Component Of Pyruvate Dehydrogenase Complex Research Articles

Cuproptosis is involved in decabromodiphenyl ether-induced ovarian dysfunction and the protective effect of melatonin

Decabromodiphenyl ether (BDE-209) has been universally detected in environmental media and animals, but its damage to ovarian function and mechanism is still unclear, and melatonin has been shown to improve mammalian ovarian function. This study aimed to investigate the toxic effects of BDE-209 on the ovary and tried to improve ovarian function with melatonin. Herein, BDE-209 was administered orally to female SD rats for 60 days. Enzyme-linked immunosorbent assay, HE staining, transcriptome analysis, qPCR and immunohistochemical staining were used to explore and verify the potential mechanism. We found that BDE-209 exposure had effects on the ovary, as shown by abnormal changes in the estrous cycle, hormone levels and ovarian reserve function in rats, while increasing the proportion of collagen fibres in ovarian tissue. In terms of mechanism, cuproptosis, a form of cell death, was identified to play a crucial role in BDE-209-induced ovarian dysfunction, with the phenotype manifested as copper salt accumulation in ovary, downregulation of glutathione pathway metabolism and copper transfer molecule (ATP7A/B), and upregulation of FDX1, lipoic acid pathway (LIAS, LIPT1), pyruvate dehydrogenase complex components (DLAT, PDHB, PDHA1), and copper transfer molecule (SLC31A1). Furthermore, possible interventions were explored. Notably, a supplement with melatonin has a repair effect on the damage to ovarian function by reversing the gene expression of cuproptosis-involved molecules. Overall, this study revealed that cuproptosis is involved in BDE-209-induced ovarian damage and the beneficial effect of melatonin on ovarian copper damage, providing evidence for the prevention and control of female reproductive damage induced by BDE-209.

Acetylation of transcription factor BpTCP20 by acetyltransferase BpPDCE23 modulates salt tolerance in birch.

Teosinte branched 1/Cycloidea/Proliferating cell factor (TCP) transcription factors function in abiotic stress responses. However, how TCPs confer salt tolerance is unclear. Here, we characterized a TCP transcription factor, BpTCP20, that responds to salt stress in birch (Betula platyphylla Suk). Plants overexpressing BpTCP20 displayed increased salt tolerance, and Bptcp20 knockout mutants displayed reduced salt tolerance relative to the wild-type (WT) birch. BpTCP20 conferred salt tolerance by mediating stomatal closure and reducing reactive oxygen species (ROS) accumulation. Chromatin immunoprecipitation sequencing showed that BpTCP20 binds to NeuroD1, T-box, and two unknown elements (termed TBS1 and TBS2) to regulate target genes. In birch, salt stress led to acetylation of BpTCP20 acetylation at lysine 259. A mutated BpTCP20 variant (abolished for acetylation, termed BpTCP20259) was overexpressed in birch, which led to decreased salt tolerance compared with plants overexpressing BpTCP20. However, BpTCP20259-overexpressing plants still displayed increased salt tolerance relative to untransformed WT plants. BpTCP20259 showed reduced binding to the promoters of target genes and decreased target gene activation, leading to decreased salt tolerance. In addition, we identified dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex (BpPDCE23), an acetyltransferase that interacts with and acetylates BpTCP20 to enhance its binding to DNA motifs. Together, these results suggest that BpTCP20 is a transcriptional regulator of salt tolerance, whose activity is modulated by BpPDCE23-mediated acetylation.

