Pyruvate Carboxylase Activates the RIG-I-like Receptor-Mediated Antiviral Immune Response by Targeting the MAVS signalosome.

When retinoic acid-inducible gene 1 protein (RIG-I)-like receptors sense viral dsRNA in the cytosol, RIG-I and melanoma differentiation-associated gene 5 (MDA5) are recruited to the mitochondria to interact with mitochondrial antiviral signaling protein (MAVS) and initiate antiviral immune responses. In this study, we demonstrate that the biotin-containing enzyme pyruvate carboxylase (PC) plays an essential role in the virus-triggered activation of nuclear factor kappa B (NF-κB) signaling mediated by MAVS. PC contributes to the enhanced production of type I interferons (IFNs) and pro-inflammatory cytokines, and PC knockdown inhibits the virus-triggered innate immune response. In addition, PC shows extensive antiviral activity against RNA viruses, including influenza A virus (IAV), human enterovirus 71 (EV71), and vesicular stomatitis virus (VSV). Furthermore, PC mediates antiviral action by targeting the MAVS signalosome and induces IFNs and pro-inflammatory cytokines by promoting phosphorylation of NF-κB inhibitor-α (IκBα) and the IκB kinase (IKK) complex, as well as NF-κB nuclear translocation, which leads to activation of interferon-stimulated genes (ISGs), including double-stranded RNA-dependent protein kinase (PKR) and myxovirus resistance protein 1 (Mx1). Our findings suggest that PC is an important player in host antiviral signaling.

Localization of inhibitory antibodies to the biotin domain of human pyruvate carboxylase.

Pyruvate carboxylase [EC 6.4.1.1] plays an important anaplerotic role in many species by catalyzing the carboxylation of pyruvate to oxaloacetate. To extend our understanding about the structure and function of pyruvate carboxylase (PC), a series of monoclonal antibodies were raised against sheep liver PC and those displaying inhibitory activity were further characterized. The binding epitopes of two monoclonal antibodies that displayed strong inhibitory activity were mapped. Six overlapping fragments of the human enzyme were expressed as thioredoxin fusion proteins in Escherichia coli and subjected to Western blot analysis. Both monoclonal antibodies (MAbs) recognized fragments encompassing the enzyme's C-terminal region, known to contain the structured biotin domain. Through deletion analysis, this domain was determined to be a minimal size of 80 amino acids. Further deletions that disrupted the conformation of the domain abolished antibody binding, indicating these antibodies recognized discontinuous epitopes. To further define the critical residues required for antibody recognition, a model of the domain was produced and an alanine scan performed on selected surface-exposed residues. Our results show that residues encompassing the biotin attachment site, but not biotin itself, are critical for the binding of both antibodies. These data provide a mechanism to explain the inhibitory activity of the antibodies.

Effects of starvation, diabetes and carbon tetrachloride intoxication on rat kidney cortex and liver pyruvate carboxylase levels.

Pyruvate carboxylase (PC) has been quantified in rat liver and kidney cortex under experimental conditions that modify the gluconeogenic response in both organs: fasting, carbon tetrachloride-induced liver degeneration and alloxan-induced diabetes. Enzymatic activity has been assayed by a 14CO2-fixation method. The amount of enzyme has been determined by competitive ELISA using antibodies raised against the purified rat kidney cortex enzyme. Purified fractions of rat-liver and rat-kidney cortex PC have been used as standards. Fasting and carbon tetrachloride administration induced a significant increase (25% to 30%) in the amount of enzyme in liver and kidney cortex. Alloxan-induced diabetes produced a nearly two-fold increase in the hepatic levels of enzyme without a significant modification in the content of the renal enzyme. These results are discussed on the basis of the different metabolic implications of both organs during the physiological or toxic treatments.

Multidrug resistance P-glycoprotein monoclonal antibody JSB-1 crossreacts with pyruvate carboxylase.

Multidrug resistance (MDR) is associated with overexpression of a 170 KD plasma membrane P-glycoprotein (P-gp), a putative energy-dependent efflux transporter that reduces intracellular accumulation of chemotherapeutic agents. For detection of P-gp expression in normal and malignant tissues, an MDR1-specific monoclonal antibody (MAb) JSB-1 has been used extensively. In this report we show that MAb JSB-1 crossreacts with a protein of M(r) approximately 130,000 present in rat liver mitochondrial inner membrane/matrix fractions. Peptide mapping and microsequencing identify this protein as pyruvate carboxylase (PC), an abundant mitochondrial enzyme. MAb JSB-1 also crossreacts with purified PC from bovine liver. Under immunoblotting conditions, this crossreactivity is partially abolished by pre-incubation of MAb JSB-1 with a 1000-fold molar excess of MAb C494 epitope-specific peptide (PNTLEGN), indicating that the epitope of MAb JSB-1 may either overlap with or be in close proximity to that of MAb C494. Immunohistochemical cross-reactivity was also demonstrated in cryosections of human skeletal muscle, a tissue known not to express P-gp. MAb JSB-1 strongly immunostained Type 1 fibers, the subtype known to contain abundant mitochondria. Use of MAb JSB-1 for detection of MDR1 P-gp expression should be approached with caution.

