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With the advent of robust and high-throughput mass spectrometric technologies and bioinformatics tools to analyze large data sets, proteomics has penetrated broadly into basic and translational life sciences research. More than 95% of FDA-approved drugs currently target proteins, and most diagnostic tests are protein-based. The introduction of proteomics to the clinic, for instance to guide patient stratification and treatment, is already ongoing. Importantly, ethical challenges come with this success, which must also be adequately addressed by the proteomics and medical communities. Consortium members of the H2020 European Union-funded proteomics initiative: European Proteomics Infrastructure Consortium-providing access (EPIC-XS) met at the Core Technologies for Life Sciences (CTLS) conference to discuss the emerging role and implementation of proteomics in the clinic. The discussion, involving leaders in the field, focused on the current status, related challenges, and future efforts required to make proteomics a more mainstream technology for translational and clinical research. Here we report on that discussion and provide an expert update concerning the feasibility of clinical proteomics, the ethical implications of generating and analyzing large-scale proteomics clinical data, and recommendations to ensure both ethical and effective implementation in real-world applications.
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Ceramides are central intermediates of sphingolipid metabolism with dual roles as mediators of cellular stress signaling and mitochondrial apoptosis. How ceramides exert their cytotoxic effects is unclear and their poor solubility in water hampers a search for specific protein interaction partners. Here, we report the application of a photoactivatable and clickable ceramide analog, pacCer, to identify ceramide binding proteins and unravel the structural basis by which these proteins recognize ceramide. Besides capturing ceramide transfer protein (CERT) from a complex proteome, our approach yielded CERT-related steroidogenic acute regulatory protein D7 (StarD7) as novel ceramide binding protein. Previous work revealed that StarD7 is required for efficient mitochondrial import of phosphatidylcholine (PC) and serves a critical role in mitochondrial function and morphology. Combining site-directed mutagenesis and photoaffinity labeling experiments, we demonstrate that the steroidogenic acute regulatory transfer domain of StarD7 harbors a common binding site for PC and ceramide. While StarD7 lacks robust ceramide transfer activity in vitro, we find that its ability to shuttle PC between model membranes is specifically affected by ceramides. Besides demonstrating the suitability of pacCer as a tool to hunt for ceramide binding proteins, our data point at StarD7 as a candidate effector protein by which ceramides may exert part of their mitochondria-mediated cytotoxic effects.
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Proteínas Portadoras/metabolismo , Ceramidas/metabolismo , Lípidos , Proteínas Portadoras/biosíntesis , Células HeLa , Humanos , Mitocondrias/metabolismoRESUMEN
The PRIME-XS consortium is a pan-European infrastructure for proteomics. As a prologue to this special issue of Molecular & Cellular Proteomics on the research activities of the PRIME-XS consortium, we, as the guest editors of this issue, provide an overview of the structure and activities of this consortium, which is funded by the European Union's 7th Framework Programme for Research and Technological Development.
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Proteómica/organización & administración , Biomarcadores/análisis , Biología Computacional , Europa (Continente) , Proteínas/genética , Proteínas/metabolismo , Proteómica/economía , Proteómica/educaciónRESUMEN
Human peptidylarginine deiminases (hPADs) have been implicated in several diseases, particularly in rheumatoid arthritis. Since hPAD2 and hPAD4 are the isotypes expressed in the inflamed joints of RA patients and protein citrullination by PADs has been proposed to play a pathophysiological role, they represent unique therapeutic targets. To facilitate the development of substrate-based PAD inhibitors the substrate specificity of hPAD2 and hPAD4 was determined. Recombinant hPADs were expressed in bacteria or mammalian cell lines and allowed to citrullinate proteins in cell lysates, as well as a series of synthetic peptides. The citrullinated residues in proteins and the efficiency of peptide citrullination were determined by mass spectrometry. In total 320 hPAD2 and 178 hPAD4 citrullination sites were characterized. Amino acid residues most commonly found in citrullination sites for both isotypes are Gly at +1 and Tyr at +3 relative to the target arginine. For hPAD4 several additional amino acids were observed to be preferred at various positions from -4 to +4. The substrate motifs determined by amino acid substitution analysis partially confirmed these preferences, although peptide context dependent differences were also observed. Taken together, our data show that the enzyme specificity for cellular substrates and synthetic peptides differs for hPAD2 and hPAD4. hPAD4 shows more restrictive substrate specificity compared to hPAD2. Consensus sequences, which can be used as the basis for the development of PAD inhibitors, were derived for the citrullination sites of both hPAD2 and hPAD4.
