Pyruvate Dehydrogenase E1 Alpha Subunit Research Articles

Insulin reduces pyroptosis-induced inflammation by PDHA1 dephosphorylation-mediated NLRP3 activation during myocardial ischemia-reperfusion injury.

Previous studies proved that pyrin domain-containing protein 3 (NLRP3)-induced pyroptosis plays an important role in Myocardial ischemia-reperfusion injury (MIRI). Insulin can inhibit the activation of NLRP3 inflammasome, although the exact mechanism remains unclear. The aim of this study was to determine whether insulin reduces NLRP3-induced pyroptosis by regulating pyruvate dehydrogenase E1alpha subunit (PDHA1) dephosphorylation during MIRI. Rat hearts were subject to 30 min global ischemia followed by 60 min reperfusion, with or without 0.5 IU/L insulin. Myocardial ischemia-reperfusion injury was evaluated by measuring myocardial enzymes release, Cardiac hemodynamics, pathological changes, infarct size, and apoptosis rate. Cardiac aerobic glycolysis was evaluated by measuring ATP, lactic acid content, and pyruvate dehydrogenase complex (PDHc) activity in myocardial tissue. Recombinant adenoviral vectors for PDHA1 knockdown were constructed. Pyroptosis-related proteins were measured by Western blotting analysis, immunohistochemistry staining, and ELISA assay, respectively. It was found that insulin significantly reduced the area of myocardial infarction, apoptosis rate, and improved cardiac hemodynamics, pathological changes, energy metabolism. Insulin inhibits pyroptosis-induced inflammation during MIRI. Subsequently, Adeno-associated virus was used to knock down cardiac PDHA1 expression. Knockdown PDHA1 not only promoted the expression of NLRP3 but also blocked the inhibitory effect of insulin on NLRP3-mediated pyroptosis in MIRI. Results suggest that insulin protects against MIRI by regulating PDHA1 dephosphorylation, its mechanism is not only to improve myocardial energy metabolism but also to reduce the NLRP3-induced pyroptosis.

PDHA1 Gene Knockout In Human Esophageal Squamous Cancer Cells Resulted In Greater Warburg Effect And Aggressive Features In Vitro And In Vivo.

BackgroundOne of the remarkable metabolic characteristics of cancer cells is that they prefer glycolysis rather than oxidative phosphorylation (OXPHOS). Pyruvate dehydrogenase E1 alpha subunit (PDHA1) is an important prerequisite for OXPHOS. Our previous studies have shown that low level of PDHA1 protein expression in esophageal squamous cell cancer (ESCC) was correlated with poor prognosis. However, the effect of PDHA1 inhibition on metabolism and biological behavior of esophageal cancer cells remains unclear.Methods And ResultsIn this study, a KYSE450 PDHA1 knockout (KO) cell line of esophageal cancer was established by CRISPR/Cas9 technology. Then, the glycose metabolism, cell proliferation and migration abilities, chemotherapeutic tolerance and angiogenesis of the PDHA1 KO cells were investigated in vitro and in vivo. In the PDHA1 KO cells, the glycolysis and the consumption of glucose and glutamine were significantly enhanced, while the OXPHOS was significantly suppressed, implying Warburg effect in the PDHA1 KO cells. Furthermore, it was also proved in vitro experiments that the PDHA1 KO cell obtained proliferation advantage, as well as significantly greater chemotherapy tolerance and migration ability. Xenograft experiments discovered not only larger tumors but also increased angiogenesis in the PDHA1 KO cell group.ConclusionInhibition of PDHA1 gene expression in human ESCC leads to metabolic reprogramming of Warburg effect and increased malignancies. Targeting ESCC metabolic reprogramming may become a potential therapeutic target.

Characterization of Mycoplasma gallisepticum pyruvate dehydrogenase alpha and beta subunits and their roles in cytoadherence.

