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1.
Cell ; 139(3): 610-22, 2009 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-19879846

RESUMO

Protein-DNA interactions (PDIs) mediate a broad range of functions essential for cellular differentiation, function, and survival. However, it is still a daunting task to comprehensively identify and profile sequence-specific PDIs in complex genomes. Here, we have used a combined bioinformatics and protein microarray-based strategy to systematically characterize the human protein-DNA interactome. We identified 17,718 PDIs between 460 DNA motifs predicted to regulate transcription and 4,191 human proteins of various functional classes. Among them, we recovered many known PDIs for transcription factors (TFs). We identified a large number of unanticipated PDIs for known TFs, as well as for previously uncharacterized TFs. We also found that over three hundred unconventional DNA-binding proteins (uDBPs)--which include RNA-binding proteins, mitochondrial proteins, and protein kinases--showed sequence-specific PDIs. One such uDBP, ERK2, acts as a transcriptional repressor for interferon gamma-induced genes, suggesting important biological roles for such proteins.


Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Interferon gama/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Transdução de Sinais , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos
2.
Environ Toxicol ; 39(6): 3512-3522, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38459654

RESUMO

BACKGROUND: The significance of regulatory T cells (Tregs) in colorectal cancer is unclear. METHODS: The single-cell sequencing data for colorectal cancer, specifically GSE132465 and GSE188711, were retrieved from the GEO database. Simultaneously, bulk transcriptome data were obtained from the UCSC Xena website. To delve into the heterogeneity of Treg cells and identify key genes at the single-cell sequencing level, we employed dimensionality reduction techniques alongside clustering and conducted differential expression gene analysis. For the bulk transcriptome data, we utilized weighted co-expression network analysis to investigate critical gene modules. Additionally, we employed COX regression and Lasso regression methodologies to construct prognostic models, thereby assessing patient outcomes. To facilitate outcome evaluation, nomograms were constructed. The integration of these diverse approaches aims to comprehensively study colorectal cancer, encompassing single-cell heterogeneity, key gene identification, and prognosis modeling using both single-cell and bulk transcriptome data. Polymerase chain reaction (PCR) experiments are used to verify mRNA expression levels of key genes. The analysis software was R software (version 4.3.2). RESULTS: Through single-cell sequencing analysis and bulk transcriptome analysis, we constructed a prognostic model composed with Treg-associated signatures. The high-risk group demonstrated significantly worse prognosis compared with the low-risk group, highlighting the clinical relevance of our models. PCR confirmed that the key gene DEAH-box helicase 15 (DHX15) was significantly overexpressed in colorectal cancer. CONCLUSIONS: The prognostic models developed in this study offer a potential tool for risk assessment, guiding treatment decisions for colorectal cancer patients.


Assuntos
Neoplasias Colorretais , Análise de Célula Única , Linfócitos T Reguladores , Transcriptoma , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/imunologia , Humanos , Linfócitos T Reguladores/imunologia , Prognóstico , Perfilação da Expressão Gênica
3.
J Rheumatol ; 50(9): 1136-1144, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37127324

RESUMO

OBJECTIVE: To expand, in an unbiased manner, our knowledge of autoantigens and autoantibodies in patients with systemic lupus erythematosus (SLE) and evaluate their associations with serological and clinical variables. METHODS: Human proteome arrays (> 21,000 proteins) were screened with serum from patients with SLE (n = 12) and healthy controls (n = 6) for IgG and IgA binding. Top hits were validated with 2 cohorts of patients with SLE (cohort 1, n = 49; cohort 2, n = 46) and other rheumatic diseases by ELISA. Clinical associations of the autoantibodies were tested. RESULTS: Ro60 was the top hit in the screen, and the 10 following proteins included 2 additional known SLE autoantigens plus 8 novel autoantigens involved in microRNA processing (Argonaute protein 1 [AGO1], AGO2, and AGO3), ribosomes (ribosomal protein lateral stalk subunit P2 and ovarian tumor deubiquitinase 5 [OTUD5]), RNA transport by the vault (major vault protein), and the immune proteasome (proteasome activator complex subunit 3). Patient serum contained IgG reactive with these proteins and IgA against the AGO proteins. Using the 95th percentile of healthy donor reactivity, 5-43% were positive for the novel antigens, with OTUD5 and AGO1 showing the highest percentages of positivity. Autoantibodies against AGO1 proteins were more prevalent in patients with oral ulcers in a statistically significant manner. IgG autoantibodies against AGO proteins were also seen in other rheumatic diseases. CONCLUSION: We discovered new autoantigens existing in cytosolic macromolecular protein assemblies containing RNA (except the proteasome) in cells. A more comprehensive list of autoantigens will allow for a better analysis of how proteins are targeted by the autoimmune response. Future research will also reveal whether specific autoantibodies have utility in the diagnosis or management of SLE.


