How phosphorylation influences E1 subunit pyruvate dehydrogenase: A computational study.

Pyruvate (PYR) dehydrogenase complex (PDC) is an enzymatic system that plays a crucial role in cellular metabolism as it controls the entry of carbon into the Krebs cycle. From a structural point of view, PDC is formed by three different subunits (E1, E2 and E3) capable of catalyzing the three reaction steps necessary for the full conversion of pyruvate to acetyl-CoA. Recent investigations pointed out the crucial role of this enzyme in the replication and survival of specific cancer cell lines, renewing the interest of the scientific community. Here, we report the results of our molecular dynamics studies on the mechanism by which posttranslational modifications, in particular the phosphorylation of three serine residues (Ser-264-α, Ser-271-α, and Ser-203-α), influence the enzymatic function of the protein. Our results support the hypothesis that the phosphorylation of Ser-264-α and Ser-271-α leads to (1) a perturbation of the catalytic site structure and dynamics and, especially in the case of Ser-264-α, to (2) a reduction in the affinity of E1 for the substrate. Additionally, an analysis of the channels connecting the external environment with the catalytic site indicates that the inhibitory effect should not be due to the occlusion of the access/egress pathways to/from the active site.

Pyruvate dehydrogenase alpha 1 as a target of omega-3 polyunsaturated fatty acids in human prostate cancer through a global phosphoproteomic analysis.

Prostate cancer is one of the leading cancers in men. Taking dietary supplements, such as fish oil (FO), which is rich in n-3 polyunsaturated fatty acids (PUFAs), has been employed as a strategy to lower prostate cancer risk and control disease progression. In this study, we investigated the global phosphoproteomic changes induced by FO using a combination of phosphoprotein-enrichment strategy and high-resolution tandem mass spectrometry. We found that FO induces many more phosphorylation changes than oleic acid when they both are compared to control group. Quantitative comparison between untreated group and FO- or oleic acid-treated groups uncovered a number of important protein phosphorylation changes induced by n-3PUFAs. This phosphoproteomic discovery study and the follow-up Western Blot validation study elucidate that phosphorylation levels of the two regulatory serine residues in pyruvate dehydrogenase alpha 1 (PDHA1), serine-232 and serine-300, are significantly decreased upon FO treatment. As expected, increased pyruvate dehydrogenase activity was also observed. This study suggests that FO-induced phosphorylation changes in PDHA1 is more likely related to the glucose metabolism pathway, and n-3 PUFAs may have a role in controlling the balance between lipid and glucose oxidation.

Role of Lipoylation of the Immunodominant Epitope of Pyruvate Dehydrogenase Complex: Toward a Peptide-Based Diagnostic Assay for Primary Biliary Cirrhosis.

Primary biliary cirrhosis is an immune-mediated chronic liver disease whose diagnosis relies on the detection of serum antimitochondrial antibodies directed against a complex set of proteins, among which pyruvate dehydrogenase complex is considered the main autoantigen. We studied the immunological role of the lipoyl domain of this protein using synthetic lipoylated peptides, showing that the lipoyl chain chirality does not affect autoantibody recognition and, most importantly, confirming that both lipoylated and unlipoylated peptides are able to recognize specific autoantibodies in patients sera. In fact, 74% of patients sera recognize at least one of the tested peptides but very few positive sera recognized exclusively the lipoylated peptide, suggesting that the lipoamide moiety plays a marginal role within the autoreactive epitope. These results are supported by a conformational analysis showing that the lipoyl moiety of pyruvate dehydrogenase complex appears to be involved in hydrophobic interactions, which may limit its exposition and thus its contribution to the complex antigenic epitope. A preliminary analysis of the specificity of the two most active peptides indicates that they could be part of a panel of synthetic antigens collectively able to mimic in a simple immunoenzymatic assay the complex positivity pattern detected in immunofluorescence.

Prolyl-hydroxylase PHD3 interacts with pyruvate dehydrogenase (PDH)-E1ß and regulates the cellular PDH activity.

Cells are frequently exposed to hypoxia in physiological and pathophysiological conditions in organisms. Control of energy metabolism is one of the critical functions of the hypoxic response. Hypoxia-Inducible Factor (HIF) is a central transcription factor that regulates the hypoxic response. HIF prolyl-hydroxylase PHDs are the enzymes that hydroxylate the α subunit of HIF and negatively regulate its expression. To further understand the physiological role of PHD3, proteomics were used to identify PHD3-interacting proteins, and pyruvate dehydrogenase (PDH)-E1ß was identified as such a protein. PDH catalyzes the conversion of pyruvate to acetyl-coA, thus playing a key role in cellular energy metabolism. PDH activity was significantly decreased in PHD3-depleted MCF7 breast cancer cells and PHD3(-/-) MEFs. PHD3 depletion did not affect the expression of the PDH-E1α, E1ß, and E2 subunits, or the phosphorylation status of E1α, but destabilized the PDH complex (PDC), resulting in less functional PDC. Finally, PHD3(-/-) cells were resistant to cell death in prolonged hypoxia with decreased production of ROS. Taken together, the study reveals that PHD3 regulates PDH activity in cells by physically interacting with PDC.

