RESUMO
The trans-translation pathway for protein tagging and ribosome release plays a critical role for viability and virulence in a wide range of pathogens but is not found in animals. To explore the use of trans-translation as a target for antibiotic development, a high-throughput screen and secondary screening assays were used to identify small molecule inhibitors of the pathway. Compounds that inhibited protein tagging and proteolysis of tagged proteins were recovered from the screen. One of the most active compounds, KKL-35, inhibited the trans-translation tagging reaction with an IC50 = 0.9 µM. KKL-35 and other compounds identified in the screen exhibited broad-spectrum antibiotic activity, validating trans-translation as a target for drug development. This unique target could play a key role in combating strains of pathogenic bacteria that are resistant to existing antibiotics.
Assuntos
Antibacterianos/biossíntese , Escherichia coli/genética , Biossíntese de Proteínas/fisiologia , RNA Bacteriano/genética , Bibliotecas de Moléculas Pequenas , Antibacterianos/farmacologia , Bioensaio , Códon de Terminação/genética , Desenho de Fármacos , Farmacorresistência Bacteriana/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Biblioteca Gênica , Humanos , Luciferases/genética , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Ribossomos/genéticaRESUMO
High-throughput screening of 700,000 small molecules has identified 235 inhibitors of the GroEL/GroES-mediated refolding cycle. Dose-response analysis of a subset of these hits revealed that 21 compounds are potent inhibitors of GroEL/GroES-mediated refolding (IC50 <10 µM). The screening results presented herein represent the first steps in a broader aim of developing molecular probes to study chaperonin biochemistry and physiology.
Assuntos
Chaperonina 10/antagonistas & inibidores , Chaperonina 60/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Dose-Resposta a Droga , Concentração Inibidora 50 , Modelos Biológicos , Dobramento de Proteína/efeitos dos fármacosRESUMO
Ectopic expression of defined transcription factors can reprogram somatic cells to induced pluripotent stem (iPS) cells, but the utility of iPS cells is hampered by the use of viral delivery systems. Small molecules offer an alternative to replace virally transduced transcription factors with chemical signaling cues responsible for reprogramming. In this report we describe a small-molecule screening platform applied to identify compounds that functionally replace the reprogramming factor Klf4. A series of small-molecule scaffolds were identified that activate Nanog expression in mouse fibroblasts transduced with a subset of reprogramming factors lacking Klf4. Application of one such molecule, kenpaullone, in lieu of Klf4 gave rise to iPS cells that are indistinguishable from murine embryonic stem cells. This experimental platform can be used to screen large chemical libraries in search of novel compounds to replace the reprogramming factors that induce pluripotency. Ultimately, such compounds may provide mechanistic insight into the reprogramming process.
Assuntos
Benzazepinas/farmacologia , Diferenciação Celular , Epigênese Genética/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Indóis/farmacologia , Células-Tronco Pluripotentes/citologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Fibroblastos/citologia , Genes Reporter , Proteínas de Homeodomínio/genética , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Luciferases/genética , Camundongos , Proteína Homeobox NanogRESUMO
The growing resistance to current first-line antimalarial drugs represents a major health challenge. To facilitate the discovery of new antimalarials, we have implemented an efficient and robust high-throughput cell-based screen (1,536-well format) based on proliferation of Plasmodium falciparum (Pf) in erythrocytes. From a screen of approximately 1.7 million compounds, we identified a diverse collection of approximately 6,000 small molecules comprised of >530 distinct scaffolds, all of which show potent antimalarial activity (<1.25 microM). Most known antimalarials were identified in this screen, thus validating our approach. In addition, we identified many novel chemical scaffolds, which likely act through both known and novel pathways. We further show that in some cases the mechanism of action of these antimalarials can be determined by in silico compound activity profiling. This method uses large datasets from unrelated cellular and biochemical screens and the guilt-by-association principle to predict which cellular pathway and/or protein target is being inhibited by select compounds. In addition, the screening method has the potential to provide the malaria community with many new starting points for the development of biological probes and drugs with novel antiparasitic activities.