Assuntos
Autoanticorpos , Lúpus Eritematoso Sistêmico , Humanos , Proteínas Ribossômicas , Complexo de Endopeptidases do Proteassoma , Proteínas Argonautas , Lúpus Eritematoso Sistêmico/diagnóstico , Autoantígenos , Imunoglobulina G , Imunoglobulina A
4.
Mol Cell Proteomics ; 19(3): 490-500, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31924693

RESUMO

Lung cancer (LC) remains the leading cause of mortality from malignant tumors worldwide. In our previous study, we surveyed both IgG and IgM-bound serological biomarkers and validated a panel of IgG-bound autoantigens for early LC diagnosis with 50% sensitivity at 90% specificity. To further improve the performance of these serological biomarkers, we surveyed HuProt arrays, comprised of 20,240 human proteins, for IgA-bound autoantigens because IgAs are a major immunoglobulin isotype in the lung. Integrating with IgG-bound autoantigens, we discovered and validated a combined biomarker panel using ELISA-format tests. Specifically, in Phase I, we obtained IgA-based autoimmune profiles of 69 early stage LC patients, 30 healthy subjects and 25 patients with lung benign lesions (LBL) on HuProt arrays and identified 28 proteins as candidate autoantigens that were significantly associated with early stage LC. In Phase II, we re-purified the autoantigens and converted them into an ELISA-format testing to profile an additional large cohort, comprised of 136 early stage LC patients, 58 healthy individuals, and 29 LBL patients. Integration of IgG autoimmune profiles allowed us to identify and validate a biomarker panel of three IgA autoantigens (i.e. BCL7A, and TRIM33 and MTERF4) and three IgG autoantigens (i.e. CTAG1A, DDX4 and MAGEC2) for diagnosis of early stage LC with 73.5% sensitivity at >85% specificity. In Phase III, the performance of this biomarker panel was confirmed with an independent cohort, comprised of 88 early stage LC patients, 18 LBL patients, and 36 healthy subjects. Finally, a blind test on 178 serum samples was conducted to confirm the performance of the biomarker panel. In summary, this study demonstrates for the first time that an integrated panel of IgA/IgG autoantigens can serve as valuable biomarkers to further improve the performance of early diagnosis of LC.


Assuntos
Autoantígenos/imunologia , Biomarcadores Tumorais/imunologia , Detecção Precoce de Câncer , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Neoplasias Pulmonares/diagnóstico , Idoso , Biomarcadores Tumorais/sangue , Feminino , Humanos , Pulmão/imunologia , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/imunologia , Masculino , Pessoa de Meia-Idade
5.
Nat Methods ; 15(5): 330-338, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29638227

RESUMO

A key component of efforts to address the reproducibility crisis in biomedical research is the development of rigorously validated and renewable protein-affinity reagents. As part of the US National Institutes of Health (NIH) Protein Capture Reagents Program (PCRP), we have generated a collection of 1,406 highly validated immunoprecipitation- and/or immunoblotting-grade mouse monoclonal antibodies (mAbs) to 737 human transcription factors, using an integrated production and validation pipeline. We used HuProt human protein microarrays as a primary validation tool to identify mAbs with high specificity for their cognate targets. We further validated PCRP mAbs by means of multiple experimental applications, including immunoprecipitation, immunoblotting, chromatin immunoprecipitation followed by sequencing (ChIP-seq), and immunohistochemistry. We also conducted a meta-analysis that identified critical variables that contribute to the generation of high-quality mAbs. All validation data, protocols, and links to PCRP mAb suppliers are available at http://proteincapture.org.


