RESUMEN
BACKGROUND: Hematopoietic progenitor kinase 1 (HPK1 or MAP4K1) has been demonstrated as a negative intracellular immune checkpoint in mediating antitumor immunity in studies with HPK1 knockout and kinase dead mice. Pharmacological inhibition of HPK1 is desirable to investigate the role of HPK1 in human immune cells with therapeutic implications. However, a significant challenge remains to identify a small molecule inhibitor of HPK1 with sufficient potency, selectivity, and other drug-like properties suitable for proof-of-concept studies. In this report, we identified a novel, potent, and selective HPK1 small molecule kinase inhibitor, compound K (CompK). A series of studies were conducted to investigate the mechanism of action of CompK, aiming to understand its potential application in cancer immunotherapy. METHODS: Human primary T cells and dendritic cells (DCs) were investigated with CompK treatment under conditions relevant to tumor microenvironment (TME). Syngeneic tumor models were used to assess the in vivo pharmacology of CompK followed by human tumor interrogation ex vivo. RESULTS: CompK treatment demonstrated markedly enhanced human T-cell immune responses under immunosuppressive conditions relevant to the TME and an increased avidity of the T-cell receptor (TCR) to recognize viral and tumor-associated antigens (TAAs) in significant synergy with anti-PD1. Animal model studies, including 1956 sarcoma and MC38 syngeneic models, revealed improved immune responses and superb antitumor efficacy in combination of CompK with anti-PD-1. An elevated immune response induced by CompK was observed with fresh tumor samples from multiple patients with colorectal carcinoma, suggesting a mechanistic translation from mouse model to human disease. CONCLUSION: CompK treatment significantly improved human T-cell functions, with enhanced TCR avidity to recognize TAAs and tumor cytolytic activity by CD8+ T cells. Additional benefits include DC maturation and priming facilitation in tumor draining lymph node. CompK represents a novel pharmacological agent to address cancer treatment resistance.
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Antineoplásicos/administración & dosificación , Neoplasias Óseas/tratamiento farmacológico , Ginsenósidos/administración & dosificación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Sarcoma/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Neoplasias Óseas/inmunología , Neoplasias Óseas/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ginsenósidos/farmacología , Humanos , Ratones , Receptores de Antígenos de Linfocitos T/metabolismo , Sarcoma/inmunología , Sarcoma/metabolismo , Resultado del Tratamiento , Microambiente Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The dysregulation of Wnt signaling is a frequent occurrence in many different cancers. Oncogenic mutations of CTNNB1/ß-catenin, the key nuclear effector of canonical Wnt signaling, lead to the accumulation and stabilization of ß-catenin protein with diverse effects in cancer cells. Although the transcriptional response to Wnt/ß-catenin signaling activation has been widely studied, an integrated understanding of the effects of oncogenic ß-catenin on molecular networks is lacking. We used affinity-purification mass spectrometry (AP-MS), label-free liquid chromatography-tandem mass spectrometry, and RNA-Seq to compare protein-protein interactions, protein expression, and gene expression in colorectal cancer cells expressing mutant (oncogenic) or wild-type ß-catenin. We generate an integrated molecular network and use it to identify novel protein modules that are associated with mutant or wild-type ß-catenin. We identify a DNA methyltransferase I associated subnetwork that is enriched in cells with mutant ß-catenin and a subnetwork enriched in wild-type cells associated with the CDKN2A tumor suppressor, linking these processes to the transformation of colorectal cancer cells through oncogenic ß-catenin signaling. In summary, multiomics analysis of a defined colorectal cancer cell model provides a significantly more comprehensive identification of functional molecular networks associated with oncogenic ß-catenin signaling.
