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Jerry Pelletier

Publications and source records attributed to Jerry Pelletier.

At least 37 records · Page 2Linked to original sources
PubMed2005

Stimulation of mammalian translation initiation factor eIF4A activity by a small molecule inhibitor of eukaryotic translation.

RNA helicases are the largest group of enzymes in eukaryotic RNA metabolism. The DEXD/H-box putative RNA helicases form the helicase superfamily II, whose members are defined by seven highly conserved amino acid motifs, making specific targeting of selected members a challenging pharmacological problem. The translation initiation factor eIF4A is the prototypical DEAD-box RNA helicase that works in conjunction with eIF4B and eIF4H and as a subunit of eIF4F to prepare the mRNA template for ribosome binding, possibly by unwinding the secondary structure proximal to the 5' m7GpppN cap structure. We report the identification and characterization of a small molecule inhibitor of eukaryotic translation initiation that acts in an unusual manner by stimulating eIF4A-associated activities. Our results suggest that proper control of eIF4A helicase activity is necessary for efficient ribosome binding and demonstrate the feasibility of selectively targeting DEAD-box RNA helicases with small molecules.

Animals↗
PubMed2005

Internal translation initiation mediated by the angiogenic factor Tie2.

Tie2 is an endothelium-specific receptor tyrosine kinase required for normal blood vessel maturation. We report that Tie2 mRNA translation is maintained under hypoxic conditions. To identify the mechanism responsible for this, we undertook structure/function analysis of the Tie2 5'-untranslated region (UTR). Transcription start site mapping indicates the existence of a several mRNA isoforms containing unusually long 5'-UTRs (>350 nucleotides) with five upstream open reading frames. We find internal ribosome binding activity that allows the Tie2 mRNA to initiate in a cap-independent fashion. Our data provide a framework for understanding how Tie2 mRNA is translated despite a cumbersome structured 5'-UTR and how its production is secured under unfavorable environmental conditions.

5' Untranslated Regions↗
PubMed2005

The complete genomes and proteomes of 27 Staphylococcus aureus bacteriophages.

Bacteriophages are the most abundant life forms in the biosphere. They play important roles in bacterial ecology, evolution, adaptation to new environments, and pathogenesis of human bacterial infections. Here, we report the complete genomic sequences, and predicted proteins of 27 bacteriophages of the Gram-positive bacterium Staphylococcus aureus. Comparative nucleotide and protein sequence analysis indicates that these phages are a remarkable source of untapped genetic diversity, encoding 2,170 predicted protein-encoding ORFs, of which 1,402 cannot be annotated for structure or function, and 522 are proteins with no similarity to other phage or bacterial sequences. Based on their genome size, organization of their gene map and comparative nucleotide and protein sequence analysis, the S. aureus phages can be organized into three groups. Comparison of their gene maps reveals extensive genome mosaicism, hinting to a large reservoir of unidentified S. aureus phage genes. Among the phages in the largest size class (178-214 kbp) that we characterized is phage Twort, the first discovered bacteriophage (responsible for the Twort-D'Herelle effect). These phage genomes offer an exciting opportunity to discern molecular mechanisms of phage evolution and diversity.

Chromosome Mapping↗
PubMed2005

Structure-activity relationships of quassinoids for eukaryotic protein synthesis.

The effect of 63 quassinoids on eukaryotic protein synthesis has been investigated. Seventeen of the tested compounds showed potent in vitro activity, with IC50s below 2 microM for inhibition in Krebs ascites translation extracts. Sixteen of these quassinoids were also potent inhibitors of in vivo protein synthesis when exposed to Hela cells for 1 h. Our results led to the following structure-activity relationships for quassinoids regarding translation inhibition. Activity is influenced by (i) the nature of the C-15 side chain, (ii) the nature of A ring modifications, (iii) the presence or absence of a sugar moiety, and (iv) the presence of an epoxymethano bridge.

Animals↗
PubMed2005

Microdeletion of target sites for insulator protein CTCF in a chromosome 11p15 imprinting center in Beckwith-Wiedemann syndrome and Wilms' tumor.

