RESUMEN
Emerging evidence suggests that RNA interference (RNAi)-related processes act both in the cytoplasm and in the nucleus. However, the process by which the RNAi machinery is transported into the nucleus remains poorly understood. The Tetrahymena Argonaute protein Twi1p localizes to the nucleus and is crucial for small RNA-directed programmed DNA elimination. In this study, we identify Giw1p, which binds to Twi1p and is required for its nuclear localization. Furthermore, the endoribonuclease (Slicer) activity of Twi1p plays a vital role in the removal of one of the two strands of Twi1p-associated small interfering RNAs (siRNAs), leading to a functionally mature Twi1p-siRNA complex. Slicer activity is also shown to be required for nuclear localization of Twi1p and for its association with Giw1p. These results suggest that Giw1p senses the state of Twi1p-associated siRNAs and selectively transports the mature Twi1p-siRNA complex into the nucleus.
Asunto(s)
Núcleo Celular/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Proteínas Protozoarias/metabolismo , ARN Interferente Pequeño/metabolismo , Tetrahymena thermophila/metabolismo , Secuencia de Aminoácidos , Conjugación Genética , Citoplasma/metabolismo , Proteínas Protozoarias/química , Tetrahymena thermophila/citología , Proteína 1 Relacionada con Twist/metabolismoRESUMEN
We present herein rPTMDetermine, an adaptive and fully automated methodology for validation of the identification of rarely occurring post-translational modifications (PTMs), using a semisupervised approach with a linear discriminant analysis (LDA) algorithm. With this strategy, verification is enhanced through similarity scoring of tandem mass spectrometry (MS/MS) comparisons between modified peptides and their unmodified analogues. We applied rPTMDetermine to (1) perform fully automated validation steps for modified peptides identified from an in silico database and (2) retrieve potential yet-to-be-identified modified peptides from raw data (that had been missed through conventional database searches). In part (1), 99 of 125 3-nitrotyrosyl-containing (nitrated) peptides obtained from a ProteinPilot search were validated and localized. Twenty nitrated peptides were falsely assigned because of incorrect monoisotopic peak assignments, leading to erroneous identification of deamidation and nitration. Five additional nitrated peptides were, however, validated after performing nonmonoisotopic peak correction. In part (2), an additional 236 unique nitrated peptides were retrieved and localized, containing 113 previously unreported nitration sites; 25 endogenous nitrated peptides with novel sites were selected and verified by comparison with synthetic analogues. In summary, we identified and confidently validated 296 unique nitrated peptides-collectively representing the largest number of endogenously identified 3-nitrotyrosyl-containing peptides from the cerebral cortex proteome of a Macaca fascicularis model of stroke. Furthermore, we harnessed the rPTMDetermine strategy to complement conventional database searching and enhance the confidence of assigning rarely occurring PTMs, while recovering many missed peptides. In a final demonstration, we successfully extended the application of rPTMDetermine to peptides featuring tryptophan oxidation.
Asunto(s)
Nitratos/metabolismo , Procesamiento Proteico-Postraduccional , Aprendizaje Automático Supervisado , Tirosina/metabolismo , Secuencia de Aminoácidos , Automatización , Análisis Discriminante , Péptidos/química , Péptidos/metabolismoRESUMEN
Equilibrative nucleoside transporters (ENTs) translocate nucleosides and nucleobases across plasma membranes, as well as a variety of anti-cancer, -viral, and -parasite nucleoside analogs. They are also key members of the purinome complex and regulate the protective and anti-inflammatory effects of adenosine. Despite their important role, little is known about the mechanisms involved in their regulation. We conducted membrane yeast 2-hybrid and coimmunoprecipitation studies and identified, for the first time to our knowledge, the existence of protein-protein interactions between human ENT1 and ENT2 (hENT1 and hENT2) proteins in human cells and the formation of hetero- and homo-oligomers at the plasma membrane and the submembrane region. The use of NanoLuc Binary Technology allowed us to analyze changes in the oligomeric status of hENT1 and hENT2 and how they rapidly modify the uptake profile for nucleosides and nucleobases and allow cells to respond promptly to external signals or changes in the extracellular environment. These changes in hENTs oligomerization are triggered by PKC activation and subsequent action of protein phosphatase 1.-Grañe-Boladeras, N., Williams, D., Tarmakova, Z., Stevanovic, K., Villani, L. A., Mehrabi, P., Siu, K. W. M., Pastor-Anglada, M., Coe, I. R. Oligomerization of equilibrative nucleoside transporters: a novel regulatory and functional mechanism involving PKC and PP1.
