RESUMO
DNA viruses are responsible for many diseases in humans. Current treatments are often limited by toxicity, as in the case of cidofovir (CDV, Vistide), a compound used against cytomegalovirus (CMV) and adenovirus (AdV) infections. CDV is a polar molecule with poor bioavailability, and its overall clinical utility is limited by the high occurrence of acute nephrotoxicity. To circumvent these disadvantages, we designed nine CDV prodrug analogues. The prodrugs modulate the polarity of CDV with a long sulfonyl alkyl chain attached to one of the phosphono oxygens. We added capping groups to the end of the alkyl chain to minimize ß-oxidation and focus the metabolism on the phosphoester hydrolysis, thereby tuning the rate of this reaction by altering the alkyl chain length. With these modifications, the prodrugs have excellent aqueous solubility, optimized metabolic stability, increased cellular permeability, and rapid intracellular conversion to the pharmacologically active diphosphate form (CDV-PP). The prodrugs exhibited significantly enhanced antiviral potency against a wide range of DNA viruses in infected human foreskin fibroblasts. Single-dose intravenous and oral pharmacokinetic experiments showed that the compounds maintained plasma and target tissue levels of CDV well above the EC50 for 24 h. These experiments identified a novel lead candidate, NPP-669. NPP-669 demonstrated efficacy against CMV infections in mice and AdV infections in hamsters following oral (p.o.) dosing at a dose of 1 mg/kg BID and 0.1 mg/kg QD, respectively. We further showed that NPP-669 at 30 mg/kg QD did not exhibit histological signs of toxicity in mice or hamsters. These data suggest that NPP-669 is a promising lead candidate for a broad-spectrum antiviral compound.
Assuntos
Infecções por Citomegalovirus , Organofosfonatos , Pró-Fármacos , Camundongos , Humanos , Animais , Antivirais/farmacocinética , Disponibilidade Biológica , Pró-Fármacos/farmacologia , Citosina , CidofovirRESUMO
During mitosis, the stacked structure of the Golgi undergoes a continuous fragmentation process. The generated mitotic fragments are evenly distributed into the daughter cells and reassembled into new Golgi stacks. This disassembly and reassembly process is critical for Golgi biogenesis during cell division, but the underlying molecular mechanism is poorly understood. In this study, we have recapitulated this process using an in vitro assay and analyzed the proteins associated with interphase and mitotic Golgi membranes using a proteomic approach. Incubation of purified rat liver Golgi membranes with mitotic HeLa cell cytosol led to fragmentation of the membranes; subsequent treatment of these membranes with interphase cytosol allowed the reassembly of the Golgi fragments into new Golgi stacks. These membranes were then used for quantitative proteomics analyses by combining the isobaric tags for relative and absolute quantification approach with OFFGEL isoelectric focusing separation and liquid chromatography-matrix assisted laser desorption ionization-tandem mass spectrometry. In three independent experiments, a total of 1,193 Golgi-associated proteins were identified and quantified. These included broad functional categories, such as Golgi structural proteins, Golgi resident enzymes, SNAREs, Rab GTPases, cargo, and cytoskeletal proteins. More importantly, the combination of the quantitative approach with Western blotting allowed us to unveil 84 proteins with significant changes in abundance under the mitotic condition compared with the interphase condition. Among these proteins, several COPI coatomer subunits (alpha, beta, gamma, and delta) are of particular interest. Altogether, this systematic quantitative proteomic study revealed candidate proteins of the molecular machinery that control the Golgi disassembly and reassembly processes in the cell cycle.
