondrial reactive oxygen species generation. Notably, DCA potentiated the antitumor effects of elesclomol, a pro-oxidative drug currently in clinical development, both by limiting cell proliferation and promoting cell death. Interestingly, this combination was also effective against BRAF V600E-mutant melanoma cells that were resistant to the BRAF inhibitor vemurafenib. Cotreatment of melanomas with DCA and elesclomol in vivo achieved a more durable response than single agent alone. Our findings offer a preclinical validation of the HIF-1/PDK3 bioenergetic pathway as a new target for therapeutic intervention in metastatic melanoma, opening the door to innovative combinations that might eradicate this disease.
Pyruvate dehydrogenase kinase (PDK) isoforms 2 and 3 were produced via co-expression with the chaperonins GroEL and GroES and purified with high specific activities in affinity tag-free forms. By using human components, we have evaluated how binding to the lipoyl domains of the dihydrolipoyl acetyltransferase (E2) produces the predominant changes in the rates of phosphorylation of the pyruvate dehydrogenase (E1) component by PDK2 and PDK3. E2 assembles as a 60-mer via its C-terminal domain and has mobile connections to an E1-binding domain and then two lipoyl domains, L2 and L1 at the N terminus. PDK3 was activated 17-fold by E2; the majority of this activation was facilitated by the free L2 domain (half-maximal activation at 3.3 microm L2). The direct activation of PDK3 by the L2 domain resulted in a 12.8-fold increase in k(cat) along with about a 2-fold decrease in the K(m) of PDK3 for E1. PDK3 was poorly inhibited by pyruvate or dichloroacetate (DCA). PDK3 activity was stimulated upon reductive acetylation of L1 and L2 when full activation of PDK3 by E2 was avoided (e.g. using free lipoyl domains or ADP-inhibited E2-activated PDK3). In marked contrast, PDK2 was not responsive to free lipoyl domains, but the E2-60-mer enhanced PDK2 activity by 10-fold. E2 activation of PDK2 resulted in a greatly enhanced sensitivity to inhibition by pyruvate or DCA; pyruvate was effective at significantly lower levels than DCA. E2-activated PDK2 activity was stimulated >/=3-fold by reductive acetylation of E2; stimulated PDK2 retained high sensitivity to inhibition by ADP and DCA. Thus, PDK3 is directly activated by the L2 domain, and fully activated PDK3 is relatively insensitive to feed-forward (pyruvate) and feed-back (acetylating) effectors. PDK2 was activated only by assembled E2, and this activated state beget high responsiveness to those effectors.
Activity of the mammalian pyruvate dehydrogenase complex is regulated by phosphorylation-dephosphorylation of three specific serine residues (site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) of the alpha subunit of the pyruvate dehydrogenase (E1) component. Phosphorylation is carried out by four pyruvate dehydrogenase kinase (PDK) isoenzymes. Specificity of the four mammalian PDKs toward the three phosphorylation sites of E1 was investigated using the recombinant E1 mutant proteins with only one functional phosphorylation site present. All four PDKs phosphorylated site 1 and site 2, however, with different rates in phosphate buffer (for site 1, PDK2 > PDK4 approximately PDK1 > PDK3; for site 2, PDK3 > PDK4 > PDK2 > PDK1). Site 3 was phosphorylated by PDK1 only. The maximum activation by dihydrolipoamide acetyltransferase was demonstrated by PDK3. In the free form, all PDKs phosphorylated site 1, and PDK4 had the highest activity toward site 2. The activity of the four PDKs was stimulated to a different extent by the reduction and acetylation state of the lipoyl moieties of dihydrolipoamide acetyltransferase with the maximum stimulation of PDK2. Substitution of the site 1 serine with glutamate, which mimics phosphorylation-dependent inactivation of E1, did not affect phosphorylation of site 2 by four PDKs and of site 3 by PDK1. Site specificity for phosphorylation of four PDKs with unique tissue distribution could contribute to the tissue-specific regulation of the pyruvate dehydrogenase complex in normal and pathophysiological states.