Asunto(s)
Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Transportador Equilibrativo 2 de Nucleósido/metabolismo , Multimerización de Proteína , Células HEK293 , Humanos , Unión Proteica , Proteína Quinasa C/metabolismo , Proteína Fosfatasa 1/metabolismoRESUMEN
We report herein the first detailed study of the mechanism of redox reactions occurring during the gas-phase dissociative electron transfer of prototypical ternary [CuII(dien)M]Ë2+ complexes (M, peptide). The two final products are (i) the oxidized non-zwitterionic π-centered [M]Ë+ species with both the charge and spin densities delocalized over the indole ring of the tryptophan residue and with a C-terminal COOH group intact, and (ii) the complementary ion [CuI(dien)]+. Infrared multiple photon dissociation (IRMPD) action spectroscopy and low-energy collision-induced dissociation (CID) experiments, in conjunction with density functional theory (DFT) calculations, revealed the structural details of the mass-isolated precursor and product cations. Our experimental and theoretical results indicate that the doubly positively charged precursor [CuII(dien)M]Ë2+ features electrostatic coordination through the anionic carboxylate end of the zwitterionic M moiety. An additional interaction exists between the indole ring of the tryptophan residue and one of the primary amino groups of the dien ligand; the DFT calculations provided the structures of the precursor ion, intermediates, and products, and enabled us to keep track of the locations of the charge and unpaired electron. The dissociative one-electron transfer reaction is initiated by a gradual transition of the M tripeptide from the zwitterionic form in [CuII(dien)M]Ë2+ to the non-zwitterionic M intermediate, through a cascade of conformational changes and proton transfers. In the next step, the highest energy intermediate is formed; here, the copper center is 5-coordinate with coordination from both the carboxylic acid group and the indole ring. A subsequent switch back to 4-coordination to an intermediate IM1, where attachment to GGW occurs through the indole ring only, creates the structure that ultimately undergoes dissociation.
Asunto(s)
Complejos de Coordinación/química , Cobre/química , Péptidos/química , Triptófano/química , Teoría Funcional de la Densidad , Transporte de Electrón , Estructura Molecular , Fotones , Espectrofotometría Infrarroja , Triptófano/análogos & derivadosRESUMEN
Collision-induced dissociation of isotopically labelled protonated pentaglycine produced two abundant [b5]+ ions, the products of the loss of water from the first and second amide groups, labelled [b5]+I and [b5]+II. IRMPD spectroscopy and DFT calculations show that these two [b5]+ ions feature N1-protonated 3,5-dihydro-4H-imidazol-4-one structures. 15N-Labelling established that some interconversion occurs between these two ions but dissociations are preferred. For both ions, DFT calculations show that the barrier to interconversion is slightly higher than those to dissociation. Dehydration of protonated hexaglycine produced three imidazolone ions. Ions [b6]+I and [b6]+II exhibit analogous CID spectra to those from [b5]+I and [b5]+II; however, the spectrum of the [b6]+III ion was dramatically different, showing losses predominantly of a further water molecule or cleavage of the second amide bond to give the glycyloxazolone (a deprotonated [b2]+ ion, labelled GlyGlyox (114 Da)) from the N-terminus. Protonated polyglycines [Glyn + H]+, where n = 7-9, all readily lose at least one water molecule. The corresponding [bn]+ ions lose either a further water molecule, an oxazolone from the N-terminus or a truncated peptide from the C-terminus. The number of amino acid residues in the latter two eliminated neutral molecules provides insight into the location of the imidazolone in the peptide chain and which oxygen was lost in the initial dehydration reaction. From this analysis, it appears that water loss from the longer protonated polyglycines is predominantly from the central residues.