Assuntos
Antimaláricos/análise , Antimaláricos/farmacologia , Biologia Computacional , Animais , Antimaláricos/química , Antimaláricos/uso terapêutico , Análise por Conglomerados , Avaliação Pré-Clínica de Medicamentos , Resistência a Medicamentos/efeitos dos fármacos , Antagonistas do Ácido Fólico/análise , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Malária/tratamento farmacológico , Modelos Moleculares , Parasitos/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Reprodutibilidade dos Testes , Relação Estrutura-Atividade , Tetra-Hidrofolato Desidrogenase/químicaRESUMO
Conformational change is a common molecular mechanism for the regulation of kinase activities. Small molecule modulators of protein conformations, including allosteric kinase inhibitors, are highly wanted as tools for the interrogation of kinase biology and as selective therapeutic agents. However, straightforward cellular assays monitoring kinase conformations in a manner conducive to high-throughput screening (HTS) are not readily available. Here we describe such an HTS-compatible conformational sensor assay for Abl based on a split luciferase construct. The Abl sensor responds to intramolecular structural rearrangements associated with intracellular Abl deactivation and small molecule inhibition. The intact regulatory CAP-SH3-SH2 domain is required for the full functionality of the sensor. Moreover, a T334I Abl mutant (T315I in Abl1a) was found to be particularly well suited for HTS purposes and mechanistic intracellular studies of T334I mutant inhibitors. We expect that the split luciferase-based conformational sensor approach might be more broadly useful to probe the intracellular activation of other kinases and enzymes in general.
Assuntos
Mutação de Sentido Incorreto , Proteínas Oncogênicas v-abl/análise , Animais , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Humanos , Luciferases , Proteínas Oncogênicas v-abl/química , Proteínas Oncogênicas v-abl/genética , Conformação Proteica/efeitos dos fármacosRESUMO
Neural precursor cell expressed, developmentally down-regulated gene 8 (NEDD8) is a recently discovered ubiquitin-like posttranslational modifier. NEDD8 acts predominantly as a regulator of ubiquitin-protein ligases and as a decoy for proteins targeted for proteasomal degradation. It thereby controls key events in cell cycle progression and embryogenesis. Deneddylase-1 (DEN1/NEDP1/SENP8) features a selective peptidase activity converting the proNEDD8 precursor to its mature form and an isopeptidase activity deconjugating NEDD8 from substrates such as cullins and p53. In this study, we describe a high-throughput screening (HTS)-compatible time-resolved fluorescent resonance energy transfer (TR-FRET) assay measuring the peptidase activity of DEN1.
Assuntos
Endopeptidases/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/química , Humanos , Proteína NEDD8 , Fatores de Tempo , Ubiquitinas/metabolismoRESUMO
Retinol-binding protein-4 (RBP4) is an emerging candidate drug target for type 2 diabetes and lipofuscin-mediated macular degeneration. The retinoic acid derivative fenretinide (N-(4-hydroxyphenyl) retinamide; HPR) exerts therapeutic effects in mouse models of obesity, diabetes, and Stargardt's disease by targeting RBP4. Fenretinide competes with retinoids for RBP4 binding, disrupts RBP4-transthyretin (TTR) complexes, and results in urinary secretion of RBP4 and systemic depletion of retinol. To enable the search for nonretinoid molecules with fenretinide-like activities we developed a HTS-compatible homogeneous TR-FRET assay monitoring the displacement of retinoic acid derivatives from RBP4 in high-density 384-well and 1536-well microtiter plate formats. The retinoid displacement assay proved to be highly sensitive and robust after miniaturization with IC(50)s for fenretinide and retinol ranging around 50 and 100 nM, respectively, and Z'-factors around 0.7. In addition, a surface plasmon resonance (SPR)-based secondary assay was developed to interrogate small molecule RBP4 binders for their ability to modulate the RBP4-TTR interaction. Finally, a 1.6 x 10(6) compound library was screened against the retinoid displacement assay. Several potent retinoid competitors were identified that also appeared to disrupt RBP4-TTR complexes. Some of these compounds could potentially serve as valuable tools to further probe RBP4 biology in the future.