Dysfunction of Drosophila mitochondrial carrier homolog (Mtch) alters apoptosis and disturbs development.

Mitochondrial carrier homologs 1 (MTCH1) and 2 (MTCH2) are orphan members of the mitochondrial transporter family SLC25. Human MTCH1 is also known as presenilin 1-associated protein, PSAP. MTCH2 is a receptor for tBid and is related to lipid metabolism. Both proteins have been recently described as protein insertases of the outer mitochondrial membrane. We have depleted Mtch in Drosophila and show here that mutant flies are unable to complete development, showing an excess of apoptosis during pupation; this observation was confirmed by RNAi in Schneider cells. These findings are contrary to what has been described in humans. We discuss the implications in view of recent reports concerning the function of these proteins.

Design, Synthesis, and Efficacy Testing of Nitroethylene- and 7-Nitrobenzoxadiazol-Based Flavodoxin Inhibitors against Helicobacter pylori Drug-Resistant Clinical Strains and in Helicobacter pylori-Infected Mice.

Helicobacter pylori (Hp) infection is the main cause of peptic ulcer and gastric cancer. Hp eradication rates have fallen due to increasing bacterial resistance to currently used broad-spectrum antimicrobials. We have designed, synthesized, and tested redox variants of nitroethylene- and 7-nitrobenzoxadiazole-based inhibitors of the essential Hp protein flavodoxin. Derivatives of the 7-nitrobenzoxadiazole lead, carrying reduced forms of the nitro group and/or oxidized forms of a sulfur atom, display high therapeutic indexes against several reference Hp strains. These inhibitors are effective against metronidazole-, clarithromycin-, and rifampicin-resistant Hp clinical isolates. Their toxicity for mice after oral administration is low, and, when administered individually at single daily doses for 8 days in a mice model of Hp infection, they decrease significantly Hp gastric colonization rates and are able to eradicate the infection in up to 60% of the mice. These flavodoxin inhibitors constitute a novel family of Hp-specific antimicrobials that may help fight the constant increase of Hp antimicrobial-resistant strains.

Inhibition of Pig Phosphoenolpyruvate Carboxykinase Isoenzymes by 3-Mercaptopicolinic Acid and Novel Inhibitors.

There exist two isoforms of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) in pig populations that differ in a single amino acid (Met139Leu). The isoenzymes have different kinetic properties, affecting more strongly the Km and Vmax of nucleotides. They are associated to different phenotypes modifying traits of considerable economic interest. In this work we use inhibitors of phosphoenolpyruvate carboxykinase activity to search for further differences between these isoenzymes. On the one hand we have used the well-known inhibitor 3-mercaptopicolinic acid. Its inhibition patterns were the same for both isoenzymes: a three-fold decrease of the Ki values for GTP in 139Met and 139Leu (273 and 873 µM, respectively). On the other hand, through screening of a chemical library we have found two novel compounds with inhibitory effects of a similar magnitude to that of 3-mercaptopicolinic acid but with less solubility and specificity. One of these novel compounds, (N'1-({5-[1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl]-2-thienyl}methylidene)-2,4-dichlorobenzene-1-carbohydrazide), exhibited significantly different inhibitory effects on either isoenzyme: it enhanced threefold the apparent Km value for GTP in 139Met, whereas in 139Leu, it reduced it from 99 to 69 µM. The finding of those significant differences in the binding of GTP reinforces the hypothesis that the Met139Leu substitution affects strongly the nucleotide binding site of PEPCK-C.

c.A2456C-substitution in Pck1 changes the enzyme kinetic and functional properties modifying fat distribution in pigs.

Cytosolic phosphoenolpyruvate carboxykinase, PCK1, is one of the main regulatory enzymes of gluconeogenesis and glyceroneogenesis. The substitution of a single amino acid (Met139Leu) in PCK1 as a consequence of a single nucleotide polymorphism (SNP), c.A2456C, is associated in the pig to a negative phenotype characterized by reduced intramuscular fat content, enhanced backfat thickness and lower meat quality. The p.139L enzyme shows reduced kcat values in the glyceroneogenic direction and enhanced ones in the anaplerotic direction. Accordingly, the expression of the p.139L isoform results in about 30% lower glucose and 9% lower lipid production in cell cultures. Moreover, the ability of this isoform to be acetylated is also compromised, what would increase its susceptibility to be degraded in vivo by the ubiquitin-proteasome system. The high frequency of the c.2456C allele in modern pig breeds implies that the benefits of including c.A2456C SNP in selection programs could be considerable.