RESUMEN
Stroke is one of the main causes of mortality and long-term disability worldwide. The pathophysiological mechanisms underlying this disease are not well understood, particularly in the chronic phase after the initial ischemic episode. In this study, a Macaca fascicularis stroke model consisting of two sample groups, as determined by MRI-quantified infarct volumes as a measure of the stroke severity 28 days after the ischemic episode, was evaluated using qualitative and quantitative proteomics analyses. By using multiple online multidimensional liquid chromatography platforms, 8790 nonredundant proteins were identified that condensed to 5223 protein groups at 1% global false discovery rate (FDR). After the application of a conservative criterion (5% local FDR), 4906 protein groups were identified from the analysis of cerebral cortex. Of the 2068 quantified proteins, differential proteomic analyses revealed that 31 and 23 were dysregulated in the elevated- and low-infarct-volume groups, respectively. Neurogenesis, synaptogenesis, and inflammation featured prominently as the cellular processes associated with these dysregulated proteins. Protein interaction network analysis revealed that the dysregulated proteins for inflammation and neurogenesis were highly connected, suggesting potential cross-talk between these processes in modulating the cytoskeletal structure and dynamics in the chronic phase poststroke. Elucidating the long-term consequences of brain tissue injuries from a cellular prospective, as well as the molecular mechanisms that are involved, would provide a basis for the development of new potentially neurorestorative therapies.
Asunto(s)
Corteza Cerebral/química , Regulación de la Expresión Génica , Proteómica/métodos , Accidente Cerebrovascular/metabolismo , Animales , Enfermedad Crónica , Modelos Animales de Enfermedad , Inflamación/genética , Macaca fascicularis , Imagen por Resonancia Magnética , Neurogénesis/genética , Mapas de Interacción de ProteínasRESUMEN
Four isomers of the radical cation of tripeptide phenylalanylglycyltryptophan, in which the initial location of the radical center is well defined, have been isolated and their collision-induced dissociation (CID) spectra examined. These ions, the π-centered [FGWπË]+, α-carbon- [FGαËW]+, N-centered [FGWNË]+ and ζ-carbon- [FζËGW]+ radical cations, were generated via collision-induced dissociation (CID) of transition metal-ligand-peptide complexes, side chain fragmentation of a π-centered radical cation, homolytic cleavage of a labile nitrogen-nitrogen single bond, and laser induced dissociation of an iodinated peptide, respectively. The π-centered and tryptophan N-centered peptide radical cations produced almost identical CID spectra, despite the different locations of their initial radical sites, which indicated that interconversion between the π-centered and tryptophan N-centered radical cations is facile. By contrast, the α-carbon-glycyl radical [FGαËW]+, and ζ-phenyl radical [FζËGW]+, featured different dissociation product ions, suggesting that the interconversions among α-carbon, π-centered (or tryptophan N-centered) and ζ-carbon-radical cations have higher barriers than those to dissociation. Density functional theory calculations have been used to perform systematic mechanistic investigations on the interconversions between these isomers and to study selected fragmentation pathways for these isomeric peptide radical cations. The results showed that the energy barrier for interconversion between [FGWπË]+ and [FGWNË]+ is only 31.1 kcal mol-1, much lower than the barriers to their dissociation (40.3 kcal mol-1). For the [FGWπË]+, [FGαËW]+, and [FζËGW]+, the barriers to interconversion are higher than those to dissociation, suggesting that interconversions among these isomers are not competitive with dissociations. The [z3 - H]Ë+ ions isolated from [FGαËW]+ and [FζËGW]+ show distinctly different fragmentation patterns, indicating that the structures of these ions are different and this result is supported by the DFT calculations.
RESUMEN
Advancements in genomics, proteomics, and bioinformatics have improved our understanding of gene/protein networks involved in intra- and intercellular communication and tumor-host interactions. Using proteomics integrated with bioinformatics, previously we reported overexpression of 14-3-3ζ in premalignant oral lesions and oral squamous cell carcinoma tissues in comparison with normal oral epithelium. 14-3-3ζ emerged as a novel molecular target for therapeutics and a potential prognostic marker in oral squamous cell carcinoma patients. However, the role of 14-3-3ζ in development and progression of oral cancer is not known yet. This study aimed to identify the 14-3-3ζ associated protein networks in oral cancer cell lines using IP-MS/MS and bioinformatics. A total of 287 binding partners of 14-3-3ζ were identified in metastatic (MDA1986) and nonmetastatic (SCC4) oral cancer cell lines including other 14-3-3 isoforms (2%), proteins involved in apoptosis (2%), cytoskeleton (9%), metabolism (16%), and maintenance of redox potential (2%). Our bioinformatics analysis revealed involvement of 14-3-3ζ in protein networks regulating cell cycle, proliferation, apoptosis, cellular trafficking, and endocytosis in oral cancer. In conclusion, our data revealed several novel protein interaction networks involving 14-3-3ζ in oral cancer progression and metastasis.