Assuntos
Ciclo Celular/fisiologia , Complexo de Golgi , Membranas Intracelulares , Proteoma/análise , Proteômica/métodos , Animais , Sistema Livre de Células , Análise por Conglomerados , Biologia Computacional , Citosol/metabolismo , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Fígado/citologia , Proteínas de Membrana/química , Proteínas de Membrana/classificação , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , RatosRESUMO
The zymogen granule is the specialized organelle in pancreatic acinar cells for digestive enzyme storage and regulated secretion and is a classic model for studying secretory granule function. Our long term goal is to develop a comprehensive architectural model for zymogen granule membrane (ZGM) proteins that would direct new hypotheses for subsequent functional studies. Our initial proteomics analysis focused on identification of proteins from purified ZGM (Chen, X., Walker, A. K., Strahler, J. R., Simon, E. S., Tomanicek-Volk, S. L., Nelson, B. B., Hurley, M. C., Ernst, S. A., Williams, J. A., and Andrews, P. C. (2006) Organellar proteomics: analysis of pancreatic zymogen granule membranes. Mol. Cell. Proteomics 5, 306-312). In the current study, a new global topology analysis of ZGM proteins is described that applies isotope enrichment methods to a protease protection protocol. Our results showed that tryptic peptides of ZGM proteins were separated into two distinct clusters according to their isobaric tag for relative and absolute quantification (iTRAQ) ratios for proteinase K-treated versus control zymogen granules. The low iTRAQ ratio cluster included cytoplasm-orientated membrane and membrane-associated proteins including myosin V, vesicle-associated membrane proteins, syntaxins, and all the Rab proteins. The second cluster having unchanged ratios included predominantly luminal proteins. Because quantification is at the peptide level, this technique is also capable of mapping both cytoplasm- and lumen-orientated domains from the same transmembrane protein. To more accurately assign the topology, we developed a statistical mixture model to provide probabilities for identified peptides to be cytoplasmic or luminal based on their iTRAQ ratios. By implementing this approach to global topology analysis of ZGM proteins, we report here an experimentally constrained, comprehensive topology model of identified zymogen granule membrane proteins. This model contributes to a firm foundation for developing a higher order architecture model of the ZGM and for future functional studies of individual ZGM proteins.
Assuntos
Proteínas de Membrana/análise , Proteínas de Membrana/química , Pâncreas/química , Vesículas Secretórias/química , Sequência de Aminoácidos , Western Blotting , Citoplasma/metabolismo , Endopeptidase K/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Proteômica , Reprodutibilidade dos Testes , Tripsina/metabolismoRESUMO
Peptides modified by pyridoxal-5'-phosphate (PLP), linked to a lysine residue via reductive amination, exhibit distinct spectral characteristics in the collision-induced dissociation (CID) tandem mass (MS/MS) spectra that are described here. The MS/MS spectra typically display two dominant peaks whose m/z values correspond to neutral losses of [H3PO4] (-98 Da) and the PLP moiety as [C8H10NO5P] (-231 Da) from the precursor peptide ion, respectively. Few other peaks are observed. Recognition of this distinct fragmentation behavior is imperative since determining sequences and sites of modifications relies on the formation of amide backbone cleavage products for subsequent interpretation via proteome database searching. Additionally, PLP-modified peptides exhibit suppressed precursor ionization efficiency which diminishes their detection in complex mixtures. Presented here is a protocol which describes an enrichment strategy for PLP-modified peptides combined with neutral loss screening and peptide mass fingerprinting to map the PLP-bonding site in a known PLP-dependent protein. This approach represents an efficient alternative to site-directed mutagenesis which has been the traditional method used for PLP-bonding site localization in proteins.
Assuntos
Mapeamento de Peptídeos/métodos , Peptídeos/química , Fosfato de Piridoxal/química , Espectrometria de Massas em Tandem/métodos , Alanina Racemase/química , Aminação , Brometo de Cianogênio/químicaRESUMO
Improvements to phosphopeptide enrichment protocols employing titanium dioxide (TiO2) are described and applied to identification of phosphorylation sites on recombinant human cyclin-dependent kinase 2 (CDK2). Titanium dioxide binds phosphopeptides under acidic conditions, and they can be eluted under basic conditions. However, some nonphosphorylated peptides, particularly acidic peptides, bind and elute under these conditions as well. These nonphosphorylated peptides contribute significantly to ion suppression of phosphopeptides and also increase sample complexity. We show here that the conversion of peptide carboxylates to their corresponding methyl esters sharply reduces nonspecific binding, improving the selectivity for phosphopeptides, just as has been reported for immobilized metal affinity chromatography (IMAC) columns. We also present evidence that monophosphorylated peptides can be effectively fractionated from multiply phosphorylated peptides, as well as acidic peptides, via stepwise elution from TiO2 using pH step gradients from pH 8.5 to pH 11.5. These approaches were applied to human CDK2 phosphorylated in vitro by yeast CAK1p in the absence of cyclin. We confirmed phosphorylation at T160, a site previously documented and shown to be necessary for CDK2 activity. However, we also discovered several novel sites of partial phosphorylation at S46, T47, T165, and Y168 when ion-suppressing nonphosphorylated peptides were eliminated using the new protocols.