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Arginina/metabolismo , Citrulina/metabolismo , Hidrolasas/metabolismo , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Arginina/química , Células COS , Chlorocebus aethiops , Citrulina/química , Escherichia coli/genética , Escherichia coli/metabolismo , Glicina/química , Glicina/metabolismo , Células HEK293 , Humanos , Hidrolasas/química , Hidrolasas/genética , Espectrometría de Masas , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Péptidos/síntesis química , Arginina Deiminasa Proteína-Tipo 4 , Desiminasas de la Arginina Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Tirosina/química , Tirosina/metabolismoAsunto(s)
Ácidos Grasos/química , Fosfolípidos/química , Proteínas/química , Animales , Células CHO , Química Clic , Cricetinae , Cricetulus , Ácidos Grasos/síntesis química , Ácidos Grasos/metabolismo , Células HeLa , Humanos , Microscopía Confocal , Fosfolípidos/metabolismo , Unión Proteica , Proteínas/metabolismo , Rayos UltravioletaRESUMEN
Citrullination, the conversion of peptidylarginine to peptidylcitrulline is catalyzed by peptidylarginine deiminases (PAD). The expression of PAD isoforms displays great variation among different tissues as demonstrated by PAD mRNA analyses. Here we have analyzed the differential expression of PAD2, PAD4 and PAD6 in mouse tissues at the protein level and by enzymatic activity assays using PAD2 and PAD4 knock-out strains. As expected, no PAD2 expression was detected in the PAD2-/- mice. In contrast, the PAD4 protein was observed in several tissues of the PAD4 knock-out mice, albeit at reduced levels in most tissues, and are therefore referred to as PAD4-low mice. In material from PAD2-/- mice, except for leukocyte lysates, hardly any PAD activity was found and no citrullinated proteins were detected after incubation in the presence of calcium. PAD activity in the PAD4-low mice was similar to that in wild-type mice. In both PAD knock-out strains the expression of PAD6 appeared to be up-regulated in all tissues analyzed, with the exception of spleen and testis. Our data demonstrate that the PAD2 protein is expressed in brain, spinal cord, spleen, skeletal muscle and leukocytes, but not detectably in liver, lung, kidney and testis. PAD4 was detected in each of these tissues, although the expression levels varied. In all tissues where PAD2 was detected, except for blood cells, this PAD isoform appeared to be responsible for virtually all peptidylarginine deiminase activity.
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Citrulina/metabolismo , Regulación de la Expresión Génica , Hidrolasas/genética , ARN Mensajero/genética , Animales , Exones , Variación Genética , Humanos , Hidrolasas/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Ratones Noqueados , Especificidad de Órganos , Arginina Deiminasa Proteína-Tipo 2 , Desiminasas de la Arginina Proteica , ARN Mensajero/metabolismoRESUMEN
Multiple sclerosis (MS) is the most common CNS-demyelinating disease of humans, showing clinical and pathological heterogeneity and a general resistance to therapy. We first discovered that abnormal myelin hypercitrullination, even in normal-appearing white matter, by peptidylarginine deiminases (PADs) correlates strongly with disease severity and might have an important role in MS progression. Hypercitrullination is known to promote focal demyelination through reduced myelin compaction. Here we report that 2-chloroacetamidine (2CA), a small-molecule, PAD active-site inhibitor, dramatically attenuates disease at any stage in independent neurodegenerative as well as autoimmune MS mouse models. 2CA reduced PAD activity and protein citrullination to pre-disease status. In the autoimmune models, disease induction uniformly induced spontaneous hypercitrullination with citrulline+ epitopes targeted frequently. 2CA rapidly suppressed T cell autoreactivity, clearing brain and spinal cord infiltrates, through selective removal of newly activated T cells. 2CA essentially prevented disease when administered before disease onset or before autoimmune induction, making hypercitrullination, and specifically PAD enzymes, a therapeutic target in MS models and thus possibly in MS.