Tyr-301 phosphorylation inhibits pyruvate dehydrogenase by blocking substrate binding and promotes the Warburg effect.

The mitochondrial pyruvate dehydrogenase complex (PDC) plays a crucial role in regulation of glucose homoeostasis in mammalian cells. PDC flux depends on catalytic activity of the most important enzyme component pyruvate dehydrogenase (PDH). PDH kinase inactivates PDC by phosphorylating PDH at specific serine residues, including Ser-293, whereas dephosphorylation of PDH by PDH phosphatase restores PDC activity. The current understanding suggests that Ser-293 phosphorylation of PDH impedes active site accessibility to its substrate pyruvate. Here, we report that phosphorylation of a tyrosine residue Tyr-301 also inhibits PDH α 1 (PDHA1) by blocking pyruvate binding through a novel mechanism in addition to Ser-293 phosphorylation. In addition, we found that multiple oncogenic tyrosine kinases directly phosphorylate PDHA1 at Tyr-301, and Tyr-301 phosphorylation of PDHA1 is common in EGF-stimulated cells as well as diverse human cancer cells and primary leukemia cells from human patients. Moreover, expression of a phosphorylation-deficient PDHA1 Y301F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at distinct serine and tyrosine residues inhibits PDHA1 through distinct mechanisms to impact active site accessibility, which act in concert to regulate PDC activity and promote the Warburg effect.

Structure-function relationships in the 2-oxo acid dehydrogenase family: substrate-specific signatures and functional predictions for the 2-oxoglutarate dehydrogenase-like proteins.

Structural relationship within the family of the thiamine diphosphate-dependent 2-oxo acid dehydrogenases was analyzed by combining different methods of sequence alignment with crystallographic and enzymological studies of the family members. For the first time, the sequence similarity of the homodimeric 2-oxoglutarate dehydrogenase to heterotetrameric 2-oxo acid dehydrogenases is established. The presented alignment of the catalytic domains of the dehydrogenases of pyruvate, branched-chain 2-oxo acids and 2-oxoglutarate unravels the sequence markers of the substrate specificity and the essential residues of the family members without the 3D structures resolved. Predicted dual substrate specificity of some of the 2-oxo acid dehydrogenases was confirmed experimentally. The results were used to decipher functions of the two hypothetical proteins of animal genomes, OGDHL and DHTKD1, similar to the 2-oxoglutarate dehydrogenase. Conservation of all the essential residues confirmed their catalytic competence. Sequence analysis indicated that OGDHL represents a previously unknown isoform of the 2-oxoglutarate dehydrogenase, whereas DHTKD1 differs from the homologs at the N-terminus and substrate binding pocket. The differences suggest changes in heterologous protein interactions and accommodation of more polar and/or bulkier structural analogs of 2-oxoglutarate, such as 2-oxoadipate, 2-oxo-4-hydroxyglutarate, or products of the carboligase reaction between a 2-oxodicarboxylate and glyoxylate or acetaldehyde. The signatures of the Ca2+-binding sites were found in the Ca2+-activated 2-oxoglutarate dehydrogenase and OGDHL, but not in DHTKD1. Mitochondrial localization was predicted for OGDHL and DHTKD1, with DHTKD1 probably localized also to nuclei. Medical implications of the obtained results are discussed in view of the possible associations of the 2-oxo acid dehydrogenases and DHTKD1 with neurodegeneration and cancer.

Glutamate 636 of the Escherichia coli pyruvate dehydrogenase-E1 participates in active center communication and behaves as an engineered acetolactate synthase with unusual stereoselectivity.