Immunocytochemical examination of neural rat and mouse primary cultures using monoclonal antibodies raised against pyruvate carboxylase.

Pyruvate carboxylase (EC 6.4.1.1; PC) catalyzes the formation of oxaloacetate by energy-dependent fixation of CO2 to pyruvate. The aim of the present work was to generate antibodies against PC and use them to localize PC in the cells of astroglia-rich and neuron-rich primary cultures derived from the brains of rats and mice. Mouse monoclonal antibodies raised against the enzyme were shown to be monospecific as indicated by immunoblotting. The staining of the cells for PC appeared in grains. These represent mitochondria, as PC is known as a mitochondrial enzyme. Immunocytochemical examination of astroglia-rich primary cultures of rat or mouse brain cells revealed a colocalization of PC with the astroglial marker glial fibrillary acidic protein (GFAP) in many cells. However, there were GFAP-positive cells showing no specific staining for PC, and vice versa. Also, in neuron-rich primary cultures PC was found only in the approximately 10% GFAP-expressing astroglial cells contaminating the neuron-rich primary culture, whereas it was absent from the neurons identified by antibodies against neuron-specific enolase. These results suggest that PC is predominantly an astroglial enzyme and that astroglial cells play an important role in the intermediary and the energy metabolism of the brain.

MDR1 gene-specific monoclonal antibody C494 cross-reacts with pyruvate carboxylase.

Overexpression of P-glycoprotein, the plasma membrane protein product of the MDR1 gene, is a major determinant in the development of resistance to a large number of cancer chemotherapeutic agents. A battery of antibodies, including the MDR1 gene-specific monoclonal antibody (mAb) C494, is used to evaluate human tissues in clinical multidrug resistance surveillance and modulation trials. In rat liver fractions, we report that mAb C494 strongly cross-reacted with a nonmembranous M(r) approximately 130,000 protein, comigrating with core-glycosylated human MDR1 on 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By immunoblotting and microsequence analysis, this protein was identified as pyruvate carboxylase (PC), an abundant mitochondrial enzyme. A search of the National Center for Biotechnology Information data base, using the epitope-specific sequence of mAb C494, revealed that PC (mouse) contains four of the five most reactive amino acids (TLEG), located near the COOH-terminal end of PC at positions 1167-1170. mAb C494 specifically reacted with PC purified from bovine liver; immunoreactivity was completely abolished by preincubating mAb C494 in the presence of excess synthetic C494 epitope-specific peptide. Furthermore, in cryosections of human skeletal muscle, a tissue known not to express P-glycoprotein, peptide-displaceable immunohistochemical staining with mAb C494 showed a distinct mitochondrial pattern specific to type 1 fibers. Variable immunostaining results were obtained with formaldehyde-fixed, paraffin-embedded muscle and isolated liver mitochondrial preparations. In summary, mAb C494 cross-reacted strongly with rat, bovine, and human PC. Caution is warranted in interpretation of immunoblots and immunohistochemical sections with this putative MDR1 gene-specific mAb.

Pyruvate carboxylase from Pseudomonas citronellolis: shape of the enzyme, and localization of its prosthetic biotin group by electron microscopic affinity labeling.

Pseudomonas citronellolis is known to contain a pyruvate carboxylase with an alpha 4 beta 4 composition. All the other pyruvate carboxylases investigated so far are made up of four seemingly identical subunits. Nevertheless, this exceptional pyruvate carboxylase exhibits a size and overall shape similar to other pyruvate carboxylases. Electron microscopic affinity labeling with avidin revealed that the prosthetic biotin groups (one per alpha beta unit, i.e. four per enzyme particle) are located close to the inter-unit junctions of pairs of alpha beta units making up the enzyme. This position of the prosthetic biotin groups is very similar to the location of the biotin in the other carboxylases.

Regulation of the synthesis and degradation of pyruvate carboxylase in 3T3-L1 cells.