Pyruvate Dehydrogenase E3 Deficiency - Heterozygous Mutation in Dihydrolipoamide Dehydrogenase (DLD) Gene Associated with Symptomatic Hypoglycaemia. A Case Report

Background: Pyruvate dehydrogenase complex deficiency (PDCD) is a mitochondrial disorder that presents with lactic acidemia and neurological manifestations. It is a very rare genetic disorder, a potentially life-threatening one with the usual presentation of hypotonia, lethargy, and developmental delay. It is commonly associated with structural brain abnormalities. We report a very rare case of transient severe symptomatic hypoglycaemia probably due to pyruvate dehydrogenase E3 deficiency; a component of pyruvate dehydrogenase complex (PDC). Case Presentation: Our patient is a 12-day-old neonate with lethargy, and vomiting presented with severe symptomatic hypoglycaemia with a critical sample suggestive of hyperinsulinism and raised lactate levels. A TMS-GCMS for metabolic abnormality screening was normal, however, a whole genome sequencing(nuclear + mitochondrial) revealed heterozygous missense variants (c.763A>C) in exon 9 of the DLD gene that results in the amino acid substitution from Methionine to Leucine at codon 255 (p.Met255Leu) and another heterozygous mutation of heterozygous missense variant (c.5277A>T) in exon 34 of the LAMB1 gene that results in the amino acid substitution from Glutamine to Histidine at codon 1759 (p.Gln1759His). Conclusion: There is no effective treatment for PDCD but reports of treatment with supplements like thiamine, biotin, and coenzyme Q may play a role in preventing the severity of the disease.

Antigen Reactivity and Clinical Significance of Autoantibodies Directed Against the Pyruvate Dehydrogenase Antigen Complex in Patients With Connective Tissue Disease.

IntroductionAntimitochondrial antibodies (AMAs) are the hallmark of primary biliary cholangitis (PBC) but can be identified also in patients with connective tissue disease, namely, systemic sclerosis (SSc). Protein immunoprecipitation (IP) and IP-Western blot (WB) can be used to confirm AMA positivity directed at the pyruvate dehydrogenase complex (PDC) subunits E1α, E1β, E2/E3, and E3BP in patients showing a cytoplasmic reticular pattern at indirect immunofluorescence when performed in a screening setting before the onset of overt cholestasis in rheumatic patients.Patients and MethodsWe studied sera from 285 patients affected by connective tissue disease [SSc, n = 144; dermato/polymyositis (DM/PM), n = 56; and undifferentiated connective tissue disease (UCTD), n = 85] by indirect immunofluorescence (IIF), protein-IP, and IP-WB to identify specific PDC subunits recognized by AMA.ResultsTwenty percent (57/285) of sera from patients with connective tissue disease had a cytoplasmic reticular pattern at IIF, and in 77% (44/57, including 20 SSc, 12 PM/DM, and 12 UCTD) of these, we detected different titers of autoantibodies against the PDC subunits, specifically against PDC-E2. Among these sera, 4 (9%) tested positive for anti-E1α, 15 (34%) for anti-E1β, and 16 (36%) for anti-E3BP. Four of the 20 AMA-positive SSc cases (20%) had been already diagnosed with PBC, and all were positive for autoantibodies against the subunits PDC-E2, E3, and E3BP.ConclusionsUsing IIF and IP, we confirm that autoantibodies against the PDC components are detected in rheumatic patients with PBC or without liver dysfunction. In view of the strong predictive value of AMA for PBC, a strict follow-up of these latter patients is warranted for an early diagnosis of the disease.

Solvent accessibility of E1α and E1β residues with known missense mutations causing pyruvate dehydrogenase complex (PDC) deficiency: Impact on PDC-E1 structure and function.

