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Author |
Parsons, A.B.; Brost, R.L.; Ding, H.; Li, Z.; Zhang, C.; Sheikh, B.; Brown, G.W.; Kane, P.M.; Hughes, T.R.; Boone, C. |
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Title |
Integration of chemical-genetic and genetic interaction data links bioactive compounds to cellular target pathways |
Type  |
Journal Article |
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Year |
2004 |
Publication |
Nature Biotechnology |
Abbreviated Journal |
Nat Biotechnol |
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Volume |
22 |
Issue |
1 |
Pages |
62-69 |
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Keywords |
Biotechnology/*methods; Cluster Analysis; Drug Industry/*methods; *Drug Resistance; Fungal Proteins/metabolism; Gene Deletion; *Gene Expression Regulation; Mutation; Pharmacogenetics; Proton-Translocating ATPases/metabolism; Saccharomyces cerevisiae/*genetics; Software |
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Abstract |
Bioactive compounds can be valuable research tools and drug leads, but it is often difficult to identify their mechanism of action or cellular target. Here we investigate the potential for integration of chemical-genetic and genetic interaction data to reveal information about the pathways and targets of inhibitory compounds. Taking advantage of the existing complete set of yeast haploid deletion mutants, we generated drug-hypersensitivity (chemical-genetic) profiles for 12 compounds. In addition to a set of compound-specific interactions, the chemical-genetic profiles identified a large group of genes required for multidrug resistance. In particular, yeast mutants lacking a functional vacuolar H(+)-ATPase show multidrug sensitivity, a phenomenon that may be conserved in mammalian cells. By filtering chemical-genetic profiles for the multidrug-resistant genes and then clustering the compound-specific profiles with a compendium of large-scale genetic interaction profiles, we were able to identify target pathways or proteins. This method thus provides a powerful means for inferring mechanism of action. |
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Call Number |
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Serial |
339 |
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Author |
Favelukes, G.; Stoppani, A.O. |
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Title |
Baker's-yeast fumarase, a thiol enzyme |
Type |
Journal Article |
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Year |
1958 |
Publication |
Biochimica et Biophysica Acta |
Abbreviated Journal |
Biochim Biophys Acta |
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Volume |
28 |
Issue |
3 |
Pages |
654-655 |
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Keywords |
*Hydro-Lyases; Saccharomyces cerevisiae/*metabolism; *Hydrases; *SACCHAROMYCES CEREVISIAE/metabolism |
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Abstract |
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Call Number |
Grinnell @ engelk @ |
Serial |
483 |
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Author |
Thacker, C.; Rose, A.M. |
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Title |
A look at the Caenorhabditis elegans Kex2/Subtilisin-like proprotein convertase family |
Type |
Journal Article |
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Year |
2000 |
Publication |
BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology |
Abbreviated Journal |
Bioessays |
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Volume |
22 |
Issue |
6 |
Pages |
545-553 |
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Keywords |
Animals; Caenorhabditis elegans/*enzymology/genetics; Genes, Helminth; Humans; Multigene Family; Mutation; Phylogeny; *Proprotein Convertases; *Saccharomyces cerevisiae Proteins; Subtilisins/chemistry/genetics/*metabolism |
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Abstract |
Significant advances have recently been made in our understanding of the mechanisms of activation of proteins that require processing. Often this involves endoproteolytic cleavage of precursor forms at basic residues, and is carried out by a group of serine endoproteinases, termed the proprotein convertases. In mammals, seven different convertases have been identified to date. These act in both the regulated secretory pathway for the processing of prohormones and proneuropeptides and in the constitutive secretory pathway, in which a variety of proproteins are activated endoproteolytically. The recently completed sequence of the nematode Caenorhabditis elegans genome affords a unique opportunity to examine the entire proprotein convertase family in a multicellular organism. Here we review the nature of the family, emphasising the structural features, characteristic of the four nematode genes, that supply all of the necessary functions unique to this group of serine endoproteinases. Studies of the C. elegans genes not only provide important information about the evaluation of this gene family but should help to illuminate the roles of these proteins in mammalian systems. BioEssays 22:545-553, 2000. |
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Call Number |
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Serial |
522 |
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Author |
Van de Ven, W.J.; Creemers, J.W.; Roebroek, A.J. |
