Biomedical subjects

Jerry Pelletier

Publications and source records attributed to Jerry Pelletier.

46 records · Page 3Linked to original sources

PubMed2004

Methionine sustituted polyamides are RNAse mimics that inhibit translation.

RNAse mimics are small molecules that can cleave RNA in a fashion similar to ribonucleases. These compounds would be very useful as gene specific reagents if their activities could be regulated and targeted. We demonstrate here that polyamides with methionine substituents show enhanced RNA cleavage activity relative to other polyamides. Conjugation of these compounds to aminoglycosides produced RNAse mimics that are capable of inhibiting eukaryotic protein synthesis. As a new class of compounds capable of interacting with nucleic acids, these novel aminoglycoside-polyamides constitute promising scaffolds for the construction of nuclease mimics with biological activity.

Aminoglycosides↗

PubMed2004

The yeast split-ubiquitin membrane protein two-hybrid screen identifies BAP31 as a regulator of the turnover of endoplasmic reticulum-associated protein tyrosine phosphatase-like B.

In the past decade, traditional yeast two-hybrid techniques have identified a plethora of interactions among soluble proteins operating within diverse cellular pathways. The discovery of associations between membrane proteins by genetic approaches, on the other hand, is less well established due to technical limitations. Recently, a split-ubiquitin system was developed to overcome this barrier, but so far, this system has been limited to the analysis of known membrane protein interactions. Here, we constructed unique split-ubiquitin-linked cDNA libraries and provide details for implementing this system to screen for binding partners of a bait protein, in this case BAP31. BAP31 is a resident integral protein of the endoplasmic reticulum, where it operates as a chaperone or cargo receptor and regulator of apoptosis. Here we describe a novel human member of the protein tyrosine phosphatase-like B (PTPLB) family, an integral protein of the endoplasmic reticulum membrane with four membrane-spanning alpha helices, as a BAP31-interacting protein. PTPLB turns over rapidly through degradation by the proteasome system. Comparisons of mouse cells with a deletion of Bap31 or reconstituted with human BAP31 indicate that BAP31 is required to maintain PTPLB, consistent with a chaperone or quality control function for BAP31 in the endoplasmic reticulum membrane.

Amino Acid Sequence↗

PubMed2004

Eukaryotic protein synthesis inhibitors identified by comparison of cytotoxicity profiles.

The National Cancer Institute (NCI) Human Tumor Cell Line Anti-Cancer Drug Screen has evaluated the cytotoxicity profiles of a large number of synthetic compounds, natural products, and plant extracts on 60 different cell lines. The data for each compound/extract can be assessed for similarity of cytotoxicity pattern, relative to a given test compound, using an algorithm called COMPARE. In applying a chemical biology approach to better understand the mechanism of eukaryotic protein synthesis, we used these resources to search for novel inhibitors of translation. The cytotoxicity profiles of 31 known protein synthesis inhibitors were used to identify compounds from the NCI database with similar activity profiles. Using this approach, two natural products, phyllanthoside and nagilactone C, were identified and characterized as novel protein synthesis inhibitors. Both compounds are specific for the eukaryotic translation apparatus, function in vivo and in vitro, and interfere with translation elongation. Our results demonstrate the feasibility of utilizing cytotoxicity profiles to identify new inhibitors of translation.

Algorithms↗

PubMed2003

WT1 is a modifier of the Pax2 mutant phenotype: cooperation and interaction between WT1 and Pax2.