Pyruvate dehydrogenase complex deficiency is a major cause of primary lactic acidemia resulting in high morbidity and mortality, with limited therapeutic options. PDHA1 mutations are responsible for >82% of cases. The E1 component of PDC is a symmetric dimer of heterodimers (αβ/α'β') encoded by PDHA1 and PDHB. We measured solvent accessibility surface area (SASA), utilized nearest-neighbor analysis, incorporated sequence changes using mutagenesis tool in PyMOL, and performed molecular modeling with SWISS-MODEL, to investigate the impact of residues with disease-causing missense variants (DMVs) on E1 structure and function. We reviewed 166 and 13 genetically resolved cases due to PDHA1 and PDHB, respectively, from variant databases. We expanded on 102 E1α and 13 E1β nonduplicate DMVs. DMVs of E1α Arg112-Arg224 stretch (exons 5-7) and of E1α Arg residues constituted 40% and 39% of cases, respectively, with invariant Arg349 accounting for 22% of arginine replacements. SASA analysis showed that 86% and 84% of residues with nonduplicate DMVs of E1α and E1β, respectively, are solvent inaccessible ("buried"). Furthermore, 30% of E1α buried residues with DMVs are deleterious through perturbation of subunit-subunit interface contact (SSIC), with 73% located in the Arg112-Arg224 stretch. E1α Arg349 represented 74% of buried E1α Arg residues involved in SSIC. Structural perturbations resulting from residue replacements in some matched neighboring pairs of amino acids on different subunits involved in SSIC at 2.9-4.0Å interatomic distance apart, exhibit similar clinical phenotype. Collectively, this work provides insight for future target-based advanced molecular modeling studies, with implications for development of novel therapeutics for specific recurrent DMVs of E1α.

Cannabinoid Receptor 1 associates to different molecular complexes during GABAergic neuron maturation.

CB1 cannabinoid receptor is widely expressed in the central nervous system of animals from late prenatal development to adulthood. Appropriate activation and signaling of CB1 cannabinoid receptors in cortical interneurons are crucial during perinatal/postnatal ages and adolescence, when long-lasting changes in brain activity may elicit subsequent appearance of disorders in the adult brain. Here we used an optimized immunoprecipitation protocol based on specific antibodies followed by shot-gun proteomics to find CB1 interacting partners in postnatal rat GABAergic cortical neurons in vitro at two different stages of maturation. Besides describing new proteins associated with CB1 like dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex (DLAT), fatty acid synthase (FASN), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), voltage-dependent anion channel 1 (VDAC1), myosin phosphatase Rho-interacting protein (MPRIP) or usher syndrome type-1C protein-binding protein 1 (USHBP1), we show that the signaling complex of CB1 is different between maturational stages. Interestingly, the CB1 signaling complex is enriched at the more immature stage in mitochondrial associated proteins and metabolic molecular functions, whereas at more mature stage, CB1 complex is increased in maturation and synaptic-associated proteins. We describe also interacting partners specifically immunoprecipitated with either N-terminal or C-terminal CB1 directed antibodies. Our results highlight new players that may be affected by altered cannabinoid signaling at this critical window of postnatal cortical development.

Integrative structure of a 10-megadalton eukaryotic pyruvate dehydrogenase complex from native cell extracts

The pyruvate dehydrogenase complex (PDHc) is a giant enzymatic assembly involved in pyruvate oxidation. PDHc components have been characterized in isolation, but the complex's quaternary structure has remained elusive due to sheer size, heterogeneity, and plasticity. Here, we identify fully assembled Chaetomium thermophilum α-keto acid dehydrogenase complexes in native cell extracts and characterize their domain arrangements utilizing mass spectrometry, activity assays, crosslinking, electron microscopy (EM), and computational modeling. We report the cryo-EM structure of the PDHc core and observe unique features of the previously unknown native state. The asymmetric reconstruction of the 10-MDa PDHc resolves spatial proximity of its components, agrees with stoichiometric data (60 E2p:12 E3BP:∼20 E1p: ≤ 12 E3), and proposes a minimum reaction path among component enzymes. The PDHc shows the presence of a dynamic pyruvate oxidation compartment, organized by core and peripheral protein species. Our data provide a framework for further understanding PDHc and α-keto acid dehydrogenase complex structure and function.

