E1 Beta Subunit Research Articles

Investigating hub genes in the relationship between septic cardiomyopathy and cuproptosis and potential Chinese herbal drug candidates with bioinformatic tools.

The aim of this study was using bioinformatic tools to identify hub genes in the relationship between septic cardiomyopathy (SCM) and cuproptosis and predict potential Chinese herbal drug candidates. SCM datasets were downloaded from the gene expression omnibus. Cuproptosis related genes were collected from a research published on Science in March, 2022. The expression profiles of genes related to cuproptosis in SCM were extracted. Differentially expressed genes (DEGs) were analyzed using R package limma. A single-sample gene set enrichment analysis was conducted to measure the correlation between DEGs and immune cell infiltration. Hub genes were screened out by random forest model. Finally, HERB database and COREMINE database were used to predict Chinese herbal drugs for hub genes and carry out molecular docking. A total of 9 DEGs were identified. Cuproptosis differential genes PDHB, DLAT, DLD, FDX1, GCSH, LIAS were significantly correlated with one or more cells and their functions in immune infiltration. The random forest model screened pyruvate dehydrogenase E1 beta subunit (PDHB) as the hub gene. PDHB was negatively correlated with Plasmacytoid dendritic cell infiltration. Pyruvic acid, rhodioloside and adenosine were predicted with PDHB as the target, and all three components are able to bind to PDHB. Cuproptosis related gene PDHB is associated with the occurrence and immune infiltration of septic cardiomyopathy. Rhodioloside and other Chinese herbal drugs may play a role in the treatment of SCM by regulating the expression of PDHB.

Characterization of pyruvate dehydrogenase complex E1 alpha and beta subunits of Mycoplasma synoviae

Mycoplasma synoviae (MS) is an important pathogen which causes huge economic losses to the poultry industry worldwide, and research on MS can provide the foundation for diagnosis, prevention, and treatment of MS infection. In this study, primers designed based on the sequences of pyruvate dehydrogenase complex (PDC) E1 alpha and beta subunit genes (pdhA and pdhB, respectively) of MS 53 strain(AE017245.1) in GenBank were used to amplify the pdhA and pdhB genes of MS WVU1853 strain through PCR. Subsequently, the prokaryotic expression vectors pET-28a(+)-pdhA and pET-28a(+)-pdhB were constructed and expressed in Escherichia coli BL21(DE3) cells. The recombinant proteins rMSPDHA and rMSPDHB were purified, and anti-rMSPDHA and anti-rMSPDHB sera were prepared by immunizing rabbits, respectively. Subcellular localization of PDHA and PDHB in MS cells, binding activity of rMSPDHA and rMSPDHB to chicken plasminogen (Plg) and human fibronectin (Fn), complement-dependent mycoplasmacidal assays, and adherence and adherence inhibition assays were accomplished. The results showed that PDHA and PDHB were distributed both on the surface membrane and within soluble cytosolic fractions of MS cells. The rMSPDHA and rMSPDHB presented binding activity with chicken Plg and human Fn. The rabbit anti-rMSPDHA and anti-rMSPDHB sera had distinct mycoplasmacidal efficacy in the presence of guinea pig complement, and the adherence of MS to DF-1 cells pretreated with Plg was effectively inhibited by treatment with anti-rMSPDHA or anti-rMSPDHB sera. These findings indicated that surface-associated MSPDHA and MSPDHB were adhesion-related factors of MS and that the binding between MSPDHA/MSPDHB and Plg/Fn contributed to MS adhesion to DF-1 cells.

Soluble (pro)renin receptor increased by hypoxia maintains oxidative metabolism in trophoblasts.