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Carcinogénesis , Neoplasias Colorrectales/patología , Perfilación de la Expresión Génica , Proteómica/métodos , beta Catenina/metabolismo , Carcinogénesis/química , Carcinogénesis/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Humanos , Vía de Señalización Wnt , beta Catenina/genéticaRESUMEN
The HIV trans-activator Tat recruits the host transcription elongation factor P-TEFb to stimulate proviral transcription. Phosphorylation of Thr-186 on the activation loop (T-loop) of cyclin-dependent kinase 9 (CDK9) is essential for its kinase activity and assembly of CDK9 and cyclin T1 (CycT1) to form functional P-TEFb. Phosphorylation of a second highly conserved T-loop site, Ser-175, alters the competitive binding of Tat and the host recruitment factor bromodomain containing 4 (BRD4) to P-TEFb. Here, we investigated the intracellular mechanisms that regulate these key phosphorylation events required for HIV transcription. Molecular dynamics simulations revealed that the CDK9/CycT1 interface is stabilized by intramolecular hydrogen bonding of pThr-186 by an arginine triad and Glu-96 of CycT1. Arginine triad substitutions that disrupted CDK9/CycT1 assembly accumulated Thr-186-dephosphorylated CDK9 associated with the cytoplasmic Hsp90/Cdc37 chaperone. The Hsp90/Cdc37/CDK9 complex was also present in resting T cells, which lack CycT1. Hsp90 inhibition in primary T cells blocked P-TEFb assembly, disrupted Thr-186 phosphorylation, and suppressed proviral reactivation. The selective CDK7 inhibitor THZ1 blocked CDK9 phosphorylation at Ser-175, and in vitro kinase assays confirmed that CDK7 activity is principally responsible for Ser-175 phosphorylation. Mutation of Ser-175 to Lys had no effect on CDK9 kinase activity or P-TEFb assembly but strongly suppressed both HIV expression and BRD4 binding. We conclude that the transfer of CDK9 from the Hsp90/Cdc37 complex induced by Thr-186 phosphorylation is a key step in P-TEFb biogenesis. Furthermore, we demonstrate that CDK7-mediated Ser-175 phosphorylation is a downstream nuclear event essential for facilitating CDK9 T-loop interactions with Tat.
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Quinasa 9 Dependiente de la Ciclina/metabolismo , VIH-1/fisiología , Factor B de Elongación Transcripcional Positiva/metabolismo , Activación Viral , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Quinasa 9 Dependiente de la Ciclina/química , Activación Enzimática , VIH-1/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Células Jurkat , Fosforilación , Unión Proteica , Serina/metabolismo , Treonina/metabolismoRESUMEN
Targeted cancer therapies, which act on specific cancer-associated molecular targets, are predominantly inhibitors of oncogenic kinases. While these drugs have achieved some clinical success, the inactivation of kinase signaling via stimulation of endogenous phosphatases has received minimal attention as an alternative targeted approach. Here, we have demonstrated that activation of the tumor suppressor protein phosphatase 2A (PP2A), a negative regulator of multiple oncogenic signaling proteins, is a promising therapeutic approach for the treatment of cancers. Our group previously developed a series of orally bioavailable small molecule activators of PP2A, termed SMAPs. We now report that SMAP treatment inhibited the growth of KRAS-mutant lung cancers in mouse xenografts and transgenic models. Mechanistically, we found that SMAPs act by binding to the PP2A Aα scaffold subunit to drive conformational changes in PP2A. These results show that PP2A can be activated in cancer cells to inhibit proliferation. Our strategy of reactivating endogenous PP2A may be applicable to the treatment of other diseases and represents an advancement toward the development of small molecule activators of tumor suppressor proteins.
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Antineoplásicos/farmacología , Activadores de Enzimas/farmacología , Proteína Fosfatasa 2/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Antineoplásicos/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Activación Enzimática , Activadores de Enzimas/química , Humanos , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Ratones Transgénicos , Unión Proteica , Proteína Fosfatasa 2/química , Transducción de Señal , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Structural characterization of proteins and their antigen complexes is essential to the development of new biologic-based medicines. Amino acid-specific covalent labeling (CL) is well suited to probe such structures, especially for cases that are difficult to examine by alternative means due to size, complexity, or instability. We present here a detailed account of carboxyl group labeling (with glycine ethyl ester (GEE) tagging) applied to a glycosylated monoclonal antibody therapeutic (mAb). The experiments were optimized to preserve the structural integrity of the mAb, and experimental conditions were varied and replicated to establish the reproducibility of the technique. Homology-based models were generated and used to compare the solvent accessibility of the labeled residues, which include aspartic acid (D), glutamic acid (E), and the C-terminus (i.e., the target probes), with the experimental data in order to understand the accuracy of the approach. Data from the mAb were compared to reactivity measures of several model peptides to explain observed variations in reactivity. Attenuation of reactivity in otherwise solvent accessible probes is documented as arising from the effects of positive charge or bond formation between adjacent amine and carboxyl groups, the latter accompanied by observed water loss. A comparison of results with previously published data by Deperalta et al using hydroxyl radical footprinting showed that 55% (32/58) of target residues were GEE labeled in this study whereas the previous study reported 21% of the targets were labeled. Although the number of target residues in GEE labeling is fewer, the two approaches provide complementary information. The results highlight advantages of this approach, such as the ease of use at the bench top, the linearity of the dose response plots at high levels of labeling, reproducibility of replicate experiments (<2% variation in modification extent), the similar reactivity of the three target probes, and significant correlation of reactivity and solvent accessible surface area.
