RESUMO
Cytosolic carboxypeptidase 5 (CCP5) is a member of a subfamily of enzymes that cleave C-terminal and/or side chain amino acids from tubulin. CCP5 was proposed to selectively cleave the branch point of glutamylated tubulin, based on studies involving overexpression of CCP5 in cell lines and detection of tubulin forms with antisera. In the present study, we examined the activity of purified CCP5 toward synthetic peptides as well as soluble α- and ß-tubulin and paclitaxel-stabilized microtubules using a combination of antisera and mass spectrometry to detect the products. Mouse CCP5 removes multiple glutamate residues and the branch point glutamate from the side chains of porcine brain α- and ß-tubulin. In addition, CCP5 excised C-terminal glutamates from detyrosinated α-tubulin. The enzyme also removed multiple glutamate residues from side chains and C termini of paclitaxel-stabilized microtubules. CCP5 both shortens and removes side chain glutamates from synthetic peptides corresponding to the C-terminal region of ß3-tubulin, whereas cytosolic carboxypeptidase 1 shortens the side chain without cleaving the peptides' γ-linked residues. The rate of cleavage of α linkages by CCP5 is considerably slower than that of removal of a single γ-linked glutamate residue. Collectively, our data show that CCP5 functions as a dual-functional deglutamylase cleaving both α- and γ-linked glutamate from tubulin.
Assuntos
Carboxipeptidases/metabolismo , Ácido Glutâmico/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Carboxipeptidases/química , Carboxipeptidases/genética , Ácido Glutâmico/química , Ácido Glutâmico/genética , Camundongos , Microtúbulos/química , Microtúbulos/genética , Paclitaxel/farmacologia , Células Sf9 , Spodoptera , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Moduladores de Tubulina/farmacologiaRESUMO
The Purkinje cell degeneration (pcd) mouse has a disruption in the gene encoding cytosolic carboxypeptidase 1 (CCP1). This study tested two proposed functions of CCP1: degradation of intracellular peptides and processing of tubulin. Overexpression (2-3-fold) or knockdown (80-90%) of CCP1 in human embryonic kidney 293T cells (HEK293T) did not affect the levels of most intracellular peptides but altered the levels of α-tubulin lacking two C-terminal amino acids (delta2-tubulin) ≥ 5-fold, suggesting that tubulin processing is the primary function of CCP1, not peptide degradation. Purified CCP1 produced delta2-tubulin from purified porcine brain α-tubulin or polymerized HEK293T microtubules. In addition, CCP1 removed Glu residues from the polyglutamyl side chains of porcine brain α- and ß-tubulin and also generated a form of α-tubulin with two C-terminal Glu residues removed (delta3-tubulin). Consistent with this, pcd mouse brain showed hyperglutamylation of both α- and ß-tubulin. The hyperglutamylation of α- and ß-tubulin and subsequent death of Purkinje cells in pcd mice was counteracted by the knock-out of the gene encoding tubulin tyrosine ligase-like-1, indicating that this enzyme hyperglutamylates α- and ß-tubulin. Taken together, these results demonstrate a role for CCP1 in the processing of Glu residues from ß- as well as α-tubulin in vitro and in vivo.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Degeneração Neural/metabolismo , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Neoplasias da Mama , Linhagem Celular Tumoral , Neoplasias do Colo , Citosol/enzimologia , Feminino , Proteínas de Ligação ao GTP/genética , Ácido Glutâmico/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Degeneração Neural/genética , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Estrutura Terciária de Proteína , Células de Purkinje/enzimologia , Células de Purkinje/patologia , D-Ala-D-Ala Carboxipeptidase Tipo Serina/genética , Suínos , Tubulina (Proteína)/químicaRESUMO
A significant challenge in proteomics biomarker research is to identify the changes that are of highest diagnostic interest, among the many unspecific aberrations associated with disease burden and inflammation. In the present study liver tissue specimens (n=18) from six experimental stages were collected from the resistant hepatocyte (RH) rat model of liver cancer and analyzed by 2D DIGE. The study included triplicates of regenerating liver, control "sham-operated" liver, three distinct premalignant stages and hepatomas. Out of 81 identified proteins two-thirds were differentially abundant in rat hepatomas compared to control rat liver and, secondly, the majority of proteins were also changed in precursor stages. This underscores the importance of adequate control samples in explorative cancer biomarker research. We confirm several proteomic changes previously identified in human hepatocellular carcinoma (HCC) and we identify novel candidate proteomic aberrations for further analysis in human HCC. In particular, increased levels of HSP70, HSP90, AKR1B1, AKR7A3, GCLM, ANXA5, VDBP, RGN and SULT1E1 were associated specifically with rat hepatomas, or with liver cancer progression in rat. In addition, we examine an integrated gel-based workflow for analysis of protein post-translational modifications (PTMs) and microtubule-association. We highlight differential PTM and localization of HSP60 as an interesting target for further analysis in liver cancer.
Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Proteômica/métodos , Animais , Carcinoma Hepatocelular/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Ratos , Ratos Endogâmicos F344 , Eletroforese em Gel Diferencial BidimensionalRESUMO
An enzyme-stabilized nucleophilic water molecule has been implicated at the transition state of Escherichia coli methylthioadenosine nucleosidase (EcMTAN) by transition state analysis and crystallography. We analyzed the EcMTAN mass in complex with a femtomolar transition state analogue to determine whether the inhibitor and nucleophilic water could be detected in the gas phase. EcMTAN-inhibitor and EcMTAN-inhibitor-nucleophilic water complexes were identified by high-resolution mass spectrometry under nondenaturing conditions. The enzyme-inhibitor-water complex is sufficiently stable to exist in the gas phase.
Assuntos
Desoxiadenosinas/metabolismo , Escherichia coli/enzimologia , N-Glicosil Hidrolases/metabolismo , Tionucleosídeos/metabolismo , Desoxiadenosinas/química , Espectrometria de Massas , Modelos Moleculares , Especificidade por Substrato , Tionucleosídeos/químicaRESUMO
MS analysis of cross-linked peptides can be used to probe protein contact sites in macromolecular complexes. We have developed a photo-cleavable cross-linker that enhances peptide enrichment, improving the signal-to-noise ratio of the cross-linked peptides in mass spectrometry analysis. This cross-linker utilizes nitro-benzyl alcohol group that can be cleaved by UV irradiation and is stable during the multiple washing steps used for peptide enrichment. The enrichment method utilizes a cross-linker that aids in eliminating contamination resulting from protein-based retrieval systems, and thus, facilitates the identification of cross-linked peptides. Homodimeric pilM protein from Pseudomonas aeruginosa 2192 (pilM) was investigated to test the specificity and experimental conditions. As predicted, the known pair of lysine side chains within 14 Å was cross-linked. An unexpected cross-link involving the protein's amino terminus was also detected. This is consistent with the predicted mobility of the amino terminus that may bring the amino groups within 19 Å of one another in solution. These technical improvements allow this method to be used for investigating protein-protein interactions in complex biological samples.