Elevated soluble (pro)renin receptor (s(P)RR) concentration in maternal blood is associated with gestational hypertension and preeclampsia. Placenta has abundant expression of (P)RR, and the binding of (P)RR with pyruvate dehydrogenase E1 beta subunit (PDHB) is reported to maintain oxidative metabolism. Thus, we hypothesized that placental hypoxia may increase (P)RR, and the increased (P)RR may preserve PDHB expression. Expression and functional analyses were performed using human placental trophoblast cells, mainly JAR cells. (P)RR co-immunoprecipitated and showed co-immunofluorescence with PDHB mainly in the mitochondria. Hypoxia treatment significantly increased intracellular s(P)RR protein expression, but secreted s(P)RR in the culture medium was decreased by hypoxia. Hypoxia treatment did not alter PDHB expression or activity in the basal condition, but when (P)RR was knocked down by siRNA, PDHB protein and activity were reduced by hypoxia. Acetyl-CoA, the product of PDH activity, was significantly reduced by hypoxia treatment with (P)RR siRNA. S(P)RR is generated from full-length PRR when cleaved by specific proteases. Protease inhibitor experiments suggested furin and site 1 protease as the enzymes generating s(P)RR in JAR cells, and only when treated by site 1 protease inhibitor, PF429242, PDHB protein showed a significant trend to decrease with hypoxia. In JAR cells, hypoxia increased intracellular s(P)RR, and (P)RR preserved the expression and function of PDHB during hypoxia. (P)RR may help maintain oxidative metabolism and efficient energy production during placental ischemia in hypertensive disorders of pregnancy.

Potentially Critical Roles of NDUFB5, TIMMDC1, and VDAC3 in the Progression of Septic Cardiomyopathy Through Integrated Bioinformatics Analysis.

Septic cardiomyopathy (SC) is a rare and harmful cardiovascular disease with decreased left ventricular (LV) output and multiple organ failure, which poses a serious threat to human life. Despite the advances in SC, its diagnostic basis and treatment methods are limited, and the specific diagnostic biomarkers and its candidate regulatory targets have not yet been fully established. In this study, the GSE79962 gene expression profile was retrieved, with 20 patients with SC and 11 healthy donors as control. Weighted gene coexpression network analysis (WGCNA) was employed to investigate gene modules that were strongly correlated with clinical phenotypes. Blue module was found to be most significantly related to SC. Moreover, Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed on the coexpression genes in blue module and showed that it was associated with metabolic pathways, oxidative phosphorylation, and cardiac muscle contraction. Furthermore, a total of 10 hub genes NDUFB5, TIMMDC1, VDAC3, COQ10A, MRPL16 (mitochondrial ribosomal protein L16), C3orf43, TMEM182, DLAT, NDUFA8, and PDHB (pyruvate dehydrogenase E1 beta subunit) in the blue module were identified at transcriptional level and further validated at translational level in myocardium of an lipopolysaccharide-induced septic cardiac dysfunction mouse model. Overall, the results of quantitative real-time polymerase chain reaction were consistent with most of the microarray analysis results. Intriguingly, we observed that the highest change was NDUFB5, TIMMDC1, and VDAC3. These identified and validated genes provided references that would advance the understanding of molecular mechanisms of SC. Taken together, using WGCNA, the hub genes NDUFB5, TIMMDC1, and VDAC3 might serve as potential biomarkers for diagnosis and/or therapeutic targets for precise treatment of SC in the future.

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.

7. Possible roles of (pro)renin receptor induced by hypoxia in trophoblasts

Objectives Elevated soluble (pro)renin receptor [s(P) RR] concentration in maternal blood is associated with gestational hypertension and preeclampsia. Placental ischemia is a presumed trigger of gestational hypertension, and because placenta has abundant expression of (P) RR, the binding of which with pyruvate dehydrogenase E1 beta subunit (PDHB) is recently reported to maintain oxidative metabolism, we hypothesized that placental (P) RR may be induced by hypoxia and have a role in the pathogenesis or maintenance of hypertensive disorders of pregnancy. Methods Expression and functional analyses were performed using human trophoblast cells, JAR and JEG3. Results Endogenous expression of (P) RR and PDHB were confirmed in two trophoblast cell lines, and (P) RR showed co-immunoprecipitation and co-immunofluorescence with PDHB mainly in mitochondria. Subjecting the cells to hypoxia in a chamber system increased s(P) RR protein expression by 4.3 ± 0.4-fold (1% O2 for 8 h, N = 6, P Conclusion Induction of s(P) RR in trophoblasts may serve to maintain oxidative metabolism and efficient energy production during hypoxia. While increased serum s(P) RR concentration in maternal blood may reflect placental ischemia, whether elevated s(P) RR leads to deleterious effects on the mother such as by activating systemic renin angiotensin system remains to be determined.