We have analyzed several cases of Beckwith-Wiedemann syndrome (BWS) with Wilms' tumor in a familial setting, which give insight into the complex controls of imprinting and gene expression in the chromosome 11p15 region. We describe a 2.2-kbp microdeletion in the H19/insulin-like growth factor 2 (IGF2)-imprinting center eliminating three target sites of the chromatin insulator protein CTCF that we believe here is necessary, but not sufficient, to cause BWS and Wilms' tumor. Maternal inheritance of the deletion is associated with IGF2 loss of imprinting and up-regulation of IGF2 mRNA. However, in at least one affected family member a second genetic lesion (a duplication of maternal 11p15) was identified and accompanied by a further increase in IGF2 mRNA levels 35-fold higher than control values. Our results suggest that the combined effects of the H19/IGF2-imprinting center microdeletion and 11p15 chromosome duplication were necessary for manifestation of BWS.

Base Sequence↗
PubMed2005

Inhibitory properties of nucleic acid-binding ligands on protein synthesis.

The use of small molecule inhibitors in the study of cellular processes is a powerful approach to understanding gene function. During the course of a high throughput screen for novel inhibitors of eukaryotic translation, we identified a number of nucleic acid binding ligands that showed activity in our assay. When tested on a panel of mRNA transcripts displaying different modes of translation initiation, these ligands showed a range of biological activities--with some inhibiting both cap-dependent and internal initiation and others preferentially blocking internal initiation. We used this information to identify a novel threading intercalator that inhibits Hepatitis C virus internal initiation.

Acridines↗
PubMed2005

High-throughput screening of G protein-coupled receptor antagonists using a bioluminescence resonance energy transfer 1-based beta-arrestin2 recruitment assay.

In this study, the authors developed HEK293 cell lines that stably coexpressed optimal amounts of beta-arrestin2-Rluc and VENUS fusions of G protein-coupled receptors (GPCRs) belonging to both class A and class B receptors, which include receptors that interact transiently or stably with beta-arrestins. This allowed the use of a bioluminescence resonance energy transfer (BRET) 1- beta-arrestin2 translocation assay to quantify receptor activation or inhibition. One of the developed cell lines coexpressing CCR5-VENUS and beta-arrestin2- Renilla luciferase was then used for high-throughput screening (HTS) for antagonists of the chemokine receptor CCR5, the primary co-receptor for HIV. A total of 26,000 compounds were screened for inhibition of the agonist-promoted beta-arrestin2 recruitment to CCR5, and 12 compounds were found to specifically inhibit the agonist-induced beta-arrestin2 recruitment to CCR5. Three of the potential hits were further tested using other functional assays, and their abilities to inhibit CCR5 agonist-promoted signaling were confirmed. This is the 1st study describing a BRET1-beta-arrestin recruitment assay in stable mammalian cells and its successful application in HTS for GPCRs antagonists.

Animals↗
PubMed2005

Ribavirin is not a functional mimic of the 7-methyl guanosine mRNA cap.

Ribavirin is a guanosine ribonucleoside analog that displays broad-spectrum anti-viral activity and is currently used for the treatment of some viral infections. Ribavirin has recently been proposed to also be a mimic of the 7-methyl guanosine cap found at the 5' end of mRNAs. To obtain supporting functional data for this hypothesis, we assessed the ability of ribavirin triphosphate to interfere with the interaction between eIF4E and 7-methyl guanosine capped mRNA. In chemical cross-linking assays, cap-affinity chromatography, and cap-dependent translation assays, ribavirin was unable to function as a cap analog.

Antiviral Agents↗
PubMed2004

Stimulation of hTAFII68 (NTD)-mediated transactivation by v-Src.

The three genes hTAF(II)68, EWS, and TLS (called the TET family) encode related RNA binding proteins containing an RNA recognition motif and three glycine-, arginine-, and proline-rich regions in the C-terminus and a degenerated repeat containing the consensus sequence Ser-Tyr-Gly-Gln-Ser in the N-terminus. In many human cancers, the N-terminal portion of hTAF(II)68, EWS, or TLS is fused to the DNA binding domain of one of several transcription factors including Fli-1, ERG, ETV1, E1AF, WT1, ATF-1, CHOP, or TEC. We have recognized the presence of several potential tyrosine phosphorylation sites within the amino-terminal domain of hTAF(II)68 and have investigated the potential effects of cytoplasmic signaling on hTAF(II)68 function. Herein, we find that hTAF(II)68 is phosphorylated on tyrosine residue(s) by ectopic expression of v-Src protein tyrosine kinase in vitro and in vivo. The hTAF(II)68 protein can associated with the SH3 domains of several cell signaling proteins, including v-Src protein tyrosine kinase. We also document that full-length v-Src can stimulate hTAF(II)68-mediated transcriptional activation, whereas deletion mutants of v-Src are unable to exert this effect. In addition, cellular Src activity appears important for hTAF(II)68 function since hTAF(II)68-mediated transactivation is reduced in a dose-dependent fashion by ectopic overexpression of a dominant-negative mutant of Src. Taken together, our results suggest that the biological activities of hTAF(II)68 are linked to the cytoplasmic Src signal transduction pathway.