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Anticuerpos Monoclonales de Origen Murino/química , Péptidos/química , Animales , Anticuerpos Monoclonales de Origen Murino/inmunología , Glicosilación , Ratones , Péptidos/inmunologíaRESUMEN
Efficient HIV transcription requires P-TEFb, an essential co-factor for Tat. In actively replicating cells, P-TEFb is incorporated into the 7SK snRNP complex together with the repressor protein HEXIM1. Using an affinity purification-tandem mass spectrometry approach to identify modification sites on HEXIM1 that regulate the sequestration of P-TEFb by 7SK snRNP, we found that HEXIM1 can be phosphorylated on adjacent residues in a region immediately upstream of the coiled-coil dimerization domain (Ser268, Thr270, Tyr271, and Tyr274). Phosphomimetic mutations of Tyr271 and Tyr274 disrupted the assembly of P-TEFb and HEXIM1 into the 7SK snRNP complex. Although Y271E/Y274E did not adversely affect the nuclear localization pattern of HEXIM1, it induced the redistribution of the CDK9 subunit of P-TEFb into the cytoplasm. By contrast, the Y271F/Y274F HEXIM1 mutant assembled normally with P-TEFb within the 7SK snRNP complex but severely reduced proviral gene expression in T cells in response to activation signals and caused a severe growth defect of Jurkat T cells. Thus, Y271F/Y274F, which cannot be phosphorylated on these residues, appears to block the exchange of active P-TEFb from the 7SK complex, thereby limiting the level of P-TEFb below the threshold required to support transcription elongation of the HIV provirus and cellular genes.
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Infecciones por VIH/metabolismo , VIH-1/fisiología , Factor B de Elongación Transcripcional Positiva/metabolismo , Provirus/metabolismo , ARN Nuclear Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Secuencia de Aminoácidos , Western Blotting , Células Cultivadas , Cromatografía Liquida , Regulación Viral de la Expresión Génica , Células HEK293 , Infecciones por VIH/genética , Infecciones por VIH/virología , Humanos , Inmunoprecipitación , Células Jurkat , Datos de Secuencia Molecular , Mutación/genética , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteómica/métodos , Proteínas de Unión al ARN/genética , Homología de Secuencia de Aminoácido , Espectrometría de Masas en Tándem , Factores de Transcripción , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
Herein, we demonstrate the efficacy of an unbiased proteomics screening approach for studying protein expression changes in the KC-Tie2 psoriasis mouse model, identifying multiple protein expression changes in the mouse and validating these changes in human psoriasis. KC-Tie2 mouse skin samples (n = 3) were compared with littermate controls (n = 3) using gel-based fractionation followed by label-free protein expression analysis. 5482 peptides mapping to 1281 proteins were identified and quantitated: 105 proteins exhibited fold-changes ≥2.0 including: stefin A1 (average fold change of 342.4 and an average p = 0.0082; cystatin A, human ortholog); slc25a5 (average fold change of 46.2 and an average p = 0.0318); serpinb3b (average fold change of 35.6 and an average p = 0.0345; serpinB1, human ortholog); and kallikrein related peptidase 6 (average fold change of 4.7 and an average p = 0.2474; KLK6). We independently confirmed mouse gene expression-based increases of selected genes including serpinb3b (17.4-fold, p < 0.0001), KLK6 (9-fold, p = 0.002), stefin A1 (7.3-fold; p < 0.001), and slc25A5 (1.5-fold; p = 0.05) using qRT-PCR on a second cohort of animals (n = 8). Parallel LC/MS/MS analyses on these same samples verified protein-level increases of 1.3-fold (slc25a5; p < 0.05), 29,000-fold (stefinA1; p < 0.01), 322-fold (KLK6; p < 0.0001) between KC-Tie2 and control mice. To underscore the utility and translatability of our combined approach, we analyzed gene and protein expression levels in psoriasis patient skin and primary keratinocytes versus healthy controls. Increases in gene expression for slc25a5 (1.8-fold), cystatin A (3-fold), KLK6 (5.8-fold), and serpinB1 (76-fold; all p < 0.05) were observed between healthy controls and involved lesional psoriasis skin and primary psoriasis keratinocytes. Moreover, slc25a5, cystatin A, KLK6, and serpinB1 protein were all increased in lesional psoriasis skin compared with normal skin. These results highlight the usefulness of preclinical disease models using readily-available mouse skin and demonstrate the utility of proteomic approaches for identifying novel peptides/proteins that are differentially regulated in psoriasis that could serve as sources of auto-antigens or provide novel therapeutic targets for the development of new anti-psoriatic treatments.