Proteomics reveals key proteins participating in growth difference between fall dormant and non-dormant alfalfa in terminal buds

To explore the molecular mechanism of growth differences between fall dormant (FD) and non-FD alfalfa, we conducted iTRAQ-based quantitative proteomics on terminal buds of Maverick (FD) and Cuf101 (non-FD) cultivars, identified differential abundance protein species (DAPS) and verified expression profiling of certain corresponding mRNA by qRT-PCR. A total of 3872 protein species were annotated. Of the 90 DAPS, 56 and 34 were respectively up- and down-accumulated in Maverick, compared to Cuf101. They were grouped into 35 functional categories and enriched in seven pathways. Of which, auxin polar transport was up-regulated, while phenylpropanoid biosynthesis, pyruvate metabolism and transportation, vitamin B1 synthesis process and flavonoid biosynthesis were down-regulated in Maverick, comparing with Cuf101. In Maverick, mRNA abundances of l-asparaginase, chalcone and stilbene synthase family protein, cinnamyl alcohol dehydrogenase-like protein, thiazole biosynthetic enzyme, pyruvate dehydrogenase E1 beta subunit, and aldo/keto reductase family oxidoreductase were significantly lower at FD than at other stages, and lower than in Cuf101. We also observed opposite mRNA profiles of thiazole biosynthetic enzyme, chalcone and stilbene synthase family protein, pyruvate dehydrogenase E1 beta subunit in both cultivars from summer to autumn. Our results suggest that these DAPS could play important roles in growth difference between FD and non-FD alfalfa. Biological significanceUp to now, as far as we know, currently the proteins related with the growth differences between FD and non-FD alfalfa cultivars in autumn have not yet been identified in terminal buds. This study identified the protein species expressed in alfalfa terminal buds, selected differentially abundant protein species in terminal buds between Maverick (FD) and Cuf101 (non-FD) cultivars in autumn and identified the important protein species participated in the growth differences. This study lays a foundation for further investigation of the molecular mechanism of the growth differences between FD and non-FD alfalfa and the cultivation of advanced alfalfa cultivars.

Immunoproteomic and bioinformatic approaches to identify secreted Leishmania amazonensis, L. braziliensis, and L. infantum proteins with specific reactivity using canine serum

Leishmania spp have a wide range of hosts, and each host can harbor several Leishmania species. Dogs, for example, are frequently infected by Leishmania infantum, where they constitute its main reservoir, but they also serve as hosts for L. braziliensis and L. amazonensis. Serological tests for antibody detection are valuable tools for diagnosis of L. infantum infection due to the high levels of antibodies induced, unlike what is observed in L. amazonensis and L. braziliensis infections. Likewise, serology-based antigen-detection can be useful as an approach to diagnose any Leishmania species infection using different corporal fluid samples. Immunogenic and secreted proteins constitute powerful targets for diagnostic methods in antigen detection. As such, we performed immunoproteomic (2-DE, western blot and mass spectrometry) and bioinformatic screening to search for reactive and secreted proteins from L. amazonensis, L. braziliensis, and L. infantum. Twenty-eight non-redundant proteins were identified, among which, six were reactive only in L. amazonensis extracts, 10 in L. braziliensis extracts, and seven in L. infantum extracts. After bioinformatic analysis, seven proteins were predicted to be secreted, two of which were reactive only in L. amazonensis extracts (52kDa PDI and the glucose-regulated protein 78), one in L. braziliensis extracts (pyruvate dehydrogenase E1 beta subunit) and three in L. infantum extracts (two conserved hypothetical proteins and elongation factor 1-beta). We propose that proteins can be suitable targets for diagnostic methods based on antigen detection.