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Citrulina/metabolismo , Hidrolasas/antagonistas & inhibidores , Esclerosis Múltiple/enzimología , Esclerosis Múltiple/patología , Amidinas/química , Amidinas/farmacología , Amidinas/uso terapéutico , Animales , Encéfalo/enzimología , Encéfalo/patología , Complejo CD3/metabolismo , Enfermedades Desmielinizantes/enzimología , Enfermedades Desmielinizantes/patología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/enzimología , Encefalomielitis Autoinmune Experimental/patología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Hidrolasas/metabolismo , Linfocitos/efectos de los fármacos , Linfocitos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Esclerosis Múltiple/tratamiento farmacológico , Nervio Óptico/efectos de los fármacos , Nervio Óptico/patología , Nervio Óptico/ultraestructura , Desiminasas de la Arginina Proteica , Bazo/efectos de los fármacos , Bazo/metabolismo , Bazo/patología , Análisis de SupervivenciaRESUMEN
Coronary atherosclerosis represents the major cause of death in Western societies. As atherosclerosis typically progresses over years without giving rise to clinical symptoms, biomarkers are urgently needed to identify patients at risk. Over the past decade, evidence has accumulated suggesting cross-talk between the diseased vasculature and cells of the innate immune system. We therefore employed proteomics to search for biomarkers associated with severe atherosclerotic coronary lumen stenosis in circulating leukocytes. In a two-phase approach, we first performed in-depth quantitative profiling of the granulocyte proteome on a small pooled cohort of patients suffering from chronic (sub)total coronary occlusion and matched control patients using stable isotope peptide labeling, two-dimensional LC-MS/MS and data-dependent decision tree fragmentation. Over 3000 proteins were quantified, among which 57 candidate biomarker proteins remained after stringent filtering. The most promising biomarker candidates were subsequently verified in the individual samples of the discovery cohort using label-free, single-run LC-MS/MS analysis, as well as in an independent verification cohort of 25 patients with total coronary occlusion (CTO) and 19 matched controls. Our data reveal bactericidal/permeability-increasing protein (BPI) as a promising biomarker for severe atherosclerotic coronary stenosis, being down-regulated in circulating granulocytes of CTO patients.
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Péptidos Catiónicos Antimicrobianos/sangre , Biomarcadores/sangre , Enfermedad de la Arteria Coronaria/sangre , Estenosis Coronaria/sangre , Granulocitos/metabolismo , Proteoma , Proteínas Sanguíneas , Separación Celular , Estudios de Cohortes , HumanosRESUMEN
Quantitative detection of phosphorylation levels is challenging and requires an expertise in both stable isotope labeling as well as enrichment of phosphorylated peptides. Recently, a microfluidic device incorporating a nanoliter flow rate reversed phase column as well as a titania (TiO(2)) enrichment column was released. This HPLC phosphochip allows excellent recovery and separation of phosphorylated peptides in a robust and reproducible manner with little user intervention. In this work, we have extended the abilities of this chip by defining the conditions required for on-chip stable isotope dimethyl labeling allowing for automated quantitation. The resulting approach will make quantitative phosphoproteomics more accessible.
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Cromatografía Líquida de Alta Presión/normas , Marcaje Isotópico/métodos , Microfluídica/normas , Fosfopéptidos/química , Automatización , Cromatografía de Gases y Espectrometría de Masas , Reproducibilidad de los Resultados , Titanio/químicaRESUMEN
Mass spectrometry has proven to be an indispensable tool for protein identification, characterization, and quantification. Among the possible methods in quantitative proteomics, stable isotope labeling by using reductive dimethylation has emerged as a cost-effective, simple, but powerful method able to compete at any level with the present alternatives. In this review, we briefly introduce experimental and software methods for proteome analysis using dimethyl labeling and provide a comprehensive overview of reported applications in the analysis of (1) differential protein expression, (2) posttranslational modifications, and (3) protein interactions.