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Pré-Albumina/análise , Retinoides/análise , Proteínas Plasmáticas de Ligação ao Retinol/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Avaliação Pré-Clínica de Medicamentos , Humanos , Estrutura Molecular , Pré-Albumina/química , Pré-Albumina/metabolismo , Ligação Proteica , Retinoides/química , Retinoides/metabolismo , Fatores de TempoRESUMO
In vivo incorporation of isotopically labeled unnatural amino acids into large proteins drastically reduces the complexity of nuclear magnetic resonance (NMR) spectra. Incorporation is accomplished by coexpressing an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid added to the media and the protein of interest with a TAG amber codon at the desired incorporation site. To demonstrate the utility of this approach for NMR studies, 2-amino-3-(4-(trifluoromethoxy)phenyl)propanoic acid (OCF 3Phe), (13)C/(15)N-labeled p-methoxyphenylalanine (OMePhe), and (15)N-labeled o-nitrobenzyl-tyrosine (oNBTyr) were incorporated individually into 11 positions around the active site of the 33 kDa thioesterase domain of human fatty acid synthase (FAS-TE). In the process, a novel tRNA synthetase was evolved for OCF 3Phe. Incorporation efficiencies and FAS-TE yields were improved by including an inducible copy of the respective aminoacyl-tRNA synthetase gene on each incorporation plasmid. Using only between 8 and 25 mg of unnatural amino acid, typically 2 mg of FAS-TE, sufficient for one 0.1 mM NMR sample, were produced from 50 mL of Escherichia coli culture grown in rich media. Singly labeled protein samples were then used to study the binding of a tool compound. Chemical shift changes in (1)H-(15)N HSQC, (1)H-(13)C HSQC, and (19)F NMR spectra of the different single site mutants consistently identified the binding site and the effect of ligand binding on conformational exchange of some of the residues. OMePhe or OCF 3Phe mutants of an active site tyrosine inhibited binding; incorporating (15)N-Tyr at this site through UV-cleavage of the nitrobenzyl-photocage from oNBTyr re-established binding. These data suggest not only robust methods for using unnatural amino acids to study large proteins by NMR but also establish a new avenue for the site-specific labeling of proteins at individual residues without altering the protein sequence, a feat that can currently not be accomplished with any other method.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Fenilalanina/análogos & derivados , Fenilpropionatos/química , Proteínas/análise , Tirosina/análogos & derivados , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Isótopos de Carbono , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Graxo Sintases/química , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Marcação por Isótopo , Isótopos de Nitrogênio , Fenilpropionatos/metabolismo , Plasmídeos/genética , Engenharia de Proteínas , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas/metabolismoRESUMO
Antibody microarray measurements show great potential for the simultaneous quantification of many proteins in small amounts of body fluids and extracts. Over the last few years, a micro-array platform centered around the concept of microarrays in microtiter wells was developed, and for the best assays we have achieved lower limits of detection in the femtomolar range using resonance light-scattering particles for the staining of biotinylated detection antibodies. Although conceptually simple, these multiplexed sandwich assays are technically challenging. Here we describe in detail our protocols and procedures for the manufacturing of antibody microarrays with up to 48 different antibodies and for performing plasma measurements.