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Modelos Animales de Enfermedad , Proteínas/metabolismo , Psoriasis/metabolismo , Piel/metabolismo , Animales , Expresión Génica , Humanos , Queratinocitos/metabolismo , Ratones , Proteínas/genética , Proteómica , Psoriasis/genética , Reproducibilidad de los ResultadosRESUMEN
Amino acid-specific covalent labeling is well suited to probe protein structure and macromolecular interactions, especially for macromolecules and their complexes that are difficult to examine by alternative means, due to size, complexity, or instability. Here we present a detailed account of carbodiimide-based covalent labeling (with GEE tagging) applied to a glycosylated monoclonal antibody therapeutic, which represents an important class of biologic drugs. Characterization of such proteins and their antigen complexes is essential to development of new biologic-based medicines. In this study, the experiments were optimized to preserve the structural integrity of the protein, and experimental conditions were varied and replicated to establish the reproducibility and precision of the technique. Homology-based models were generated and used to compare the solvent accessibility of the labeled residues, which include D, E, and the C-terminus, against the experimental surface accessibility data in order to understand the accuracy of the approach in providing an unbiased assessment of structure. Data from the protein were also compared to reactivity measures of several model peptides to explain sequence or structure-based variations in reactivity. The results highlight several advantages of this approach. These include: the ease of use at the bench top, the linearity of the dose response plots at high levels of labeling (indicating that the label does not significantly perturb the structure of the protein), the high reproducibility of replicate experiments (<2 % variation in modification extent), the similar reactivity of the 3 target probe residues (as suggested by analysis of model peptides), and the overall positive and significant correlation of reactivity and solvent accessible surface area (the latter values predicted by the homology modeling). Attenuation of reactivity, in otherwise solvent accessible probes, is documented as arising from the effects of positive charge or bond formation between adjacent amine and carboxyl groups, the latter accompanied by observed water loss. The results are also compared with data from hydroxyl radical-mediated oxidative footprinting on the same protein, showing that complementary information is gained from the 2 approaches, although the number of target residues in carbodiimide/GEE labeling is fewer. Overall, this approach is an accurate and precise method for assessing protein structure of biologic drugs.
RESUMEN
To determine a molecular basis for prognostic differences in glioblastoma multiforme (GBM), we employed a combinatorial network analysis framework to exhaustively search for molecular patterns in protein-protein interaction (PPI) networks. We identified a dysregulated molecular signature distinguishing short-term (survival<225 days) from long-term (survival>635 days) survivors of GBM using whole genome expression data from The Cancer Genome Atlas (TCGA). A 50-gene subnetwork signature achieved 80% prediction accuracy when tested against an independent gene expression dataset. Functional annotations for the subnetwork signature included "protein kinase cascade," "IκB kinase/NFκB cascade," and "regulation of programmed cell death" - all of which were not significant in signatures of existing subtypes. Finally, we used label-free proteomics to examine how our subnetwork signature predicted protein level expression differences in an independent GBM cohort of 16 patients. We found that the genes discovered using network biology had a higher probability of dysregulated protein expression than either genes exhibiting individual differential expression or genes derived from known GBM subtypes. In particular, the long-term survivor subtype was characterized by increased protein expression of DNM1 and MAPK1 and decreased expression of HSPA9, PSMD3, and CANX. Overall, we demonstrate that the combinatorial analysis of gene expression data constrained by PPIs outlines an approach for the discovery of robust and translatable molecular signatures in GBM.