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Marcaje Isotópico/métodos , Proteínas/química , Proteómica/métodos , Animales , Humanos , Metilación , Proteínas/metabolismoRESUMEN
INTRODUCTION: Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation of the joints and the presence of autoantibodies directed against proteins containing the non-standard arginine-derived amino acid citrulline. The protein fibrinogen, which has an essential role in blood clotting, is one of the most prominent citrullinated autoantigens in RA, particularly because it can be found in the inflamed tissue of affected joints. Here, we set out to analyze the presence of citrullinated endogenous peptides in the synovial fluid of RA and arthritic control patients. METHODS: Endogenous peptides were isolated from the synovial fluid of RA patients and controls by filtration and solid phase extraction. The peptides were identified and quantified using high-resolution liquid chromatography-mass spectrometry. RESULTS: Our data reveal that the synovial fluid of RA patients contains soluble endogenous peptides, derived from fibrinogen, containing significant amounts of citrulline residues and, in some cases, also phosphorylated serine. Several citrullinated peptides are found to be more abundantly present in the synovial fluid of RA patients compared to patients suffering from other inflammatory diseases affecting the joints. CONCLUSIONS: The increased presence of citrullinated peptides in RA patients points toward a possible specific role of these peptides in the immune response at the basis of the recognition of citrullinated peptides and proteins by RA patient autoantibodies.
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Artritis Reumatoide/inmunología , Autoantígenos/análisis , Fibrinógeno/análisis , Péptidos/análisis , Líquido Sinovial/química , Artritis Reumatoide/metabolismo , Cromatografía Liquida , Citrulina/inmunología , Citrulina/metabolismo , Fibrinógeno/metabolismo , Humanos , Espectrometría de Masas , Líquido Sinovial/metabolismoRESUMEN
The aminoacyl-tRNA synthetases are ubiquitously expressed enzymes that catalyze the esterification of amino acids to their cognate tRNAs. Autoantibodies against several aminoacyl-tRNA synthetases are found in autoimmune polymyositis and dermatomyositis patients. Because necrosis is often found in skeletal muscle biopsies of these patients, we hypothesized that cell-death-induced protein modifications may help in breaking immunological tolerance. Since cell death is associated with oxidative stress, the effect of oxidative stress on the main myositis-specific autoantibody target Jo-1 (histidyl-tRNA synthetase; HisRS) was studied in detail. The exposure of Jurkat cells to hydrogen peroxide resulted in the detection of several oxidized methionines and one oxidized tryptophan residue in the HisRS protein, as demonstrated by mass spectrometry. Unexpectedly, the tRNA aminoacylation activity of HisRS appeared to be increased upon oxidative modification. The analysis of myositis patient sera did not lead to the detection of autoantibodies that are specifically reactive with the modified HisRS protein. The results of this study demonstrate that the Jo-1/HisRS autoantigen is modified under oxidative stress conditions. The consequences of these modifications for the function of HisRS and its autoantigenicity are discussed.
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Histidina-ARNt Ligasa/metabolismo , Estrés Oxidativo , Aminoacilación de ARN de Transferencia , Secuencia de Aminoácidos , Especificidad de Anticuerpos , Apoptosis , Autoanticuerpos/sangre , Autoanticuerpos/metabolismo , Dermatomiositis/sangre , Dermatomiositis/inmunología , Activación Enzimática , Humanos , Peróxido de Hidrógeno/farmacología , Immunoblotting , Células Jurkat , Metionina/metabolismo , Datos de Secuencia Molecular , Polimiositis/sangre , Polimiositis/inmunología , Espectrometría de Masas en Tándem , Triptófano/metabolismoRESUMEN
Quantitative methodologies for the global in-depth comparison of proteomes are frequently based on chemical derivatization of peptides with isotopically distinguishable labeling agents. In the present work, we set out to study the feasibility of the dimethyl labeling method in combination with ZIC-cHILIC (zwitterionic hydrophilic interaction liquid chromatography) technology for quantitative proteomics. We first addressed the potential issue of isotope effects perturbing the essential coelution of differently labeled peptides under ZIC-cHILIC separation. The deuterium incorporation-induced effect can be largely eliminated by favoring the mixed-mode ZIC-cHILIC separation based on combined hydrophilic and ionic interactions. Then, we evaluated the performance and applicability of this strategy using a sample consisting of human cell lysate. We demonstrate that our approach is suitable to perform unbiased quantitative proteome analysis, still quantifying more than 2500 proteins when analyzing only a few micrograms of sample.
