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Biomedical subjects

Peter G Schultz

Publications and source records attributed to Peter G Schultz.

At least 109 records · Page 6Linked to original sources
PubMed2003

Progress toward an expanded eukaryotic genetic code.

Expanding the eukaryotic genetic code to include unnatural amino acids with novel properties would provide powerful tools for manipulating protein function in eukaryotic cells. Toward this goal, a general approach with potential for isolating aminoacyl-tRNA synthetases that incorporate unnatural amino acids with high fidelity into proteins in Saccharomyces cerevisiae is described. The method is based on activation of GAL4-responsive HIS3, URA3, or lacZ reporter genes by suppression of amber codons in GAL4. The optimization of GAL4 reporters is described, and the positive and negative selection of active Escherichia coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNA(CUA) is demonstrated. Importantly, both selections can be performed on a single cell and with a range of stringencies. This method will facilitate the isolation of a range of aminoacyl-tRNA synthetase (aaRS)/tRNA(CUA) activities from large libraries of mutant synthetases.

Amino Acids↗
PubMed2003

Development and characterization of nonpeptidic small molecule inhibitors of the XIAP/caspase-3 interaction.

Elevated expression of inhibitor of apoptosis protein (IAP) family members in various types of cancers is thought to provide a survival advantage to these cells. Thus, antiapoptotic functions of IAPs, and their potential as novel anticancer targets have attracted considerable interest. Among the IAPs, the X chromosome-linked inhibitor of apoptosis protein (XIAP) is regarded as the most potent suppressor of mammalian apoptosis through direct binding and inhibition of caspases. A high-throughput biochemical screen of a combinatorial chemical library led to the discovery of a novel nonpeptidic small molecule that has the ability to disrupt the XIAP/caspase-3 interaction. The activity of this nonpeptidic small molecule inhibitor of the XIAP/caspase-3 interaction has been characterized both in vitro and in cells. Molecules of this type can be used to conditionally inhibit the cellular function of XIAP and may provide insights into the development of therapeutic agents that act by modulating apoptotic pathways.

Apoptosis↗
PubMed2003

Searching for interferon-induced genes that inhibit hepatitis B virus replication in transgenic mouse hepatocytes.

We have previously shown that alpha/beta interferon (IFN-alpha/beta) and IFN-gamma inhibit hepatitis B virus (HBV) replication noncytopathically in the livers of HBV transgenic mice and in hepatocyte cell lines derived from these mice. The present study was designed to identify transcriptionally controlled hepatocellular genes that are tightly associated with the inhibition of HBV replication and that might, therefore, mediate the antiviral effect of these cytokines. Twenty-nine genes were identified, many of which have known or potential antiviral activity. Notably, multiple components of the immunoproteasome and ubiquitin-like proteins were strongly induced by both IFN-alpha/beta and IFN-gamma, as were a number of GTP-binding proteins, including GTPases with known antiviral activity, chemokines, signaling molecules, and miscellaneous genes associated with antigen processing, DNA-binding, or cochaperone activity and several expressed sequence tags. The results suggest that one or more members of this relatively small subset of genes may mediate the antiviral effect of IFN-alpha/beta and IFN-gamma against HBV. We have already exploited this information by demonstrating that the antiviral activity of IFN-alpha/beta and IFN-gamma is proteasome dependent.

Animals↗
PubMed2002

Addition of the keto functional group to the genetic code of Escherichia coli.

Although the keto group is the most versatile of the functional groups in organic chemistry, it is absent in the genetically encoded amino acids. To overcome this natural limitation on protein biosynthesis, we have evolved an orthogonal tRNA-synthetase pair that makes possible the efficient incorporation of a keto amino acid, p-acetyl-l-phenylalanine, into proteins in E. coli with high translational fidelity in response to the amber nonsense codon. To demonstrate the utility of this keto amino acid, we have used it to modify a protein selectively with a small molecule fluorophore and biotin derivative. This additional genetically encoded amino acid should greatly expand our ability to manipulate protein structure and function both in vitro and in living cells.