The differentiation of mouse 3T3-L1 cells is characterized by an accumulation of cytosolic triglyceride and marked increase in many enzymatic activities involved in triglyceride biosynthesis. The specific activity of one such enzyme, pyruvate carboxylase, increases at least 20-fold and is due to a parallel increase in the intracellular concentration of the protein. Pulse-labeling experiments demonstrated that the increase in the specific activity of pyruvate carboxylase was due to an increase in the rate of enzyme synthesis. In the differentiated cell, pyruvate carboxylase represented 1.9% of the total cellular protein and 1% of the protein radiolabeled during a 1-h pulse. This was 35-and 28-fold higher than in the undifferentiated cell, respectively. The turnover of pyruvate carboxylase in the differentiated cell was similar to that in the undifferentiated cell with the enzyme having a half-life of 28-35 h. The half-life of apopyruvate carboxylase in avidin-treated 3T3-L1 cells was 24 h, indicating that the turnover of the apoenzyme was not significantly different than that of the holoenzyme. Radiolabeling pyruvate carboxylase with [14C]biotin and [3H]leucine demonstrated that the turnover of biotin associated with the enzyme was identical to the turnover of the enzymatic protein.

A simple method for the preparation of antibodies to the mitochondrial biotin-dependent carboxylases.

Heterologous antiserum to the 3 biotin-dependent carboxylases was prepared by selective removal of these enzymes from human liver on an avidin-sepharose column. A carboxylase-avidin-sepharose matrix was used as an antigen to produce anti-carboxylase antibodies. The resultant antisera can be used to purify the specific carboxylases, to prepare monoclonal antibodies to these enzymes or to study inherited carboxylase deficiencies and biotin-dependent intermediary metabolism.

Effect of streptozotocin-induced diabetes mellitus on the turnover of rat liver pyruvate carboxylase and pyruvate dehydrogenase.

Immunochemical techniques were used to study the effect of streptozotocin-induced diabetes on the amounts of pyruvate carboxylase and pyruvate dehydrogenase and on their rates of synthesis and degradation. Livers from diabetic rats had twice the pyruvate carboxylase activity of livers from normal rats when expressed in terms of DNA or body weight. The changes in catalytic activity closely paralleled changes in immunoprecipitable enzyme protein. Relative rates of synthesis determined by pulse-labelling studies showed that the ratio of synthesis of pyruvate carboxylase to that of average mitochondrial protein was increased 2.0-2.5 times in diabetic animals over that of control animals. Other radioisotopic studies indicated that the rate of degradation of this enzyme was not altered significantly in diabetic rats, suggesting that the increase in this enzyme was due to an increased rate of synthesis. Similar experiments with pyruvate dehydrogenase, the first component of the pyruvate dehydrogenase complex, showed that livers from diabetic rats had approximately the same amount of immunoprecipitable enzyme protein as the control animals, but a larger proportion of the enzyme was in its inactive state. The rates of synthesis and degradation of pyruvate dehydrogenase were not affected significantly by diabetes.

Induction of pyruvate carboxylase apoenzyme and holoenzyme in 3T3-L1 cells during differentiation.

The specific activity of pyruvate carboxylase [pyruvate:carbon-dioxide ligase (ADP-forming); EC 6.4.1.1] in 3T3-L1 cells increases approximately 20-fold when these cells differentiate to an adipocyte-like form [Mackall, J. C. & Lane, M. D. (1977) Biochem. Biophys. Res. Commun. 79, 720-725]. A specific antibody to the purified rat liver enzyme quantitatively precipitated pyruvate carboxylase from 3T3-L1 crude homogenates. Use of this immunological technique permitted us to demonstrate that the increase in pyruvate carboxylase activity is due to an increase in the intracellular concentration of the enzyme. The content of pyruvate carboxylase in differentiated 3T3-L1 cells is sufficiently high (1-2% of total protein) that the increase in this large protein (subunit M(r) = 130,000) can be visualized when 3T3-L1 crude extracts are subjected to electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. When 3T3-L1 cells differentiated in the presence of avidin, they contained less than 5% of the pyruvate carboxylase activity of cells that differentiated in the absence of avidin. However, the immunoprecipitable pyruvate carboxylase content of the avidin-treated cells was essentially the same as that of cells that differentiated without avidin. Full activity of the enzyme was rapidly restored in the avidin-treated cells upon the addition of excess biotin. The recovery of activity was closely correlated with the incorporation of [(14)C]biotin into immunoprecipitable pyruvate carboxylase. The rapidity with which the activity was restored and the insensitivity of the process to inhibitors of protein synthesis strongly suggest that the apoenzyme of pyruvate carboxylase accumulates during differentiation in the presence of avidin.