Assuntos
Anticorpos/química , Análise Serial de Proteínas/métodos , Animais , Antígenos/química , Biotinilação , Proteínas Sanguíneas/química , Calibragem , Primers do DNA/química , Haptenos/química , Humanos , Imunoglobulinas/química , Luz , Oligonucleotídeos/química , Análise Serial de Proteínas/instrumentação , Reprodutibilidade dos Testes , Espalhamento de RadiaçãoAssuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Inibidores de Proteínas Quinases/química , Fatores de Transcrição SOXB1/metabolismo , Quinases da Família src/antagonistas & inibidores , Animais , Dasatinibe , Doxiciclina/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Fatores de Transcrição SOXB1/genética , Tiazóis/química , Tiazóis/farmacologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
HIV-1 integrase (HIV-IN) is a well-validated antiviral drug target catalyzing a multistep reaction to incorporate the HIV-1 provirus into the genome of the host cell. Small molecule inhibitors of HIV-1 integrase that specifically target the strand transfer step have demonstrated efficacy in the suppression of virus propagation. However, only few specific strand transfer inhibitors have been identified to date, and the need to screen for novel compound scaffolds persists. Here, the authors describe 2 homogeneous time-resolved fluorescent resonance energy transfer-based assays for the measurement of HIV-1 integrase 3'-processing and strand transfer activities. Both assays were optimized for high-throughput screening formats, and a diverse library containing more than 1 million compounds was screened in 1536-well plates for HIV-IN strand transfer inhibitors. As a result, compounds were found that selectively affect the enzymatic strand transfer reaction over 3beta processing. Moreover, several bioactive molecules were identified that inhibited HIV-1 reporter virus infection in cellular model systems. In conclusion, the assays presented herein have proven their utility for the identification of mechanistically interesting and biologically active inhibitors of HIV-1 integrase that hold potential for further development into potent antiviral drugs.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Integrase de HIV/genética , Integrase de HIV/metabolismo , Antivirais/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Clonagem Molecular , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/instrumentação , Inibidores de Integrase de HIV/farmacologia , Humanos , Concentração Inibidora 50 , Modelos Genéticos , Fosfatidilcolinas/farmacologia , Fatores de TempoRESUMO
The efficacy of most marketed antimalarial drugs has been compromised by evolution of parasite resistance, underscoring an urgent need to find new drugs with new mechanisms of action. We have taken a high-throughput approach toward identifying novel antimalarial chemical inhibitors of prioritized drug targets for Plasmodium falciparum, excluding targets which are inhibited by currently used drugs. A screen of commercially available libraries identified 5655 low molecular weight compounds that inhibit growth of P. falciparum cultures with EC(50) values below 1.25µM. These compounds were then tested in 384- or 1536-well biochemical assays for activity against nine Plasmodium enzymes: adenylosuccinate synthetase (AdSS), choline kinase (CK), deoxyuridine triphosphate nucleotidohydrolase (dUTPase), glutamate dehydrogenase (GDH), guanylate kinase (GK), N-myristoyltransferase (NMT), orotidine 5'-monophosphate decarboxylase (OMPDC), farnesyl pyrophosphate synthase (FPPS) and S-adenosylhomocysteine hydrolase (SAHH). These enzymes were selected using TDRtargets.org, and are believed to have excellent potential as drug targets based on criteria such as their likely essentiality, druggability, and amenability to high-throughput biochemical screening. Six of these targets were inhibited by one or more of the antimalarial scaffolds and may have potential use in drug development, further target validation studies and exploration of P. falciparum biochemistry and biology.
Assuntos
Antimaláricos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Plasmodium falciparum/efeitos dos fármacos , Antimaláricos/isolamento & purificação , Inibidores Enzimáticos/isolamento & purificação , Enzimas/metabolismo , Concentração Inibidora 50 , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/antagonistas & inibidoresRESUMO
Myotonic dystrophy type 1 (DM1) is a genetic disorder characterized by muscle wasting, myotonia, cataracts, cardiac arrhythmia, hyperinsulinism and intellectual deficits, and is caused by expansion of a CTG repeat in the 3'UTR of the Dystrophia Myotonica-Protein Kinase (DMPK) gene. The DMPK transcripts containing expanded CUG repeats accumulate in nuclear foci and ultimately cause mis-splicing of secondary genes through the dysregulation of RNA-binding proteins including Muscleblind 1 (MBNL1) and CUG binding protein 1 (CUGBP1). Correction of mis-splicing of genes such as the Skeletal muscle-specific chloride channel 1 (CLCN1), Cardiac troponin T (TNNT2), Insulin receptor (INSR) and Sarcoplasmic/endoplasmic reticulum Ca(2+)ATPase 1 (SERCA1) may alleviate some of the symptoms of DM1; hence identification of small molecule modulators is an important step towards a therapy for DM1 patients. Here we describe the generation of immortalized myoblast cell lines derived from healthy (DMPK CTG(5)) and DM1 patient (DMPK CTG(1000)) fibroblasts by constitutive overexpression of human telomerase reverse transcriptase (hTERT) and inducible overexpression of the Myoblast determination factor (MYOD). MBNL1-containing nuclear foci, mis-splicing events and defective myotube differentiation defects characteristic of DM1 were observed in these cells. A CLCN1 luciferase minigene construct (CLCN1-luc) was stably introduced to monitor intron 2 retention in the DM1 cellular context (a reported splicing defect in DM1). The assay was validated by performing a high-throughput screen (HTS) of ~13,000 low molecular weight compounds against the CLCN1-luc DM1 myoblast cell line, providing an ideal system for conducting HTS to better understand and treat DM1.