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Title |
Furin: the prototype mammalian subtilisin-like proprotein-processing enzyme. Endoproteolytic cleavage at paired basic residues of proproteins of the eukaryotic secretory pathway |
Type |
Journal Article |
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Year |
1991 |
Publication |
Enzyme |
Abbreviated Journal |
Enzyme |
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Volume |
45 |
Issue |
5-6 |
Pages |
257-270 |
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Keywords |
Animals; Binding Sites; Catalysis; Cloning, Molecular; Drosophila melanogaster; Furin; Humans; Invertebrate Hormones/genetics/metabolism; Mice; Models, Molecular; Multigene Family; Protein Conformation; Protein Precursors/*metabolism; *Protein Processing, Post-Translational; Sequence Homology, Amino Acid; Substrate Specificity; Subtilisins/genetics/*metabolism |
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Abstract |
Furin, the translational product of the recently discovered fur gene, appears to be the first known mammalian member of the subtilisin family of serine proteases and the first known mammalian proprotein-processing enzyme with cleavage selectivity for paired basic amino acid residues. Structurally and functionally, it resembles the prohormone-processing enzyme, kexin (EC 3.4.21.61), which is encoded by the KEX2 gene of yeast Saccharomyces cerevisiae. Most likely, furin is primarily involved in the processing of precursors of proteins that are secreted via the constitutive secretory pathway. Here, we review the discovery of the fur gene and describe the isolation of cDNA clones corresponding to human and mouse fur and to two fur-like genes of Drosophila melanogaster, Dfur1 and Dfur2. We also compare the structural organization of the various deduced furin proteins to that of yeast kexin, and of other members of the subtilisin family of serine proteases. Furthermore, the biosynthesis of biologically active human and mouse furin is evaluated. Finally, the cleavage specificity for paired basic amino acid residues of human and mouse furin is demonstrated by the correct processing of the precursor for von Willebrand factor. |
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Call Number |
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Serial |
524 |
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Author |
Heinisch, J.J. |
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Title |
Baker's yeast as a tool for the development of antifungal kinase inhibitors--targeting protein kinase C and the cell integrity pathway |
Type |
Journal Article |
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Year |
2005 |
Publication |
Biochimica et Biophysica Acta |
Abbreviated Journal |
Biochim Biophys Acta |
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Volume |
1754 |
Issue |
1-2 |
Pages |
171-182 |
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Keywords |
Antifungal Agents/*chemistry/metabolism/pharmacology; Cell Cycle/*drug effects; Cell Wall/drug effects/metabolism; Enzyme Inhibitors/*chemistry/metabolism/pharmacology; Humans; MAP Kinase Signaling System/drug effects; Models, Biological; Protein Kinase C/*antagonists & inhibitors/chemistry/drug effects/metabolism; Protein Kinases/genetics/metabolism; Recombinant Fusion Proteins/chemistry/*metabolism; Saccharomyces cerevisiae/chemistry/enzymology/*metabolism; Saccharomyces cerevisiae Proteins/*antagonists & inhibitors/chemistry/drug effects/metabolism |
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Abstract |
Today, the yeast Saccharomyces cerevisiae is probably the best-studied eukaryotic organism. This review first focuses on the signaling process which is mediated by the unique yeast protein kinase C (Pkc1p) and a downstream mitogen-activated protein kinase (MAPK) cascade. This pathway ensures cellular integrity by sensing cell surface stress and controlling cell wall biosynthesis and progression through the cell cycle. The domain structure of Pkc1p is conserved from yeast to humans. A yeast system for heterologous expression of specific domains in a chimeric yeast/mammalian PKC enzyme (“domain shuffling”) is depicted. It is also proposed how this system could be employed for the study of protein kinase inhibitors in high-throughput screens. Moreover, a reporter assay that allows a quantitative readout of the activity of the cell integrity signaling pathway is introduced. Since a variety of protein kinases take part in the signal transduction, this broadens the range of targets for potential inhibitors. |
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Call Number |
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Serial |
554 |
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Author |
Mazzoni, C.; Falcone, C. |
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Title |
Caspase-dependent apoptosis in yeast |
Type |
Journal Article |
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Year |
2008 |
Publication |
Biochimica et Biophysica Acta |
Abbreviated Journal |
Biochim Biophys Acta |
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Volume |
1783 |
Issue |
7 |
Pages |
1320-1327 |
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Keywords |
Apoptosis--genetics, physiology; Apoptosis Regulatory Proteins--metabolism; Caspases--metabolism; Mitochondria--metabolism; Saccharomyces cerevisiae--genetics, physiology; Saccharomyces cerevisiae Proteins--metabolism; Signal Transduction |