Assuntos
Proteínas de Bactérias/química , Reagentes de Ligações Cruzadas/química , Peptídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sequência de Aminoácidos , Cristalografia por Raios X , Dimerização , Modelos Biológicos , Dados de Sequência Molecular , Estrutura Molecular , Ligação Proteica , Pseudomonas aeruginosa/químicaRESUMO
Differential detergent fractionation (DDF) is frequently used to partition fresh cells and tissues into distinct compartments. We have tested whether DDF can reproducibly extract and fractionate cellular protein components from frozen tissues. Frozen kidneys were sequentially extracted with three different buffer systems. Analysis of the three fractions with liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 1693 proteins, some of which were common to all fractions and others of which were unique to specific fractions. Normalized spectral index (SI(N)) values obtained from these data were compared to evaluate both the reproducibility of the method and the efficiency of enrichment. SI(N) values between replicate fractions demonstrated a high correlation, confirming the reproducibility of the method. Correlation coefficients across the three fractions were significantly lower than those for the replicates, supporting the capability of DDF to differentially fractionate proteins into separate compartments. Subcellular annotation of the proteins identified in each fraction demonstrated a significant enrichment of cytoplasmic, cell membrane, and nuclear proteins in the three respective buffer system fractions. We conclude that DDF can be applied to frozen tissue to generate reproducible proteome coverage discriminating subcellular compartments. This demonstrates the feasibility of analyzing cellular compartment-specific proteins in archived tissue samples with the simple DDF method.
Assuntos
Secções Congeladas , Proteoma/análise , Proteômica/métodos , Frações Subcelulares/química , Animais , Fracionamento Celular/métodos , Detergentes/química , Congelamento , Camundongos , Camundongos Endogâmicos C57BL , Espectrometria de Massas em TandemRESUMO
A broad range of mass spectrometers are used in mass spectrometry (MS)-based proteomics research. Each type of instrument possesses a unique design, data system and performance specifications, resulting in strengths and weaknesses for different types of experiments. Unfortunately, the native binary data formats produced by each type of mass spectrometer also differ and are usually proprietary. The diverse, nontransparent nature of the data structure complicates the integration of new instruments into preexisting infrastructure, impedes the analysis, exchange, comparison and publication of results from different experiments and laboratories, and prevents the bioinformatics community from accessing data sets required for software development. Here, we introduce the 'mzXML' format, an open, generic XML (extensible markup language) representation of MS data. We have also developed an accompanying suite of supporting programs. We expect that this format will facilitate data management, interpretation and dissemination in proteomics research.
Assuntos
Sistemas de Gerenciamento de Base de Dados , Bases de Dados Factuais , Disseminação de Informação/métodos , Armazenamento e Recuperação da Informação/métodos , Espectrometria de Massas/métodos , Proteômica/métodos , Interface Usuário-Computador , Armazenamento e Recuperação da Informação/normas , Espectrometria de Massas/normas , Proteoma/análise , Proteoma/química , Proteoma/classificação , Proteômica/normas , SoftwareRESUMO
Chronic overnutrition, for instance, high-fat diet (HFD) feeding, is a major cause of rapidly growing incidence of metabolic syndromes. However, the mechanisms underlying HFD-induced adverse effects on human health are not clearly understood. HFD-fed C57BL6/J mouse has been a popular model employed to investigate the mechanisms. Yet, there is no systematic and comprehensive study of the impact of HFD on the protein profiles of the animal. Here, we present a proteome-wide study of the consequences of long-term HFD feeding. Utilizing a powerful technology, stable isotope labeling of mammals, we detected and quantitatively compared 965 proteins extracted from livers of chow-diet-fed and HFD-fed mice. Among which, 122 proteins were significantly modulated by HFD. Fifty-four percent of those 122 proteins are involved in metabolic processes and the majority participate in lipid metabolism. HFD up-regulates proteins that play important roles in fatty acid uptake and subsequent oxidation and are linked to the transcription factors PPARα and PGC-1α. HFD suppresses lipid biosynthesis-related proteins that play major roles in de novo lipogenesis and are linked to SREBP-1 and PPARγ. These data suggest that HFD-fed mice tend to develop enhanced fat utilization and suppressed lipid biosynthesis, understandably a self-protective mechanism to counteract to excessive fat loading, which causes liver steatosis. Enhanced fatty acid oxidation increases reactive oxygen species and inhibits glucose oxidation, which are associated with hyperglycemia and insulin resistance. This proteomics study provides molecular understanding of HFD-induced pathology and identifies potential targets for development of therapeutics for metabolic syndromes.