Insights into the planktonic to sessile transition in a marine biofilm-forming Pseudoalteromonas isolate using comparative proteomic analysis

Bacterial biofilms play an important role in marine biofouling. The formation of a biofilm starts when marine bacterial cells transition from a planktonic to an attached state. However, the molecular mechanisms involved in this transition are poorly understood. Here, 51 strains of marine bacteria were isolated from natural biofilms growing on submerged artificial surfaces (glass slides, epoxy panels, and bridge pillars) and evaluated for their biofilm-forming capacity. Eleven strains formed relatively strong biofilms and 16S rRNA gene sequence analysis indicated that they belonged to the genera Leisingera, Roseobacter, Pseudoalteromonas, Alteromonas, Tenacibaculum, Vibrio, Chryseobacterium, Aquimarina, and Acinetobacter. Strain Pseudoalteromonas sp. W-7 showed efficient and rapid attachment and was therefore chosen for further study. An iTRAQ-based comparative proteomic analysis of planktonic and attached strain W-7 cells was carried out. A total of 3468 proteins were identified, of which 163 showed significant differential expression (120 down-regulated and 43 up-regulated in attached cells relative to planktonic cells). KEGG (Kyoto encyclopedia of genes and genomes) analysis indicated that pyruvate metabolism, carbon fixation, and carbon metabolism were significantly affected in attached cells. Up-regulated proteins such as UTP-glucose-1-phosphate uridylyltransferase, acetyltransferase component of pyruvate dehydrogenase complex, OmpA-like protein, and acetyl-coenzyme A synthetase may be important during initial adhesion. Our findings provide a deeper understanding of the planktonic to sessile transition of marine fouling bacteria.

Mitochondrial proteomics investigation of frontal cortex in an animal model of depression: Focus on chronic antidepressant drugs treatment.

Alteration in the brain mitochondrial functions have been suggested to participate, as a relevant factor, in the development of mental disorders. Therefore, the brain mitochondria may be a crucial therapeutic target in the course of depression. Our goal was to find out the impact of two antidepressant drugs with various mechanisms of action - imipramine and fluoxetine, on the frontal cortex mitochondria-enriched fraction in an animal model of depression based on the prenatal stress procedure. Our results confirmed that the prenatal stress caused depressive-like disturbances in the adult offspring rats, which were normalized by the chronic imipramine and fluoxetine administration. For the first time, using 2D-LC-MS/MS, we demonstrated nine differentially expressed proteins after the imipramine administration. Of these proteins, the up-regulation of the 2',3'-cyclic-nucleotide 3'-phosphodiesterase enzyme and down-regulation of the Hypoxanthine-guanine phosphoribosyltransferase (HPRT), Ras-related proteins (Rap-1A and Rap-1B) and Transgelin-3 (NP25) were the most striking. In contrast, after the chronic fluoxetine treatment, we observed differential expression in five proteins, including the enhanced expression of component of pyruvate dehydrogenase complex and diminished of Glutathione S-transferase P (Gstp-1), as well as Maleylacetoacetate isomerase. These results overcome the interesting data that brain mitochondria in the frontal cortex may constitute the target for pharmacotherapy. The multifaceted profile of both antidepressant drugs action makes difficult to elucidate the exact mechanism of imipramine and fluoxetine action in the brain mitochondria. Further study of mitochondrial dysfunction in psychiatric disorders will be base to know the possible biological consequences of our observations.

Pyrene degradation by Chlorella sp. MM3 in liquid medium and soil slurry: Possible role of dihydrolipoamide acetyltransferase in pyrene biodegradation