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Abstract |
Damaging environment, certain intracellular defects or heterologous expression of pro-apoptotic genes induce death in yeast cells exhibiting typical markers of apoptosis. In mammals, apoptosis can be directed by the activation of groups of proteases, called caspases, that cleave specific substrates and trigger cell death. In addition, in plants, fungi, Dictyostelium and metazoa, paracaspases and metacaspases have been identified that share some homologies with caspases but showing different substrate specificity. In the yeast Saccharomyces cerevisiae, a gene (MCA1/YCA1) has been identified coding for a metacaspase involved in the induction of cell death. Metacaspases are not biochemical, but sequence and functional homologes of caspases, as deletion of them rescues entirely different death scenarios. In this review we will summarize the current knowledge in S. cerevisiae on apoptotic processes, induced by internal and external triggers, which are dependent on the metacaspase gene YCA1. |
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Call Number |
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Serial |
850 |
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Author |
Weaver, T.; Lees, M.; Zaitsev, V.; Zaitseva, I.; Duke, E.; Lindley, P.; McSweeny, S.; Svensson, A.; Keruchenko, J.; Keruchenko, I.; Gladilin, K.; Banaszak, L. |
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Title |
Crystal structures of native and recombinant yeast fumarase |
Type |
Journal Article |
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Year |
1998 |
Publication |
Journal of Molecular Biology |
Abbreviated Journal |
J Mol Biol |
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Volume |
280 |
Issue |
3 |
Pages |
431-442 |
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Keywords |
Binding Sites; Crystallography, X-Ray; Fumarate Hydratase/*chemistry; Fungal Proteins/*chemistry; Models, Molecular; Polymers/chemistry; *Protein Conformation; Saccharomyces cerevisiae/*enzymology; Water/chemistry |
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Abstract |
Crystal structures for both native and recombinant forms of yeast fumarase from Saccharomyces cerevisiae have been completed to moderate resolution by two separate laboratories. The recombinant form was obtained by the construction of an expression plasmid for Escherichia coli. Despite a high level of amino acid sequence similarity, purification of the eukaryotic enzyme from the wild-type prokaryotic enzyme was feasible. The crystal structure of the native form, NY-fumarase, encompasses residues R22 through M484, while the recombinant form, RY-fumarase, consists of residues S27 through L485. Both crystal structures lack the N-terminal translocation segment. Each subunit of the homo-tetrameric protein has three domains. The active site is formed by segments from each of three polypeptide chains. The results of these studies on the eukaryotic proteins are unique, since the recombinant form was done in the absence of dicarboxylic acid and has an unoccupied active site. As a comparison, native fumarase was crystallized in the presence of the competitive inhibitor, meso-tartrate. Meso-tartrate occupies a position close to that of the bound citrate molecule found in the active site of the E. coli enzyme. This inhibitor participates in hydrogen bonding to an active-site water molecule. The independent determination of the two structures provides further evidence that an active-site water molecule may play an active role in the fumarase-catalyzed reaction. |
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Call Number |
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Serial |
1178 |
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Author |
McPheeters, D.S.; Wise, J.A. |
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Title |
Measurement of in vivo RNA synthesis rates |
Type |
Journal Article |
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Year |
2013 |
Publication |
Methods in Enzymology |
Abbreviated Journal |
Methods Enzymol |
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Volume |
530 |
Issue |
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Pages |
117-135 |
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Keywords |
Gene Expression Regulation, Fungal; RNA, Fungal/*genetics; Saccharomyces cerevisiae/*genetics; Schizosaccharomyces/*genetics; Transcription, Genetic; Immobilized DNA/RNA; Immobilized probes; In vivo RNA synthesis rates; Labeled RNA; Nascent transcripts |
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Abstract |
