Gérard Loison

Human lamin B receptor exhibits sterol C14-reductase activity in Saccharomyces cerevisiae.

Lamin B receptor (LBR), a nuclear protein of avian and mammalian cells, contains an hydrophobic domain that shares extensive structural similarities with the members of the sterol reductase family. To test if the sterol-reductase-like domain of LBR could be enzymatically competent, several sterol reductase-defective strains of Saccharomyces cerevisiae were transformed with a human-LBR expressing vector. LBR production did not change the ergosterol biosynthesis defect in an erg4 mutant impaired in sterol C24(28) reductase. In contrast, the sterol C14 reduction step and ergosterol prototrophy were restored in LBR-producing erg24 transformants which lack endogenous sterol C14 reductase. To test the effects of C14 reductase inhibitors on LBR activity, we constructed EMY54, an ergosterol-requiring strain that is devoid of both sterol C8-C7 isomerase and sterol C14 reductase activities. EMY54 cells recovered the capability of synthesizing ergost-8-en-3beta-ol upon transformation with a vector that expressed either yeast sterol C14 reductase or hLBR. In addition, growth in sterol-free medium was restored in these transformants. Sterol biosynthesis and proliferation of LBR-producing cells were found to be highly susceptible to fenpropimorph and tridemorph, but only moderately susceptible to SR 31747. Our results strongly suggest that hLBR is a sterol C14 reductase.

Emopamil-binding Protein, a Mammalian Protein That Binds a Series of Structurally Diverse Neuroprotective Agents, Exhibits Δ8-Δ7 Sterol Isomerase Activity in Yeast

Δ8-Δ7 sterol isomerase is an essential enzyme on the sterol biosynthesis pathway in eukaryotes. This endoplasmic reticulum-resident membrane protein catalyzes the conversion of Δ8-sterols to their corresponding Δ7-isomers. No sequence data for high eukaryote sterol isomerase being available so far, we have cloned a murine sterol isomerase-encoding cDNA by functional complementation of the corresponding deficiency in the yeast Saccharomyces cerevisiae. The amino acid sequence deduced from the cDNA open reading frame is highly similar to human emopamil-binding protein (EBP), a protein of unknown function that constitutes a molecular target for neuroprotective drugs. A yeast strain in which the sterol isomerase coding sequence has been replaced by that of human EBP or its murine homologue recovers the ability to convert Δ8-sterol into Δ7-sterol, both in vivo and in vitro. In these recombinant strains, both cell proliferation and the sterol isomerization reaction are inhibited by the high affinity EBP ligand trifluoperazine, as is the case in mammalian cells but not in wild type yeast cell. In contrast, the recombinant strains are much less susceptible to the sterol inhibition effect of haloperidol and fenpropimorph, as compared with wild type yeast strains. Our results strongly suggest that EBP and Δ8-Δ7 sterol isomerase are identical proteins in mammals.

The M r 18,000 Subunit of the Peripheral-type Benzodiazepine Receptor Exhibits Both Benzodiazepine and Isoquinoline Carboxamide Binding Sites in the Absence of the Voltage-dependent Anion Channel or of the Adenine Nucleotide Carrier

The peripheral type benzodiazepine receptor (PBR) binds benzodiazepines such as RO5-4864 and isoquinoline carboxamide derivatives such as PK11195. This receptor includes anM r 18,000 isoquinoline-binding subunit predominantly located in mitochondrial mem- branes. This protein has been found to copurify with two other mitochondrial proteins, namely the outer membrane voltage-dependent anion channel (VDAC), also known as mitochondrial porin, and the inner membrane adenine nucleotide carrier. In vitro reconstitution experiments suggested that the PBR was a multimeric complex in which the isoquinoline binding site was on the M r18,000 subunit, denoted pk18, whereas the benzodiazepine binding site required the association of this subunit with VDAC to be expressed. Untransformed cells of the yeast Saccharomyces cerevisiaeare devoid of specific binding sites for isoquinolines and benzodiazepines, whereas yeast cells transformed with a pk18-expressing vector exhibit RO5-4864 and PK11195 binding sites that are pharmacologically identical to those of the PBR. To clarify the role of VDAC and of the adenine nucleotide carrier, if any, in the constitution of the benzodiazepine binding site, yeast host strains were constructed in which the corresponding genes had been knocked out. Mitochondria prepared from pk18-producing cells devoid of either VDAC or adenine nucleotide carrier exhibit both benzodiazepine and isoquinoline carboxamide binding sites with little or no change in theK d values as compared with the wild-type background. These results rule out the contention that VDAC is indispensable for establishing the benzodiazepine binding site and are in agreement with the hypothesis that the M r18,000 subunit carries both the isoquinoline carboxamide and benzodiazepine binding domains.