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Neoplasias Encefálicas/genética , Redes Reguladoras de Genes , Glioblastoma/genética , Sobrevivientes , Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/patología , Perfilación de la Expresión Génica , Glioblastoma/enzimología , Glioblastoma/patología , Humanos , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , ProteómicaRESUMEN
The HIV transactivator protein, Tat, enhances HIV transcription by recruiting P-TEFb from the inactive 7SK snRNP complex and directing it to proviral elongation complexes. To test the hypothesis that T-cell receptor (TCR) signaling induces critical post-translational modifications leading to enhanced interactions between P-TEFb and Tat, we employed affinity purification-tandem mass spectrometry to analyze P-TEFb. TCR or phorbal ester (PMA) signaling strongly induced phosphorylation of the CDK9 kinase at Ser175. Molecular modeling studies based on the Tat/P-TEFb X-ray structure suggested that pSer175 strengthens the intermolecular interactions between CDK9 and Tat. Mutations in Ser175 confirm that this residue could mediate critical interactions with Tat and with the bromodomain protein BRD4. The S175A mutation reduced CDK9 interactions with Tat by an average of 1.7-fold, but also completely blocked CDK9 association with BRD4. The phosphomimetic S175D mutation modestly enhanced Tat association with CDK9 while causing a 2-fold disruption in BRD4 association with CDK9. Since BRD4 is unable to compete for binding to CDK9 carrying S175A, expression of CDK9 carrying the S175A mutation in latently infected cells resulted in a robust Tat-dependent reactivation of the provirus. Similarly, the stable knockdown of BRD4 led to a strong enhancement of proviral expression. Immunoprecipitation experiments show that CDK9 phosphorylated at Ser175 is excluded from the 7SK RNP complex. Immunofluorescence and flow cytometry studies carried out using a phospho-Ser175-specific antibody demonstrated that Ser175 phosphorylation occurs during TCR activation of primary resting memory CD4+ T cells together with upregulation of the Cyclin T1 regulatory subunit of P-TEFb, and Thr186 phosphorylation of CDK9. We conclude that the phosphorylation of CDK9 at Ser175 plays a critical role in altering the competitive binding of Tat and BRD4 to P-TEFb and provides an informative molecular marker for the identification of the transcriptionally active form of P-TEFb.
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Linfocitos T CD4-Positivos/metabolismo , Quinasa 9 Dependiente de la Ciclina/metabolismo , Infecciones por VIH/metabolismo , VIH/metabolismo , Factor B de Elongación Transcripcional Positiva/metabolismo , Provirus/metabolismo , Transcripción Genética , Biomarcadores/metabolismo , Linfocitos T CD4-Positivos/virología , Proteínas de Ciclo Celular , Ciclina T/genética , Ciclina T/metabolismo , Quinasa 9 Dependiente de la Ciclina/genética , Femenino , VIH/genética , Infecciones por VIH/genética , Humanos , Masculino , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación/genética , Factor B de Elongación Transcripcional Positiva/genética , Unión Proteica/genética , Provirus/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Pluripotent stem cells are capable of differentiating into all cell types of the body and therefore hold tremendous promise for regenerative medicine. Despite their widespread use in laboratories across the world, a detailed understanding of the molecular mechanisms that regulate the pluripotent state is currently lacking. Mouse embryonic (mESC) and epiblast (mEpiSC) stem cells are two closely related classes of pluripotent stem cells, derived from distinct embryonic tissues. Although both mESC and mEpiSC are pluripotent, these cell types show important differences in their properties suggesting distinct pluripotent ground states. To understand the molecular basis of pluripotency, we analyzed the nuclear proteomes of mESCs and mEpiSCs to identify protein networks that regulate their respective pluripotent states. Our study used label-free LC-MS/MS to identify and quantify 1597 proteins in embryonic and epiblast stem cell nuclei. Immunoblotting of a selected protein subset was used to confirm that key components of chromatin regulatory networks are differentially expressed in mESCs and mEpiSCs. Specifically, we identify differential expression of DNA methylation, ATP-dependent chromatin remodeling and nucleosome remodeling networks in mESC and mEpiSC nuclei. This study is the first comparative study of protein networks in cells representing the two distinct, pluripotent states, and points to the importance of DNA and chromatin modification processes in regulating pluripotency. In addition, by integrating our data with existing pluripotency networks, we provide detailed maps of protein networks that regulate pluripotency that will further both the fundamental understanding of pluripotency as well as efforts to reliably control the differentiation of these cells into functional cell fates.
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Cromatina/metabolismo , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica , Estratos Germinativos/metabolismo , Células Madre Pluripotentes/metabolismo , Mapas de Interacción de Proteínas/genética , Animales , Diferenciación Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Cromatina/genética , Ensamble y Desensamble de Cromatina , Cromatografía Liquida , ADN/genética , ADN/metabolismo , Metilación de ADN , Embrión de Mamíferos , Células Madre Embrionarias/citología , Estratos Germinativos/citología , Ratones , Células Madre Pluripotentes/citología , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Histidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C(2)-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK(a) values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR), an enzyme proposed to undergo multiple conformational changes during catalysis. METHODOLOGY/PRINCIPAL FINDINGS: Using His-HDX-MS, the pK(a) values and the half-lives (t(1/2)) of HDX reactions of five histidine residues of apo-DHFR, DHFR in complex with methotrexate (DHFR-MTX), DHFR in complex with MTX and NADPH (DHFR-MTX-NADPH), and DHFR in complex with folate and NADP+ (DHFR-folate-NADP+) were determined. The results showed that the two parameters (pK(a) and t(1/2)) are sensitive to the changes of the microenvironment around the histidine residues. Although four of the five histidine residues are located far from the active site, ligand binding affected their pK(a), t(1/2) or both. This is consistent with previous observations of ligand binding-induced distal conformational changes on DHFR. Most of the observed pK(a) and t(1/2) changes could be rationalized using the X-ray structures of apo-DHFR, DHFR-MTX-NADPH, and DHFR-folate-NADP+. The availability of the neutron diffraction structure of DHFR-MTX enabled us to compare the protonation states of histidine imidazole rings. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins.