Microalgae inhabiting the real contaminated sites are capable of degrading organic pollutants. In the present study, the potential of a microalga, Chlorella ssp. MM3, a soil isolate from a former cattle dip site, was assessed in degrading pyrene both in aqueous medium and soil slurry. Strain MM3 can grow on pyrene in culture medium at concentrations as high as 250μM. When grown in presence of 50μM pyrene, the cell density increased from 1.1×105cellsmL−1 to 16.45×105cellsmL−1 within 7days. With an initial cell density of 3×107cellsmL−1, nearly 70% of 50μM pyrene was degraded after 7days of incubation. When compared with Triton X-100, Tween 80 was a better non-ionic surfactant for pyrene biodegradation. Nearly 20% increase in degradation of pyrene was observed with the use of 0.005% Tween 80. Differential protein expression in pyrene-grown cells of the microalga resulted in distinct accumulation of dihydrolipoamide acetyltransferase (or dihydrolipoyl transacetylase), one of the three components of pyruvate dehydrogenase complex, indicating a possible role of this enzyme in microalgal degradation of pyrene. The microalgal cells immobilized in calcium alginate completely degraded 50μM of pyrene within 10days in nonsterile soil slurry treated with 0.005% Tween 80. Our results clearly indicate that the strain MM3 has a great potential for its use in remediating soils contaminated with pyrene.

Identification of a novel mitochondrial interacting protein of C1QBP using subcellular fractionation coupled with CoIP-MS.

The study of protein-protein interactions is an essential process to understand the biological functions of proteins and the underlying mechanisms. Co-immunoprecipitation coupled with mass spectrometry (CoIP-MS) is one of the most extensively used high-throughput techniques to discover novel protein-protein interactions. However, the traditional CoIP process uses whole cell lysate, disrupts cellular organization, and leads to potential false positives by inducing artificial protein-protein interactions. Here, we have developed a strategy by combining subcellular fractionation with CoIP-MS to study the interacting proteins of the complement component 1, q subcomponent binding protein (C1QBP) in the mitochondria. Using this method, a novel C1QBP interacting protein, dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial (DLAT) was identified and validated. Furthermore, the activity of the pyruvate dehydrogenase (PDH) was found to be affected by the expression level of C1QBP. These results provide novel insights regarding the mitochondrial function of C1QBP in the regulation of cellular energy metabolism. This method could also be used to analyze the subcellular protein-protein interactions for other proteins of interest.

Specific inhibition by synthetic analogs of pyruvate reveals that the pyruvate dehydrogenase reaction is essential for metabolism and viability of glioblastoma cells.

The pyruvate dehydrogenase complex (PDHC) and its phosphorylation are considered essential for oncotransformation, but it is unclear whether cancer cells require PDHC to be functional or silenced. We used specific inhibition of PDHC by synthetic structural analogs of pyruvate to resolve this question. With isolated and intramitochondrial PDHC, acetyl phosphinate (AcPH, KiAcPH = 0.1 μM) was a much more potent competitive inhibitor than the methyl ester of acetyl phosphonate (AcPMe, KiAcPMe = 40 μM). When preincubated with the complex, AcPH also irreversibly inactivated PDHC. Pyruvate prevented, but did not reverse the inactivation. The pyruvate analogs did not significantly inhibit other 2-oxo acid dehydrogenases. Different cell lines were exposed to the inhibitors and a membrane-permeable precursor of AcPMe, dimethyl acetyl phosphonate, which did not inhibit isolated PDHC. Using an ATP-based assay, dependence of cellular viability on the concentration of the pyruvate analogs was followed. The highest toxicity of the membrane-permeable precursor suggested that the cellular action of charged AcPH and AcPMe requires monocarboxylate transporters. The relevant cell-specific transcripts extracted from Gene Expression Omnibus database indicated that cell lines with higher expression of monocarboxylate transporters and PDHC components were more sensitive to the PDHC inhibitors. Prior to a detectable antiproliferative action, AcPH significantly changed metabolic profiles of the investigated glioblastoma cell lines. We conclude that catalytic transformation of pyruvate by pyruvate dehydrogenase is essential for the metabolism and viability of glioblastoma cell lines, although metabolic heterogeneity causes different cellular sensitivities and/or abilities to cope with PDHC inhibition.

Characterization of the chaperonin GroEL in Mycoplasma gallisepticum.