Asunto(s)
Proteínas 14-3-3/metabolismo , Mapas de Interacción de Proteínas/fisiología , Proteoma/análisis , Proteoma/metabolismo , Proteómica/métodos , Proteínas 14-3-3/análisis , Proteínas 14-3-3/química , Línea Celular Tumoral , Humanos , Unión Proteica , Proteoma/química , Transducción de SeñalRESUMEN
Intramolecular hydrogen atom transfer (HAT) was examined in homocysteine (Hcy) thiyl radical/alkali metal ion complexes in the gas phase by combination of experimental techniques (ion-molecule reactions and infrared multiple photon dissociation spectroscopy) and theoretical calculations. The experimental results unequivocally show that metal ion complexation (as opposed to protonation) of the regiospecifically generated Hcy thiyl radical promotes its rapid isomerisation into an α-carbon radical via HAT. Theoretical calculations were employed to calculate the most probable HAT pathway and found that in alkali metal ion complexes the activation barrier is significantly lower, in full agreement with the experimental data. This is, to our knowledge, the first example of a gas-phase thiyl radical thermal rearrangement into an α-carbon species within the same amino acid residue and is consistent with the solution phase behaviour of Hcy radical.
Asunto(s)
Aminoácidos/química , Homocisteína/química , Metales Alcalinos/química , Radicales Libres/química , Hidrógeno/química , Modelos Moleculares , Teoría CuánticaRESUMEN
Peptide radical cations that contain an aromatic amino acid residue cleave to give [zn - H]Ë⺠ions with [b2 - H - 17]Ë⺠and [c1 - 17](+) ions, the dominant products in the dissociation of [zn - H]Ëâº, also present in lower abundance in the CID spectra. Isotopic labeling in the aromatic ring of [YπËGG](+) establishes that in the formation of [b2 - H - 17]Ë⺠ions a hydrogen from the δ-position of the Y residue is lost, indicating that nucleophilic substitution on the aromatic ring has occurred. A preliminary DFT investigation of nine plausible structures for the [c1 - 17](+) ion derived from [Y(π)ËGG](+) shows that two structures resulting from attack on the aromatic ring by oxygen and nitrogen atoms from the peptide backbone have significantly better energies than other isomers. A detailed study of [Y(π)ËGG](+) using two density functionals, B3LYP and M06-2X, with a 6-31++G(d,p) basis set gives a higher barrier for attack on the aromatic ring of the [zn - H]Ë⺠ion by nitrogen than by the carbonyl oxygen. However, subsequent rearrangements involving proton transfers are much higher in energy for the oxygen-substituted isomer leading to the conclusion that the [c1 - 17](+) ions are the products of nucleophilic attack by nitrogen, protonated 2,7-dihydroxyquinoline ions. The [b2 - H - 17]Ë⺠ions are formed by loss of glycine from the same intermediates involved in the formation of the [c1 - 17](+) ions.
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Amidas/química , Nitrógeno/química , Iones , Espectrometría de Masas , Estructura MolecularRESUMEN
Macrocyclization is commonly observed in large bn(+) (n≥ 4) ions and as a consequence can lead to incorrect protein identification due to sequence scrambling. In this work, the analogous [b5- H]Ë(+) radical cations derived from aliphatic hexapeptides (GA5Ë(+)) also showed evidence of macrocyclization under CID conditions. However, the major fragmentation for [b5- H]Ë(+) ions is the loss of CO2 and not CO loss, which is commonly observed in closed-shell bn(+) ions. Isotopic labeling using CD3 and (18)O revealed that more than one common structure underwent dissociations. Theoretical studies found that the loss of CO2 is radical-driven and is facilitated by the radical being located at the Cα atom immediately adjacent to the oxazolone ring. Comparable energy barriers against macrocyclization, hydrogen-atom transfer, and fragmentations are found by DFT calculations and the results are consistent with the experimental observations that a variety of dissociation products are observed in the CID spectra.