Assuntos
Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Força Próton-Motriz , Titânio/química , Sequência de Aminoácidos , Sítios de Ligação , Ácidos Carboxílicos/química , Ácidos Carboxílicos/metabolismo , Fracionamento Químico , Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/metabolismo , Esterificação , Humanos , Fosforilação , Especificidade por Substrato , Espectrometria de Massas em TandemRESUMO
Hypotension is the dose-limiting side effect of the radio-protective drug Amifostine and results from relaxation of the vascular smooth muscle, which is directly mediated by the active metabolite, WR-1065, of Amifostine. The route of administration (currently FDA-approved only for intravenous administration) and the rapid metabolic conversion of Amifostine combine to yield high systemic levels of WR-1065 and facilitate the onset of hypotension. Research efforts aiming to optimize the delivery of WR-1065 to maintain efficacy while reducing its peak, systemic concentration below levels that induce hypotension are underway. To fully characterize the effect of reduced dose levels and alternative routes of administration of Amifostine on systemic WR-1065 concentrations, improved analytical techniques are needed. We have developed and evaluated a highly sensitive method for measuring WR-1065 in rat plasma that employs chemical derivatization, protein precipitation and UPLC-MS/MS analysis. The method exhibits a limit of quantification (LOQ) of 7.4â¯nM in plasma, which is a significant improvement over conventional approaches that utilize LC-electrochemical detection (ECD) (LOQ 150â¯nM or higher). The method was assessed in a pharmacokinetics study in rats administered Amifostine intravenously and via direct jejunal injection (10â¯mg/kg each route). The bioavailability of WR-1065 was 61.5% after direct jejunal injection indicating rapid conversion and absorption of the metabolite in the intestinal tract. This demonstrates that an oral formulation of Amifostine designed for site-specific release of the drug in the upper GI tract can deliver systemic absorption/conversion to WR-1065, provided that the formulation protects the therapeutic from gastric decomposition in the stomach.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Mercaptoetilaminas/sangue , Protetores contra Radiação/análise , Espectrometria de Massas em Tandem/métodos , Animais , Estabilidade de Medicamentos , Modelos Lineares , Masculino , Mercaptoetilaminas/química , Mercaptoetilaminas/farmacocinética , Protetores contra Radiação/química , Protetores contra Radiação/farmacocinética , Ratos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Regulation of the redox state of protein disulfide isomerase (PDI) is critical for its various catalytic functions. Here we describe a procedure utilizing isotope-coded affinity tag (ICAT) technology and mass spectrometry that quantitates relative changes in the dynamic thiol and disulfide states of human PDI. Human PDI contains six cysteine residues, four present in two active sites within the a and a' domains, and two present in the b' domain. ICAT labeling of human PDI indicates a difference between the redox state of the two active sites. Furthermore, under auto-oxidation conditions an approximately 80% decrease in available thiols within the a domain was detected. Surprisingly, the redox state of one of the two cysteines, Cys-295, within the b' domain was altered between the fully reduced and the auto-oxidized state of PDI while the other b' domain cysteine remained fully reduced. An interesting mono- and dioxidation modification of an invariable tryptophan residue, Trp-35, within the active site was also mapped by tandem mass spectrometry. Our findings indicate that ICAT methodology in conjunction with mass spectrometry represents a powerful tool to monitor changes in the redox state of individual cysteine residues within PDI under various conditions.
Assuntos
Marcação por Isótopo/métodos , Isomerases de Dissulfetos de Proteínas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Compostos de Sulfidrila/química , Sequência de Aminoácidos , Cisteína/química , Humanos , Dados de Sequência Molecular , Oxirredução , Mapeamento de Peptídeos , Proteínas Recombinantes/químicaRESUMO
The fragmentation characteristics of peptides derivatized at the side-chain epsilon-amino group of lysyl residues via reductive amination with benzaldehyde have been examined using collision-induced dissociation (CID) tandem mass spectrometry. The resulting MS/MS spectra exhibit peaks representing product ions formed from two independent fragmentation pathways. One pathway results in backbone fragmentation and commonly observed sequence ion peaks. The other pathway corresponds to the unsymmetrical, heterolytic cleavage of the C(zeta)-N(epsilon) bond that links the benzyl derivative to the side-chain lysyl residue. This results in the elimination of the derivative as a benzylic or tropylium carbocation and a (n - 1)(+)-charged peptide product (where n is the precursor ion charge state). The frequency of occurrence of the elimination pathway increases with increasing charge of the precursor ion. For the benzyl-modified tryptic peptides analyzed in this study, peaks representing products from both of these pathways are observed in the MS/MS spectra of doubly-charged precursor ions, but the carbocation elimination pathway occurs almost exclusively for triply-charged precursor ions. The experimental evidence presented herein, combined with molecular orbital calculations, suggests that the elimination pathway is a charge-directed reaction contingent upon protonation of the secondary epsilon-amino group of the benzyl-derivatized lysyl side chain. If the secondary epsilon-amine is protonated, the elimination of the carbocation is observed. If the precursor is not protonated at the secondary epsilon-amine, backbone fragmentation persists. The application of appropriately substituted benzyl analogs may allow for selective control over the relative abundance of product ions generated from the two pathways.