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Fígado/metabolismo , Proteoma/metabolismo , Animais , Fígado Gorduroso/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologiaRESUMO
Despite the clinical success of microtubule-interacting agents (MIA), a significant challenge for oncologists is the inability to predict the response of individual patients with cancer to these drugs. In the present study, six cell lines were compared by 2D DIGE proteomics to investigate cellular resistance to the class of MIAs known as microtubule-stabilizing agents (MSA). The human lung cancer cell line A549 was compared with two drug-resistant daughter cell lines, a taxol-resistant cell line (AT12) and an epothilone B (EpoB)-resistant cell line (EpoB40). The ovarian cancer cell line Hey was compared with two drug-resistant daughter cell lines, an EpoB-resistant cell line (EpoB8) and an ixabepilone-resistant cell line (Ixab80). All 2D DIGE results were validated by Western blot analyses. A variety of cytoskeletal and cytoskeleton-associated proteins were differentially expressed in drug-resistant cells. Differential abundance of 14-3-3σ, galectin-1 and phosphorylation of stathmin are worthy of further studies as candidate predictive biomarkers for MSAs. This is especially true for galectin-1, a ß-galactose-binding lectin that mediates tumor invasion and metastasis. Galectin-1 was greatly increased in EpoB- and ixabepilone-resistant cells and its suppression caused an increase in drug sensitivity in both drug-sensitive and -resistant Hey cells. Furthermore, the growth medium from resistant Hey cells contained higher levels of galectin-1, suggesting that galectin-1 could play a role in resistance to MSAs.
Assuntos
Proteínas do Citoesqueleto/biossíntese , Resistencia a Medicamentos Antineoplásicos/genética , Microtúbulos/efeitos dos fármacos , Proteínas de Neoplasias/biossíntese , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/genética , Eletroforese em Gel Bidimensional , Epotilonas/administração & dosagem , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Neoplasias/genética , Paclitaxel/administração & dosagem , ProteômicaRESUMO
The tumor microenvironment including glial cells and their inflammatory products regulates brain tumor development and progression. We have previously established that human glioma cells are exquisitely sensitive to IL-1 stimulation leading us to undertake a comparative analysis of the secretome of unstimulated and cytokine (IL-1)-stimulated glioblastoma cells. We performed label-free quantitative proteomic analysis and detected 190 proteins which included cytokines, chemokines, growth factors, proteases, cell adhesion molecules, extracellular matrix (ECM) and related proteins. Measuring area under the curve (AUC) of peptides for quantitation, the IL-1-induced secretome contained 13 upregulated and 5 downregulated extracellular proteins (p<0.05) compared to controls. Of these, IL-8, CCL2, TNC, Gal-1 and PTX3 were validated as upregulated and SERPINE1, STC2, CTGF and COL4A2 were validated as downregulated factors by immunochemical methods. A major representation of the ECM and related proteins in the glioblastoma secretome and their modulation by IL-1 suggested that IL-1 induces its effect in part by altering TGFß expression, activity and signaling. These findings enhance our understanding of IL-1-induced modulation of glioma microenvironment, with implications for increased tumor invasion, migration and angiogenesis. They further provide novel targets for the glioblastoma intervention. BIOLOGICAL SIGNIFICANCE: Present study is on an unbiased screening of the glioblastoma secretome stimulated by IL-1 which triggers neuroinflammatory cascades in the central nervous system. Network of secreted proteins were shown to be regulated revealing their possible contribution to glioma progression. Label free quantitative proteomics has provided unique novel targets for potential glioblastoma intervention.