RESUMEN
Starch branching enzyme IIb (SBEIIb) plays a crucial role in amylopectin biosynthesis in maize endosperm by defining the structural and functional properties of storage starch and is regulated by protein phosphorylation. Native and recombinant maize SBEIIb were used as substrates for amyloplast protein kinases to identify phosphorylation sites on the protein. A multidisciplinary approach involving bioinformatics, site-directed mutagenesis, and mass spectrometry identified three phosphorylation sites at Ser residues: Ser(649), Ser(286), and Ser(297). Two Ca(2+)-dependent protein kinase activities were partially purified from amyloplasts, termed K1, responsible for Ser(649) and Ser(286) phosphorylation, and K2, responsible for Ser(649) and Ser(297) phosphorylation. The Ser(286) and Ser(297) phosphorylation sites are conserved in all plant branching enzymes and are located at opposite openings of the 8-stranded parallel ß-barrel of the active site, which is involved with substrate binding and catalysis. Molecular dynamics simulation analysis indicates that phospho-Ser(297) forms a stable salt bridge with Arg(665), part of a conserved Cys-containing domain in plant branching enzymes. Ser(649) conservation appears confined to the enzyme in cereals and is not universal, and is presumably associated with functions specific to seed storage. The implications of SBEIIb phosphorylation are considered in terms of the role of the enzyme and the importance of starch biosynthesis for yield and biotechnological application.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/química , Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Amilopectina/biosíntesis , Endospermo/enzimología , Zea mays/enzimología , Enzima Ramificadora de 1,4-alfa-Glucano/antagonistas & inhibidores , Enzima Ramificadora de 1,4-alfa-Glucano/genética , Secuencia de Aminoácidos , Sitios de Unión , Calcio/metabolismo , Inhibidores Enzimáticos/farmacología , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Péptidos/farmacología , Fosforilación , Conformación Proteica , Proteínas Quinasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The collision-induced dissociation (CID) of [b5 - H]Ë(+) ions containing four alanine residues and one tryptophan give identical spectra regardless of the initial location of the tryptophan indicating that, as proposed for b5(+) ions, sequence scrambling occurs prior to dissociation. Cleavage occurs predominantly at the peptide bonds and at the N-Cα bond of the alanine residue that is attached to the N-terminus of the tryptophan residue. The product of the latter pathway, an ion at m/z 240, is the base peak in all the mass spectra. With the exception of one minor channel giving a b3(+) ion, the product ions retain both the tryptophan residue and the radical. Experiments with one trideuterated alanine established the sequences of loss of alanine residues. Formation of identical products implies a common intermediate, a [b5 - H]Ë(+) ion that has a 'linear' structure in which the tryptophan residue is present as an α-radical located in the oxazolone ring, structure Ie. Density functional theory calculations show this structure to be at the global minimum, 14.6 kcal mol(-1) below the macrocyclic structure, ion II. Loss of CO from the [b5 - H]Ë(+) ions is inhibited by the presence of the radical centre in the oxazolone ring and migration of the proton from the oxazolone ring onto the peptide backbone induces cleavage of an N-Cα or peptide bond. Three calculated structures for the ion at m/z 240 all have an oxazolone ring. Two of these structures may be formed from Ie, depending upon which proton migrates onto the peptide chain prior to the dissociation. The barrier to interconversion between these two structures requires a 1,3-hydrogen atom shift and is high (51.0 kcal mol(-1)), but both can convert into a third isomer that readily loses CO2 (barrier 38.7 kcal mol(-1)). The lowest barrier to the loss of CO, the usual fragmentation path observed for protonated oxazolones, is 47.0 kcal mol(-1).
Asunto(s)
Alanina/química , Protones , Triptófano/química , Radicales Libres/química , Oxazolona/química , Péptidos/química , Teoría Cuántica , TermodinámicaRESUMEN
Metastatic renal cell carcinoma (RCC) is one of the most treatment-resistant malignancies, and patients have a dismal prognosis, with a <10% five-year survival rate. The identification of markers that can predict the potential for metastases will have a great effect in improving patient outcomes. In this study, we used differential proteomics with isobaric tags for relative and absolute quantitation (iTRAQ) labeling and LC-MS/MS analysis to identify proteins that are differentially expressed in metastatic and primary RCC. We identified 1256 non-redundant proteins, and 456 of these were quantified. Further analysis identified 29 proteins that were differentially expressed (12 overexpressed and 17 underexpressed) in metastatic and primary RCC. Dysregulated protein expressions of profilin-1 (Pfn1), 14-3-3 zeta/delta (14-3-3ζ), and galectin-1 (Gal-1) were verified on two independent sets of tissues by means of Western blot and immunohistochemical analysis. Hierarchical clustering analysis showed that the protein expression profile specific for metastatic RCC can distinguish between aggressive and non-aggressive RCC. Pathway analysis showed that dysregulated proteins are involved in cellular processes related to tumor progression and metastasis. Furthermore, preliminary analysis using a small set of tumors showed that increased expression of Pfn1 is associated with poor outcome and is a potential prognostic marker in RCC. In addition, 14-3-3ζ and Gal-1 also showed higher expression in tumors with poor prognosis than in those with good prognosis. Dysregulated proteins in metastatic RCC represent potential prognostic markers for kidney cancer patients, and a greater understanding of their involved biological pathways can serve as the foundation of the development of novel targeted therapies for metastatic RCC.