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Medición de Intercambio de Deuterio/métodos , Histidina/química , Espectrometría de Masas/métodos , Tetrahidrofolato Deshidrogenasa/química , Dominio Catalítico , Cristalografía/métodos , Deuterio/química , Deuterio/metabolismo , Ambiente , Escherichia coli/química , Escherichia coli/enzimología , Histidina/metabolismo , Hidrógeno/química , Hidrógeno/metabolismo , Cinética , Modelos Biológicos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
Myosin II motor proteins play important roles in cell migration. Although myosin II filament assembly plays a key role in the stabilization of focal contacts at the leading edge of migrating cells, the mechanisms and signaling pathways regulating the localized assembly of lamellipodial myosin II filaments are poorly understood. We performed a proteomic analysis of myosin heavy chain (MHC) phosphorylation sites in MDA-MB 231 breast cancer cells to identify MHC phosphorylation sites that are activated during integrin engagement and lamellar extension on fibronectin. Fibronectin-activated MHC phosphorylation was identified on novel and previously recognized consensus sites for phosphorylation by protein kinase C and casein kinase II (CK-II). S1943, a CK-II consensus site, was highly phosphorylated in response to matrix engagement, and phosphoantibody staining revealed phosphorylation on myosin II assembled into leading-edge lamellae. Surprisingly, neither pharmacological reduction nor small inhibitory RNA reduction in CK-II activity reduced this stimulated S1943 phosphorylation. Our data demonstrate that S1943 phosphorylation is upregulated during lamellar protrusion, and that CK-II does not appear to be the kinase responsible for this matrix-induced phosphorylation event.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Movimiento Celular/fisiología , Miosina Tipo II/metabolismo , Proteoma/análisis , Animales , Células COS , Quinasa de la Caseína II/química , Quinasa de la Caseína II/genética , Quinasa de la Caseína II/metabolismo , Línea Celular Tumoral , Chlorocebus aethiops , Femenino , Fibronectinas/metabolismo , Células HeLa , Humanos , Modelos Moleculares , Cadenas Pesadas de Miosina/química , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo II/química , Miosina Tipo II/genética , Fosforilación , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Seudópodos/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Proteins and protein networks associated with cochlear pathogenesis in the Ames waltzer (av) mouse, a model for deafness in Usher syndrome 1F (USH1F), were identified. Cochlear protein from wild-type and av mice at postnatal day 30, a time point in which cochlear pathology is well established, was analyzed by quantitative 2D gel electrophoresis followed by mass spectrometry (MS). The analytic gel resolved 2270 spots; 69 spots showed significant changes in intensity in the av cochlea compared with the control. The cochlin protein was identified in 20 peptide spots, most of which were up-regulated, while a few were down-regulated. Analysis of MS sequence data showed that, in the av cochlea, a set of full-length isoforms of cochlin was up-regulated, while isoforms missing the N-terminal FCH/LCCL domain were down-regulated. Protein interaction network analysis of all differentially expressed proteins was performed with Metacore software. That analysis revealed a number of statistically significant candidate protein networks predicted to be altered in the affected cochlea. Quantitative PCR (qPCR) analysis of select candidates from the proteomic and bioinformatic investigations showed up-regulation of Coch mRNA and those of p53, Brn3a and Nrf2, transcription factors linked to stress response and survival. Increased mRNA of Brn3a and Nrf2 has previously been associated with increased expression of cochlin in human glaucomatous trabecular meshwork. Our report strongly suggests that increased level of cochlin is an important etiologic factor leading to the degeneration of cochlear neuroepithelia in the USH1F model.
