O-GlcNAcylation mediates the control of cytosolic phosphoenolpyruvate carboxykinase activity via Pgc1a

PGC1a is a coactivator of many transcription factors and cytosolic phosphoenolpyruvate carboxykinase (PCK1) is a key enzyme for gluconeogenesis. PGC1a interacts with the transcription factor PPAR¿ to stimulate PCK1 expression and thus de novo glucose synthesis. These proteins are not only important for central energy metabolism but also for supplying intermediates for other metabolic pathways, including lipidogenesis and protein synthesis and might therefore be important factors in the ethiopathogenesis of metabolic disorders like diabetes but also in other pathologies like cancer. Since polymorphisms in these proteins have been related to some phenotypic traits in animals like pigs and PGC1a G482S polymorphism increases fat deposition in humans, we have investigated the molecular basis of such effects focusing on a commonly studied polymorphism in pig Pgc1a, which changes a cysteine at position 430 (WT) of the protein to a serine (C430S). Biochemical analyses show that Pgc1a WT stimulates higher expression of human PCK1 in HEK293T and HepG2 cells. Paradoxically, Pgc1a WT is less stable than Pgc1a p.C430S in HEK293T cells. However, the study of different post-translational modifications shows a higher O-GlcNAcylation level of Pgc1a p.C430S. This higher O-GlcNAcylation level significantly decreases the interaction between Pgc1a and PPAR¿ demonstrating the importance of post-translational glycosylation of PGC1a in the regulation of PCK1 activity. This, furthermore, could explain at least in part the observed epistatic effects between PGC1a and PCK1 in pigs

Kinetic and functional properties of human mitochondrial phosphoenolpyruvate carboxykinase

The cytosolic form of phosphoenolpyruvate carboxykinase (PCK1) plays a regulatory role in gluconeogenesis and glyceroneogenesis. The role of the mitochondrial isoform (PCK2) remains unclear. We report the partial purification and kinetic and functional characterization of human PCK2. Kinetic properties of the enzyme are very similar to those of the cytosolic enzyme. PCK2 has an absolute requirement for Mn2+ ions for activity; Mg2+ ions reduce the Km for Mn2+ by about 60 fold. Its specificity constant is 100 fold larger for oxaloacetate than for phosphoenolpyruvate suggesting that oxaloacetate phosphorylation is the favored reaction in vivo. The enzyme possesses weak pyruvate kinase-like activity (kcat=2.7 s-1). When overexpressed in HEK293T cells it enhances strongly glucose and lipid production showing that it can play, as the cytosolic isoenzyme, an active role in glyceroneogenesis and gluconeogenesis

Benzbromarone, quercetin, and folic acid inhibit amylin aggregation

Human Amylin, or islet amyloid polypeptide (hIAPP), is a small hormone secreted by pancreatic ß-cells that forms aggregates under insulin deficiency metabolic conditions, and it constitutes a pathological hallmark of type II diabetes mellitus. In type II diabetes patients, amylin is abnormally increased, self-assembled into amyloid aggregates, and ultimately contributes to the apoptotic death of ß-cells by mechanisms that are not completely understood. We have screened a library of approved drugs in order to identify inhibitors of amylin aggregation that could be used as tools to investigate the role of amylin aggregation in type II diabetes or as therapeutics in order to reduce ß-cell damage. Interestingly, three of the compounds analyzed—benzbromarone, quercetin, and folic acid—are able to slow down amylin fiber formation according to Thioflavin T binding, turbidimetry, and Transmission Electron Microscopy assays. In addition to the in vitro assays, we have tested the effect of these compounds in an amyloid toxicity cell culture model and we have found that one of them, quercetin, has the ability to partly protect cultured pancreatic insulinoma cells from the cytotoxic effect of amylin. Our data suggests that quercetin can contribute to reduce oxidative damage in pancreatic insulinoma ß cells by modulating the aggregation propensity of amylin

Estudios In Vitro de Cementos de α-Fosfato Tricálcico Modificados con β-Silicato Dicálcico