Asunto(s)
Carcinoma de Células Renales/genética , Neoplasias Renales/genética , Proteínas de Neoplasias/análisis , Proteoma/análisis , Proteínas 14-3-3/metabolismo , Biomarcadores de Tumor/análisis , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/mortalidad , Carcinoma de Células Renales/secundario , Cromatografía Liquida , Progresión de la Enfermedad , Galectina 1/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Renales/metabolismo , Neoplasias Renales/mortalidad , Metástasis de la Neoplasia , Profilinas/metabolismo , Pronóstico , Proteómica , Espectrometría de Masas en TándemRESUMEN
Early detection of oral lesions (OLs) at high risk of cancer development is of utmost importance for intervention. There is an urgent unmet clinical need for biomarkers that allow identification of high-risk OLs. Recently, we identified and verified a panel of five candidate protein biomarkers namely S100A7, prothymosin alpha, 14-3-3ζ, 14-3-3σ and heterogeneous nuclear ribonucleoprotein K using proteomics to distinguish OLs with dysplasia and oral cancers from normal oral tissues. The objective of our study was to evaluate the potential of these candidate protein biomarkers for identification of oral dysplastic lesions at high risk of cancer development. Using immunohistochemistry, we analyzed expressions of these five candidate protein biomarkers in 110 patients with biopsy-proven oral dysplasia and known clinical outcome and determined their correlations with p16 expression and HPV 16/18 status. Kaplan-Meier survival analysis showed reduced oral cancer-free survival (OCFS) of 68.6 months (p = 0.007) in patients showing cytoplasmic S100A7 overexpression when compared to patients with weak or no S100A7 immunostaining in cytoplasm (mean OCFS = 122.8 months). Multivariate Cox regression analysis revealed cytoplasmic S100A7 overexpression as the most significant candidate marker associated with cancer development in dysplastic lesions (p = 0.041, hazard ratio = 2.36). In conclusion, our study suggested the potential of S100A7 overexpression in identifying OLs with dysplasia at high risk of cancer development.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/patología , Transformación Celular Neoplásica/patología , Leucoplasia Bucal/patología , Neoplasias de la Boca/patología , Lesiones Precancerosas/patología , Proteínas S100/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/metabolismo , Transformación Celular Neoplásica/metabolismo , Femenino , Estudios de Seguimiento , Humanos , Técnicas para Inmunoenzimas , Leucoplasia Bucal/metabolismo , Masculino , Persona de Mediana Edad , Neoplasias de la Boca/metabolismo , Estadificación de Neoplasias , Lesiones Precancerosas/metabolismo , Pronóstico , Estudios Retrospectivos , Factores de Riesgo , Proteína A7 de Unión a Calcio de la Familia S100RESUMEN
Metastatic renal cell carcinoma (mRCC) is a devastating disease with a 5-year survival rate of approximately 9 % and low response to chemotherapy and radiotherapy. Targeted therapies have slightly improved patient survival, but are only effective in a small subset of patients, who eventually develop resistance. A better understanding of pathways contributing to tumor progression and metastasis will allow for the development of novel targeted therapies and accurate prognostic markers. We performed extensive bioinformatics coupled with experimental validation on proteins dysregulated in mRCC. Gene ontology analysis showed that many proteins are involved in oxidation reduction, metabolic processes, and signal transduction. Pathway analysis showed metabolic pathways are altered in mRCC including glycolysis and pyruvate metabolism, the citric acid cycle, and the pentose phosphate pathway. RT-qPCR analysis showed that genes involved in the citric acid cycle were downregulated in metastatic RCC while genes of the pentose phosphate pathway were overexpressed. Protein-protein interaction analysis showed that most of the 198 proteins altered in mRCC clustered together and many were involved in glycolysis and pyruvate metabolism. We identified 29 reported regions of chromosomal aberrations in metastatic disease that correlate with the direction of protein dysregulation in mRCC. Furthermore, 36 proteins dysregulated in mRCC are predicted to be targets of metastasis-related miRNAs. A more comprehensive understanding of the pathways dysregulated in metastasis can be useful for the development of new therapies and novel prognostic markers. Also, multileveled analyses provide a unique "snapshot" of the molecular "environment" in RCC with prognostic and therapeutic implications.