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Cromatografía Liquida , Deuterio/análisis , Fragmentos de Péptidos/análisis , Proteoma/análisis , Proteómica , Animales , Bovinos , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Marcaje Isotópico , Fragmentos de Péptidos/aislamiento & purificación , Albúmina Sérica Bovina/análisisRESUMEN
Methionine oxidation in the ubiquitous calcium signaling protein calmodulin (CaM) is known to disrupt downstream signaling and target CaM for proteasomal degradation. The susceptibility of CaM to oxidation in the different conformations that are sampled during calcium signaling is currently not well defined. Using an integrative mass spectrometry (MS) approach, applying both native MS and LC/MS/MS, we unravel molecular details of CaM methionine oxidation in the context of its interaction with the Ca(2+)/CaM-dependent protein kinase II (CaMKII). Sensitivity to methionine oxidation in CaM was found to vary according to the conformational state. Three methionine residues (Met71, 72, 145) show increased reactivity in calcium-saturated CaM (holo-CaM) compared to calcium-free CaM (apo-CaM), which has important consequences for oxidation-targeted proteasomal degradation. In addition, all four methionines in the C-terminal lobe (Met109, 124, 144 and 145) are found to be protected from oxidation in a peptide-based model of the CaMKII-bound conformation (cbp-CaM). We furthermore demonstrate that the oxidation of Met144 and 145 inhibits the interaction of CaM with CaMKII. cbp-CaM, in contrast to apo- and holo-CaM, maintains its ability to bind CaMKII under simulated conditions of oxidative stress and is also protected from oxidation-induced unfolding. Thus, we show that the susceptibility towards oxidation of specific residues in CaM is tightly linked to its signaling state and conformation, which has direct implications for calcium/CaM-CaMKII related signaling.
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Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calmodulina/metabolismo , Metionina/metabolismo , Animales , Bovinos , Cromatografía Liquida , Oxidación-Reducción , Unión Proteica , Espectrometría de Masas en TándemRESUMEN
A major problem in the analysis of mass spectrometry-based proteomics data is the vast growth of data volume, caused by improvements in sequencing speed of mass spectrometers. This growth affects analysis times and storage requirements so severely that many analysis tools are no longer able to cope with the increased file sizes. We present a tool, RockerBox, to address size problems for search results obtained from the widely used Mascot search engine. RockerBox allows for a fast evaluation of large result files by means of a number of commonly accepted metrics that can often be viewed through charts. Moreover, result files can be filtered without altering their informative content, based on a number of FDR calculation methods. File sizes can be reduced dramatically, often to a tenth of their original size, thus relaxing the need for storage and computation power, and boosting analysis of current and future proteomics experiments.
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Biología Computacional/métodos , Espectrometría de Masas/métodos , Proteómica/métodos , Programas Informáticos , Bases de Datos de Proteínas , Células HEK293 , Humanos , Espectrometría de Masas/instrumentación , Curva ROCRESUMEN
INTRODUCTION: Rheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullinated fibrinogen is commonly found in inflamed synovial tissue and is a frequent target of autoantibodies in RA patients. To obtain insight into the B-cell response to citrullinated fibrinogen in RA, its autoepitopes were systematically mapped using a new methodology. METHODS: Human fibrinogen was citrullinated in vitro by peptidylarginine deiminases (PAD), subjected to proteolysis and the resulting peptides were fractionated by ion exchange chromatography. The peptide composition of the citrullinated peptide-containing fractions was determined by high resolution tandem mass spectrometry. The recognition of these fractions by patient sera was subsequently analyzed by imaging surface plasmon resonance on microarrays. RESULTS: In total about two-thirds of the 81 arginines of human fibrinogen were found to be susceptible to citrullination by the human PAD2, the human PAD4 or the rabbit PAD2 enzymes. Citrullination sites were found in all three polypeptide chains of fibrinogen, although the α-chain appeared to contain most of them. The analysis of 98 anti-citrullinated protein antibody-positive RA sera using the new methodology allowed the identification of three major citrullinated epitope regions in human fibrinogen, two in the α- and one in the ß-chain. CONCLUSIONS: A comprehensive overview of citrullination sites in human fibrinogen was generated. The multiplex analysis of peptide fractions derived from a post-translationally modified protein, characterized by mass spectrometry, with patient sera provides a versatile system for mapping modified amino acid-containing epitopes. The citrullinated epitopes of human fibrinogen most efficiently recognized by RA autoantibodies are confined to three regions of its polypeptides.