The combination of in situ self-setting and biocompatibility, makes calcium phosphate cements highly promising materials for a wide range of clinical applications. However, its low strength limits their use only to lowstress applications. β-Dicalcium silicate (β-C2S) is a Portland cement component, able to react with water to form a hydrated phase that enhance mechanical strength of material. Different authors reported the bioactive capacity of this compound. In this investigation, α-TCP cements modified with β-C2S were prepared. The α-tricalcium phosphate (αTCP) powder was prepared through acid-base method, and β-C2S was synthesized by sol-gel method. Materials were characterized both chemically and physically. Biodegradability was studied by soaking the materials in simulated body fluid for various time periods. The results showed that a cement with 20 % of β-C2S exhibited greater compressive strength and pH values (19,8 MPa and 8,09 respectively).La capacidad que presentan los cementos de fosfato de calcio de fraguar en condiciones fisiológicas, así como su excelente biocompatibilidad, hacen estos materiales factibles para diferentes aplicaciones clínicas. Sin embargo sus bajas propiedades mecánicas limitan dichas aplicaciones a zonas de menores esfuerzos físicos. El βsilicato dicálcico (β-C2S) es un componente del cemento Portland, que reacciona con agua formando una fase hidratada de elevada resistencia mecánica. Diferentes autores han demostrado la capacidad bioactiva de este compuesto. En el presente trabajo fueron preparados cementos de α-fosfato tricálcico (α-TCP) modificados con β-C2S.El polvo de α-TCP fue obtenido por reacción ácido-base y el β-C2S vía sol-gel. Los materiales fueron caracterizados físico-químicamente, además de estudiar la biodegradación de los mismos a través de su inmersión en solución fisiológica simulada. Los mayores valores de resistencia a la compresión y pH correspondieron al cemento con 20% de β-C2S (19,8 MPa and 8,09 respectivamente

Pharmacology and preclinical validation of a novel anticancer compound targeting PEPCK-M

Background: Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the decarboxylation of oxaloacetate to phosphoenolpyruvate. The mitochondrial isozyme, PEPCK-M is highly expressed in cancer cells, where it plays a role in nutrient stress response. To date, pharmacological strategies to target this pathway have not been pursued. Methods: A compound embodying a 3-alkyl-1,8-dibenzylxanthine nucleus (iPEPCK-2), was synthesized and successfully probed in silico on a PEPCK-M structural model. Potency and target engagement in vitro and in vivo were evaluated by kinetic and cellular thermal shift assays (CETSA). The compound and its target were validated in tumor growth models in vitro and in murine xenografts. Results: Cross-inhibitory capacity and increased potency as compared to 3-MPA were confirmed in vitro and in vivo. Treatment with iPEPCK-2 inhibited cell growth and survival, especially in poor-nutrient environment, consistent with an impact on colony formation in soft agar. Finally, daily administration of the PEPCK-M inhibitor successfully inhibited tumor growth in two murine xenograft models as compared to vehicle, without weight loss, or any sign of apparent toxicity. Conclusion: We conclude that iPEPCK-2 is a compelling anticancer drug targeting PEPCK-M, a hallmark gene product involved in metabolic adaptations of the tumor.We acknowledge the skillful technical support by the Scientific and Technical Services at the University of Barcelona, Bellvitge Campus, and to the “Consorci de Serveis Universitaris de Catalunya” (CSUC) for computational facilities

The C-terminal Region of Mitochondrial Single-subunit RNA Polymerases Contains Species-specific Determinants for Maintenance of Intact Mitochondrial Genomes

Functional conservation of mitochondrial RNA polymerases was investigated in vivo by heterologous complementation studies in yeast. It turned out that neither the full-length mitochondrial RNA polymerase of Arabidopsis thaliana, nor a set of chimeric fusion constructs from plant and yeast RNA polymerases can substitute for the yeast mitochondrial core enzyme Rpo41p when expressed in Δrpo41 yeast mutants. Mitochondria from mutant cells, expressing the heterologous mitochondrial RNA polymerases, were devoid of any mitochondrial genomes. One important exception was observed when the carboxyl-terminal domain of Rpo41p was exchanged with its plant counterpart. Although this fusion protein could not restore respiratory function, stable maintenance of mitochondrial petite genomes (ρ(−))(−) was supported. A carboxyl-terminally truncated Rpo41p exhibited a comparable activity, in spite of the fact that it was found to be transcriptionally inactive. Finally, we tested the carboxyl-terminal domain for complementation in trans. For this purpose the last 377 amino acid residues of yeast mitochondrial Rpo41p were fused to its mitochondrial import sequence. Coexpression of this fusion protein with C-terminally truncated Rpo41p complemented the Δrpo41 defect. These data reveal the importance of the carboxyl-terminal extension of Rpo41p for stable maintenance of intact mitochondrial genomes and for distinct species-specific intramolecular protein–protein interactions