Assuntos
Anticorpos Monoclonais/imunologia , Análise Serial de Proteínas/métodos , Fatores de Transcrição/metabolismo , Animais , Clonagem Molecular , Bases de Dados Factuais , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Reprodutibilidade dos Testes
6.
Dig Dis Sci ; 64(5): 1390-1391, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30852771

RESUMO

The original version of the article unfortunately contained errors in Materials and Methods section, Figure 3 and Figure 4.

7.
Dig Dis Sci ; 64(2): 447-455, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30370491

RESUMO

BACKGROUND: Calcitonin gene-related peptide (CGRP) has antioxidant and anti-inflammatory activities on the pathological damage of acute pancreatitis. However, its molecular mechanism on severe acute pancreatitis (SAP) remains unknown. AIMS: To evaluate the influence of CGRP-mediated p38MAPK signaling pathway in rats with SAP. METHODS: SD rats were randomly divided into Sham group, SAP group, CGRP group (SAP rats injected with CGRP), SB203580 group (rats injected with p38MAPK pathway inhibitor SB203580), and CGRP8-37 group (SAP rats injected with CGRP8-37). Serum amylase and lipase activities were determined. Histopathological observations were evaluated, and the expression of inflammatory cytokines and oxidative stress-related indexes were measured. RESULTS: Compared with Sham group, SAP rats were increased in the activities of serum amylase and lipase, the pathologic assessment of pancreatic tissue, the levels of TNF-α, IL-1ß, IL-6, and IL-8, the content of MDA and MPO, and the expressions of CGRP, and p-p38MAPK protein, but they were decreased in SOD activity and GSH content. The above alterations were aggravated in the CGRP8-37 group when compared with SAP group. Besides, in comparison with SAP group, rats in the CGRP and SB203580 groups presented a reduction in the activities of serum amylase and lipase, the levels of inflammatory cytokines, the content of MDA and MPO, and the expressions of p-p38MAPK protein, while showed an elevation in SOD activity and GSH content. CONCLUSION: Pretreatment with CGRP alleviated oxidative stress and inflammatory response of SAP rats possibly by suppressing the activity of p38MAPK pathway, and thereby postponing the disease progression.


Assuntos
Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Pâncreas/efeitos dos fármacos , Pancreatite/patologia , Fragmentos de Peptídeos/farmacologia , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Doença Aguda , Amilases/sangue , Amilases/efeitos dos fármacos , Animais , Peptídeo Relacionado com Gene de Calcitonina/efeitos dos fármacos , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Citocinas/efeitos dos fármacos , Citocinas/imunologia , Progressão da Doença , Inflamação , Interleucina-1beta/efeitos dos fármacos , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Interleucina-8/efeitos dos fármacos , Interleucina-8/imunologia , Lipase/sangue , Lipase/efeitos dos fármacos , Malondialdeído/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Pâncreas/imunologia , Pâncreas/patologia , Pancreatite/imunologia , Peroxidase/efeitos dos fármacos , Peroxidase/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Índice de Gravidade de Doença , Transdução de Sinais , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
8.
Mol Cell Proteomics ; 16(12): 2069-2078, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29021294

RESUMO

Lung cancer (LC) remains the leading cause of mortality from malignant tumors worldwide. Currently, a lack of serological biomarkers for early LC diagnosis is a major roadblock for early intervention and prevention of LC. To undertake this challenge, we employed a two-phase strategy to discover and validate a biomarker panel using a protein array-based approach. In Phase I, we obtained serological autoimmune profiles of 80 LC patients and 20 healthy subjects on HuProt arrays, and identified 170 candidate proteins significantly associated with LC. In Phase II, we constructed a LC focused array with the 170 proteins, and profiled a large cohort, comprised of 352 LC patients, 93 healthy individuals, and 101 patients with lung benign lesions (LBL). The comparison of autoimmune profiles between the early stage LC and the combined group of healthy and LBL allowed us to identify and validate a biomarker panel of p53, HRas, and ETHE1 for diagnosis of early stage LC with 50% sensitivity at >90% specificity. Finally, the performance of this biomarker panel was confirmed in ELISA tests. In summary, this study represents one of the most comprehensive proteome-wide surveys with one of the largest (i.e. 1,101 unique samples) and most diverse (i.e. nine disease groups) cohorts, resulting in a biomarker panel with good performance.