RESUMO
One therapeutic approach to Duchenne Muscular Dystrophy (DMD) recently entering clinical trials aims to convert DMD phenotypes to that of a milder disease variant, Becker Muscular Dystrophy (BMD), by employing antisense oligonucleotides (AONs) targeting splice sites, to induce exon skipping and restore partial dystrophin function. In order to search for small molecule and genetic modulators of AON-dependent and independent exon skipping, we screened approximately 10,000 known small molecule drugs, >17,000 cDNA clones, and >2,000 kinase- targeted siRNAs against a 5.6 kb luciferase minigene construct, encompassing exon 71 to exon 73 of human dystrophin. As a result, we identified several enhancers of exon skipping, acting on both the reporter construct as well as endogenous dystrophin in mdx cells. Multiple mechanisms of action were identified, including histone deacetylase inhibition, tubulin modulation and pre-mRNA processing. Among others, the nucleolar protein NOL8 and staufen RNA binding protein homolog 2 (Stau2) were found to induce endogenous exon skipping in mdx cells in an AON-dependent fashion. An unexpected but recurrent theme observed in our screening efforts was the apparent link between the inhibition of cell cycle progression and the induction of exon skipping.
Assuntos
Distrofina/genética , Éxons/genética , Ensaios de Triagem em Larga Escala/métodos , Oligonucleotídeos Antissenso/farmacologia , Bibliotecas de Moléculas Pequenas/análise , Processamento Alternativo/efeitos dos fármacos , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , DNA Complementar/genética , Elementos Facilitadores Genéticos/genética , Ensaios Enzimáticos , Genes Reporter , Genoma Humano/genética , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Luciferases/metabolismo , Camundongos , Índice Mitótico , Distrofia Muscular de Duchenne/genética , Fosfotransferases/metabolismo , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/farmacologia , Moduladores de Tubulina/farmacologiaRESUMO
BACKGROUND: The increasing number of kinases as potential drug targets, in combination with the need to screen large compound collections, demands the kinase assays to be homogeneous, non-radioactive, robust, sensitive, easy to miniaturize, and high-throughput. OBJECTIVE: This review will focus on some of the chief biochemical and cellular kinase assay technologies and their applications in tyrosine kinase drug discovery. METHODS: Recent literatures on these tyrosine kinase assay technologies are reviewed, each assay principle, advantages and drawbacks, as well as their potential utilities in tyrosine kinase drug discovery are discussed. RESULTS/CONCLUSION: There is no perfect assay yet; the choice of assay technology relies on the consideration of many factors including the intrinsic properties of the assay, intended application, cost, timeline, synergy with other in-house assay technologies and the expertise, familiarity and comfort level with certain technologies.
RESUMO
High-throughput cellular profiling has successfully stimulated early drug discovery pipelines by facilitating targeted as well as opportunistic lead finding, hit annotation and SAR analysis. While automation-friendly universal assay formats exist to address most established drug target classes like GPCRs, NHRs, ion channels or Tyr-kinases, no such cellular assay technology is currently enabling an equally broad and rapid interrogation of the Ser/Thr-kinase space. Here we present the foundation of an emerging cellular Ser/Thr-kinase platform that involves a) coexpression of targeted kinases with promiscuous peptide substrates and b) quantification of intracellular substrate phosphorylation by homogeneous TR-FRET. Proof-of-concept data is provided for cellular AKT, B-RAF and CamK2delta assays. Importantly, comparable activity profiles were found for well characterized B-Raf inhibitors in TR-FRET assays relying on either promiscuous peptide substrates or a MEK1(WT) protein substrate respectively. Moreover, IC(50)-values correlated strongly between cellular TR-FRET assays and a gold standard Ba/F3 proliferation assay for B-Raf activity. Finally, we expanded our initial assay panel by screening a kinase-focused cDNA library and identified starting points for >20 cellular Ser/Thr-kinase assays.