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Artritis Reumatoide/inmunología , Autoantígenos/inmunología , Mapeo Epitopo/métodos , Epítopos de Linfocito B/inmunología , Fibrinógeno/inmunología , Resonancia por Plasmón de Superficie/métodos , Autoantígenos/metabolismo , Western Blotting , Cromatografía Liquida , Citrulina/inmunología , Fibrinógeno/metabolismo , Humanos , Espectrometría de Masas , Análisis por MicromatricesRESUMEN
Understanding of protease specificity is essential in proteomics strategies to correctly compare peptide fragmentation spectra with spectra predicted from data extracted from sequence databases. Here we evaluate the specificities of two proteolytic enzymes Lys-C and Lys-N, which cleave peptide-bonds at, respectively, the C- and N-terminal side of a lysine residue.
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Lisina/química , Péptido Hidrolasas/metabolismo , Péptidos/química , Péptido Hidrolasas/química , Proteómica , Especificidad por SustratoRESUMEN
Inhibitors of phosphodiesteraseâ 5 (PDE5) are widely used for the treatment of erectile dysfunction and pulmonary hypertension. The commercially available inhibitors are effective, well-tolerated drugs, but differ in their phosphodiesterase specificity. To explore and manipulate the specificity of PDE5 inhibitors, a small library of four inhibitors was synthesized using the structure of known PDE5 inhibitors as a scaffold. Their inhibitory potency towards PDE5 and related family members was evaluated. Next, they were immobilized on a matrix to perform affinity pull-down assays in rat testis tissue, followed by mass spectrometric (MS) analysis. By using unique peptide spectral counts of identified proteins in the MS analysis, we were able to assess the relative binding of these inhibitors to a large set of proteins, allowing the determination of their selectivity profiles in vitro. For selected proteins of interest, the results were verified using quantitative isotopic dimethyl labeling and immunoblotting, and isothermal titration calorimetry (ITC). For the PDE5 inhibitors, our data reveal that even slight chemical modifications can bias their selectivity significantly towards other interacting proteins, opening up the potential of these compounds to be used as scaffolds for the development of inhibitors for new protein targets. In a broad sense, we demonstrate that the combination of chemical proteomics and unique peptide spectral counting allows for the confident and facile analysis of the differential interactome of bioactive small molecules.
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Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Proteómica/métodos , Animales , Calorimetría , Cromatografía Liquida , Immunoblotting , Espectrometría de Masas , Inhibidores de Fosfodiesterasa/síntesis química , Inhibidores de Fosfodiesterasa/química , Inhibidores de Fosfodiesterasa/clasificación , Ratas , Relación Estructura-ActividadRESUMEN
The exosome is an exoribonuclease complex involved in the degradation and maturation of a wide variety of RNAs. The nine-subunit core of the eukaryotic exosome is catalytically inactive and may have an architectural function and mediate substrate binding. In Saccharomyces cerevisiae, the associated Dis3 and Rrp6 provide the exoribonucleolytic activity. The human exosome-associated Rrp6 counterpart contributes to its activity, whereas the human Dis3 protein is not detectably associated with the exosome. Here, a proteomic analysis of immunoaffinity-purified human exosome complexes identified a novel exosome-associated exoribonuclease, human Dis3-like exonuclease 1 (hDis3L1), which was confirmed to associate with the exosome core by co-immunoprecipitation. In contrast to the nuclear localization of Dis3, hDis3L1 exclusively localized to the cytoplasm. The hDis3L1 isolated from transfected cells degraded RNA in an exoribonucleolytic manner, and its RNB domain seemed to mediate this activity. The siRNA-mediated knockdown of hDis3L1 in HeLa cells resulted in elevated levels of poly(A)-tailed 28S rRNA degradation intermediates, indicating the involvement of hDis3L1 in cytoplasmic RNA decay. Taken together, these data indicate that hDis3L1 is a novel exosome-associated exoribonuclease in the cytoplasm of human cells.
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Exorribonucleasas/metabolismo , Exosomas/metabolismo , Subunidades de Proteína/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Línea Celular , Citoplasma/enzimología , Exorribonucleasas/genética , Complejo Multienzimático de Ribonucleasas del Exosoma , Humanos , Datos de Secuencia Molecular , Subunidades de Proteína/genética , ARN/genética , ARN/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The composition of 20S mouse proteasome complexes isolated from mice heart, kidney, liver, lung, thymus and spleen was compared using quantitative mass spectrometry. Significant variety was observed in hybrid classes of immunoproteasomes which may have implications for the use of proteasome targeted inhibitors.