Identification of Novel Immunogenic Proteins from Mycoplasma bovis and Establishment of an Indirect ELISA Based on Recombinant E1 Beta Subunit of the Pyruvate Dehydrogenase Complex

The pathogen Mycoplasma bovis (M. bovis) is a major cause of respiratory disease, mastitis, and arthritis in cattle. Screening the key immunogenic proteins and updating rapid diagnostic techniques are necessary to the prevention and control of M. bovis infection. In this study, 19 highly immunogenic proteins from M. bovis strain PD were identified using 2-dimensional gel electrophoresis, immunoblotting and MALDI-TOF/TOF MS. Of these 19 proteins, pyruvate dehydrogenase E1 component beta subunit (PDHB) showed excellent immune reactivity and repeatability. PDHB was found to be conserved in different M. bovis isolates, as indicated by Western blot analysis. On the basis of these results, a rPDHB-based indirect ELISA (iELISA) was established for the detection of serum antibodies using prokaryotically expressed recombinant PDHB protein as the coating antigen. The specificity analysis result showed that rPDHB-based iELISA did not react with other pathogens assessed in our study except M. agalactiae (which infects sheep and goats). Moreover, 358 serum samples from several disease-affected cattle feedlots were tested using this iELISA system and a commercial kit, which gave positive rates of 50.8% and 39.9%, respectively. The estimated Kappa agreement coefficient between the two methods was 0.783. Notably, 39 positive serum samples that had been missed by the commercial kit were all found to be positive by Western blot analysis. The detection rate of rPDHB-based iELISA was significantly higher than that of the commercial kit at a serum dilution ratio of 1∶5120 to 1∶10,240 (P<0.05). Taken together, these results provide important information regarding the novel immunogenic proteins of M. bovis. The established rPDHB-based iELISA may be suitable for use as a new method of antibody detection in M. bovis.

The E1 beta-subunit of pyruvate dehydrogenase is surface-expressed in Lactobacillus plantarum and binds fibronectin

Lactobacillus plantarum is among the species with a probiotic activity. Adhesion of probiotic bacteria to host tissues is an important principle for strain selection, because it represents a crucial step in the colonization process of either pathogens or commensals. Most bacterial adhesins are proteins, and a major target for them is fibronectin, an extracellular matrix glycoprotein. In this study we demonstrate that PDHB, a component of the pyruvate dehydrogenase complex, is a factor contributing to fibronectin-binding in L. plantarum LM3. By means of fibronectin overlay immunoblotting assay, we identified a L. plantarum LM3 surface protein with apparent molecular mass of 35kDa. Mass spectrometric analysis shows that this protein is the pyruvate dehydrogenase E1 beta-subunit (PDHB). The corresponding pdhB gene is located in a 4-gene cluster encoding pyruvate dehydrogenase. In LM3-B1, carrying a null mutation in pdhB, the 35kDa adhesin was not anymore detectable by immunoblotting assay. Nevertheless, the pdhB null mutation did not abolish pdhA, pdhC, and pdhD transcription in LM3-B1. By adhesion assays, we show that LM3-B1 cells bind to immobilized fibronectin less efficiently than wild type cells. Moreover, we show that pdhB expression is negatively regulated by the CcpA protein and is induced by bile.

Protein profiles in human ovarian cancer cell lines correspond to their metabolic activity and to metabolic profiles of respective tumor xenografts

Many solid tumors show a large variability in glycolytic activity and lactate accumulation, which has been correlated with different metastatic spread, radioresistance and patient survival. To investigate potential differences in protein profiles underlying these metabolic variances, the highly glycolytic human ovarian cancer cell line OC316 was investigated and compared with the less glycolytic line IGROV-1. Extracellular acidification and oxygen consumption were analyzed with an extracellular flux analyzer. Glycolysis-associated proteins, including specific membrane transporters, were quantified through in-cell western analyses. Metabolic properties of corresponding tumor xenografts were assessed via induced metabolic bioluminescence imaging. Extracellular flux analyses revealed elevated bioenergetics of OC316 cells. Hexokinase II, pyruvate kinase, pyruvate dehydrogenase E1 beta subunit and pyruvate dehydrogenase kinase 1, as well as the glucose transporter 1 and the monocarboxylate transporter 4, were overexpressed in these cells compared with IGROV-1. When generating tumor xenografts in SCID mice, cells maintained their glycolytic behavior, i.e. OC316 showed higher lactate concentrations than IGROV-1 tumors. In summary, a congruency between protein profiles and metabolic properties has been demonstrated in the human ovarian cancer lines investigated. Also, a perpetuation of glycolytic characteristics during the transition from in vitro to the in vivo situation has been documented. This model system could be useful for systematic studies on therapeutic intervention by manipulation of tumor glycolysis and associated pathways.