Mycoplasma gallisepticum is a causative agent of chronic respiratory disease in chickens, typically causing great economic losses. Cytoadherence is the critical stage for mycoplasma infection, and the associated proteins are important for mycoplasma pathogenesis. Many glycolytic enzymes are localized on the cell surface and can bind the extracellular matrix of host cells. In this study, the M. gallisepticum pyruvate dehydrogenase E1 alpha subunit (PDHA) and beta subunit (PDHB) were expressed in Escherichia coli, and their enzymatic activities were identified based on 2,6-dichlorophenol indophenol reduction. When recombinant PDHA (rPDHA) and recombinant PDHB (rPDHB) were mixed at a 1:1 molar ratio, they exhibited strong enzymatic activity. Alone, rPDHA and rPDHB exhibited no or weak enzymatic activity. Further experiments indicated that both PDHA and PDHB were surface-exposed immunogenic proteins of M. gallisepticum. Bactericidal assays showed that the mouse anti-rPDHA and anti-rPDHB sera killed 48.0% and 75.1% of mycoplasmas respectively. A combination of rPDHA and rPDHB antisera had a mean bactericidal rate of 65.2%, indicating that rPDHA and rPDHB were protective antigens, and combining the two sera did not interfere with bactericidal activity. Indirect immunofluorescence and surface display assays showed that both PDHA and PDHB adhered to DF-1 chicken embryo fibroblast cells and adherence was significantly inhibited by antisera against PDHA and PDHB. Adherence inhibition of M. gallisepticum to DF-1 chicken embryo fibroblast cells was 30.2% for mouse anti-rPDHA serum, 45.1% for mouse anti-rPDHB serum and 72.5% for a combination of rPDHA and rPDHB antisera, suggesting that rPDHA and rPDHB antisera may have synergistically interfered with M. gallisepticum cytoadherence. Plasminogen (Plg)-binding assays further demonstrated that both PDHA and PDHB were Plg-binding proteins, which may have contributed to bacterial colonization. Our results clarified the enzymatic activity of M. gallisepticum PDHA and PDHB and demonstrated these compounds as Plg-binding proteins involved in cytoadherence.

Abstract 1695: Quantification of mitochondria in MCF-10A, MDA-MB-231, and SUM149 cells to understand potential defects in oxidative phosphorylation in cancer.

Abstract Cancer cells are known to exhibit altered metabolism. The Warburg effect, whereby cancer cells preferentially use glycolysis instead of oxidative phosphorylation to produce ATP, plays an important role in the tumorigenic capacity of breast cancer. Previously, we found that SUM149 (inflammatory breast cancer) and MDA-MB-231 (invasive breast cancer) cells have decreased oxidative phosphorylation. Since mitochondria are the powerhouses of oxidative phosphorylation, examining their concentrations, phenotypes, and the distribution of their key enzymes can provide crucial information. These data can be used to further clarify whether the observed metabolic discrepancies stem from abundance differences or from other sources, such as defective enzymes and/or intermediate participation in alternate metabolic pathways. The purpose of our research was to elucidate if differential mitochondria concentrations accounted for the observed marked decrease in oxidative phosphorylation in cancer cells. Specifically, we focused on quantifying the number of mitochondria in two aggressive cancer cell lines, MDA-MB-231 and SUM149, and a normal-like breast cell line, MCF-10A. In order to measure the number of mitochondria in the cancerous and noncancerous cells, a commercially available antibody conjugated to green fluorescent protein (GFP) and directed against the E1-alpha subunit of pyruvate dehydrogenase was used in each cell line. The resulting cells were imaged under high-resolution confocal microscopy and their relative fluorescence assessed. The average intensity of GFP in each cell was calculated and used as an indicator for the amount of mitochondria in each cell line. Additionally, these results were compared to transmission electron microscopy (TEM) images of each of these cell lines to validate the immunofluorescent quantifications. Our results showed that there is no significant difference among the number of mitochondria in MDA-MB-231, SUM149, and MCF-10A cells. Additionally, the distribution of mitochondria among all three cell lines was relatively uniform, with a slightly greater abundance of mitochondria around the nuclei. These findings are surprising given that the published research on the Warburg effect, as well as our own prior observations on these three specific cell lines, suggested that cancer cells may have different numbers of mitochondria, although the previous work did not offer definitive proof of this interpretation. Therefore, these results contradict that hypothesis, and direct further studies to identify qualitative functional defects within mitochondria such as mutated or silenced mitochondrial genes, and to explore alternate pathways that breast cancer cells use to derive energy. Our investigation highlights the importance of taking a multi-facetted approach to studying cancer cell metabolism. Citation Format: Sepideh Ashrafzadeh, Lauren D. Van Wassenhove, Sofia D. Merajver. Quantification of mitochondria in MCF-10A, MDA-MB-231, and SUM149 cells to understand potential defects in oxidative phosphorylation in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1695. doi:10.1158/1538-7445.AM2013-1695