Amino Acid Substitution↗
PubMed2002

A potent and highly selective sulfotransferase inhibitor.

In the present work, we have used a newly developed, fluorescence-based assay to screen a library of >30 000 compounds as potential beta-arylsulfotransferase-IV inhibitors. A total of 11 inhibitors were discovered. Most of the compounds discovered showed low micromolar inhibition, but one of the compounds showed potent inhibition (Ki = 96 nM). The most potent of these inhibitors was tested against a variety of other purine binding enzymes and showed remarkable specificity.

Adenosine Diphosphate↗
PubMed2002

Enzymatic phosphorylation of unnatural nucleosides.

In an effort to expand the genetic alphabet, a number of unnatural, predominantly hydrophobic, nucleoside analogues have been developed which pair selectively in duplex DNA and during enzymatic synthesis. Significant progress has been made toward the efficient in vitro replication of DNA containing these base pairs. However, the in vivo expansion of the genetic alphabet will require that the unnatural nucleoside triphosphates be available within the cell at sufficient concentrations for DNA replication. We report our initial efforts toward the development of an unnatural in vivo nucleoside phosphorylation pathway that is based on nucleoside salvage enzymes. The first step of this pathway is catalyzed by the D. melanogaster nucleoside kinase, which catalyzes the phosphorylation of nucleosides to the corresponding monophosphates. We demonstrate that each unnatural nucleoside is phosphorylated with a rate that should be sufficient for the in vivo replication of DNA.

Animals↗
PubMed2002

A small molecule with osteogenesis-inducing activity in multipotent mesenchymal progenitor cells.

Purmorphamine, which is a 2,6,9-trisubstituted purine compound, was discovered through cell-based high-throughput screening from a heterocycle combinatorial library. It differentiates multipotent mesenchymal progenitor cells into an osteoblast lineage. It will serve as a unique chemical tool to study the molecular mechanisms of osteogenesis of stem cells and bone development.

Alkaline Phosphatase↗
PubMed2002

The chemistry of the antibody molecule.

Immunochemistry has traditionally focused on understanding the molecular basis of antibody binding affinity and specificity. In the last 20 years a major shift has occurred in the field. We are now asking how we can exploit the remarkable properties of the antibody molecule and the immune response in chemistry-from testing basic theories of enzymatic catalysis to the synthesis of new catalysts. Moreover, the concept of molecular diversity, which underlies these experiments, is now being applied to a large number of other problems-from libraries of biopolymers to libraries of solid-state materials. Importantly, theses studies are providing fundamental molecular insights into many biological, chemical, and physical phenomena, as well as changing the way we approach the synthesis of new molecular function.

Antibodies↗
PubMed2002

A novel silver(i)-mediated DNA base pair.

We have generated a novel silver(I)-mediated unnatural DNA base pair consisting of two 2,6-bis(ethylthiomethyl)pyridine nucleobases SPy. This metallo-base pair has a remarkably high pairing stability and selectivity which rivals that of the natural base pairs dA:dT and dC:dG. UV-melting experiments revealed that the dSPy:dSPy self-pair can replace natural base pairs at multiple sites and still form stable DNA duplexes.

Base Pairing↗
PubMed2002

Genomic analysis of the host response to hepatitis C virus infection.

We have examined the progression of hepatitis C virus (HCV) infections by gene expression analysis of liver biopsies in acutely infected chimpanzees that developed persistent infection, transient viral clearance, or sustained clearance. Both common responses and outcome-specific changes in expression were observed. All chimpanzees showed gene expression patterns consistent with an IFN-alpha response that correlated with the magnitude and duration of infection. Transient and sustained viral clearance were uniquely associated with induction of IFN-gamma-induced genes and other genes involved in antigen processing and presentation and the adaptive immune response. During the early stages of infection, host genes involved in lipid metabolism were also differentially regulated. We also show that drugs that affect these biosynthetic pathways can regulate HCV replication in HCV replicon systems. Our results reveal genome-wide transcriptional changes that reflect the establishment, spread, and control of infection, and they reveal potentially unique antiviral programs associated with clearance of HCV infection.