Assuntos
Detecção Precoce de Câncer/métodos , Neoplasias Pulmonares/diagnóstico , Proteínas Mitocondriais/imunologia , Proteínas de Transporte Nucleocitoplasmático/imunologia , Análise Serial de Proteínas/métodos , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Proteína Supressora de Tumor p53/imunologia , Idoso , Autoanticorpos/análise , Autoimunidade , Biomarcadores Tumorais/imunologia , Estudos de Casos e Controles , Feminino , Humanos , Neoplasias Pulmonares/imunologia , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade
9.
Anal Chem ; 90(18): 10958-10966, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30106562

RESUMO

Bacterial meningitis in neonates and infants is an acute lethal disease and occurs in response to microbial exploitation of the blood-brain barrier (BBB), resulting in the intracranial inflammation. Several pathogens, such as Escherichia coli ( E. coli), can cause this devastating disease; however, the underlying molecular mechanisms by which these pathogens exploit the BBB remain incompletely understood. To identify important players on both the pathogen and host sides that govern the E. coli-BBB cell interactions, we took advantage of the E. coli and human proteome microarrays (i.e., HuProt) as an unbiased, proteome-wide tool for identification of important players on both sides. Using the E. coli proteome microarrays, we developed a unique high throughput chip-based cell probing assay to probe with fluorescent live human brain microvascular endothelial cells (HBMEC, which constitute the BBB). We identified several transmembrane proteins, which effectively bound to live HBMEC. We focused on YojI protein for further study. By probing the HuProt arrays with YojI, interferon-alpha receptor (IFNAR2) was identified as one of its binding proteins. The importance of YojI and IFNAR2 involved in E. coli-HBMEC interactions was characterized using the YojI knockout bacteria and IFNAR2-knock down HBMEC and further confirmed by E. coli binding assay in HBMEC. This study represents a new paradigm (dual-microarray technology) that enables rapid, unbiased discovery of both pathogen and host players that are involved in pathogen-host interactions for human infectious diseases in a high throughput manner.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Barreira Hematoencefálica/microbiologia , Infecções por Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Interações Hospedeiro-Patógeno , Proteômica/instrumentação , Receptor de Interferon alfa e beta/metabolismo , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Linhagem Celular , Desenho de Equipamento , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Humanos , Dispositivos Lab-On-A-Chip
10.
J Transl Med ; 16(1): 82, 2018 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-29606147

RESUMO

BACKGROUND: Immune checkpoint inhibitors (anti-CTLA-4, anti-PD-1, or the combination) enhance anti-tumor immune responses, yielding durable clinical benefit in several cancer types, including melanoma. However, a subset of patients experience immune-related adverse events (irAEs), which can be severe and result in treatment termination. To date, no biomarker exists that can predict development of irAEs. METHODS: We hypothesized that pre-treatment antibody profiles identify a subset of patients who possess a sub-clinical autoimmune phenotype that predisposes them to develop severe irAEs following immune system disinhibition. Using a HuProt human proteome array, we profiled baseline antibody levels in sera from melanoma patients treated with anti-CTLA-4, anti-PD-1, or the combination, and used support vector machine models to identify pre-treatment antibody signatures that predict irAE development. RESULTS: We identified distinct pre-treatment serum antibody profiles associated with severe irAEs for each therapy group. Support vector machine classifier models identified antibody signatures that could effectively discriminate between toxicity groups with > 90% accuracy, sensitivity, and specificity. Pathway analyses revealed significant enrichment of antibody targets associated with immunity/autoimmunity, including TNFα signaling, toll-like receptor signaling and microRNA biogenesis. CONCLUSIONS: Our results provide the first evidence supporting a predisposition to develop severe irAEs upon immune system disinhibition, which requires further independent validation in a clinical trial setting.