Cell Line↗
PubMed2004

Survival signalling by Akt and eIF4E in oncogenesis and cancer therapy.

Evading apoptosis is considered to be a hallmark of cancer, because mutations in apoptotic regulators invariably accompany tumorigenesis. Many chemotherapeutic agents induce apoptosis, and so disruption of apoptosis during tumour evolution can promote drug resistance. For example, Akt is an apoptotic regulator that is activated in many cancers and may promote drug resistance in vitro. Nevertheless, how Akt disables apoptosis and its contribution to clinical drug resistance are unclear. Using a murine lymphoma model, we show that Akt promotes tumorigenesis and drug resistance by disrupting apoptosis, and that disruption of Akt signalling using the mTOR inhibitor rapamycin reverses chemoresistance in lymphomas expressing Akt, but not in those with other apoptotic defects. eIF4E, a translational regulator that acts downstream of Akt and mTOR, recapitulates Akt's action in tumorigenesis and drug resistance, but is unable to confer sensitivity to rapamycin and chemotherapy. These results establish Akt signalling through mTOR and eIF4E as an important mechanism of oncogenesis and drug resistance in vivo, and reveal how targeting apoptotic programmes can restore drug sensitivity in a genotype-dependent manner.

Animals↗
PubMed2004

Activation of the WT1 tumor suppressor gene promoter by Pea3.

Gene array profiling of RNA from cells engineered to express a dominant-negative version of the ETS family member transcription factor Pea3 (polyomavirus enhancer activator 3) identified WT1 as a candidate downstream gene. Given the co-expression of WT1 and Pea3 in developing kidney and breast tissue undergoing mesenchymal to epithelial transitions, we further characterized this potential gene hierarchy. Analysis of the human WT1 promoter revealed several potential binding sites for Pea3. Pea3 transactivated the WT1 promoter in transient transfection assays and bound to specific sites within the WT1 promoter in vitro. Our results position Pea3 upstream of WT1 and define a gene hierarchy important for mesenchymal-epithelial transitions.

Adenoviridae↗
PubMed2004

Insights into the physiological role of WT1 from studies of genetically modified mice.

The identification of WT1 gene mutations in children with WAGR and Denys-Drash syndromes pointed toward a role for WT1 in genitourinary system development. Biochemical analysis of the different WT1 protein isoforms showed that WT1 is a transcription factor and also has the ability to bind RNA. Analysis of WT1 complexes identified several target genes and protein partners capable of interacting with WT1. Some of these studies placed WT1, its downstream targets, and protein partners in a transcriptional regulatory network that controls urogenital system development. We review herein studies on WT1 knockout and transgenic models that have been instrumental in defining a physiological role for WT1 in normal and abnormal urogenital development.

Animals↗
PubMed2004

Inhibitors of protein synthesis identified by a high throughput multiplexed translation screen.

The use of small molecule inhibitors of cellular processes is a powerful approach to understanding gene function that complements the genetic approach. We have designed a high throughput screen to identify new inhibitors of eukaryotic protein synthesis. We used a bicistronic mRNA reporter to multiplex our assay and simultaneously screen for inhibitors of cap-dependent initiation, internal initiation and translation elongation/termination. Functional screening of >90 000 compounds in an in vitro translation reaction identified 36 inhibitors, 14 of which are known inhibitors of translation and 18 of which are nucleic acid-binding ligands. Our results indicate that intercalators constitute a large class of protein synthesis inhibitors. Four non-intercalating compounds were identified, three of which block elongation and one of which inhibits initiation. The novel inhibitor of initiation affects 5' end-mediated initiation, as well as translation initiated from picornaviral IRESs, but does not significantly affect internal initiation from the hepatitis C virus 5'-untranslated region. This compound should be useful for delineating differences in mechanism of initiation among IRESs.