A quantitative mass spectrometry-based approach for identifying protein kinase clients and quantifying kinase activity

The Homo sapiens and Arabidopsis thaliana genomes are believed to encode more than 500 and 1000 protein kinases, respectively. Despite this abundance, few bona fide kinase-client relationships have been described in detail. Here we describe a quantitative mass spectrometry (MS)-based approach for identifying kinase-client proteins. During method development, we used the dedicated kinase pyruvate dehydrogenase kinase (PDK) for the in vitro assays. As kinase substrate, we used synthetic peptide cocktails and, in the process, demonstrated that the assay is both sensitive and specific. The method is also useful for characterizing protein kinase-substrate kinetics once the peptide substrate is detected. Applying a label-free spectral counting method, the activity of PDK was determined using the peptide substrate YHGH(292)SMSDPGSTYR derived from the pyruvate dehydrogenase E1alpha subunit sequence. The utility of spectral counting was further validated by studying the negative effect of Met oxidation on peptide phosphorylation. We also measured the activity of the unrelated calcium-dependent protein kinase 3 (CPK3), demonstrating the utility of the method in protein kinase screening applications.

AAV3-mediated transfer and expression of the pyruvate dehydrogenase E1 alpha subunit gene causes metabolic remodeling and apoptosis of human liver cancer cells

Most cancers rely disproportionately on glycolysis for energy even in the presence of adequate oxygen supply, a condition known as “aerobic glycolysis”, or the Warburg effect. Pharmacological reversal of the Warburg effect has been shown to cause selective apoptosis of tumor cells, presumably by stimulating mitochondrial respiratory chain activity and production of reactive oxygen species that, in turn, induce a caspase-mediated series of reactions leading to cell death. We reasoned that a similar effect on tumor cells might result from up-regulation of the E1α subunit gene ( pda1) of the pyruvate dehydrogenase complex (PDC) that catalyzes the rate-limiting step in aerobic glucose oxidation and thus plays a major role in the control of oxidative phosphorylation. To test this postulate, we employed a self-complementary adeno-associated virus (scAAV)-based delivery and expression system for targeting pda1 to the mitochondria of primary cultures of human hepatoblastoma (HB) and hepatocellular carcinoma (HCC) cells. Serotypes 1–10 scAAV vectors that included enhanced green fluorescent ( egfp) reporter gene driven by either cytomegalovirus (CMV) or chicken beta-actin (CBA) promoters were analyzed for transduction ability of HB (Huh-6) and HCC (Huh-7 and HepG2) cell lines and primary cultures of normal human hepatocytes. Serotype 3 scAAV- egfp (scAAV3 -egfp) vector was the most efficient and transduced up to 90% of cells. We limited the transgene expression primarily to liver cancer cells by generating scAAV3 vectors that contained the human alpha-fetoprotein promoter (AFP)-driven reporter gene (scAAV3.AFP -egfp) and the potentially therapeutic gene scAAV3.AFP -pda1. Infection of Huh-6 cells by the scAAV3.AFP -pda1 vector increased protein expression of E1α, PDC catalytic activity, and late-stage apoptotic cell death. Apoptosis was also associated with increased protein expression of Bcl-X/S, an early marker of apoptosis, and release of cytochrome c into the cytosol of infected HB cells . These data indicate that molecular targeting of mitochondrial oxidative metabolism in liver cancer cells by AAV3-mediated delivery of pda1 holds promise as a novel and effective therapeutic approach for human hepatic tumors.