Assuntos
Anticorpos Antineoplásicos/sangue , Imunoterapia/efeitos adversos , Melanoma/imunologia , Melanoma/terapia , Idoso , Feminino , Humanos , Masculino , Melanoma/sangue , Proteômica , Reprodutibilidade dos Testes
11.
J Biol Chem ; 289(43): 29631-41, 2014 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-25164819

RESUMO

D-Serine, an endogenous co-agonist for the glycine site of the synaptic NMDA glutamate receptor, regulates synaptic plasticity and is implicated in schizophrenia. Serine racemase (SR) is the enzyme that converts L-serine to D-serine. In this study, we demonstrate that SR interacts with the synaptic proteins, postsynaptic density protein 95 (PSD-95) and stargazin, forming a ternary complex. SR binds to the PDZ3 domain of PSD-95 through the PDZ domain ligand at its C terminus. SR also binds to the C terminus of stargazin, which facilitates the cell membrane localization of SR and inhibits its activity. AMPA receptor activation internalizes SR and disrupts its interaction with stargazin, therefore derepressing SR activity, leading to more D-serine production and potentially facilitating NMDA receptor activation. These interactions regulate the enzymatic activity as well as the intracellular localization of SR, potentially coupling the activities of NMDA and AMPA receptors. This shuttling of a neurotransmitter synthesizing enzyme between two receptors appears to be a novel mode of synaptic regulation.


Assuntos
Canais de Cálcio/metabolismo , Ácido Glutâmico/metabolismo , Guanilato Quinases/metabolismo , Proteínas de Membrana/metabolismo , N-Metilaspartato/metabolismo , Racemases e Epimerases/metabolismo , Transmissão Sináptica , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/metabolismo , Animais , Biocatálise , Membrana Celular/metabolismo , Proteína 4 Homóloga a Disks-Large , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Modelos Biológicos , Ligação Proteica , Ratos , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo
12.
Mol Syst Biol ; 9: 655, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23549483

RESUMO

The landscape of human phosphorylation networks has not been systematically explored, representing vast, unchartered territories within cellular signaling networks. Although a large number of in vivo phosphorylated residues have been identified by mass spectrometry (MS)-based approaches, assigning the upstream kinases to these residues requires biochemical analysis of kinase-substrate relationships (KSRs). Here, we developed a new strategy, called CEASAR, based on functional protein microarrays and bioinformatics to experimentally identify substrates for 289 unique kinases, resulting in 3656 high-quality KSRs. We then generated consensus phosphorylation motifs for each of the kinases and integrated this information, along with information about in vivo phosphorylation sites determined by MS, to construct a high-resolution map of phosphorylation networks that connects 230 kinases to 2591 in vivo phosphorylation sites in 652 substrates. The value of this data set is demonstrated through the discovery of a new role for PKA downstream of Btk (Bruton's tyrosine kinase) during B-cell receptor signaling. Overall, these studies provide global insights into kinase-mediated signaling pathways and promise to advance our understanding of cellular signaling processes in humans.


Assuntos
Linfócitos B/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/genética , Tirosina Quinase da Agamaglobulinemia , Algoritmos , Sequência de Aminoácidos , Linfócitos B/citologia , Teorema de Bayes , Proteínas Quinases Dependentes de AMP Cíclico/genética , Humanos , Dados de Sequência Molecular , Fosforilação , Análise Serial de Proteínas , Mapas de Interação de Proteínas , Proteínas Tirosina Quinases/genética , Receptores de Antígenos de Linfócitos B/genética , Tirosina/metabolismo
13.
Mol Cell Proteomics ; 11(6): O111.016253, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22307071

RESUMO

To broaden the range of tools available for proteomic research, we generated a library of 16,368 unique full-length human ORFs that are expressible as N-terminal GST-His(6) fusion proteins. Following expression in yeast, these proteins were then individually purified and used to construct a human proteome microarray. To demonstrate the usefulness of this reagent, we developed a streamlined strategy for the production of monospecific monoclonal antibodies that used immunization with live human cells and microarray-based analysis of antibody specificity as its central components. We showed that microarray-based analysis of antibody specificity can be performed efficiently using a two-dimensional pooling strategy. We also demonstrated that our immunization and selection strategies result in a large fraction of monospecific monoclonal antibodies that are both immunoblot and immunoprecipitation grade. Our data indicate that the pipeline provides a robust platform for the generation of monoclonal antibodies of exceptional specificity.