Animals↗
PubMed2004

Antimicrobial drug discovery through bacteriophage genomics.

Over evolutionary time bacteriophages have developed unique proteins that arrest critical cellular processes to commit bacterial host metabolism to phage reproduction. Here, we apply this concept of phage-mediated bacterial growth inhibition to antibiotic discovery. We sequenced 26 Staphylococcus aureus phages and identified 31 novel polypeptide families that inhibited growth upon expression in S. aureus. The cellular targets for some of these polypeptides were identified and several were shown to be essential components of the host DNA replication and transcription machineries. The interaction between a prototypic pair, ORF104 of phage 77 and DnaI, the putative helicase loader of S. aureus, was then used to screen for small molecule inhibitors. Several compounds were subsequently found to inhibit both bacterial growth and DNA synthesis. Our results suggest that mimicking the growth-inhibitory effect of phage polypeptides by a chemical compound, coupled with the plethora of phages on earth, will yield new antibiotics to combat infectious diseases.

Anti-Bacterial Agents↗
PubMed2004

Flavonoid glycosides from Adina racemosa and their inhibitory activities on eukaryotic protein synthesis.

From the dried leaves, flowers, and twigs of Adina racemosa, five new flavonoid glycosides, quercetin 3-O-alpha-l-rhamnopyranosyl(1-->6)-(3-O-trans-p-coumaroyl)-beta-d-galactopyranoside (1), quercetin 3-O-alpha-l-rhamnopyranosyl(1-->6)-[(4-O-trans-p-coumaroyl)-alpha-l-rhamnopyranosyl(1-->2)]-(4-O-trans-p-coumaroyl)-beta-d-galactopyranoside (2), kaempferol 3-O-alpha-l-rhamnopyranosyl(1-->6)-[(4-O-trans-p-coumaroyl)-alpha-l-rhamnopyranosyl(1--> 2)]-(4-O-trans-p-coumaroyl)-beta-d-galactopyranoside (3), quercetin 3-O-alpha-l-rhamnopyranosyl(1-->6)-[(4-O-trans-p-coumaroyl)-alpha-l-rhamnopyranosyl(1-->2)]-(3-O-trans-p-coumaroyl)-beta-d-galactopyranoside (4), and quercetin 3-O-alpha-l-rhamnopyranosyl(1-->6)-[(4-O-trans-caffeoyl)-alpha-l-rhamnopyranosyl(1-->2)]-(3-O-trans-p-coumaroyl)-beta-d-galactopyranoside (5), and eight known compounds were isolated. The structures of the new compounds were determined by spectroscopic and chemical means. Their inhibitory activities on protein synthesis were assessed. The new glycosides were found to be inhibitors of eukayrotic, but not prokaryotic, protein synthesis.

Eukaryotic Cells↗
PubMed2004

A new antimalarial quassinoid from Simaba orinocensis.

A new antimalarial quassinoid, namely, orinocinolide (1), was isolated from the root bark of Simaba orinocensis, together with the previously reported simalikalactone D (2). The structure of 1 was determined primarily from 1D and 2D NMR analysis, as well as by chemical derivatization. Compound 1 was found to be as equally potent as 2 against Plasmodium falciparum clones D6 and W2 (IC(50) 3.27 and 8.53 ng/mL vs 3.0 and 3.67 ng/mL, respectively), but was 4- and 28-fold less toxic than 2 against VERO cells (IC(50) 10 vs 2.3 microg/mL) and HL-60 (IC(50) 0.7 vs 0.025 microg/mL), respectively. In addition, 2 was >46- and >31-fold more potent than pentamidine and amphotericin B (IC(50) 0.035 vs 1.6 and 1.1 microg/mL) against Leishmania donovani, while 1 was inactive. Orinocinolide (1) inhibited growth of human cancer cells SK-MEL, KB, BT-549, and SK-OV-3, but was less potent than 2 (IC(50) 0.8-1.9 vs 0.3-1.0 microg/mL) against these cells.

Animals↗