Functional comparison of citrate synthase isoforms from S. cerevisiae

In this study, the product of the CIT3 gene has been identified as a dual specificity mitochondrial citrate and methylcitrate synthase and that of the CIT1 gene as a specific citrate synthase. Recombinant Cit1p had catalytic activity only with acetyl-CoA whereas Cit3p had similar catalytic efficiency with both acetyl-CoA and propionyl-CoA. Deletion of CIT1 dramatically shifted the ratio of these two activities in whole cell extracts towards greater methylcitrate synthase. Deletion of CIT3 had little effect on either citrate or methylcitrate synthase activities. A Δ cit2Δcit3 strain showed no methylcitrate synthase activity, suggesting that Cit2p, a peroxisomal isoform, may also have methylcitrate synthase activity. Although wild-type strains of Saccharomyces cerevisiae did not grow with propionate as a sole carbon source, deletion of CIT2 allowed growth on propionate, suggesting a toxic production of methylcitrate in the peroxisomes of wild-type cells. The Δ cit2Δcit3 double mutant did not grow on propionate, providing further evidence for the role of Cit3p in propionate metabolism. 13C NMR analysis showed the metabolism of 2- 13C-propionate to acetate, pyruvate, and alanine in wild-type, Δ cit1 and Δ cit2 cells, but not in the Δ cit3 mutant. 13C NMR and GC–MS analysis of pyruvate metabolism revealed an accumulation of acetate and of isobutanol in the Δ cit3 mutant, suggesting a metabolic alteration possibly resulting from inhibition of the lipoamide acetyltransferase subunit of the pyruvate dehydrogenase complex by propionyl-CoA. In contrast to Δ cit3, pyruvate metabolism in a Δ pda1 (pyruvate dehydrogenase E1 alpha subunit) mutant strain was only shifted towards accumulation of acetate.

855. Optimization of Delivery and Expression of the Pyruvate Dehydrogenase E1 Alpha Subunit Gene Using Self-Complementary Adeno-Associated Virus Serotype Vectors

855. Optimization of Delivery and Expression of the Pyruvate Dehydrogenase E1 Alpha Subunit Gene Using Self-Complementary Adeno-Associated Virus Serotype Vectors

Clinical and molecular survey in 124 Chinese patients with Leigh or Leigh‐like syndrome

Leigh syndrome is the most common mitochondrial disorder in children characterized by necrotic lesions in the central nervous system. Both mitochondrial DNA (mtDNA) and nuclear DNA defects in the mitochondrial respiratory chain can lead to this disease. To characterize the clinical and genetic traits of Leigh or Leigh-like syndrome patients in China, 124 unrelated cases were collected between 1992 and 2005. Seventy-seven cases (62.1%) met the typical criteria of Leigh syndrome, including symmetrical bilateral abnormal signals in the basal ganglia, thalamus and brain stem, etc. Other cases (37.9%) belonged to Leigh-like syndrome with atypical clinical or radiological manifestations. Late-onset patients accounted for 20.2%, which is more than previously reported. Movement disorder was the most common symptoms in our patients. Thirty-two patients (25.8%) were confirmed to carry mutant genes. Among them, six cases (4.8%) have been demonstrated to have point mutations in mitochondrial DNA. Two separate patients were detected to have mutations on A8344G and A3243G. The T8993G point mutation was identified in one patient and T8993C in one other patient. SURF1 mutations associated with cytochrome-c oxidase deficiency were identified in 25 patients (20.2%). Four unreported variations have been identified in SURF1 gene from three patients. G604C was found in 22 patients. Only one patient had C214T mutation in the pyruvate dehydrogenase E1alpha subunit gene. In the remaining 92 patients (74.2%), a specific molecular dysfunction or underlying metabolic abnormality could not be identified.