Pluripotency-associated genes in human nasopharyngeal carcinoma CNE-2 cells are reactivated by a unique epigenetic sub-microenvironment

BackgroundThere is increasing evidence that cancers contain their own stem-like cells, and particular attention has been paid to one subset of cancer-stem cells termed side population (SP). Stem cells under normal physical conditions are tightly controlled by their microenvironment, however, the regulatory role of the microenvironment surrounding cancer stem cells is not well characterized yet. In this study we found that the phenotype of SP can be "generated" by macrophage-like cells under conditioned culture. Furthermore the gene regulation pathway involved in cellular reprogramming process was investigated.MethodsThe selection and identification of SP in 50 CNE-2 single cell clones were performed by flow cytometry. The transwell assay and immunofluorescence staining were used to measure migration and cancer stem cell characters of non-SP single clone cells cultured with conditioned medium respectively. The subtraction suppression hybridization (SSH) technique and northern blotting analysis was applied to explore the pluripotency-associated genes under a unique epigenetic sub-microenvironment.ResultsAmong 50 clones, only one did not possess SP subpopulation while others did. The non-SP cells induced by macrophage-like cells showed more aggressive characters, which increased cell migration compared with the control cells and showed some fraction of SP phenotype. These cells expressed distinguished level of pluripotency-associated genes such as ADP-ribosylation factor-like 6 interacting protein (ARMER), poly (rC) binding protein 1 (PCBP1) and pyruvate dehydrogenase E1-β subunit (PDHB) when subjected to the environment.ConclusionTo our knowledge, this is the first study to demonstrate that non-SP single-clone cells can be induced to generate a SP phenotype when they are cultured with conditioned medium of macrophage-like cells, which is associated with the reactivation of pluripotency-associated genes.

Thiamine responsive pyruvate dehydrogenase deficiency in an adult with peripheral neuropathy and optic neuropathy

Pyruvate dehydrogenase complex (PDHc) is a thiamine dependent key enzyme for energy production which catalyses decarboxylation of pyruvate into acetyl CoA for the Krebs cycle.1 PDHc deficiency is generally (80%)...

Mapping phosphoproteins in Mycoplasma genitalium and Mycoplasma pneumoniae.

BackgroundLittle is known regarding the extent or targets of phosphorylation in mycoplasmas, yet in many other bacterial species phosphorylation is known to play an important role in signaling and regulation of cellular processes. To determine the prevalence of phosphorylation in mycoplasmas, we examined the CHAPS-soluble protein fractions of Mycoplasma genitalium and Mycoplasma pneumoniae by two-dimensional gel electrophoresis (2-DE), using a combination of Pro-Q Diamond phosphoprotein stain and 33P labeling. Protein spots that were positive for phosphorylation were identified by peptide mass fingerprinting using MALDI-TOF-TOF mass spectrometry.ResultsWe identified a total of 24 distinct phosphoproteins, about 3% and 5% of the total protein complement in M. pneumoniae and M. genitalium, respectively, indicating that phosphorylation occurs with prevalence similar to many other bacterial species. Identified phosphoproteins include pyruvate dehydrogenase E1 alpha and beta subunits, enolase, heat shock proteins DnaK and GroEL, elongation factor Tu, cytadherence accessory protein HMW3, P65, and several hypothetical proteins. These proteins are involved in energy metabolism, carbohydrate metabolism, translation/transcription and cytadherence. Interestingly, fourteen of the 24 phosphoproteins we identified (58%) were previously reported as putatively associated with a cytoskeleton-like structure that is present in the mycoplasmas, indicating a potential regulatory role for phosphorylation in this structure.ConclusionThis study has shown that phosphorylation in mycoplasmas is comparable to that of other bacterial species. Our evidence supports a link between phosphorylation and cytadherence and/or a cytoskeleton-like structure, since over half of the proteins identified as phosphorylated have been previously associated with these functions. This opens the door to further research into the purposes and mechanisms of phosphorylation for mycoplasmas.