Asunto(s)
Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Redes y Vías Metabólicas/fisiología , Proteómica/métodos , Biomarcadores de Tumor/análisis , Carcinoma de Células Renales/secundario , Humanos , Neoplasias Renales/patología , Metástasis de la Neoplasia , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Residue-specific phosphorylation is a protein post-translational modification that regulates cellular functions. Experimental determination of the exact sites of protein phosphorylation provides an understanding of the signaling and processes at work for a given cellular state. Any experimental artifact that involves migration of the phosphate group during measurement is a concern, as the outcome can lead to erroneous conclusions that may confound studies on cellular signal transduction. Herein, we examine computationally the mechanism by which a phosphate group migrates from one serine residue to another serine in monoprotonated pentapeptides [BA-pSer-Gly-Ser-BB + H]+ â [BA-Ser-Gly-pSer-BB + H]+ (where BA and BB are different combinations of the three basic amino acids, histidine, lysine, and arginine). In addition to moving the phosphate group, the overall mechanism involves transferring a proton from the N-terminal amino acid, BA, to the C-terminal amino acid, BB. This is not a synchronous process, and there is a key high-energy intermediate, structure C, that is zwitterionic with both the basic amino acids protonated and the phosphate group attached to both serine residues and carrying a negative charge. The barriers to moving the phosphate group are calculated to be in the range of 219-274 kJ mol-1 at the B3LYP/6-31G(d) level. These barriers are systematically slightly lower and in good agreement with single-point energy calculations at both M06-2X/6-311++G(d,p) and MP2/6-31++G(d,p) levels. The competitive reaction, loss of phosphoric acid from the protonated pentapeptides, has a barrier in the range of 176-202 kJ mol-1 at the B3LYP/6-31G(d) level. Extension of the theory to M06-2X/6-311++G(d,p)//B3LYP/6-31G(d) and MP2/6-31++G(d,p)// B3LYP/6-31G(d) gives higher values for the loss of phosphoric acid, falling in the range of 196-226 kJ mol-1; these are comparable to the barriers against phosphate migration at the same levels of theory. For larger peptides His-pSer-(Gly)n-Ser-His, where n has values from 2 to 5, the barriers against the loss of phosphoric acid are higher than those against the phosphate group migration. This difference is most pronounced and significant when n = 4 and 5 (the differences are approximately 80 kJ mol-1 under the single-point energy calculations at the M06-2X and MP2 levels). Energy differences using two more recent functionals, M08-HX and MN15, on His-pSer-(Gly)n-Ser-His, where n = 1 and 5, are in good agreement with the M06-2X and MP2 calculations. These results provide the mechanistic rationale for phosphate migration versus other competing reactions in the gas phase under tandem mass spectrometry conditions.
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Fosfatos , Fosfopéptidos , Protones , Serina/química , Ácidos Fosfóricos , ArgininaRESUMEN
Using proteomics in tandem with bioinformatics, the secretomes of nonaggressive and aggressive thyroid carcinoma (TC) cell lines were analyzed to detect potential biomarkers for tumor aggressiveness. A panel of nine proteins, activated leukocyte cell adhesion molecule (ALCAM/CD166), tyrosine-protein kinase receptor (AXL), amyloid beta A4 protein, amyloid-like protein 2, heterogeneous nuclear ribonucleoprotein K, phosphoglycerate kinase 1, pyruvate kinase isozyme M2, phosphatase 2A inhibitor (SET), and protein kinase C inhibitor protein 1 (14-3-3 zeta) was chosen to confirm their expression in TC patients' sera and tissues. Increased presurgical circulating levels of ALCAM were associated with aggressive tumors (p = 0.04) and presence of lymph node metastasis (p = 0.018). Increased serum AXL levels were associated with extrathyroidal extension (p = 0.027). Furthermore, differential expression of amyloid beta A4 protein, AXL, heterogeneous nuclear ribonucleoprotein K, phosphoglycerate kinase 1, pyruvate kinase muscle isozyme M2, and SET was observed in TC tissues compared to benign nodules. Decreased nuclear expression of AXL can detect malignancy with 90% specificity and 100% sensitivity (AUC = 0.995, p < 0.001). In conclusion, some of these proteins show potential for future development as serum and/or tissue-based biomarkers for TC and warrant further investigation in a large cohort of patients.