Short-Term Feeding of Fish Oil Down-Regulates the Expression of Pyruvate Dehydrogenase E1 Alpha Subunit mRNA in Mouse Brain

Previous studies have suggested that docosahexaenoic acid (DHA), contained in fish oil, prevents brain disease. In the current study, the effect of fish oil feeding on gene expression in the brain was investigated by suppression subtractive hybridization. We found that pyruvate dehydrogenase E1 alpha (PDHE1alpha) mRNA expression is down-regulated by fish oil feeding. We examined whether the expression of PDHE1alpha mRNA is altered by DHA treatment in differentiated PC12 cells. PDHE1alpha mRNA was reduced by supplementation of DHA with a significant decrease in cellular ATP level. These results indicate that fish oil feeding might modulate energy metabolism in the brain.

Phosphorylation of Formate Dehydrogenase in Potato Tuber Mitochondria

Two highly phosphorylated proteins were detected after two-dimensional (blue native/SDS-PAGE) gel electrophoretic separation of the matrix fraction isolated from potato tuber mitochondria. These two phosphoproteins were identified by mass spectrometry as formate dehydrogenase (FDH) and the E1alpha-subunit of pyruvate dehydrogenase (PDH). Isoelectric focusing/SDS-PAGE two-dimensional gels separated FDH and PDH and resolved several different phosphorylated forms of FDH. By using combinations of matrix-assisted laser desorption/ionization mass spectrometry and electrospray ionization tandem mass spectrometry, several phosphorylation sites were identified for the first time in FDH and PDH. FDH was phosphorylated on Thr76 and Thr333, whereas PDH was phosphorylated on Ser294. Both Thr76 and Thr333 in FDH were accessible to protein kinases, as demonstrated by protein structure homology modeling. The extent of phosphorylation of both FDH and PDH was strongly decreased by NAD+, formate, and pyruvate, indicating that reversible phosphorylation of FDH and PDHs was regulated in a similar fashion. At low oxygen concentrations inside the intact potato tubers, FDH activity was strongly increased relative to cytochrome c oxidase activity pointing to a possible involvement of FDH in hypoxic metabolism. Computational sequence analysis indicated that a conserved local sequence motif of pyruvate formate-lyase is found in the Arabidopsis thaliana genome, and this enzyme might be the source of formate for FDH in plants.

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.

ZMPP2, a novel type-2C protein phosphatase from maize

A cDNA clone was selected as a candidate for the catalytic subunit of phospho-pyruvate dehydrogenase phosphatase (PDP) by screening a Zea mays expressed sequence tag database with the bovine PDP deduced amino acid sequence. Both strands of the cDNA were completely sequenced. The maize clone contains an open reading frame of 1098 base pairs that encodes a polypeptide of 40 127 Da, ZMPP2. The deduced amino acid sequence of ZMPP2 contains the five PP2C signature domains, as does PDP. However, the expression pattern of ZMPP2, determined by reverse transcriptase-polymerase chain reaction, was different from those of the maize pyruvate dehydrogenase E1 alpha subunit and pyruvate dehydrogenase kinase. Additionally, the predicted subcellular location of ZMPP2 is cytoplasmic, while the pyruvate dehydrogenase complex, regulated by reversible phosphorylation, is mitochondrial. Thus, ZMPP2 is a PP2C-type protein phosphatase related to but distinct from PDP.

Recombinant adeno-associated virus vector-based gene transfer for defects in oxidative metabolism.

Defects in oxidative metabolism may be caused by mutations either in nuclear genes or in mitochondrial DNA (mtDNA). We tested the hypothesis that recombinant adeno-associated virus (rAAV) could be used to complement mtDNA mutations. AAV vector constructs were designed to express the reporter gene encoding green fluorescent protein (GFP), fused to a targeting presequence that directed GFP to be translocated into mitochondria. These vectors mediated expression of mitochondrial-localized GFP, as indicated by fluorescence microscopy and electron microscopy, in respiring human embryonic kidney 293 cells and nonrespiring mtDNA-deficient (rho 0) cells. However, when sequences encoding hydrophobic segments of proteins normally encoded by mtDNA were inserted between the presequence and GFP, mitochondrial import failed to occur. In similar experiments, a fusion was created between pyruvate dehydrogenase (PDH) E1 alpha subunit, a nuclear-encoded mitochondrial gene with its own targeting presequence, and GFP. With this construct, expression of GFP was observed in mitochondria in vitro and in vivo. We conclude that the hydrophobicity of mtDNA-encoded proteins limits their ability to be transported from the cytoplasm. However, rAAV-based gene therapy may hold promise for gene therapy of PDH deficiency, the most common biochemically proven cause of congenital lactic acidosis.