Pyruvate dehydrogenase (PDH), the first component of the human pyruvate dehydrogenase complex, has two isoenzymes, somatic cell-specific PDH1 and testis-specific PDH2 with 87% sequence identity in the alpha subunit of alpha(2) beta(2) PDH. The presence of functional testis-specific PDH2 is important for sperm cells generating nearly all their energy from carbohydrates via pyruvate oxidation. Kinetic and regulatory properties of recombinant human PDH2 and PDH1 were compared in this study. Site-specific phosphorylation/dephosphorylation of the three phosphorylation sites by four PDH kinases (PDK1-4) and two PDH phosphatases (PDP1-2) were investigated by substituting serines with alanine or glutamate in PDHs. PDH2 was found to be very similar to PDH1 as follows: (i) in specific activities and kinetic parameters as determined by the pyruvate dehydrogenase complex assay; (ii) in thermostability at 37 degrees C; (iii) in the mechanism of inactivation by phosphorylation of three sites; and (iv) in the phosphorylation of sites 1 and 2 by PDK3. In contrast, the differences for PDH2 were indicated as follows: (i) by a 2.4-fold increase in binding affinity for the PDH-binding domain of dihydrolipoamide acetyltransferase as measured by surface plasmon resonance; (ii) by possible involvement of Ser-264 (site 1) of PDH2 in catalysis as evident by its kinetic behavior; and (iii) by the lower activities of PDK1, PDK2, and PDK4 as well as PDP1 and PDP2 toward PDH2. These differences between PDH2 and PDH1 are less than expected from substitution of 47 amino acids in each PDH2 alpha subunit. The multiple substitutions may have compensated for any drastic alterations in PDH2 structure thereby preserving its kinetic and regulatory characteristics largely similar to that of PDH1.
Pyruvate dehydrogenase kinase (PDK) isoforms are molecular switches that downregulate the pyruvate dehydrogenase complex (PDC) by reversible phosphorylation in mitochondria. We have determined structures of human PDK1 or PDK3 bound to the inhibitors AZD7545, dichloroacetate (DCA), and radicicol. We show that the trifluoromethylpropanamide end of AZD7545 projects into the lipoyl-binding pocket of PDK1. This interaction results in inhibition of PDK1 and PDK3 activities by aborting kinase binding to the PDC scaffold. Paradoxically, AZD7545 at saturating concentrations robustly increases scaffold-free PDK3 activity, similar to the inner lipoyl domain. Good DCA density is present in the helix bundle in the N-terminal domain of PDK1. Bound DCA promotes local conformational changes that are communicated to both nucleotide-binding and lipoyl-binding pockets of PDK1, leading to the inactivation of kinase activity. Finally, radicicol inhibits kinase activity by binding directly to the ATP-binding pocket of PDK3, similar to Hsp90 and Topo VI from the same ATPase/kinase superfamily.
Pyruvate dehydrogenase kinase (PDK) isoforms 2 and 3 were produced via co-expression with the chaperonins GroEL and GroES and purified with high specific activities in affinity tag-free forms. By using human components, we have evaluated how binding to the lipoyl domains of the dihydrolipoyl acetyltransferase (E2) produces the predominant changes in the rates of phosphorylation of the pyruvate dehydrogenase (E1) component by PDK2 and PDK3. E2 assembles as a 60-mer via its C-terminal domain and has mobile connections to an E1-binding domain and then two lipoyl domains, L2 and L1 at the N terminus. PDK3 was activated 17-fold by E2; the majority of this activation was facilitated by the free L2 domain (half-maximal activation at 3.3 microm L2). The direct activation of PDK3 by the L2 domain resulted in a 12.8-fold increase in k(cat) along with about a 2-fold decrease in the K(m) of PDK3 for E1. PDK3 was poorly inhibited by pyruvate or dichloroacetate (DCA). PDK3 activity was stimulated upon reductive acetylation of L1 and L2 when full activation of PDK3 by E2 was avoided (e.g. using free lipoyl domains or ADP-inhibited E2-activated PDK3). In marked contrast, PDK2 was not responsive to free lipoyl domains, but the E2-60-mer enhanced PDK2 activity by 10-fold. E2 activation of PDK2 resulted in a greatly enhanced sensitivity to inhibition by pyruvate or DCA; pyruvate was effective at significantly lower levels than DCA. E2-activated PDK2 activity was stimulated >/=3-fold by reductive acetylation of E2; stimulated PDK2 retained high sensitivity to inhibition by ADP and DCA. Thus, PDK3 is directly activated by the L2 domain, and fully activated PDK3 is relatively insensitive to feed-forward (pyruvate) and feed-back (acetylating) effectors. PDK2 was activated only by assembled E2, and this activated state beget high responsiveness to those effectors.
Protein participating in biochemical reactions in which carbohydrates are involved. Carbohydrate is a general term for sugars and related compounds with the general formula Cn(H2O)n. The smallest are monosaccharides (e.g. glucose); polysaccharides (e.g. starch, cellulose, glycogen) can be large and vary in length.
Protein which catalyzes the phosphorylation of serine or threonine residues on target proteins by using ATP as phosphate donor. Such phosphorylation may cause changes in the function of the target protein. Protein kinases share a conserved catalytic core common to both serine/ threonine and tyrosine protein kinases.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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