The residue Glu636 is located near the thiamine diphosphate (ThDP) binding site of the Escherichia coli pyruvate dehydrogenase complex E1 subunit (PDHc-E1), and to probe its function two variants, E636A and E636Q were created with specific activities of 2.5 and 26% compared with parental PDHc-E1. According to both fluorescence binding and kinetic assays, the E636A variant behaved according to half-of-the-sites mechanism with respect to ThDP. In contrast, with the E636Q variant a K(d,ThDP) = 4.34 microM and K(m,ThDP) = 11 microM were obtained with behavior more reminiscent of the parental enzyme. The CD spectra of both variants gave evidence for formation of the 1',4'-iminopyrimidine tautomer on binding of phosphonolactylthiamine diphosphate, a stable analog of the substrate-ThDP covalent complex. Rapid formation of optically active (R)-acetolactate by both variants, but not by the parental enzyme, was observed by CD and NMR spectroscopy. The acetolactate configuration produced by the Glu636 variants is opposite that produced by the enzyme acetolactate synthase and the Asp28-substituted variants of yeast pyruvate decarboxylase, suggesting that the active centers of the two sets of enzymes exhibit different facial selectivity (re or si) vis à vis pyruvate. The tryptic peptide map (mass spectral analysis) revealed that the Glu636 substitution changed the mobility of a loop comprising amino acid residues from the ThDP binding fold. Apparently, the residue Glu636 has important functions both in active center communication and in protecting the active center from undesirable "carboligase" side reactions.

Organization of the cores of the mammalian pyruvate dehydrogenase complex formed by E2 and E2 plus the E3-binding protein and their capacities to bind the E1 and E3 components.

The subunits of the dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex can form a 60-mer via association of the C-terminal I domain of E2 at the vertices of a dodecahedron. Exterior to this inner core structure, E2 has a pyruvate dehydrogenase component (E1)-binding domain followed by two lipoyl domains, all connected by mobile linker regions. The assembled core structure of mammalian pyruvate dehydrogenase complex also includes the dihydrolipoyl dehydrogenase (E3)-binding protein (E3BP) that binds the I domain of E2 by its C-terminal I' domain. E3BP similarly has linker regions connecting an E3-binding domain and a lipoyl domain. The composition of E2.E3BP was thought to be 60 E2 plus approximately 12 E3BP. We have prepared homogenous human components. E2 and E2.E3BP have s(20,w) values of 36 S and 31.8 S, respectively. Equilibrium sedimentation and small angle x-ray scattering studies indicate that E2.E3BP has lower total mass than E2, and small angle x-ray scattering showed that E3 binds to E2.E3BP outside the central dodecahedron. In the presence of saturating levels of E1, E2 bound approximately 60 E1 and maximally sedimented 64.4 +/- 1.5 S faster than E2, whereas E1-saturated E2.E3BP maximally sedimented 49.5 +/- 1.4 S faster than E2.E3BP. Based on the impact on sedimentation rates by bound E1, we estimate fewer E1 (approximately 12) were bound by E2.E3BP than by E2. The findings of a smaller E2.E3BP mass and a lower capacity to bind E1 support the smaller E3BP substituting for E2 subunits rather than adding to the 60-mer. We describe a substitution model in which 12 I' domains of E3BP replace 12 I domains of E2 by forming 6 dimer edges that are symmetrically located in the dodecahedron structure. Twelve E3 dimers were bound per E248.E3BP12 mass, which is consistent with this model.

Antisense inhibition of mitochondrial pyruvate dehydrogenase E1alpha subunit in anther tapetum causes male sterility.

We hypothesized that cytoplasmic male sterility (CMS) in sugar beet may be the consequence of mitochondrial dysfunctions affecting normal anther development. To test the hypothesis, we attempted to mimic the sugar beet CMS phenotype by inhibiting the expression of mitochondrial pyruvate dehydrogenase (PDH), which is essential for the operation of the tricarboxylic acid (TCA) cycle. Screening with a cDNA library of sugar beet flower buds allowed the identification of two PDH E1alpha subunit genes (bvPDH_E1alpha-1 and bvPDH_E1alpha-2). bvPDH_E1alpha-1 was found to be highly expressed in tap roots, whereas bvPDH_E1alpha-2 was expressed most abundantly in flower buds. Green fluorescent protein (GFP) fusion of bvPDH_E1alpha revealed mitochondrial targeting properties. A 300-bp bvPDH_E1alpha-1 cDNA sequence (from +620 to +926) was connected to a tapetum-specific promoter in the antisense orientation and then introduced into tobacco. Antisense expression of bvPDH_E1alpha-1 resulted in conspicuously decreased endogenous bvPDH_E1alpha-1 transcripts and male sterility. The tapetum in the male-sterile anthers showed swelling or abnormal vacuolation. It is also worth noting that in the sterile anthers, cell organelles, such as elaioplasts, tapetosomes and orbicules were poorly formed and microspores exhibited aberrant exine development. These features are shared by sugar beet CMS. The results thus clearly indicate that inhibition of PDH activity in anther tapetum is sufficient to cause male sterility, a phenocopy of the sugar beet CMS.