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Biología Computacional , Proteínas/genética , Proteómica , Regulación hacia Arriba , Síndromes de Usher/genética , Animales , Modelos Animales de Enfermedad , Electroforesis en Gel Bidimensional , Proteínas de la Matriz Extracelular , Femenino , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Marcadores Genéticos , Humanos , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas/química , Proteínas/metabolismo , Síndromes de Usher/metabolismoRESUMEN
Protein nitration occurs as a result of oxidative stress induced by reactive oxygen (ROS) and reactive nitrogen species (RNS). Therefore, protein nitration serves as a hallmark for protein oxidation in vivo. We have previously reported on age dependent protein nitration in cardiac tissue of Fisher 344 BN-F1 rats analyzed by two-dimensional gel electrophoresis; however, only one specific nitration site was identified [Kanski, J., Behring, A., Pelling, J., Schöneich, C., 2005a. Proteomic identification of 3-nitrotyrosine-containing rat cardiac proteins: effects of biological aging. Am. J. Physiol. Heart Circ. Physiol. 288, H371-381]. In the present report, we used solution phase isoelectric focusing (IEF) followed by nanoHPLC-ESI-MS/MS that allowed us to obtain good MS/MS data to identify specific sites of protein nitration in cardiac tissue. As expected, more nitrated proteins were detected in cardiac tissue of old rats, including myosin heavy chain, neurofibromin, tropomyosin and nebulin-related anchoring protein. The post-translational modification of these cytoskeletal proteins may provide some rationale for the age-dependent functional decline of the heart.
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Envejecimiento/fisiología , Corazón/fisiología , Proteínas/fisiología , Alquilación , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión/métodos , Electroforesis en Gel de Poliacrilamida , Corazón/crecimiento & desarrollo , Focalización Isoeléctrica , Espectrometría de Masas , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Nitratos/metabolismo , Estrés Oxidativo/fisiología , Proteínas/química , Proteínas/metabolismo , Proteoma , RatasRESUMEN
3-Nitrotyrosine (3-NT) is a useful biomarker of increasing oxidative stress and protein nitration during biological aging. The proteomic analysis of cerebellar homogenate from Fisher 344/Brown Norway (BN/F1) rats shows an age-dependent increase in protein nitration, monitored by western-blot analysis after two-dimensional gel electrophoresis (2DE), mainly in the acidic region. Analysis of in-gel digests by nanoelectrospray (NSI)-MS/MS resulted in the identification of 16 putatively nitrated proteins. The selective isolation of nitrated proteins using immunoprecipitation, followed by SDS-PAGE and in-gel digest/NSI-MS/MS analysis led to the identification of 22 putatively nitrated proteins, of which 7 were identical to those detected after 2DE. When proteins were separated by solution isoelectrofocusing and analyzed by NSI MS/MS, we obtained MS/MS spectra of 3-NT containing peptides of four proteins - similar to ryanodine receptor 3, low density lipoprotein related receptor 2, similar to nebulin-related anchoring protein isoform C and 2,3 cyclic nucleotide 3-phosphodiesterase. Although the functional consequences of protein nitration for these targets are not yet known, our proteomic experiments serve as a first screen for the more targeted analysis of nitrated proteins from aging cerebellum for functional characterization.
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Envejecimiento/metabolismo , Cerebelo/metabolismo , Proteínas/metabolismo , Proteómica/métodos , Secuencia de Aminoácidos , Animales , Western Blotting , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Ratas , Ratas Endogámicas F344 , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
In vivo glucose sensor nitric oxide (NO) release is a means of mediating the inflammatory response that may cause sensor/tissue interactions and degraded sensor performance. The NO release (NOr) sensors were prepared by doping the outer polymeric membrane coating of previously reported needle-type electrochemical sensors with suitable lipophilic diazeniumdiolate species. The Clarke error grid correlation of sensor glycemia estimates versus blood glucose measured in Sprague-Dawley rats yielded 99.7% of the points for NOr sensors and 96.3% of points for the control within zones A and B (clinically acceptable) on Day 1, with a similar correlation for Day 3. Histological examination of the implant site demonstrated that the inflammatory response was significantly decreased for 100% of the NOr sensors at 24 h. The NOr sensors also showed a reduced run-in time of minutes versus hours for control sensors. NO evolution does increase protein nitration in tissue surrounding the sensor, which may be linked to the suppression of inflammation. This study further emphasizes the importance of NO as an electroactive species that can potentially interfere with glucose (peroxide) detection. The NOr sensor offers a viable option for in vivo glucose sensor development.