Assuntos
Anticorpos Monoclonais Murinos/imunologia , Especificidade de Anticorpos , Proteoma/imunologia , Proteínas Recombinantes de Fusão/imunologia , Animais , Antígenos/química , Antígenos/imunologia , Linhagem Celular Tumoral , Humanos , Hibridomas , Proteínas Imobilizadas/química , Proteínas Imobilizadas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Análise Serial de Proteínas , Proteoma/química , Proteínas Recombinantes de Fusão/química
14.
Anal Chem ; 85(17): 8046-54, 2013 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-23941274

RESUMO

To facilitate high-throughput biochemical analyses of membrane proteins, we have developed a novel display technology in a microarray format. Both single-pass (cluster of differentiation 4, CD4) and multiple-pass (G protein-coupled receptor 77, GPR77) human transmembrane proteins were engineered to be displayed in the membrane envelop of herpes simplex virions. These viruses produce large spherical virions displaying multiple copies of envelop proteins. Our aim was to engineer this virus to express these human proteins during the virus productive cycle and incorporate the human proteins into the virion during the assembly process. Another strategy presented includes engineering a fusion of glycoprotein C (gC), a major constituent of herpes simplex virus type 1 (HSV-1) virions, by hijacking the cis-acting signals to direct incorporation of the chimeric protein into the virion. The expression of the human proteins in infected cells, at the cell surface and in purified virions, is in the correct transmembrane orientation, and the proteins are biochemically functional. Purified virions printed on glass slides form a high-density Virion Display (VirD) Array, and the displayed proteins were demonstrated to retain their native conformations and interactions on the VirD Array judging by similar assays, such as antibody staining, as well as lectin and ligand binding. This method can be readily scaled or tailored for different modalities including a high-content, high-throughput platform for screening ligands and drugs of human membrane proteins.


Assuntos
Membrana Celular/genética , Proteínas de Membrana/genética , Vírion/genética , Animais , Membrana Celular/química , Chlorocebus aethiops , Humanos , Masculino , Proteínas de Membrana/análise , Análise Serial de Proteínas/métodos , Células Vero , Vírion/química
15.
J Virol ; 86(9): 5179-91, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22379092

RESUMO

The Kaposi's sarcoma-associated herpesvirus (KSHV) LANA protein functions in latently infected cells as an essential participant in KSHV genome replication and as a driver of dysregulated cell growth. To identify novel LANA protein-cell protein interactions that could contribute to these activities, we performed a proteomic screen in which purified, adenovirus-expressed Flag-LANA protein was incubated with an array displaying 4,192 nonredundant human proteins. Sixty-one interacting cell proteins were consistently detected. LANA interactions with high-mobility group AT-hook 1 (HMGA1), HMGB1, telomeric repeat binding factor 1 (TRF1), xeroderma pigmentosum complementation group A (XPA), pygopus homolog 2 (PYGO2), protein phosphatase 2A (PP2A)B subunit, Tat-interactive protein 60 (TIP60), replication protein A1 (RPA1), and RPA2 proteins were confirmed in coimmunoprecipitation assays. LANA-associated TIP60 retained acetyltransferase activity and, unlike human papillomavirus E6 and HIV-1 TAT proteins, LANA did not reduce TIP60 stability. The LANA-bound PP2A B subunit was associated with the PP2A A subunit but not the catalytic C subunit, suggesting a disruption of PP2A phosphatase activity. This is reminiscent of the role of simian virus 40 (SV40) small t antigen. Chromatin immunoprecipitation (ChIP) assays showed binding of RPA1 and RPA2 to the KSHV terminal repeats. Interestingly, LANA expression ablated RPA1 and RPA2 binding to the cell telomeric repeats. In U2OS cells that rely on the alternative mechanism for telomere maintenance, LANA expression had minimal effect on telomere length. However, LANA expression in telomerase immortalized endothelial cells resulted in telomere shortening. In KSHV-infected cells, telomere shortening may be one more mechanism by which LANA contributes to the development of malignancy.


Assuntos
Antígenos Virais/metabolismo , Histona Acetiltransferases/metabolismo , Proteínas Nucleares/metabolismo , Proteína Fosfatase 2/metabolismo , Encurtamento do Telômero , Antígenos Virais/genética , Linhagem Celular , Expressão Gênica , Proteínas HMGA/metabolismo , Proteína HMGB1/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisina Acetiltransferase 5 , Proteínas Nucleares/genética , Análise Serial de Proteínas , Ligação Proteica , Proteína de Replicação A/metabolismo , Telômero/metabolismo , Encurtamento do Telômero/genética , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo
16.
Cancers (Basel) ; 15(8)2023 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-37190187