Assuntos
Hemoglobina Fetal/metabolismo , Bibliotecas de Moléculas Pequenas/química , Anemia Falciforme/tratamento farmacológico , Linhagem Celular Tumoral , Hemoglobina Fetal/química , Ensaios de Triagem em Larga Escala , Humanos , Purinas/química , Fator de Transcrição STAT3/metabolismo , Bibliotecas de Moléculas Pequenas/uso terapêuticoRESUMO
A series of dihydroxyphenylpyrazole compounds were identified as a unique class of reversible Hsp90 inhibitors. The crystal structures for two of the identified compounds complexed with the N-terminal ATP binding domain of human Hsp90alpha were determined. The dihydroxyphenyl ring of the compounds fits deeply into the adenine binding pocket with the C2 hydroxyl group forming a direct hydrogen bond with the side chain of Asp93. The pyrazole ring forms hydrogen bonds to the backbone carbonyl of Gly97, the hydroxyl group of Thr184 and to a water molecule, which is present in all of the published HSP90 structures. One of the identified compounds (G3130) demonstrated cellular activities (in Her-2 degradation and activation of Hsp70 promoter) consistent with the inhibition of cellular Hsp90 functions.
Assuntos
Proteínas de Choque Térmico HSP90/química , Pirazóis/química , Linhagem Celular , Linhagem Celular Tumoral , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Estrutura Terciária de Proteína , Pirazóis/farmacologia , Relação Estrutura-Atividade , Fatores de TempoRESUMO
Four different immunoassay and antibody microarray methods performed at four different sites were used to measure the levels of a broad range of proteins (N = 323 assays; 39, 88, 168, and 28 assays at the respective sites; 237 unique analytes) in the human serum and plasma reference specimens distributed by the Plasma Proteome Project (PPP) of the HUPO. The methods provided a means to (1) assess the level of systematic variation in protein abundances associated with blood preparation methods (serum, citrate-anticoagulated-plasma, EDTA-anticoagulated-plasma, or heparin-anticoagulated-plasma) and (2) evaluate the dependence on concentration of MS-based protein identifications from data sets using the HUPO specimens. Some proteins, particularly cytokines, had highly variable concentrations between the different sample preparations, suggesting specific effects of certain anticoagulants on the stability or availability of these proteins. The linkage of antibody-based measurements from 66 different analytes with the combined MS/MS data from 18 different laboratories showed that protein detection and the quality of MS data increased with analyte concentration. The conclusions from these initial analyses are that the optimal blood preparation method is variable between analytes and that the discovery of blood proteins by MS can be extended to concentrations below the ng/mL range under certain circumstances. Continued developments in antibody-based methods will further advance the scientific goals of the PPP.
Assuntos
Proteínas Sanguíneas/química , Coleta de Amostras Sanguíneas/métodos , Imunoensaio/métodos , Espectrometria de Massas/métodos , Proteômica/métodos , Calibragem , Ácido Cítrico/farmacologia , Ácido Edético/farmacologia , Heparina/farmacologia , Humanos , Análise em Microsséries , Análise Serial de Proteínas , Padrões de Referência , Valores de Referência , Manejo de Espécimes/métodos , Estatística como AssuntoRESUMO
The organization of myosin into motile cellular structures requires precise temporal and spatial regulation. Proteins containing a UCS (UNC-45/CRO1/She4p) domain are necessary for the incorporation of myosin into the contractile ring during cytokinesis and into thick filaments during muscle development. We report that the carboxyl-terminal regions of UNC-45 bound and exerted chaperone activity on the myosin head. The amino-terminal tetratricopeptide repeat domain of UNC-45 bound the molecular chaperone Hsp90. Thus, UNC-45 functions both as a molecular chaperone and as an Hsp90 co-chaperone for myosin, which can explain previous findings of altered assembly and decreased accumulation of myosin in UNC-45 mutants of Caenorhabditis elegans.