Animals↗
PubMed2002

A versatile linkage strategy for solid-phase synthesis of N,N-dimethyltryptamines and beta-carbolines.

Various tryptamines are captured by a vinylsulfonylmethyl polystyrene resin, generating a safety-catch linkage. Beta-carbolines can be formed from 4 by a Pictet-Spengler reaction with the introduction of R(1). Tryptamine 4 can also be derivatized by acylation or copper-mediated coupling to introduce R(2). If X = Br, Suzuki coupling can be used to introduce R(3). After derivatization, the indole derivatives are activated with methyl iodide and released under mild basic condition. [reaction: see text]

Carbolines↗
PubMed2002

An efficient system for the evolution of aminoacyl-tRNA synthetase specificity.

A variety of strategies to incorporate unnatural amino acids into proteins have been pursued, but all have limitations with respect to technical accessibility, scalability, applicability to in vivo studies, or site specificity of amino acid incorporation. The ability to selectively introduce unnatural functional groups into specific sites within proteins, in vivo, provides a potentially powerful approach to the study of protein function and to large-scale production of novel proteins. Here we describe a combined genetic selection and screen that allows the rapid evolution of aminoacyl-tRNA synthetase substrate specificity. Our strategy involves the use of an "orthogonal" aminoacyl-tRNA synthetase and tRNA pair that cannot interact with any of the endogenous synthetase-tRNA pairs in Escherichia coli. A chloramphenicol-resistance (Cm(r)) reporter is used to select highly active synthetase variants, and an amplifiable fluorescence reporter is used together with fluorescence-activated cell sorting (FACS) to screen for variants with the desired change in amino acid specificity. Both reporters are contained within a single genetic construct, eliminating the need for plasmid shuttling and allowing the evolution to be completed in a matter of days. Following evolution, the amplifiable fluorescence reporter allows visual and fluorimetric evaluation of synthetase activity and selectivity. Using this system to explore the evolvability of an amino acid binding pocket of a tyrosyl-tRNA synthetase, we identified three new variants that allow the selective incorporation of amino-, isopropyl-, and allyl-containing tyrosine analogs into a desired protein. The new enzymes can be used to produce milligram-per-liter quantities of unnatural amino acid-containing protein in E. coli.

Amino Acids↗
PubMed2002

Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline.

Structural genomics is emerging as a principal approach to define protein structure-function relationships. To apply this approach on a genomic scale, novel methods and technologies must be developed to determine large numbers of structures. We describe the design and implementation of a high-throughput structural genomics pipeline and its application to the proteome of the thermophilic bacterium Thermotoga maritima. By using this pipeline, we successfully cloned and attempted expression of 1,376 of the predicted 1,877 genes (73%) and have identified crystallization conditions for 432 proteins, comprising 23% of the T. maritima proteome. Representative structures from TM0423 glycerol dehydrogenase and TM0449 thymidylate synthase-complementing protein are presented as examples of final outputs from the pipeline.

Cloning, Molecular↗
PubMed2002

Gene expression during the priming phase of liver regeneration after partial hepatectomy in mice.

Understanding the gene-expression patterns during liver regeneration may help to reveal how regenerative processes are initiated and controlled as well as shed new light onto processes that lead to liver disease. Using high-density oligonucleotide arrays, we have examined the gene-expression program in the livers of mice after partial hepatectomy. A time course was constructed for gene expression between 0 and 4 h after partial hepatectomy, corresponding to the priming phase of liver regeneration. The genomic program for liver regeneration involves transcription-factor generation, stress and inflammatory responses, cytoskeletal and extracellular matrix modification, and regulation of cell-cycle entry. The genome-wide changes that are observed provide a detailed and comprehensive map of the initial priming stage of liver regeneration.

Animals↗