Proteins complexed to the P1 adhesin of Mycoplasma pneumoniae

Adherence of Mycoplasma pneumoniae to host cells requires several mycoplasmal membrane proteins and cytoskeleton-like proteins in addition to the adhesin P1, a transmembrane protein of 170 kDa. To analyse interactions of the P1 adhesin with other membrane proteins or with cytoskeleton-like proteins, cross-linking studies were performed in vivo using the permeant reagent paraformaldehyde. The cross-linked protein complex was isolated by immunoaffinity chromatography, and proteins complexed to the P1 protein were identified by immunoblot analysis followed by high mass accuracy tryptic peptide mapping using matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). In addition to the P1 protein and a truncated form of the same protein, the adhesin-related 30 kDa protein, two membrane proteins of 40 and 90 kDa, the cytoskeleton-associated 65 kDa protein and two cytoskeleton-forming proteins, HMW1 and HMW3, were found to be components of the isolated protein complex. Furthermore, the cross-linked complex contained the chaperone DnaK and the E1alpha subunit of pyruvate dehydrogenase. In summary, it was shown that cytadherence-associated membrane proteins are located in close proximity to cytoskeleton-like proteins, suggesting a functional interaction between membrane and cytoskeleton-like proteins. DnaK might be involved in translocation of proteins from the cytoplasm to the membrane and pyruvate dehydrogenase might be a structural protein of the attachment organelle.

Reg1p targets protein phosphatase 1 to dephosphorylate hexokinase II in Saccharomyces cerevisiae: characterizing the effects of a phosphatase subunit on the yeast proteome.

Protein phosphatase 1 (Glc7p) and its binding protein Reg1p are essential for the regulation of glucose repression pathways in Saccharomyces cerevisiae. In order to identify physiological substrates for the Glc7p-Reg1p complex, we examined the effects of deletion of the REG1 gene on the yeast phosphoproteome. Analysis by two-dimensional phosphoprotein mapping identified two distinct proteins that were greatly increased in phosphate content in reg1Delta mutants. Mixed peptide sequencing identified these proteins as hexokinase II (Hxk2p) and the E1alpha subunit of pyruvate dehydrogenase. Consistent with increased phosphorylation of Hxk2p in response to REG1 deletion, fractionation of yeast extracts by anion-exchange chromatography identified Hxk2p phosphatase activity in wild-type strains that was selectively lost in the reg1Delta mutant. The phosphorylation state of Hxk2p and Hxk2p phosphatase activity was restored to wild-type levels in the reg1Delta mutant by expression of a LexA-Reg1p fusion protein. In contrast, expression of LexA-Reg1p containing mutations at phenylalanine in the putative PP-1C-binding site motif (K/R)(X)(I/V)XF was unable to rescue Hxk2p dephosphorylation in intact yeast or restore Hxk2p phosphatase activity. These results demonstrate that Reg1p targets PP-1C to dephosphorylate Hxk2p in vivo and that the motif (K/R)(X) (I/V)XF is necessary for its PP-1 targeting function.

Coordinate Induction of Energy Gene Expression in Tissues of Mitochondrial Disease Patients