Mycoplasma gallisepticum (MG) is a common and widespread cause of chronic respiratory disease in poultry. In this study, antigenic proteins were identified from MG membrane using two-dimensional gel electrophoresis (2-DE) analysis followed by Western blot and matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), including translation elongation factor Tu, dihydrolipoamide acetyltransferase (E2) component of pyruvate dehydrogenase complex, trigger factor, chaperone protein DnaK, heat shock protein GroEL and so on. Furthermore, recombinant MG GroEL protein was successfully expressed in E. coli BL21 (DE3) with pET-28a (+) vector and found to possess ATPase activity and contributed to the refolding of recombinant MG PrpC protein. Complement-dependent bactericidal assay indicated that the rabbit antisera against MG rGroEL had satisfactory bactericidal effect, which is similar to the chicken antisera induced by MG-inactivated vaccine, suggesting MG GroEL is a protective antigen, could be used as a novel vaccine candidate. This study is the first report of the biological characterization of chaperone GroEL protein in MG.

2D DIGE proteomic analysis of multidrug resistant and susceptible clinical Mycobacterium tuberculosis isolates

Tuberculosis (TB) is the leading cause of infectious disease related mortality worldwide. Infection of Mycobacterium tuberculosis (Mtb) leads to nearly 3 million deaths every year due to tuberculosis. Rifampicin and Isoniazid (RH) are the key drugs to being used for the treatment of tuberculosis. Reports in recent years indicate that the increasing emergence of resistance to these drugs. The resistance to these drugs severely affects options for effective treatment. The current vaccine for tuberculosis has variable protective efficacy and there is no commercially available serodiagnostic test for this disease with acceptable sensitivity and specificity for routine laboratory use, especially in case of multidrug resistance. In order to develop a new diagnostic tool for detection of Mtb, multidrug resistant Mtb as well and improve the tuberculosis vaccine, it is necessary to indentify novel antigenic candidates, especially in identification of multidrug resistant associated protein antigens. Here, we present a 2-D gel-based proteomic survey of the changes in RH resistant Mtb. The proteins extracted from RH resistant and susceptible Mtb clinical isolates were analyzed by two-dimensional differential in gel electrophoresis (2D-DIGE). Protein intensities of 41 spots were found to be regulated in RH resistant isolates. A total of 28 proteins were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Twelve proteins of interest are NADH-dependent enoyl-[acyl-carrier-protein] reductase, 60 kDa chaperonin 2, Chaperone protein DnaK, 3-oxoacyl-(Acyl-carrier-protein0 reductase, Probable acetyl-CoA acyltransferase FadA2, two Acetyl/propionyl-CoA carboxylase, alpha subunit, Universal stress protein Rv1636/MT1672, Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, Glutamine synthetase 1 and two uncharacterized proteins (Rv2557 and Rv1505c).

Proteomic differences between white and brown adipocytes

Although a series of protein levels from several protein pathways have been shown to differ between white (WA) and brown (BA) adipocytes, proteomic work on this subject with the exception of mitochondrial protein differences is limited. It was, therefore, the aim of the study to compare WA with BA soluble protein levels. Proteins were extracted from WA and BA and the soluble fraction was run on two-dimensional gel electrophoresis. Quantification of spot volume was carried out and protein spots, statistically different between groups (P < 0.01), were in-gel digested with trypsin and peptides were identified using nano-LC-ESI-MS/MS in the CID and ETD mode. Differences between selected proteins were evaluated by immunoblotting. A network was generated using the ingenuity pathway analysis. Five proteins, protein DJ-1, dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, isocitrate dehydrogenase subunit alpha, electron transfer flavoprotein subunit alpha and immunoglobulin-binding protein 1, were increased in BA based on a gel-based proteomic method and differential expression was verified by immunoblotting. These individual proteins were represented by one spot each and sequence coverages were between 28 and 65%. A network generated based on these results indicated a link to ubiquitination. Differential protein levels between WA and BA allow interpretation of previous work on adipocyte biochemistry and form the basis for future studies with genetic or pharmacological inhibition of these proteins accompanied by work on phenotype and adipocyte function.