Branched chain α-keto acid dehydrogenase, E1-β subunit gene is associated with premature ovarian failure

Genetic variants of the human branched chain alpha-keto acid dehydrogenase, E1-beta subunit (BCKDHB) gene were identified and they have been associated with premature ovarian failure (POF). Reconstructed haplotype from these variants was also associated with POF.

Proteomic survey of the pathogenic Mycoplasma hyopneumoniae strain 7448 and identification of novel post-translationally modified and antigenic proteins

Mycoplasma hyopneumoniae is an important pathogen for pigs, being the causative agent of enzootic pneumonia. Recently, the genome sequences of three strains, J, 7448 and 232 have been reported. Here, we describe the results of a proteomic analysis, based on two-dimensional gel electrophoresis of soluble protein extracts, immunoblot and mass spectrometry, which was carried out aiming the identification of gene products and antigenic proteins from the M. hyopneumoniae pathogenic strain 7448. A preliminary M. hyopneumoniae proteome map in two pH ranges (3–10 and 4–7) was produced. A total of 31 different coding DNA sequences (CDSs), including three hypothetical ones, were experimentally verified with the identification of the corresponding protein products by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. According to the Clusters of Orthologous Groups (COG) functional classification, the identified proteins were assigned to the groups of metabolism (13), cellular processes (5) and information and storage processing (4). Nine of the identified proteins were not classifiable by COG, including some related to cytoadherence and possibly involved in pathogenicity. Moreover, at least five highly antigenic proteins of M. hyopneumoniae were identified by immunoblots, including four novel ones (a heat shock protein 70, an elongation factor Tu, a pyruvate dehydrogenase E1-beta subunit and the P76 membrane protein). The now available proteome map is expected to serve as a reference for comparative analyses between M. hyopneumoniae pathogenic and non-pathogenic strains, and for methabolic studies based on cells cultured under modified conditions.

Apparent presence of Ser133‐phosphorylated cyclic AMP response element binding protein (pCREB) in brain mitochondria is due to cross‐reactivity of pCREB antibodies with pyruvate dehydrogenase

Cyclic AMP response element binding protein (CREB) is a constitutive transcription factor that activates transcription following stimulus-dependent phosphorylation at Ser133, implicated in synaptic plasticity and neuronal survival pathways. The prevailing view that CREB is exclusively nuclear has been questioned by several studies, and, for example, mitochondrial localization has been reported. Using subcellular fractionation of rat brain cortex coupled with western immunoblotting with Ser133-phospho-CREB (pCREB) antibodies, we found a robust pCREB immunoreactivity (IR) in mitochondria-enriched fractions. The pCREB antibodies also stained the mitochondria, in addition to nuclei, of glial cells in primary cortical cultures. However, two CREB antibodies against different epitopes and gel shift assay detected the CREB protein mainly in the nuclear fraction. The two-dimensional electrophoretic mobility of mitochondrial pCREB IR differed markedly from the nuclear CREB/pCREB IR, indicating that the pCREB antibody cross-reacts with another mitochondrial protein. Immunoprecipitation of the mitochondrial pCREB IR produced three bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as E2, E1 alpha-subunit, and E1 beta-subunit of pyruvate dehydrogenase complex. The cross-reacting epitope was identified as phospho-Ser300 of the alpha-subunit. In conclusion, this study confirms the presence of pCREB-like IR in brain mitochondria that, after careful scrutiny, turned out to be pyruvate dehydrogenase rather than authentic CREB.

The delta subunit of RNA polymerase functions in sporulation.

