Expression Of Pyruvate Dehydrogenase Complex Research Articles

Restoration of fitness lost due to dysregulation of the pyruvate dehydrogenase complex is triggered by ribosomal binding site modifications.

SUMMARYPyruvate dehydrogenase complex (PDC) functions as the main determinant of the respiro-fermentative balance because it converts pyruvate to acetyl-coenzyme A (CoA), which then enters the TCA (tricarboxylic acid cycle). PDC is repressed by the pyruvate dehydrogenase complex regulator (PdhR) in Escherichia coli. The deletion of the pdhR gene compromises fitness in aerobic environments. We evolve the E. coli pdhR deletion strain to examine its achievable growth rate and the underlying adaptive strategies. We find that (1) optimal proteome allocation to PDC is critical in achieving optimal growth rate; (2) expression of PDC in evolved strains is reduced through mutations in the Shine-Dalgarno sequence; (3) rewiring of the TCA flux and increased reactive oxygen species (ROS) defense occur in the evolved strains; and (4) the evolved strains adapt to an efficient biomass yield. Together, these results show how adaptation can find alternative regulatory mechanisms for a key cellular process if the primary regulatory mode fails.

Abstract 5419: Thiamine deficiency induces HIF-1 α stabilization and target gene expression.

Abstract Introduction: Thiamine deficiencies have been reported in a number of common pathologies including diabetes, neurodegenerative disorders and in particular, cancer. Deficiencies can arise due to drug mediated reductions in cellular availability of thiamine or due to limited diffusion into solid tumors. Biochemically, a metabolic consequence of thiamine deficiency is a reduction in enzyme activity and expression of pyruvate dehydrogenase complex (PDH), which converts pyruvate to acetyl-CoA. Evidence suggests that trans-activation of hypoxia inducible factor 1-alpha (HIF-1α) regulated genes is mediated through stoichiometric changes in the metabolic intermediate pyruvate, independent of low oxygen termed pseudo-hypoxia. The resulting dysfunction of PDH and accumulation of pyruvate during TD may facilitate a pseudo-hypoxic state and mediate trans-activation of HIF-1α regulated genes. Therefore, we hypothesize that thiamine deficiency induces a pseudo-hypoxic state resulting in a stabilization of HIF-1α. Methods: The impact of TD treatments on cellular pyruvate levels was determined after 48h of TD treatment using a pyruvate assay. Protein expression of HIF-1α was assessed in nuclear lysates of SK-N-BE cells treated with 1% O2 and TD for 12, 24, and 48h by Western blot. Expression of the known HIF-1α regulated gene, GLUT1 was measured by qRTPCR after 12, 24 and 48h hypoxia (1% O2) and thiamine depletion. Results: An approximately 2 fold increase in cellular pyruvate was observed after 48h of TD exposure. A corresponding increase in HIF-1α protein expression in both 1% O2 and TD was found after 48h exposure. SK-N-BE cells demonstrated a 6 and 10 fold increase in expression of GLUT1 and SLC19A3, respectively after 48h of 1% O2 exposure. TD treatment exhibited an approximately 2 fold increase in expression of both GLUT1 and SLC19A3 after 48h treatments. Conclusions: An increase in pyruvate production and the induction of HIF-1α protein expression after TD treatment suggests the role of TD acting as a feed forward mechanism for HIF-1α stabilization and gene regulation. These results demonstrate nutritional regulation of the pro-oncogenic transcription factor, HIF-1α and an important connection of cellular thiamine to adaptive glycolytic metabolism. Citation Format: Rebecca Sweet, Lauren Lim, Jason Zastre. Thiamine deficiency induces HIF-1 α stabilization and target gene expression. [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 5419. doi:10.1158/1538-7445.AM2013-5419

Proapoptotic protein bim and its upstream activator Foxo3A are overexpressed in primary biliary cirrhosis but not in primary sclerosing cholangitis

IntroductionApoptosis, with subsequent aberrant expression of pyruvate dehydrogenase complex (PDC-E2) on cell surface has been postulated to play an important role in the pathogenesis of Primary Biliary Cirrhosis (PBC). More...

Liver proteome analysis of adaptive response in rat immediately after partial hepatectomy

The extraordinary ability of the liver to regenerate after resection continues to be an important fascination to mammalian liver researchers. However, at present, there are still several central questions regarding the process of liver regeneration that are not clear. In our study, we try to clarify how the liver is able to maintain its functions as well as to initiate liver regeneration after a significant loss of two-thirds. Here differentially expressed proteins in rat livers at 1 h after partial hepatectomy (PHx) and sham operation were analyzed using 2-DE combined with MALDI-TOF/TOF MS. After the analysis, 24 significantly changed spots (ratio> or =2, p<0.05) were identified. Those proteins are involved in important liver functions such as metabolism, detoxification, and inflammation. Based on the changes in the protein levels found in our data, we identified two aspects of remnant liver immediately after PHx, which focused on the hepatic adaptation and the inflammatory response associated with the initiation of liver regeneration after PHx. For the first time, the differential expression of pyruvate dehydrogenase complex (PDHX), paraoxonase 1 (PON1), thyroid hormone receptor beta, GAP43 (where GAP stands for growth-associated protein), and interleukin-2 (IL2), after PHx, were validated by Western blot.