Elementary Flux Modes Analysis of Functional Domain Networks Allows a Better Metabolic Pathway Interpretation

Metabolic network analysis is an important step for the functional understanding of biological systems. In these networks, enzymes are made of one or more functional domains often involved in different catalytic activities. Elementary flux mode (EFM) analysis is a method of choice for the topological studies of these enzymatic networks. In this article, we propose to use an EFM approach on networks that encompass available knowledge on structure-function. We introduce a new method that allows to represent the metabolic networks as functional domain networks and provides an application of the algorithm for computing elementary flux modes to analyse them. Any EFM that can be represented using the classical representation can be represented using our functional domain network representation but the fine-grained feature of functional domain networks allows to highlight new connections in EFMs. This methodology is applied to the tricarboxylic acid cycle (TCA cycle) of Bacillus subtilis, and compared to the classical analyses. This new method of analysis of the functional domain network reveals that a specific inhibition on the second domain of the lipoamide dehydrogenase (pdhD) component of pyruvate dehydrogenase complex leads to the loss of all fluxes. Such conclusion was not predictable in the classical approach.

NMR studies of the N‐terminal domains of E2 component of human pyruvate dehydrogenase complex.

Dihydrolipoamide acetyltransferase (E2) is the second component of pyruvate dehydrogenase complex (PDHc). PDHc catalyzes oxidative decarboxylation of pyruvate, linking glycolysis to the tricarboxylic acid cycle and has an important role in glucose homeostasis. It is composed of multiple copies of three enzymes E1, E2 and E3. PDHc is regulated by four pyruvate dehydrogenase kinases (PDK) and two phosphatases (PDP). The E2 component has gained particular attention because it provides structural and organizational framework to the entire complex in addition to its role in catalysis. It is comprised of three domains, two consecutive lipoyl domains (L1, L2) at the amino terminus, an interim E1/E3 binding domain (S) and carboxy terminal core domain (C) that are linked by alanine, proline rich flexible linkers. We used solution Nuclear Magnetic Resonance (NMR) methods for sequence‐specific backbone assignment of the E2 fragments (L2S and L1L2S domains). In order to understand the role of these proteins in substrate specificity, catalytic function, as well as their interactions with PDKs and other components in the entire PDHc multienzyme complex, we applied NMR and isothermal titration calorimetric (ITC) methods. One of the goals of the project is to develop new interventions to type 2 diabetes, by designing effective inhibitors for this most important glucose‐metabolizing pathway. Supported by NIHGM‐050380.

The role of natural killer (NK) and NK T cells in the loss of tolerance in murine primary biliary cirrhosis

One of the major obstacles in dissecting the mechanism of pathology in human primary biliary cirrhosis (PBC) has been the absence of animal models. Our laboratory has focused on a model in which mice, following immunization with a xenobiotic chemical mimic of the immunodominant autoepitope of the E2 component of pyruvate dehydrogenase complex (PDC-E2), develop autoimmune cholangitis. In particular, following immunization with 2-octynoic acid (a synthetic chemical mimic of lipoic acid-lysine located within the inner domain of PDC-E2) coupled to bovine serum albumin (BSA), several strains of mice develop typical anti-mitochondrial autoantibodies and portal inflammation. The role of innate immune effector cells, such as natural killer (NK) cells and that NK T cells, was studied in this model based on the hypothesis that early events during immunization play an important role in the breakdown of tolerance. We report herein that, following in-vivo depletion of NK and NK T cells, there is a marked suppression of anti-mitochondrial autoantibodies and cytokine production from autoreactive T cells. However, there was no change in the clinical pathology of portal inflammation compared to controls. These data support the hypothesis that there are probably multiple steps in the natural history of PBC, including a role of NK and NK T cells in initiating the breakdown of tolerance. However, the data suggest that adaptive autoimmune effector mechanisms are required for the progression of clinical disease.