A technique is described to directly measure ongoing transcription from individual genes in permeabilized cells of either the budding yeast Saccharomyces cerevisiae or the fission yeast Schizosaccharomyces pombe. Transcription run-on (TRO) analysis is used to compare the relative rates of synthesis for specific transcripts in cells grown under different environmental conditions or harvested at different stages of development. As the amount of an individual RNA species present at any given time is determined by its net rate of synthesis and degradation, an accurate picture of transcription per se can be obtained only by directly measuring de novo synthesis of RNA (if you are interested in RNA degradation, see Method for measuring mRNA decay rate in Saccharomyces cerevisiae). Most techniques employed to measure changes in the relative levels of individual transcripts present under different conditions, including Northern analysis (see Northern blotting), RT-PCR (see Reverse-transcription PCR (RT-PCR)), nuclease protection assays (see Explanatory Chapter: Nuclease Protection Assays), and genome-wide assays, such as microarray analysis and high throughput RNA sequencing, measure changes in the steady-state level of a transcript, which may or may not reflect the actual changes in transcription of the gene. Recent studies carried out in fission yeast have demonstrated that increases in the steady-state level (accumulation) of many individual mRNAs occur without any significant changes in transcription rates (McPheeters et al., 2009), highlighting the important role of regulated RNA stability in determining gene expression programs (Harigaya et al., 2006). |
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Call Number |
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Serial |
1345 |
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Author |
Leskovac, V.; Trivic, S.; Anderson, B.M. |
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Title |
Use of competitive dead-end inhibitors to determine the chemical mechanism of action of yeast alcohol dehydrogenase |
Type |
Journal Article |
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Year |
1998 |
Publication |
Molecular and Cellular Biochemistry |
Abbreviated Journal |
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Volume |
178 |
Issue |
1-2 |
Pages |
219-227 |
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Keywords |
yeast; alcohol; dehydrogenase; dead-end inhibitors; mechanism of action; dehydrogenases |
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Abstract |
In this work, we have postulated a comprehensive and unified chemical mechanism of action for yeast alcohol dehydrogenase (EC 1.1.1.1, constitutive, cytoplasmic), isolated from Saccharomyces cerevisiae. The chemical mechanism of yeast enzyme is based on the integrity of the proton relay system: His-51....NAD+....Thr-48....R.CH2OH(H2>O)....Zn<math>++, stretching from His-51 on the surface of enzyme to the active site zinc atom in the substrate-binding site of enzyme. Further, it is based on extensive studies of steady-state kinetic properties of enzyme which were published recently. In this study, we have reported the pH-dependence of dissociation constants for several competitive dead-end inhibitors of yeast enzyme from their binary complexes with enzyme, or their ternary complexes with enzyme and NAD+ or NADH; inhibitors include: pyrazole, acetamide, sodium azide, 2-fluoroethanol, and 2,2,2-trifluorethanol. The unified mechanism describes the structures of four dissociation forms of apoenzyme, two forms of the binary complex E.NAD+, three forms of the ternary complex E.NAD+.alcohol, two forms of the ternary complex E.NADH.aldehyde and three binary complexes E.NADH. Appropriate pKa values have been ascribed to protonation forms of most of the above mentioned complexes of yeast enzyme with coenzymes and substrates. |
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Call Number |
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Serial |
1414 |
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Author |
Schenone, M.; Dancik, V.; Wagner, B.K.; Clemons, P.A. |
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Title |
Target identification and mechanism of action in chemical biology and drug discovery |
Type |
Journal Article |
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Year |
2013 |
Publication |
Nature Chemical Biology |
Abbreviated Journal |
Nat Chem Biol |
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Volume |
9 |
Issue |
4 |
Pages |
232-240 |
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Keywords |
Animals; Biomarkers, Pharmacological/chemistry/*metabolism; *Drug Discovery; *Drug Evaluation, Preclinical; *High-Throughput Screening Assays; Humans; Isotope Labeling; Mass Spectrometry; Molecular Targeted Therapy; Phenotype; RNA Interference; Reverse Genetics; Saccharomyces cerevisiae/drug effects/genetics/metabolism; Small Molecule Libraries/chemistry/*metabolism/pharmacology; Validation Studies as Topic |
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Abstract |
Target-identification and mechanism-of-action studies have important roles in small-molecule probe and drug discovery. Biological and technological advances have resulted in the increasing use of cell-based assays to discover new biologically active small molecules. Such studies allow small-molecule action to be tested in a more disease-relevant setting at the outset, but they require follow-up studies to determine the precise protein target or targets responsible for the observed phenotype. Target identification can be approached by direct biochemical methods, genetic interactions or computational inference. In many cases, however, combinations of approaches may be required to fully characterize on-target and off-target effects and to understand mechanisms of small-molecule action. |
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Call Number |
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Serial |
1592 |
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