Purification and Characterization of the Human SR 31747A-binding Protein A NUCLEAR MEMBRANE PROTEIN RELATED TO YEAST STEROL ISOMERASE

SR 31747A, defined as a sigma ligand, is a novel immunosuppressive agent that blocks proliferation of human and mouse lymphocytes. Using a radiolabeled chemical probe, we here purified a target of SR 31747A and called it SR 31747A-binding protein (SR-BP). Purified SR-BP retained its binding properties and migrated on SDS-polyacrylamide gel as a M r 28,000 protein. Cloning of the cDNA encoding human SR-BP shows an open reading frame for a 223-amino acid protein, which is homologous to the recently cloned sigma 1 receptor. Interestingly, the deduced amino acid sequence was found to be related to fungal C8-C7 sterol isomerase, encoded by the ERG2 gene. The ERG2 gene product has been identified recently as the molecular target of SR 31747A that mediates antiproliferative effects of the drug in yeast. Northern blot analysis of SR-BP gene expression revealed a single transcript of 2 kilobases which was widely expressed among organs, with the highest abundance in liver and the lowest abundance in brain. Subcellular localization analysis in various cells, using a specific monoclonal antibody raised against SR-BP, demonstrated that this protein was associated with the nuclear envelope. When studying the binding of SR 31747A on membranes from yeast expressing SR-BP, we found a pharmacological profile of sigma 1 receptors; binding was displaced by (+)-pentazocine, haloperidol, and (+)-SKF 10,047, with (+)-SKF 10,047 being a more potent competitor than (−)-SKF 10,047. Scatchard plot analysis revealed K d values of 7.1 nm and 0.15 nm for (+)-pentazocine and SR 31747A, respectively, indicating an affinity of SR-BP 50-fold higher for SR 31747A than for pentazocine. Additionally, we showed that pentazocine, a competitive inhibitor of SR 31747A binding, also prevents the immunosuppressive effect of SR 31747A. Taken together, these findings strongly suggest that SR-BP represents the molecular target for SR 31747A in mammalian tissues, which could be critical for T cell proliferation.

Sterol metabolism and ERG2 gene regulation in the yeast Saccharomyces cerevisiae

Certain exogenously‐supplied sterols, like ergost‐8‐enol, are efficiently converted into ergosterol in yeast. We have taken advantage of this property to study the regulation of the Δ8‐Δ7‐sterol isomerase‐encoding ERG2 gene in an ergosterol auxotrophic mutant devoid of squalene‐synthase activity. Ergosterol starvation leads to an 8–16‐fold increase in ERG2 gene expression. Such an increase was also observed in wild‐type cells either grown anaerobically or treated with SR31747A a sterol isomerase inhibitor. Exogenously‐supplied zymosterol is entirely transformed into ergosterol, which represses ERG2 transcription. By contrast, exogenously‐supplied ergosterol has little or no effect on ERG2 transcription.

Cloning and characterization of theeapB andeapC genes ofCryphonectria parasitica encoding two new acid proteinases, and disruption ofeapC

Two new proteinases secreted byCryphonectria parasitica, namely EapB and EapC, have been purified. The corresponding structural genes were isolated by screening a cosmid library, and sequenced. Comparison of genomic and cDNA sequences revealed that theeapB andeapC genes contain three and two introns, respectively. The products of theeapB andeapC genes as deduced from the nucleotide sequences, are 268 and 269 residues long, respectively. N-terminal amino acid sequencing data indicates that EapC is synthesized as a zymogen, which yields a mature 206-amino acid enzyme after cleavage of the prepro sequence. Similarly, sequence alignment studies suggest that EapB is secreted as a 203-residue form which shares extensive similarities not only with EapC but also with two other acid fungal proteinases. However, they display distinct structural features; for example, no cysteine residue is found in EapC. TheeapC gene was mutated using a two-step gene replacement strategy which allowed the specific introduction of several stop codons at the beginning of theeapC coding sequence in an endothiapepsin-deficient (EapA+)C. parasitica strain. Although the resulting strain did not secrete EapC, it still exhibited residual extracellular proteolytic activity, which could be due to EapB.