Role of acetyl-coenzyme A synthetase in leaves of Arabidopsis thaliana

Acetyl-coenzyme A synthetase (ACS) is a plastidic enzyme that forms acetyl-coenzyme A (acetyl-CoA) from acetate and coenzyme A using the energy from ATP. Traditionally it has been thought to be the major source for the production of acetyl-CoA destined for fatty acid formation. Recent work suggested that the accumulation of lipids in developing Arabidopsis seeds was more closely correlated with the expression of pyruvate dehydrogenase complex than with the expression of ACS, suggesting that most of the carbon for fatty acid formation in the plastids of seeds comes from pyruvate rather than from acetate. To explore the role of this enzyme, Arabidopsis plants with altered amounts of ACS were generated by overexpressing its cDNA in either the sense or the antisense configuration. The resulting plants had in vitro enzyme activities that ranged from about 5% to over 400% of wild-type levels. The rate of [1- 14C]acetate conversion into fatty acids was closely related to the in vitro ACS activity, showing that the amount of enzyme clearly limited the capacity of leaves to convert exogenous acetate to fatty acids. There was, however, no relationship between the ACS level and the capacity of the plants to incorporate 14CO 2 into 14C-labeled fatty acids. These data strongly support the idea that, although plants can convert acetate into fatty acids, relatively little carbon moves through this pathway under normal conditions.

Pyruvate dehydrogenase and the regulation of glucose oxidation in hypertrophied rat hearts.

Coupling of glucose oxidation to glycolysis is lower in hypertrophied than in non-hypertrophied hearts, contributing to the compromised mechanical performance of hypertrophied hearts. Here, we describe studies to test the hypothesis that low coupling of glucose oxidation to glycolysis in hypertrophied hearts is due to reduced activity and/or expression of the pyruvate dehydrogenase complex (PDC). We examined the effects of dichloroacetate (DCA), an inhibitor of PDC kinase, and of alterations in exogenous palmitate supply on coupling of glucose oxidation to glycolysis in isolated working hypertrophied and control hearts from aortic-constricted and sham-operated male Sprague-Dawley rats. It was anticipated that the addition of DCA or the absence of palmitate would promote PDC activation and consequently normalize coupling between glycolysis and glucose oxidation in hypertrophied hearts if our hypothesis was correct. Addition of DCA or removal of palmitate improved coupling of glucose oxidation to glycolysis in control and hypertrophied hearts. However, coupling remained substantially lower in hypertrophied hearts. PDC activity in extracts of hypertrophied hearts was similar to or higher than in extracts of control hearts under all perfusion conditions. No differences were observed between hypertrophied and control hearts with respect to expression of PDC, PDC kinase, or PDC phosphatase. Low coupling of glucose oxidation to glycolysis in hypertrophied hearts is not due to a reduction in PDC activity or subunit expression indicating that other mechanism(s) are responsible.

Secretory autoantibodies in primary biliary cirrhosis (PBC).

It is unclear how breakdown in immune tolerance to the ubiquitous self-antigen pyruvate dehydrogenase complex (PDC), seen in the autoimmune liver disease PBC, gives rise to tissue damage with such a limited distribution (restricted to the liver and salivary and lachrymal glands). One property shared by these tissues is the ability to export secretory IgA by the process of transcytosis. The aim of this study was to address whether active transcytosis of anti-PDC IgA occurs across epithelial surfaces in PBC, a finding that might implicate mucosal specific immune mechanisms in the pathogenesis of this disease. Parotid saliva was collected from PBC patients (n = 44), normal controls (n = 28) and PBC patients post-liver transplantation (n = 11). IgA and secretory component-positive antibodies specific for human PDC were quantified by ELISA and immunoblotting. PBC patients (but not control subjects) had anti-PDC IgA in their saliva. The strong correlation seen between titres detected using anti-IgA and anti-secretory component antibodies suggests that this is predominantly secretory IgA reaching the saliva by the active process of epithelial transcytosis. Titres of anti-PDC IgA remain high in PBC patients saliva post-liver transplant. Findings from studies of IgA in viral infection models raise the possibility that anti-PDC IgA could, whilst undergoing transcytosis, bind to newly translated PDC components in the cytoplasm of the epithelial cells transporting them out of the cell and inducing metabolic damage. This model would, if correct, help to explain the mechanism and tropism of tissue damage in PBC and the aberrant pattern of expression of PDC on the apical surface of biliary and salivary epithelial cells reported in this disease.

Biliary epithelial expression of pyruvate dehydrogenase complex in primary biliary cirrhosis: An immunohistochemical and immunoelectron microscopic study

It has been reported recently that there is a unique distribution of the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2) on biliary epithelial cells in patients with primary biliary cirrhosis (PBC) but not primary sclerosing cholangitis. This distribution has been demonstrated using a mouse monoclonal antibody, coined C355.1. The epitope recognized by C355.1 is near the lipoic acid binding site of PDC-E2. C355.1 inhibits PDC-E2 activity in vitro and, unlike a panel of other monoclonal antibodies against different regions of PDC-E2, appears to bind not only to mitochondria but also to a unique antigen expressed predominantly on the luminal side of biliary epithelial cells in PBC. We have extended these observations by studying the subcellular reactivity of C355.1 using postembedding immunoelectron microscopy on the intrahepatic small bile ducts of PBC livers, extrahepatic biliary obstruction (EBO) livers, and normal livers. We report that the reactivity of C355.1 can be classified into two categories. The first category is characterized by small foci of reaction products that were randomly dispersed in cytoplasm, particularly in supranuclear areas; the ultrastructural characterization of these foci was impossible to define but was similar in PBC and EBO. However, of particular interest was the second category of reactivity, which was characterized by deposition of reaction products around the biliary lumen, including microvilli and adjacent subluminal ectoplasm and secretory substances in the biliary lumen. This staining pattern was frequent in PBC livers, only occasionally evident in EBO livers, and not found in normal livers. These data further define and highlight the unique subcellular distribution of PDC-E2 around the biliary lumen in PBC livers and suggest that this abnormality is related to the pathogenesis of bile duct lesions.