Metanephric kidney development requires an inductive interaction between the ureteric bud and progenitor mesenchyme, where the early expression of two genes, Wilms' tumour 1 (WT1) and paired box 2 (Pax2), establishes critical but unknown developmental pathways. Indeed, transgenic mice with deregulated overexpression of Pax2 exhibit structural kidney defects and impaired renal function, as do mice harboring targeted disruptions and/or spontaneous mutations of either the Pax2 or WT1 genes. WT1 and Pax2 are thought to regulate each other's expression during renal development. To better define the relationship between WT1 and Pax2, we generated mouse embryos containing heterozygous mutations in both genes. WT1(+/-)/Pax2(1Neu/+) kidneys were 50% smaller than wild-type kidneys. They were characterized by severe attenuation of the renal medulla, and reduced development of calyces and the renal pelvis. Renal cortex development in compound heterozygotes culminated in fewer nephrons than in WT1(+/-), Pax2(1Neu/+) or wild-type mice. Only minor variations in the mesenchymal expression pattern of Pax2 protein, and the mRNA expression levels of Pax2 and WT1, were noted in mutant kidneys. We show that WT1 and Pax2 proteins interact in vitro and in vivo, demonstrating that WT1 and Pax2 can form a molecular complex. Our data suggest that WT1 is a modifier of the Pax2 mutant phenotype.

Animals↗

PubMed2002

An upstream open reading frame impedes translation of the huntingtin gene.

Expansion of a CAG tract within the huntingtin gene, leading to the production of a protein with an expanded polyglutamine tract, is responsible for Huntington's disease. We show here that the 5' untranslated region (UTR) of the huntingtin gene plays an important role in controlling the synthesis of huntingtin. In particular, the 5' UTR contains an upstream open reading frame (uORF) encoding a 21 amino acid peptide. We demonstrate that the presence of this uORF negatively influences expression from the huntingtin mRNA. Our results suggest a role for the uORF in limiting ribosomal access to downstream initiation sites. Mechanisms involving the post-transcriptional regulation of huntingtin are not well understood, and this may be an important way of regulating huntingtin protein levels.

5' Untranslated Regions↗

PubMed2002

Inhibition of Wilms tumor 1 transactivation by bone marrow zinc finger 2, a novel transcriptional repressor.

The Wilms tumor suppressor gene, wt1, encodes a zinc finger transcription factor that has been implicated in the regulation of a number of genes. Protein-protein interactions are known to modulate the transcription regulatory functions of Wilms tumor (WT1) and have also implicated WT1 in splicing. In this report, we identify a novel WT1-interacting protein, bone marrow zinc finger 2 (BMZF2), by affinity chromatography utilizing immobilized WT1 protein. BMZF2 is a potential transcription factor with 18 zinc fingers. The BMZF2 mRNA is mainly expressed in fetal tissues, and the protein is predominantly nuclear. Co-immunoprecipitation experiments are consistent with an in vivo association between WT1 and BMZF2. Glutathione S-transferase pulldown assays and far Western blots revealed that zinc fingers VI-X (amino acids 231-370) are required for interaction with the zinc finger region of WT1. Functionally, BMZF2 inhibits transcriptional activation by WT1. Moreover, a chimeric protein generated by fusion of BMZF2 to the GAL4 DNA-binding domain significantly decreases promoter activity of a reporter containing GAL4 DNA-binding sites, suggesting the presence of an active repressor domain within BMZF2. Our results suggest that BMZF2 interferes with the transactivation potential of WT1.

Amino Acid Sequence↗

PubMed2002

Nucleotide binding and nucleotide hydrolysis properties of the ABC transporter MRP6 (ABCC6).