RESUMO

Due to poor compliance and uptake of LDCT screening among high-risk populations, lung cancer is often diagnosed in advanced stages where treatment is rarely curative. Based upon the American College of Radiology's Lung Imaging and Reporting Data System (Lung-RADS) 80-90% of patients screened will have clinically "non-actionable" nodules (Lung-RADS 1 or 2), and those harboring larger, clinically "actionable" nodules (Lung-RADS 3 or 4) have a significantly greater risk of lung cancer. The development of a companion diagnostic method capable of identifying patients likely to have a clinically actionable nodule identified during LDCT is anticipated to improve accessibility and uptake of the paradigm and improve early detection rates. Using protein microarrays, we identified 501 circulating targets with differential immunoreactivities against cohorts characterized as possessing either actionable (n = 42) or non-actionable (n = 20) solid pulmonary nodules, per Lung-RADS guidelines. Quantitative assays were assembled on the Luminex platform for the 26 most promising targets. These assays were used to measure serum autoantibody levels in 841 patients, consisting of benign (BN; n = 101), early-stage non-small cell lung cancer (NSCLC; n = 245), other early-stage malignancies within the lung (n = 29), and individuals meeting United States Preventative Screening Task Force (USPSTF) screening inclusion criteria with both actionable (n = 87) and non-actionable radiologic findings (n = 379). These 841 patients were randomly split into three cohorts: Training, Validation 1, and Validation 2. Of the 26 candidate biomarkers tested, 17 differentiated patients with actionable nodules from those with non-actionable nodules. A random forest model consisting of six autoantibody (Annexin 2, DCD, MID1IP1, PNMA1, TAF10, ZNF696) biomarkers was developed to optimize our classification performance; it possessed a positive predictive value (PPV) of 61.4%/61.0% and negative predictive value (NPV) of 95.7%/83.9% against Validation cohorts 1 and 2, respectively. This panel may improve patient selection methods for lung cancer screening, serving to greatly reduce the futile screening rate while also improving accessibility to the paradigm for underserved populations.

17.
Nat Med ; 29(4): 888-897, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-37012549

RESUMO

B7 homolog 3 (B7-H3; CD276), a tumor-associated antigen and possible immune checkpoint, is highly expressed in prostate cancer (PCa) and is associated with early recurrence and metastasis. Enoblituzumab is a humanized, Fc-engineered, B7-H3-targeting antibody that mediates antibody-dependent cellular cytotoxicity. In this phase 2, biomarker-rich neoadjuvant trial, 32 biological males with operable intermediate to high-risk localized PCa were enrolled to evaluate the safety, anti-tumor activity and immunogenicity of enoblituzumab when given before prostatectomy. The coprimary outcomes were safety and undetectable prostate-specific antigen (PSA) level (PSA0) 1 year postprostatectomy, and the aim was to obtain an estimate of PSA0 with reasonable precision. The primary safety endpoint was met with no notable unexpected surgical or medical complications, or surgical delay. Overall, 12% of patients experienced grade 3 adverse events and no grade 4 events occurred. The coprimary endpoint of the PSA0 rate 1 year postprostatectomy was 66% (95% confidence interval 47-81%). The use of B7-H3-targeted immunotherapy in PCa is feasible and generally safe and preliminary data suggest potential clinical activity. The present study validates B7-H3 as a rational target for therapy development in PCa with larger studies planned. The ClinicalTrials.gov identifier is NCT02923180.


Assuntos
Antineoplásicos , Neoplasias da Próstata , Masculino , Humanos , Antígeno Prostático Específico/uso terapêutico , Terapia Neoadjuvante , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/cirurgia , Neoplasias da Próstata/patologia , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Antígenos B7
18.
J Proteome Res ; 11(11): 5301-10, 2012 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-23025254