Assuntos
Movimento Celular , Glioblastoma/metabolismo , Interleucina-1/farmacologia , Proteínas de Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Proteoma/metabolismo , Microambiente Tumoral , Linhagem Celular Tumoral , Glioblastoma/patologia , Humanos , Invasividade Neoplásica/patologia , Neovascularização Patológica/patologiaRESUMO
Chronic over-nutrition is a major contributor to the spread of obesity and its related metabolic disorders. Development of therapeutics has been slow compared to the speedy increase in occurrence of these metabolic disorders. We have identified a natural compound, mangiferin (MGF) (a predominant component of the plants of Anemarrhena asphodeloides and Mangifera indica), that can protect against high fat diet (HFD) induced obesity, hyperglycemia, insulin resistance and hyperlipidemia in mice. However, the molecular mechanisms whereby MGF exerts these beneficial effects are unknown. To understand MGF mechanisms of action, we performed unbiased quantitative proteomic analysis of protein profiles in liver of mice fed with HFD utilizing 15N metabolically labeled liver proteins as internal standards. We found that out of 865 quantified proteins 87 of them were significantly differentially regulated by MGF. Among those 87 proteins, 50% of them are involved in two major processes, energy metabolism and biosynthesis of metabolites. Further classification indicated that MGF increased proteins important for mitochondrial biogenesis and oxidative activity including oxoglutarate dehydrogenase E1 (Dhtkd1) and cytochrome c oxidase subunit 6B1 (Cox6b1). Conversely, MGF reduced proteins critical for lipogenesis such as fatty acid stearoyl-CoA desaturase 1 (Scd1) and acetyl-CoA carboxylase 1 (Acac1). These mass spectrometry data were confirmed and validated by western blot assays. Together, data indicate that MGF upregulates proteins pivotal for mitochondrial bioenergetics and downregulates proteins controlling de novo lipogenesis. This novel mode of dual pharmacodynamic actions enables MGF to enhance energy expenditure and inhibit lipogenesis, and thereby correct HFD induced liver steatosis and prevent adiposity. This provides a molecular basis supporting development of MGF or its metabolites into therapeutics to treat metabolic disorders.
Assuntos
Fármacos Antiobesidade/farmacologia , Dieta Hiperlipídica/efeitos adversos , Fígado/efeitos dos fármacos , Obesidade/tratamento farmacológico , Xantonas/farmacologia , Animais , Fármacos Antiobesidade/uso terapêutico , Células Cultivadas , Metabolismo Energético/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/sangue , Fígado/patologia , Redes e Vias Metabólicas , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Obesidade/sangue , Obesidade/etiologia , Tamanho do Órgão/efeitos dos fármacos , Proteoma/metabolismo , Xantonas/uso terapêuticoRESUMO
UNLABELLED: Epigenetic gene regulation has emerged as a major mechanism for gene regulation in all eukaryotes. Histones are small, basic proteins that constitute the major protein component of chromatin, and posttranslational modifications (PTM) of histones are essential for epigenetic gene regulation. The different combinations of histone PTM form the histone code for an organism, marking functional units of chromatin that recruit macromolecular complexes that govern chromatin structure and regulate gene expression. To characterize the repertoire of Toxoplasma gondii histone PTM, we enriched histones using standard acid extraction protocols and analyzed them with several complementary middle-down and bottom-up proteomic approaches with the high-resolution Orbitrap mass spectrometer using collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and/or electron transfer dissociation (ETD) fragmentation. We identified 249 peptides with unique combinations of PTM that comprise the T. gondii histone code. T. gondii histones share a high degree of sequence conservation with human histones, and many modifications are conserved between these species. In addition, T. gondii histones have unique modifications not previously identified in other species. Finally, T. gondii histones are modified by succinylation, propionylation, and formylation, recently described histone PTM that have not previously been identified in parasitic protozoa. The characterization of the T. gondii histone code will facilitate in-depth analysis of how epigenetic regulation affects gene expression in pathogenic apicomplexan parasites and identify a new model system for elucidating the biological functions of novel histone PTM. IMPORTANCE: Toxoplasma gondii is among the most common parasitic infections in humans. The transition between the different stages of the T. gondii life cycle are essential for parasite virulence and survival. These differentiation events are accompanied by significant changes in gene expression, and the control mechanisms for these transitions have not been elucidated. Important mechanisms that are involved in the control of gene expression are the epigenetic modifications that have been identified in several eukaryotes. T. gondii has a full complement of histone-modifying enzymes, histones, and variants. In this paper, we identify over a hundred PTM and a full repertoire of PTM combinations for T. gondii histones, providing the first large-scale characterization of the T. gondii histone code and an essential initial step for understanding how epigenetic modifications affect gene expression and other processes in this organism.
