Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
1.
Eur Respir J ; 59(3)2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34561290

RESUMO

BACKGROUND: Immune cells contain a specialised type of proteasome, i.e. the immunoproteasome, which is required for intracellular protein degradation. Immunoproteasomes are key regulators of immune cell differentiation, inflammatory activation and autoimmunity. Immunoproteasome function in peripheral immune cells might be altered by smoking and in chronic obstructive pulmonary disease (COPD), thereby affecting immune cell responses. METHODS: We analysed the expression and activity of proteasome complexes in peripheral blood mononuclear cells (PBMCs) isolated from healthy male young smokers as well as from patients with severe COPD and compared them with matching controls. RESULTS: Proteasome expression was upregulated in COPD patients as assessed by quantitative reverse transcriptase-PCR and mass spectrometry-based proteomic analysis. Proteasome activity was quantified using activity-based probes and native gel analysis. We observed distinct activation of immunoproteasomes in the peripheral blood cells of young male smokers and severely ill COPD patients. Native gel analysis and linear regression modelling confirmed robust activation and elevated assembly of 20S proteasomes, which correlated significantly with reduced lung function parameters in COPD patients. The immunoproteasome was distinctly activated in COPD patients upon inflammatory cytokine stimulation of PBMCs in vitro. Inhibition of the immunoproteasome reduced pro-inflammatory cytokine expression in COPD-derived blood immune cells. CONCLUSIONS: Given the crucial role of chronic inflammatory signalling and the emerging involvement of autoimmune responses in COPD, therapeutic targeting of the immunoproteasome might represent a novel therapeutic concept for COPD.


Assuntos
Complexo de Endopeptidases do Proteassoma , Doença Pulmonar Obstrutiva Crônica , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteômica , Fumantes
2.
Am J Respir Crit Care Med ; 193(11): 1230-41, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-26756824

RESUMO

RATIONALE: Patients with chronic obstructive pulmonary disease (COPD) and in particular smokers are more susceptible to respiratory infections contributing to acute exacerbations of disease. The immunoproteasome is a specialized type of proteasome destined to improve major histocompatibility complex (MHC) class I-mediated antigen presentation for the resolution of intracellular infections. OBJECTIVES: To characterize immunoproteasome function in COPD and its regulation by cigarette smoke. METHODS: Immunoproteasome expression and activity were determined in bronchoalveolar lavage (BAL) and lungs of human donors and patients with COPD or idiopathic pulmonary fibrosis (IPF), as well as in cigarette smoke-exposed mice. Smoke-mediated alterations of immunoproteasome activity and MHC I surface expression were analyzed in human blood-derived macrophages. Immunoproteasome-specific MHC I antigen presentation was evaluated in spleen and lung immune cells that had been smoke-exposed in vitro or in vivo. MEASUREMENTS AND MAIN RESULTS: Immunoproteasome and MHC I mRNA expression was reduced in BAL cells of patients with COPD and in isolated alveolar macrophages of patients with COPD or IPF. Exposure of immune cells to cigarette smoke extract in vitro reduced immunoproteasome activity and impaired immunoproteasome-specific MHC I antigen presentation. In vivo, acute cigarette smoke exposure dynamically regulated immunoproteasome function and MHC I antigen presentation in mouse BAL cells. End-stage COPD lungs showed markedly impaired immunoproteasome activities. CONCLUSIONS: We here show that the activity of the immunoproteasome is impaired by cigarette smoke resulting in reduced MHC I antigen presentation. Regulation of immunoproteasome function by cigarette smoke may thus alter adaptive immune responses and add to prolonged infections and exacerbations in COPD and IPF.


Assuntos
Imunoproteínas/fisiologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Fumaça/efeitos adversos , Fumar/fisiopatologia , Idoso , Idoso de 80 Anos ou mais , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Nicotiana
3.
Proc Natl Acad Sci U S A ; 110(7): 2472-7, 2013 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-23359682

RESUMO

Rhomboid proteases are evolutionary conserved intramembrane serine proteases. Because of their emerging role in many important biological pathways, rhomboids are potential drug targets. Unfortunately, few chemical tools are available for their study. Here, we describe a mass spectrometry-based assay to measure rhomboid substrate cleavage and inhibition. We have identified isocoumarin inhibitors and developed activity-based probes for rhomboid proteases. The probes can distinguish between active and inactive rhomboids due to covalent, reversible binding of the active-site serine and stable modification of a histidine residue. Finally, the structure of an isocoumarin-based inhibitor with Escherichia coli rhomboid GlpG uncovers an unusual mode of binding at the active site and suggests that the interactions between the 3-substituent on the isocoumarin inhibitor and hydrophobic residues on the protease reflect S' subsite binding. Overall, these probes represent valuable tools for rhomboid study, and the structural insights may facilitate future inhibitor design.