Cloning and mutation of the gene encoding endothiapepsin from Cryphonectria parasitica

SummaryEndothiapepsin is an aspartic protease secreted by Cryphonectria parasitica. It has a milk-clotting activity and is used in the cheese industry. The eapA gene encoding endothiapepsin has been cloned and sequenced. An open reading frame of 419 codons, which encodes a precursor differing from mature endothiapepsin by the presence of an 89 aa residue prepro-sequence, was found. The eapA gene is interrupted by three introns. C. parasitica mutant strains deficient in the production of endothiapepsin (eapA-) were constructed using a gene-replacement strategy. Two nonsense mutations were introduced at the beginning of the coding sequence by PCR-induced mutagenesis. The mutated DNA fragment was introduced in C. parasitica by co-transformation with a benomyl-resistant (benR) selection plasmid. Transformants which have the eapA- phenotype were obtained. Protein analysis confirmed that they secreted no detectable amount of endothiapepsin. No ectopic integration of the mutated eapA gene occurred in the eapA- transformants. Moreover, after one conidiation step, eapA- transformants yielded benomyl-sensitive (benS) segregants which were analyzed by Southern blotting experiments. The results revealed no difference with the wildtype strain, suggesting that the eapA-, benS segregants differed from the non-transformed strain only by the presence of the two nonsense mutations in the eapA locus.

Transformation of Aspergillus flavus: construction of urate oxidase-deficient mutants by gene disruption

SummaryA transformation procedure based on the complementation of a genetic defect was developed using a nitrate reductase-deficient mutant of Aspergillus flavus. The initial transformation efficiency was improved 40-fold by combining factors in a planned experimental program. Although low, this transformation rate was sufficient to obtain transformants in which the urate oxidase-encoding gene (uaZ) was disrupted in a gene replacement experiment. These new uaZ- strains were unable to utilize uric acid as the unique nitrogen source and could be reversed directly to the wild-type phenotype in second order transformation experiments using a urate oxidase-expressing vector.

Self-cloning in filamentous fungi: Application to the construction of endothiapepsin overproducers in Cryphonectria parasitica

The filamentous ascomycete fungus Cryphonectria parasitica naturally secretes endothiapepsin, an aspartic proteinase. It is cultured on a commercial scale as a source of the milk-clotting enzyme for cheese making. Our objective was to increase enzyme production of an industrial C. parasitica strain by a new technique of self-cloning; it consisted in the screening for transformants producing higher levels of endothiapepsin and having integrated only the DNA fragment of interest. Such genetically improved strains that are devoid of any foreign genes should be more readily acceptable for the production of food-grade enzymes.

Differential Virtual Screening (DVS) with Active and Inactive Molecular Models for Finding and Profiling GPCR Modulators: Case of the CCK1 Receptor

We discovered a constitutively activating mutation (CAM) V308E for the neurotensin NT1 receptor. Molecular dynamics (MD) performed for the CAM NT1‐V308E exhibiting a high spontaneous activity, and for the wild‐type NT1 without basal activity, show dramatic conformational changes for the CAM. To test if the two MD models could be valuable active and inactive templates for building molecular models for other class‐A GPCR, supposed active and inactive models were built by homology for the cholecystokinin CCK1 receptor. Virtual screening of a corporate library with 250 000 compounds was performed with the two CCK1 models, and a differential virtual screening analysis (DVS), led us to isolate 250 predicted agonists and 250 predicted antagonists. The two sets were merged and the compounds were tested in CCK1 agonist and antagonist cellular assays. An excellent correlation was obtained between predictions and biological results. The effective profiling provided by DVS with active and inactive molecular models, opens new perspectives for finding agonists and antagonists for other class‐A GPCR, notably for orphan GPCRs for which no ligands are known.