Early growth response 1 (EGR-1) is a transcriptional regulator of mitochondrial carrier homolog 1 (MTCH 1)/presenilin 1-associated protein (PSAP).

Attempts to elucidate the cellular function of MTCH1 (mitochondrial carrier homolog 1) have not yet rendered a clear insight into the function of this outer mitochondrial membrane protein. Classical biochemical and cell biology approaches have not produced the expected outcome. In vitro experiments have indicated a likely role in the regulation of cell death by apoptosis, and its reported interaction with presenilin 1 suggests a role in the cellular pathways in which this membrane protease participates, nevertheless in vivo data are missing. In an attempt to identify cellular pathways in which this protein might participate, we have studied its promoter looking for transcriptional regulators. We have identified several putative binding sites for EGR-1 (Early growth response 1; a protein involved in growth, proliferation and differentiation), in the proximal region of the MTCH1 promoter. Chromatin immunoprecipitation showed an enrichment of these sequences in genomic DNA bound to EGR-1 and transient overexpression of EGR-1 in cultured HEK293T cells induces an increase of endogenous MTCH1 levels. We also show that MTCH1 levels increase in response to treatment of cells with doxorubicin, an apoptosis inducer through DNA damage. The endogenous levels of MTCH1 decrease when EGR-1 levels are lowered by RNA interference. Our results indicate that EGR-1 is a transcriptional regulator of MTCH1 and give some clues about the cellular processes in which MTCH1 might participate.

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.

The closed conformation of the LDL receptor is destabilized by the low Ca(++) concentration but favored by the high Mg(++) concentration in the endosome.

The LDL receptor (LDLR) internalizes LDL and VLDL particles. In the endosomes, it adopts a closed conformation important for recycling, by interaction of two modules of the ligand binding domain (LR4-5) and a ß-propeller motif. Here, we investigate by SPR the interactions between those two modules and the ß-propeller. Our results indicate that the two modules cooperate to bind the ß-propeller. The binding is favored by low pH and by high [Ca(++)]. Our data show that Mg(++), at high concentration in the endosome, favors the formation of the closed conformation by replacing the structuring effect of Ca(++) in LR5. We propose a sequential model of LDL release where formation of the close conformation follows LDL release.

Extending in silico mechanism-of-action analysis by annotating targets with pathways: application to cellular cytotoxicity readouts.

BACKGROUND: An in silico mechanism-of-action analysis protocol was developed, comprising molecule bioactivity profiling, annotation of predicted targets with pathways and calculation of enrichment factors to highlight targets and pathways more likely to be implicated in the studied phenotype. RESULTS: The method was applied to a cytotoxicity phenotypic endpoint, with enriched targets/pathways found to be statistically significant when compared with 100 random datasets. Application on a smaller apoptotic set (10 molecules) did not allowed to obtain statistically relevant results, suggesting that the protocol requires modification such as analysis of the most frequently predicted targets/annotated pathways. CONCLUSION: Pathway annotations improved the mechanism-of-action information gained by target prediction alone, allowing a better interpretation of the predictions and providing better mapping of targets onto pathways.

Identification of specific pluripotent stem cell death--inducing small molecules by chemical screening.