RESUMO

Although significant effort is expended on identifying transcripts/proteins that are up-regulated in cancer, there are few reports on systematic elucidation of transcriptional mechanisms underlying such druggable cancer-specific targets. The mesothelin (MSLN) gene offers a promising subject, being expressed in a restricted pattern normally, yet highly overexpressed in almost one-third of human malignancies and a target of cancer immunotherapeutic trials. CanScript, a cis promoter element, appears to control MSLN cancer-specific expression; its related genomic sequences may up-regulate other cancer markers. CanScript is a 20-nt bipartite element consisting of an SP1-like motif and a consensus MCAT sequence. The latter recruits TEAD (TEA domain) family members, which are universally expressed. Exploration of the active CanScript element, especially the proteins binding to the SP1-like motif, thus could reveal cancer-specific features having diagnostic or therapeutic interest. The efficient identification of sequence-specific DNA-binding proteins at a given locus, however, has lagged in biomarker explorations. We used two orthogonal proteomics approaches--unbiased SILAC (stable isotope labeling by amino acids in cell culture)/DNA affinity-capture/mass spectrometry survey (SD-MS) and a large transcription factor protein microarray (TFM)--and functional validation to explore systematically the CanScript interactome. SD-MS produced nine candidates, and TFM, 18. The screens agreed in confirming binding by TEAD proteins and by newly identified NAB1 and NFATc. Among other identified candidates, we found functional roles for ZNF24, NAB1 and RFX1 in MSLN expression by cancer cells. Combined interactome screens yield an efficient, reproducible, sensitive, and unbiased approach to identify sequence-specific DNA-binding proteins and other participants in disease-specific DNA elements.


Assuntos
Proteínas Ligadas por GPI/metabolismo , Neoplasias/metabolismo , Sequência de Bases , Western Blotting , Linhagem Celular , DNA , Humanos , Luciferases/genética , Mesotelina , Dados de Sequência Molecular , Neoplasias/diagnóstico , Neoplasias/terapia , Espectrometria de Massas em Tandem
19.
Cell Mol Life Sci ; 68(10): 1657-68, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21207099

RESUMO

Sequence-specific protein-DNA interactions (PDIs) are critical for regulating many cellular processes, including transcription, DNA replication, repair, and rearrangement. We review recent experimental advances in high-throughput technologies designed to characterize PDIs and discuss recent studies that use these tools, including ChIP-chip/seq, SELEX-based approaches, yeast one-hybrid, bacterial one-hybrid, protein binding microarray, and protein microarray. The results of these studies have challenged some long-standing concepts of PDI and provide valuable insights into the complex transcriptional regulatory networks.


Assuntos
Proteínas de Ligação a DNA/análise , DNA/química , Imunoprecipitação da Cromatina , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Análise Serial de Proteínas/métodos , Técnica de Seleção de Aptâmeros , Técnicas do Sistema de Duplo-Híbrido
20.
Contrast Media Mol Imaging ; 2022: 5468317, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36304773

RESUMO

Taijiquan training emphasizes the relaxation of the mind and the body, pay attention to maintain the peace of mind, and minimize the impact of external interference on the body so as to make the mind more comfortable. This study mainly explores the influence of Taijiquan practice on emotion regulation based on intelligent medical health big data analysis. The significance of Taijiquan in developing and improving the positive emotions of middle-aged people and maintaining physical and mental health is expounded. There are two methods of data collection: full collection and incremental collection. In this study, when the psychological testing equipment was launched, a full amount of historical data was collected; after the psychological testing equipment was online, the collection method was generally carried out in the way of incremental collection. The subjects exercised Taijiquan three times a week, one hour each time, and the exercise content was the 24-style Taijiquan designated by the workstation. At the same time, the subjects were asked not to engage in other regular physical exercise projects in their spare time. By longitudinal tracking and comparison of the Taijiquan intervention group after participating in the Taijiquan exercise intervention, the differences in the state of mind and emotion regulation strategies, and 12 subjects were selected voluntarily to participate in the emotional Stroop (the color words used in the classic Stroop paradigm were replaced with emotional and nonemotional words written in different colors, and the subjects were still tasked with responding to colors) experimental paradigm. In this paper, the moderate-intensity Taijiquan project is selected, which is in line with the effective value threshold theory of exercise load. It studies the effects of exercise on the body shape, cardiopulmonary function, flexibility, and balance ability of the body according to the metabolism theory and the movement balance theory of the human body adapting to the environment. Before the experiment, there was no significant difference between the Taijiquan training group and the control group, but after the experiment, there was a significant difference between the Taijiquan training group and the control group (P<0.05). Taijiquan has a significant effect on improving students' body shape, cardiopulmonary function, flexibility, balance, and mood.


Assuntos
Regulação Emocional , Tai Chi Chuan , Pessoa de Meia-Idade , Humanos , Análise de Dados
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