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Biomarcadores de Tumor/análisis , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/metabolismo , Neoplasias de la Tiroides/metabolismo , Adulto , Biomarcadores de Tumor/sangre , Western Blotting , Línea Celular Tumoral , Biología Computacional , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/sangre , Proteínas de Neoplasias/aislamiento & purificación , Proteómica , Espectrometría de Masas en Tándem , Glándula Tiroides/química , Neoplasias de la Tiroides/sangre , Neoplasias de la Tiroides/química , Neoplasias de la Tiroides/patologíaRESUMEN
The Stk1/Stp1 and GraSR signal-transduction pathways are two distinct pathways in Staphylococcus aureus that rely on a reversible phosphorylation process in transducing external stimuli intracellularly. Stk1/Stp1 is an eukaryote-like Ser/Thr kinase phosphatase pair involved in purine biosynthesis, cell-wall metabolism, and autolysis. GraSR is a two-component system involved in resistance to cationic antimicrobial peptides. Both systems are implicated in S. aureus virulence and resistance to cell-wall inhibitors. Our study shows that the response regulator protein GraR undergoes phosphorylation by Stk1 at three threonine residues in the DNA-binding domain. Phosphorylation by Stk1 depends on the structural integrity of GraR as well as the amino acid sequences flanking the phosphorylation sites. Its homologue in Bacillus subtilis , BceR, which harbors two of the three phosphorylation sites in GraR, does not undergo Stk1-dependent phosphorylation. GraR is involved in regulation of the dltABCD operon, the gene products of which add the d-Ala on wall teichoic acid (WTA). Investigation of WTA isolated from the S. aureus RN6390 ΔgraR strain by NMR spectroscopy showed a clear negative effect that graR deletion has on the d-Ala content of WTA. Moreover, complementation of ΔgraR mutant with graR lacking the Stk1 phosphorylation sites mirrors this effect. These findings provide evidence that GraR is a target of Stk1 in vivo and suggest that modification of WTA by d-Ala is modulated by Stk1. The crosstalk between these two otherwise independent signaling pathways may facilitate S. aureus interaction with its environment to modulate processes such as cell growth and division and virulence.
Asunto(s)
Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Staphylococcus aureus/metabolismo , Factores de Virulencia/metabolismo , Modelos Biológicos , Fosforilación , Transducción de Señal/fisiologíaRESUMEN
Gas-phase complexes of five metal ions with the dipeptide HisGly have been characterized by DFT computations and by infrared multiple photon dissociation spectroscopy (IRMPD) using the free electron laser FELIX. Fine agreement is found in all five cases between the predicted IR spectral features of the lowest energy structures and the observed IRMPD spectra in the diagnostic region 1500-1800 cm(-1), and the agreement is largely satisfactory at longer wavelengths from 1000 to 1500 cm(-1). Weak-binding metal ions (K(+), Ba(2+), and Ca(2+)) predominantly adopt the charge-solvated (CS) mode of chelation involving both carbonyl oxygens, an imidazole nitrogen of the histidine side chain, and possibly the amino nitrogen. Complexes with Mg(2+) and Ni(2+) are found to adopt iminol (Im) binding, involving the deprotonated amide nitrogen, with tetradentate chelation. This tetradentate coordination of Ni(II) is the preferred binding mode in the gas phase, against the expectation under condensed-phase conditions that such binding would be sterically unfavorable and overshadowed by other outcomes such as metal ion hydration and formation of dimeric complexes. The HisGly results are compared with corresponding results for the PheAla, PheGly, and PhePhe ligands, and parallel behavior is seen for the dipeptides with N-terminal Phe versus His residues. An exception is the different chelation pattern determined for PhePhe versus HisGly, reflecting the intercalation-type cation binding pocket of the PhePhe ligand. The complexes group into three well-defined spectroscopic patterns: nickel and magnesium, calcium and barium, and potassium. Factors leading to differentiation of these distinct spectroscopic categories are (1) differing propensities for choosing the iminol binding pattern, and (2) single versus double charge on the metal center. Nickel and magnesium ions show similar gas-phase binding behavior, contrasting with their quite different patterns of peptide interaction in condensed phases.