Assuntos
Epigênese Genética , Código das Histonas , Processamento de Proteína Pós-Traducional , Toxoplasma/química , Toxoplasma/fisiologia , Sequência de Aminoácidos , Técnicas de Química Analítica , Sequência Conservada , Proteoma/análise , Proteínas de Protozoários/análiseRESUMO
BACKGROUND: Female genital tract secretions are bactericidal for Escherichia (E.) coli ex vivo. However, the intersubject variability and molecules that contribute to this activity have not been defined. METHODS: The bactericidal activity and concentration of immune mediators in cervicovaginal lavage (CVL) collected from 99 healthy women were determined. RESULTS: CVL reduced the number of E. coli colonies by 68% [-26, 100] (median [range]). CVL were active against laboratory and clinical isolates of E. coli, but were inactive against Lactobacillus species. Bactericidal activity correlated with the concentration of protein recovered (p<0.001), but not with cytokines, chemokines or antimicrobial peptides. Four CVL with>90% inhibitory activity (active) and two with<30% activity were subjected to MS/MS proteomic analysis. 215 proteins were identified and six were found exclusively in active samples. Four of these corresponded to Lactobacillus crispatus or jensenii proteins. Moreover, culture supernatants from Lactobacillus jensenii were bactericidal for E. coli. CONCLUSION: Both host and commensal microbiota proteins contribute to mucosal defense. Identification of these proteins will facilitate the development of strategies to maintain a healthy vaginal microbiome and prevent colonization with pathogenic bacteria such as E. coli that increase the risk for urinary tract infections, preterm labor and perinatal infection.
Assuntos
Colo do Útero/microbiologia , Escherichia coli/metabolismo , Vagina/microbiologia , Adolescente , Adulto , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Colo do Útero/patologia , Endopeptidase K/metabolismo , Feminino , Genitália Feminina/microbiologia , Humanos , Lactobacillus/metabolismo , Pessoa de Meia-Idade , Proteômica/métodos , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodos , Irrigação Terapêutica/métodos , Infecções Urinárias/microbiologia , Vagina/patologia , Vaginose Bacteriana/microbiologiaRESUMO
While investigating whether proteins retrieved by cervicovaginal lavages (CVL) from women with cervical intraepithelial neoplasia (CIN) might correlate with risk of progression to invasive cervical cancer, we unexpectedly identified HIV gag and env glycoprotein in CVL from women with HIV-negative serology. HIV antigens were consistently identified by mass spectrometry (MS) in CVL from 4 women but were absent in CVL from the remaining 16 women. HIV serologies of all 20 patients were negative for both HIV-1 and HIV-2 antibodies. To validate the unexpected MS findings we performed Western blot (WB) and immunoaffinity chromatography (IC) analysis of CVL for HIV proteins, viral load assays of paired CVL and blood samples, and immunohistochemical HIV p24 expression in cervical biopsy specimens. WB analysis of CVL for prostate-specific antigen (PSA) was performed to exclude semen contamination as the source of HIV proteins. WB and IC results demonstrated the presence of HIV-1 gp41 and p24 antigens in four CVL that were identified by MS to have the HIV proteins. Despite negative serology, HIV RNA in CVL and HIV p24 in cervix biopsies were detected in patients with HIV antigen-positive CVL. HIV p24-positive CVL were PSA negative. All 20 subjects remained HIV seronegative throughout the study. Women with HIV proteins and RNA were comparatively older. Our findings suggest that CVL HIV proteins in women with CIN could be markers for unrecognized HIV exposure or subclinical infection. Proteomic screening of cervical secretions may be useful in identifying seronegative women exposed to HIV and/or at risk for AIDS.