Abstract 997: Oncogenic tyrosine kinases are localized to mitochondria and regulate cancer metabolism by phosphorylating key components of pyruvate dehydrogenase kinase complex

Abstract The Warburg effect describes the observation that cancer cells take up more glucose than normal tissue and favor aerobic glycolysis. Many tumor cells rely on aerobic glycolysis instead of mitochondrial oxidative phosphorylation for their continued proliferation and survival. This may be, in part, due to attenuated mitochondrial function, which is suggested to be achieved through inhibition of pyruvate dehydrogenase complex (PDC) in cancer cells. Pyruvate dehydrogenase (PDH) is the first and most important enzyme component of PDC, which catalyzes the conversion of pyruvate to acetyl-CoA that subsequently enters into the Krebs cycle to produce ATP and electron donors including NADH. PDH activity is negatively regulated by phosphorylation at several serine sites. Phosphorylation of PDH by PDH kinase (PDHK) results in the inactivation of PDC, while dephosphorylation by pyruvate dehydrogenase phosphatase (PDP) restores PDC activity. Thus, PDHK and PDP coordinate to regulate the phosphorylation and consequently activation status of PDH. In cancer cells, c-Myc and HIF-1alpha are believed to promote inhibition of PDC by, in part, upregulating gene expression of PDHK. However, how oncogenic signals regulate PDH, and/or coordinate PDHK and PDP activity to inhibit PDH and ultimately attenuate PDC activity in cancer metabolism still remains unclear. We recently reported that, surprisingly, functional PDC can form in mitochondria outside of matrix in some cancer cells. Moreover, several oncogenic tyrosine kinases including FGFR1, FGFR1 fusion tyrosine kinase, and FLT3-ITD mutant, as well as BCR-ABL and JAK2 V617F mutants, are localized to different mitochondrial compartments including outer membrane and matrix, respectively, in cancer cells, where they phosphorylate PDHK1, PDP1 and PDHA. Our studies demonstrate that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding, which promotes cancer cell metabolism, proliferation and tumor growth. In consonance with these findings, we found that tyrosine phosphorylation attenuates PDP1 phosphatase activity but inhibits PDHA enzyme activity. These data suggest that mitochondrial oncogenic tyrosine kinases regulate PDC activity by inhibiting PDHA1 enzyme activity directly through tyrosine phosphorylation and indirectly by enhancing and attenuating PDHK1 and PDP1 activity, respectively. These findings represent an acute mechanism underlying the Warburg effect, in addition to the long-term regulation that is believed to be regulated by c-Myc and HIF-1alpha. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 997. doi:1538-7445.AM2012-997

Hippocampal proteomic profile in temporal lobe epilepsy

In this study we used proteomics approaches to obtain the protein profile of human epileptic hippocampi (N=6) and control hippocampi obtained from autopsy (N=6). We used two-dimensional gel electrophoresis (2-D) coupled to HPLC and Mass spectroscopy (MALDI-TOF) to identify proteins differentially expressed. Nine proteins were differentially expressed comparing the hippocampus of patients with the hippocampus of control. Proteins that were increased in the hippocampus of patients with TLE were: isoform 1 of serum albumin, HSP 70, dihydropyrimidinase-related protein 2, isoform 1 of myelin basic protein, proton ATPase catalytic subunit A, and dihydrolipoyllysine-residue acethyltransferase component of pyruvate dehydrogenase complex. The expression of isoform 3 of spectrin alpha chain (fodrin) was down-regulated while the proteins glutathione S-transferase P and the DJ-1 (PARK7) were detected only in the hippocampus of patients with TLE. Taken together, our results provide evidence supporting a direct link between metabolic disturb and oxidative damage related to pathophysiology of TLE. Besides, indicates biomarkers for further investigations as therapies targeted to epilepsy.