Purified RNA polymerase from Bacillus subtilis and other Gram-positive organisms contains a novel subunit designated delta encoded by the rpoE gene. There is no distinctive phenotype of strains with a disruption of this gene, so the function of delta is very subtle or redundant. We have found, however, that suppression of a block in sporulation of B. subtilis at early stage III owing to disruption of the pdhC gene encoding the E2 subunit of pyruvate dehydrogenase (PDH) was attributable to a Tn10 insertion in the rpoE gene. An independent disruption of this gene also caused suppression. An earlier sporulation block due to absence of the E1beta subunit of PDH was not suppressed. This specific suppression indicates that the delta subunit does have some direct or indirect role in sporulation, probably in the transcription of selected genes at stage II-III of sporulation, which is critical but only when there is functional E2.

Structural and Biochemical Basis for Novel Mutations in Homozygous Israeli Maple Syrup Urine Disease Patients: A PROPOSED MECHANISM FOR THE THIAMIN-RESPONSIVE PHENOTYPE

Maple syrup urine disease (MSUD) results from mutations affecting different subunits of the mitochondrial branched-chain alpha-ketoacid dehydrogenase complex. In this study, we identified seven novel mutations in MSUD patients from Israel. These include C219W-alpha (TGC to TGG) in the E1alpha subunit; H156Y-beta (CAT to TAT), V69G-beta (GTT to GGT), IVS 9 del[-7:-4], and 1109 ins 8bp (exon 10) in the E1beta subunit; and H391R (CAC to CGC) and S133stop (TCA to TGA) affecting the E2 subunit of the branched-chain alpha-ketoacid dehydrogenase complex. Recombinant E1 proteins carrying the C219W-alpha or H156Y-beta mutation show no catalytic activity with defective subunit assembly and reduced binding affinity for cofactor thiamin diphosphate. The mutant E1 harboring the V69G-beta substitution cannot be expressed, suggesting aberrant folding caused by this mutation. These E1 mutations are ubiquitously associated with the classic phenotype in homozygous-affected patients. The H391R substitution in the E2 subunit abolishes the key catalytic residue that functions as a general base in the acyltransfer reaction, resulting in a completely inactive E2 component. However, wild-type E1 activity is enhanced by E1 binding to this full-length mutant E2 in vitro. We propose that the augmented E1 activity is responsible for robust thiamin responsiveness in homozygous patients carrying the H391R E2 mutation and that the presence of a full-length mutant E2 is diagnostic of this MSUD phenotype. The present results offer a structural and biochemical basis for these novel mutations and will facilitate DNA-based diagnosis for MSUD in the Israeli population.

Differential effect of DCA treatment on the pyruvate dehydrogenase complex in patients with severe PDHC deficiency.

Dichloroacetate (DCA) is a structural analog of pyruvate that has been recommended for the treatment of primary lactic acidemia, particularly in patients with pyruvate dehydrogenase (PDHC) deficiency. Recent reports have demonstrated that the response to DCA may depend on the type of molecular abnormality. In this study, we investigated the response to DCA in various PDHC-deficient cell lines and tried to determine the mechanism involved. The effect of chronic 3-d DCA treatment on PDHC activity was assessed in two PDHC-deficient cell lines, each with a different point mutation in the E1alpha subunit gene (R378C and R88C), and one cell line in which an 8-bp tandem repeat was deleted (W383 del). Only two (R378C and R88C) of the three PDHC-deficient cell lines with very low levels of PDHC activity and unstable polypeptides were sensitive to chronic DCA treatment. In these cell lines, DCA treatment resulted in an increase in PDHC activity by 125 and 70%, respectively, with concomitant increases of 121 and 130% in steady-state levels of immunoreactive E1alpha. DCA treatment reduced the turnover of the E1alpha subunit in R378C and R88C mutant cells with no significant effect on the E1beta subunit. Chronic DCA treatment significantly improved the metabolic function of PDHC in digitonin-permeabilized R378C and R88C fibroblasts. The occurrence of DCA-sensitive mutations suggests that DCA treatment is potentially useful as an adjuvant to ketogenic and vitamin treatment in PDHC-deficient patients.