Mutations in the MRP gene family member MRP6 cause pseudoxanthoma elasticum (PXE) in humans, a disease affecting elasticity of connective tissues. The normal function of MRP6, including its physiological substrate(s), remains unknown. To address these issues, recombinant rat Mrp6 (rMrp6) was expressed in the methylotrophic yeast Pichia pastoris. The protein was expressed in the membrane fraction as a stable 170 kDa protein. Its nucleotide binding and hydrolysis properties were investigated using the photoactive ATP analogue 8-azido-[alpha-(32)P]ATP and compared to those of the drug efflux pump MRP1. rMrp6 can bind 8-azido-[alpha-(32)P]ATP in a Mg(2+)-dependent and EDTA-sensitive fashion. Co(2+), Mn(2+), and Ni(2+) can also support 8-azido-[alpha-(32)P]ATP binding by rMrp6 while Ca(2+), Cd(2+), and Zn(2+) cannot. Under hydrolysis conditions (at 37 degrees C), the phosphate analogue beryllium fluoride (BeF(x)()) can stimulate trapping of the 8-azido-[alpha-(32)P]adenosine nucleotide in rMrp6 (and in MRP1) in a divalent cation-dependent and temperature-sensitive fashion. This suggests active ATPase activity, followed by trapping and photo-cross-linking of the 8-azido-[alpha-(32)P]ADP to the protein. By contrast to MRP1, orthovanadate-stimulated nucleotide trapping in rMrp6 does not occur in the presence of Mg(2+) but can be detected with Ni(2+) ions, suggesting structural and/or functional differences between the two proteins. The rMrp6 protein can be specifically photolabeled by a fluorescent photoactive drug analogue, [(125)I]-IAARh123, with characteristics similar to those previously reported for MRP1 (1), and this photolabeling of rMrp6 can be modulated by several structurally unrelated compounds. The P. pastoris expression system has allowed demonstration of ATP binding and ATP hydrolysis by rMrp6. In addition to providing large amounts of active protein for detailed biochemical studies, this system should also prove useful to identify potential rMrp6 substrates in [(125)I]-IAARh123 photolabeling competition studies, as well as to study the molecular basis of PXE mutations, which are most often found in the NBD2 of MRP6.

Adenosine Diphosphate↗

PubMed2002

Forced engagement of a RNA/protein complex by a chemical inducer of dimerization to modulate gene expression.

A general strategy is described for forcing the engagement of an RNA/protein complex by using small-molecule ligands. A bivalent molecule was created by linking a protein-binding ligand to an RNA-binding ligand. On presentation of the chemical inducer of dimerization to the RNA by the protein, cooperative binding ensued, resulting in higher-affinity complexes. When the chemical inducer of dimerization was used to target the protein to an mRNA template, the resulting RNA/protein complex was sufficiently stable to inhibit mRNA translation. This approach provides a logic to modulate gene expression by using small-molecule ligands to recruit protein surfaces to mRNAs.

3' Untranslated Regions↗

PubMed2002

Inhibition of translation by RNA-small molecule interactions.

Small molecule ligand-RNA interactions have the potential to influence gene expression at a variety of steps and in a number of ways. Here, we demonstrate that such interactions are sufficiently stable to inhibit translation of eukaryotic mRNAs in vitro and in vivo. Inhibition is only observed when the 5' UTR of the mRNA is targeted, and the response is proportional to the number of binding sites within this region. We find that small molecule ligand-RNA interactions can be sufficiently stable to prevent 80S ribosome assembly on an mRNA template. The ability to specifically ablate expression of a defined mRNA with a small molecule ligand demonstrates proof of principle for pharmacological targeting aimed at controlling translation of specific mRNAs.

3' Untranslated Regions↗

PubMed2002

Inhibition of protein synthesis by aminoglycoside-arginine conjugates.

Inhibition of translation by small molecule ligands has proven to be a useful tool for understanding this complex cellular mechanism, as well as providing drugs of significant medical importance. Many small molecule ligands inhibit translation by binding to RNA or RNA/protein components of the ribosomal subunits and usurping their function. A class of peptidomimetics [aminoglycoside-arginine conjugates (AAC)] has recently been designed to inhibit HIV TAR/tat interaction and in experiments aimed at assessing the inhibitory effects of AACs on TAR-containing transcripts, we found that AACs are general inhibitors of translation. Experiments reported herein aim at characterizing these novel properties of AACs. We find that AACs are inhibitors of eukaryotic and prokaryotic translation and exert their effects by blocking peptide chain elongation. Structure/activity relationship studies suggest that inhibition of translation by AACs is directly related to the number of arginine groups present on the aminoglycoside backbone and to the nature of the core aminoglycoside. AACs are therefore attractive tools for understanding and probing ribosome function.

Aminoglycosides↗