Assuntos
Anticorpos Anti-HIV/sangue , Antígenos HIV/isolamento & purificação , Infecções por HIV/virologia , HIV/isolamento & purificação , RNA Viral/isolamento & purificação , Displasia do Colo do Útero/complicações , Feminino , Humanos , Vagina/virologia , Ducha VaginalRESUMO
Rat organic anion transporting protein 1a1 (oatp1a1), a hepatocyte basolateral plasma membrane protein, mediates transport of various amphipathic compounds. Our previous studies indicated that serine phosphorylation of a single tryptic peptide inhibits its transport activity without changing its cell surface content. The site of phosphorylation is unknown and was the subject of the present study. Following immunoaffinity chromatographic purification from rat liver, oatp1a1 was subjected to trypsin digestion and MALDI-TOF. Except for predicted N-glycosylated peptides, 97% of oatp1a1 tryptic peptides were observed. A single tryptic phosphopeptide was found in the C-terminus (aa 626-647), existing in unphosphorylated or singly or doubly phosphorylated forms and sensitive to alkaline phosphatase treatment. The beta-elimination reaction resulted in a mass loss of 98 or 196 Da from this peptide, and subsequent Michael addition with cysteamine increased masses by the predicated 77 and 154 Da, indicating that oatp1a1 can be singly or doubly phosphorylated at serine or threonine residues in the C-terminal sequence SSATDHT (aa 634-640). Subsequent tandem MS/MS analysis revealed that phosphorylation at S634 accounted for all singly phosphorylated peptide, while phosphorylation at S634 and S635 accounted for all doubly phosphorylated peptide. These findings identify the site of oatp1a1 phosphorylation and demonstrate that it is an ordered process, in which phosphorylation at S634 precedes that at S635. The mechanism by which phosphorylation results in loss of transport activity in hepatocytes remains to be established. Whether phosphorylation near the C-terminus inhibits C-terminal oligomerization of oatp1a1, required for normal transport function, can be speculated upon but is as yet unknown.
Assuntos
Transportadores de Ânions Orgânicos Sódio-Independentes/química , Fosfopeptídeos/química , Fosfatase Alcalina/metabolismo , Fosfatase Alcalina/farmacologia , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Fígado/metabolismo , Masculino , Metionina/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Transportadores de Ânions Orgânicos Sódio-Independentes/isolamento & purificação , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Oxirredução , Mapeamento de Peptídeos , Fosforilação , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Isoniazid (INH) is an essential drug used to treat tuberculosis. The mycobactericidal agents are INH adducts [INH-NAD(P)] of the pyridine nucleotide coenzymes, which are generated in vivo after INH activation and which bind to, and inhibit, essential enzymes. The NADH-dependent enoyl-ACP reductase (InhA) and the NADPH-dependent dihydrofolate reductase (DfrA) have both been shown to be inhibited by INH-NAD(P) adducts with nanomolar affinity. In this paper, we profiled the Mycobacterium tuberculosis proteome using both the INH-NAD and INH-NADP adducts coupled to solid supports and identified, in addition to InhA and DfrA, 16 other proteins that bind these adducts with high affinity. The majority of these are predicted to be pyridine nucleotide-dependent dehydrogenases/reductases. They are involved in many cellular processes, including S-adenosylmethionine-dependent methyl transfer reactions, pyrimidine and valine catabolism, the arginine degradative pathway, proton and potassium transport, stress response, lipid metabolism, and riboflavin biosynthesis. The targeting of multiple enzymes could, thus, account for the pleiotropic effects of, and powerful mycobactericidal properties of, INH.