We have examined the transcript levels of a variety of oxidative phosphorylation (OXPHOS) and associated bioenergetic genes in tissues of a patient carrying the myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) A3243G mitochondrial DNA (mtDNA) mutation and the skeletal muscles of 14 patients harboring other pathogenic mtDNA mutations. The patients' tissues, which harbored 88% or more mutant mtDNA, had increased levels of mtDNA transcripts, increased nuclear OXPHOS gene transcripts including the ATP synthase beta subunit and the heart-muscle isoform of the adenine nucleotide translocator, and increased ancillary gene transcripts including muscle mitochondrial creatine phosphokinase, muscle glycogen phosphorylase, hexokinase I, muscle phosphofructokinase, the E1alpha subunit of pyruvate dehydrogenase, and the ubiquinone oxidoreductase. A similar coordinate induction of bioenergetic genes was observed in the muscle biopsies of severe pathologic mtDNA mutations. The more significant coordinated expression was found in muscle from patients with the MELAS, myoclonic epilepsy with ragged red fibers, and chronic progressive external ophthalmoplegia deletion syndromes, with ragged red muscle fibers and mitochondrial paracrystalline inclusions. High levels of mutant mtDNAs were linked to a high induction of the mtDNA and nuclear OXPHOS genes and of several associated bioenergetic genes. These observations suggest that human tissues attempt to compensate for OXPHOS defects associated with mtDNA mutations by stimulating mitochondrial biogenesis, possibly mediated through redox-sensitive transcription factors.

Stabilization of the pyruvate dehydrogenase E1alpha subunit by dichloroacetate.

To test the effects of dichloroacetate (DCA) treatment on the rate of turnover of pyruvate dehydrogenase (PDH) subunits. PDH deficiency is a nuclear-encoded mitochondrial disorder and a major recognized cause of neonatal encephalomyopathies associated with primary lactic acidosis. DCA has been used for its treatment. The primary mechanism of action of DCA has been thought to increase the proportion of enzyme in the activated, dephosphorylated state. However, this mechanism does not readily account for responses to treatment with mutations that do not obviously affect regulation of the enzyme complex. PDH subunit turnover rates were measured using pulse-chase methods in a normal fibroblastic cell line before and after chronic (5-day) treatment with 5 mM DCA. Chronic DCA treatment causes a more than twofold decrease in the apparent first-order rate constant for degradation of the PDH E1alpha subunit (kE1alpha(pre-DCA) = 0.025 +/- 0.006 hr(-1), n = 6; kE1alpha(post-DCA) = 0.011 /- 0.002 hr(-1), n = 3; p < 0.01) and a selective, progressive increase in the total cell PDH activity by 150 +/- 5% (p < 0.0005). These results suggest an additional novel mechanism of action for the chronic DCA treatment of lactic acidemia; namely, inhibition of mitochondrial E1alpha subunit degradation leading to an increase in maximal PDH complex activity.

Mouse mutants carrying deletions that remove the genes mutated in Coffin-Lowry syndrome and lactic acidosis.

The mouse X-linked mutants lined and stripey are associated with lethality of affected males in utero and a striping of the coat in carrier females. We demonstrate that the underlying mutations are nested deletions which lie in the Phex-Amelx chromosomal segment conserved between man and mouse. The lined deletion contains less than approximately 0.7 cM of genetic material and includes the growth factor-regulated protein kinase gene, Rsk2. Stripey carries a larger deletion which removes approximately 2.0 cM of genetic material, including Rsk2 and the pyruvate dehydrogenase E1alpha subunit gene, Pdha1 . Since Coffin-Lowry syndrome and neonatal lactic acidosis are associated with mutations in the human homologues of Rsk2 and Pdha1 respectively, lined and stripey provide models for gene deficiencies in these disorders.

Transfection screening for primary defects in the pyruvate dehydrogenase E1alpha subunit gene.

In a significant number of patients with biochemical evidence of a defect in the E1 (pyruvate dehydrogenase) component of the pyruvate dehydrogenase complex, it has not proved possible to identify a mutation in the gene coding regions. To assess the need for more extensive genetic analysis in these patients and to establish a test system in which to study the biochemical consequences of mutations in the E1alpha subunit gene (which is responsible for the great majority of defined cases of pyruvate dehydrogenase deficiency), we have developed a method to screen for E1alpha gene defects based on complementation of the enzyme deficiency in transformed fibroblast cell lines following transfection and expression of the normal cDNA. Using this system, cell lines from patients with a variety of different defined mutations in the E1alpha gene show restoration of enzyme activity. A number of patients have been identified in whom deficient enzyme activity is not restored by expression of the normal cDNA indicating that an alternative explanation for the enzyme defect must be sought.