A potential application of embryonic and inducible pluripotent stem cells for the therapy of degenerative diseases involves pure somatic cells, free of tumorigenic undifferentiated embryonic and inducible pluripotent stem cells. In complex collections of chemicals with pharmacological potential we expect to find molecules able to induce specific pluripotent stem cell death, which could be used in some cell therapy settings to eliminate undifferentiated cells. Therefore, we have screened a chemical library of 1120 small chemicals to identify compounds that induce specifically apoptotic cell death in undifferentiated mouse embryonic stem cells (ESCs). Interestingly, three compounds currently used as clinically approved drugs, nortriptyline, benzethonium chloride and methylbenzethonium chloride, induced differential effects in cell viability in ESCs versus mouse embryonic fibroblasts (MEFs). Nortriptyline induced apoptotic cell death in MEFs but not in ESCs, whereas benzethonium and methylbenzethonium chloride showed the opposite effect. Nortriptyline, a tricyclic antidepressant, has also been described as a potent inhibitor of mitochondrial permeability transition, one of two major mechanisms involved in mitochondrial membrane permeabilization during apoptosis. Benzethonium chloride and methylbenzethonium chloride are quaternary ammonium salts used as antimicrobial agents with broad spectrum and have also been described as anticancer agents. A similar effect of benzethonium chloride was observed in human induced pluripotent stem cells (hiPSCs) when compared to both primary human skin fibroblasts and an established human fibroblast cell line. Human fibroblasts and hiPSCs were similarly resistant to nortriptyline, although with a different behavior. Our results indicate differential sensitivity of ESCs, hiPSCs and fibroblasts to certain chemical compounds, which might have important applications in the stem cell-based therapy by eliminating undifferentiated pluripotent stem cells from stem cell-derived somatic cells to prevent tumor formation after transplantation for therapy of degenerative diseases.

Allelic incidence in several pig breeds of a missense variant of pig melanocortin-4 receptor (MC4R) gene associated with carcass and productive traits; its relation to IGF2 genotype.

MC4R, melanocortin-4 receptor, is involved in feed intake regulation. A mutation in a single base of MC4R, a G/A substitution in position 1426, has been linked to enhanced backfat thickness, average daily gain and daily feed intake. We present in this work a method to diagnose this mutation using real time PCR (RT-PCR) which allows rapid, cheap and reliable analysis of hundreds of samples in just 2h after DNA extraction. We have used this RT-PCR based assay to study the incidence of the mutation in several pig breeds or crosses (Iberian, Duroc, Pietrain, Large White, Large White×Pietrain) and wild boars. IGF2, insuline like growth factor 2, a gene with well demonstrated effects on carcass composition, of all these animals has also been analyzed and we show, using linkage disequilibrium analysis that both genes are independent. The implications of our results for pig selection toward fatty or lean carcasses are discussed.

Incidence in diverse pig populations of an IGF2 mutation with potential influence on meat quality and quantity: An assay based on real time PCR (RT-PCR).

IGF2, insulin-like growth factor 2, is implicated in myogenesis and lean meat content. A mutation in a single base (A for G substitution) of the gene for IGF2 (position 3072 in intron 3) has been recently described as the cause of a major QTL effect on muscle growth in pigs [Van Laere, A. S, Nguyen, M., Braunschweig, M., Nezer, C., Collete, C., & Moreau, L. et al. (2003). Nature, 425, 832-836]. We describe here a rapid assay based on real time PCR (RT-PCR) to detect this mutation. We have evaluated the incidence of the mutation in commercial pig crosses, in three populations of purebred Iberian or Iberian×Duroc crosses, and in cured meat products and wild boars. The incidence of the mutation varies among these groups. Penetrance of the A mutation is about 80% in the commercial population. Purebred Iberian pigs were all homozygous G/G whereas crosses of Iberian pigs were heterozygous (90%) or homozygous A/A (10%). The implications of this gene for the selection of Iberian pigs are discussed.

A real time PCR (RT-PCR) alternative assay to detect the T/C mutation in position 1843 of the ryanodine receptor gene.

The discovery of the causal mutation of malignant hyperthermia in pigs, a T for C substitution in base 1843 of the ryanodine receptor gene, opened the door to selection procedures based on the analysis of ryanodine receptor genotype based on PCR amplification of the region containing base 1843, subsequent digestion with specific restriction enzymes of the amplified DNA fragment, and electrophoretic analysis of the resulting bands. In this paper, we describe an assay that allows analysis of the three possible genotypes of the ryanodine receptor gene using real time PCR to amplify and detect them in a single step. Results obtained with the RT-PCR assay described in this work match 100% with those obtained using traditional PCR methods. RT-PCR methods are cheaper and faster than traditional ones allowing one to genotype up to 384 samples in a single run.