Assuntos
Compostos de Benzil/química , Gases/química , Lisina/química , Peptídeos/química , Tripsina/química , Aminas/química , Transição de Fase , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodosAssuntos
Proteoma/análise , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/análise , Saccharomyces cerevisiae/química , Aerobiose , Anaerobiose , Focalização Isoelétrica/métodos , Proteoma/isolamento & purificação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Coloração e Rotulagem/métodosAssuntos
Cromatografia de Fase Reversa/métodos , Proteoma/isolamento & purificação , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Saccharomyces cerevisiae/química , Aerobiose , Anaerobiose , Proteoma/análise , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/análise , Coloração e Rotulagem/métodosAssuntos
Proteoma/análise , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/análise , Saccharomyces cerevisiae/química , Coloração e Rotulagem/métodos , Aerobiose , Anaerobiose , Proteoma/isolamento & purificação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/isolamento & purificaçãoRESUMO
The zymogen granule (ZG) is the specialized organelle in pancreatic acinar cells for digestive enzyme storage and regulated secretion and has been a model for studying secretory granule functions. In an initial effort to comprehensively understand the functions of this organelle, we conducted a proteomic study to identify proteins from highly purified ZG membranes. By combining two-dimensional gel electrophoresis and two-dimensional LC with tandem mass spectrometry, 101 proteins were identified from purified ZG membranes including 28 known ZG proteins and 73 previously unknown proteins, including SNAP29, Rab27B, Rab11A, Rab6, Rap1, and myosin Vc. Moreover several hypothetical proteins were identified that represent potential novel proteins. The ZG localization of nine of these proteins was further confirmed by immunocytochemistry. To distinguish intrinsic membrane proteins from soluble and peripheral membrane proteins, a quantitative proteomic strategy was used to measure the enrichment of intrinsic membrane proteins through the purification process. The iTRAQ ratios correlated well with known or Transmembrane Hidden Markov Model-predicted soluble or membrane proteins. By combining subcellular fractionation with high resolution separation and comprehensive identification of proteins, we have begun to elucidate zymogen granule functions through proteomic and subsequent functional analysis of its membrane components.
Assuntos
Precursores Enzimáticos , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Proteínas de Membrana/análise , Pâncreas/citologia , Proteômica , Vesículas Secretórias/metabolismo , Animais , Proteínas de Membrana/isolamento & purificação , Transporte Proteico , Ratos , Vesículas Secretórias/químicaRESUMO
Methionine (Met) S-methyltransferase (MMT) catalyzes the synthesis of S-methyl-Met (SMM) from Met and S-adenosyl-Met (Ado-Met). SMM can be reconverted to Met by donating a methyl group to homocysteine (homo-Cys), and concurrent operation of this reaction and that mediated by MMT sets up the SMM cycle. SMM has been hypothesized to be essential as a methyl donor or as a transport form of sulfur, and the SMM cycle has been hypothesized to guard against depletion of the free Met pool by excess Ado-Met synthesis or to regulate Ado-Met level and hence the Ado-Met to S-adenosylhomo-Cys ratio (the methylation ratio). To test these hypotheses, we isolated insertional mmt mutants of Arabidopsis and maize (Zea mays). Both mutants lacked the capacity to produce SMM and thus had no SMM cycle. They nevertheless grew and reproduced normally, and the seeds of the Arabidopsis mutant had normal sulfur contents. These findings rule out an indispensable role for SMM as a methyl donor or in sulfur transport. The Arabidopsis mutant had significantly higher Ado-Met and lower S-adenosylhomo-Cys levels than the wild type and consequently had a higher methylation ratio (13.8 versus 9.5). Free Met and thiol pools were unaltered in this mutant, although there were moderate decreases (of 30%-60%) in free serine, threonine, proline, and other amino acids. These data indicate that the SMM cycle contributes to regulation of Ado-Met levels rather than preventing depletion of free Met.