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Técnicas Biosensibles , Glucemia/análisis , Óxido Nítrico/metabolismo , Animales , Calibración , Electroquímica , Inflamación/metabolismo , Ratas , Ratas Sprague-Dawley , Tirosina/análogos & derivados , Tirosina/análisisRESUMEN
A rapid, homogeneous aptamer-based bioanalysis is reported for the sensitive detection of immunoglobulin E (IgE) using fluorescence polarization (FP). 5'-End-labeled D17.4 DNA aptamer was used for IgE detection based on the anisotropy differences of the labeled ligand. Two different fluorophores, fluorescein and Texas Red, were used to analyze IgE in the low-nanomolar range with high specificity. Measurable anisotropy changes were observed with a short equilibration time. Analysis of the binding data reveals a possible cooperative binding process in solution. The nature of the fluorophore clearly influences the sensitivity of the analysis more than the tether length used for the dye conjugation. The local fluorophore motion is seen to influence the sensitivity of the FP probe significantly. Texas Red is seen to be relatively more sensitive for this approach and has apparently favorable dye-DNA interactions, and a limit of detection of 350 pM was obtained. Significant temperature dependence of the FP responses has been observed in this work. Ionic composition of the binding buffer also influences the assay sensitivity. The results confirm the promise and potential of similar homogeneous assays for aptamer-based bioanalysis.
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Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Polarización de Fluorescencia/métodos , Inmunoglobulina E/análisis , Fluoresceína/química , Colorantes Fluorescentes/química , Inmunoglobulina E/metabolismo , Sensibilidad y Especificidad , Temperatura , Xantenos/químicaRESUMEN
We report the picosecond time-scale fluorescence dynamics of a dye-labeled DNA oligonucleotide or "aptamer" designed to bind specifically to Immunoglobulin E. Comparison of the photophysics of Texas Red (TR), fluorescein and 5'-carboxytetramethylrhodamine (TAMRA)-labeled aptamers reveals surprising differences with significant implications for measurements of oligonucleotide structure and dynamics. The fluorescence decay of the TR-aptamer is a simple single exponential with a weak temperature dependence. The fluorescence decay of the fluorescein-aptamer (fl-aptamer) is pH dependent and displays a complex temperature dependence with significant changes on melting of the aptamer tertiary structure. Despite its similarities to TR, TAMRA is strongly quenched when conjugated to the aptamer and displays complex fluorescence kinetics best described by a distributed rate model. Using the maximum entropy method, we have discovered two highly temperature-dependent fluorescence lifetimes for the TAMRA-aptamer. One of these lifetimes is similar to that of free TAMRA and displays the same temperature dependence. The other lifetime is quenched and displays a temperature dependence characteristic of a charge transfer reaction. These data set TR apart as an attractive alternative to TAMRA and fluorescein for studies such as fluorescence polarization and fluorescence resonance energy transfer, where environmental sensitivity of the probe is not desired.
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ADN/química , Fluoresceína/química , Oligonucleótidos/química , Rodaminas/química , Xantenos/química , Transferencia de Energía , Fluorescencia , Polarización de Fluorescencia , Transferencia Resonante de Energía de Fluorescencia , TemperaturaRESUMEN
We report the picosecond and nanosecond timescale rotational dynamics of a dye-labeled DNA oligonucleotide or "aptamer" designed to bind specifically to immunoglobulin E. Rotational dynamics in combination with fluorescence lifetime measurements provide information about dye-DNA interactions. Comparison of Texas Red (TR), fluorescein, and tetramethylrhodamine (TAMRA)-labeled aptamers reveals surprising differences with significant implications for biophysical studies employing such conjugates. Time-resolved anisotropy studies demonstrate that the TR- and TAMRA-aptamer anisotropy decays are dominated by the overall rotation of the aptamer, whereas the fluorescein-aptamer anisotropy decay displays a subnanosecond rotational correlation time much shorter than that expected for the overall rotation of the aptamer. Docking and molecular dynamics simulations suggest that the low mobility of TR is a result of binding in the groove of the DNA helix. Additionally, associated anisotropy analysis of the TAMRA-aptamer reveals both quenched and unquenched states that experience significant coupling to the DNA motion. Therefore, quenching of TAMRA by guanosine must depend on the configuration of the dye bound to the DNA. The strong coupling of TR to the rotational dynamics of the DNA aptamer, together with the absence of quenching of its fluorescence by DNA, makes it a good probe of DNA orientational dynamics. The understanding of the nature of dye-DNA interactions provides the basis for the development of bioconjugates optimized for specific biophysical measurements and is important for the sensitivity of anisotropy-based DNA-protein interaction studies employing such conjugates.