Assuntos
Proteínas de Ligação a DNA/química , Endopeptidases/química , Proteínas de Escherichia coli/química , Proteínas de Membrana/química , Modelos Moleculares , Sondas Moleculares/química , Proteólise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Química Click , Cristalização , Proteínas de Ligação a DNA/metabolismo , Endopeptidases/metabolismo , Proteínas de Escherichia coli/metabolismo , Isocumarinas/química , Proteínas de Membrana/metabolismo , Sondas Moleculares/metabolismo , Estrutura Molecular , Especificidade por Substrato
4.
Chem Biol ; 22(1): 129-38, 2015 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-25556945

RESUMO

MALT1 paracaspase is activated upon antigen receptor stimulation to promote lymphocyte activation. In addition, deregulated MALT1 protease activity drives survival of distinct lymphomas such as the activated B cell type of diffuse large B cell lymphoma (ABC-DLBCL). Here, we designed fluorophore or biotin-coupled activity based-probes (ABP) that covalently modify the active center of MALT1. MALT1-ABPs are exclusively labeling an active modified full length form of MALT1 upon T cell stimulation. Further, despite the CARMA1 requirement for initial MALT1 activation, the MALT1-ABPs show that protease activity is not confined to the high-molecular CARMA1-BCL10-MALT1 (CBM) complex. Using biotin-coupled ABPs, we developed a robust assay for sensitive and selective detection of active MALT1 in cell lines, primary lymphocytes, and DLBCL tumor biopsies. Taken together, MALT1-ABPs represent powerful chemical tools to measure cellular MALT1 activation, determine efficacy of small molecule inhibitors, and classify lymphomas based on MALT1 activity status.


Assuntos
Caspases/metabolismo , Linfoma Difuso de Grandes Células B/enzimologia , Linfoma Difuso de Grandes Células B/patologia , Sondas Moleculares/química , Proteínas de Neoplasias/metabolismo , Linfócitos T/enzimologia , Biotina/química , Western Blotting , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Linhagem Celular , Química Click , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Guanilato Ciclase/metabolismo , Humanos , Células Jurkat , Sondas Moleculares/síntese química , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Proteínas de Neoplasias/antagonistas & inibidores , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Rodaminas/química , Linfócitos T/citologia , Linfócitos T/imunologia
5.
Sci Rep ; 5: 10230, 2015 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-25989070

RESUMO

Impaired immune function contributes to the development of chronic obstructive pulmonary disease (COPD). Disease progression is further exacerbated by pathogen infections due to impaired immune responses. Elimination of infected cells is achieved by cytotoxic CD8(+) T cells that are activated by MHC I-mediated presentation of pathogen-derived antigenic peptides. The immunoproteasome, a specialized form of the proteasome, improves generation of antigenic peptides for MHC I presentation thereby facilitating anti-viral immune responses. However, immunoproteasome function in the lung has not been investigated in detail yet. In this study, we comprehensively characterized the function of immunoproteasomes in the human and murine lung. Parenchymal cells of the lung express low constitutive levels of immunoproteasomes, while they are highly and specifically expressed in alveolar macrophages. Immunoproteasome expression is not altered in whole lung tissue of COPD patients. Novel activity-based probes and native gel analysis revealed that immunoproteasome activities are specifically and rapidly induced by IFNγ treatment in respiratory cells in vitro and by virus infection of the lung in mice. Our results suggest that the lung is potentially capable of mounting an immunoproteasome-mediated efficient adaptive immune response to intracellular infections.


Assuntos
Infecções por Herpesviridae/imunologia , Interferon gama/imunologia , Pulmão/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Imunidade Adaptativa/imunologia , Animais , Linhagem Celular , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Feminino , Humanos , Pulmão/metabolismo , Pulmão/virologia , Macrófagos Alveolares/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Rhadinovirus/imunologia , Linfócitos T Citotóxicos/imunologia , Infecções Tumorais por Vírus/imunologia
6.
PLoS One ; 8(8): e72307, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23991088

RESUMO

Rhomboids are intramembrane serine proteases that play diverse biological roles, including some that are of potential therapeutical relevance. Up to date, rhomboid inhibitor assays are based on protein substrate cleavage. Although rhomboids have an overlapping substrate specificity, substrates cannot be used universally. To overcome the need for substrates, we developed a screening assay using fluorescence polarization activity-based protein profiling (FluoPol ABPP) that is compatible with membrane proteases. With FluoPol ABPP, we identified new inhibitors for the E. coli rhomboid GlpG. Among these was a structural class that has not yet been reported as rhomboid inhibitors: ß-lactones. They form covalent and irreversible complexes with the active site serine of GlpG. The presence of alkyne handles on the ß-lactones also allowed activity-based labeling. Overall, these molecules represent a new scaffold for future inhibitor and activity-based probe development, whereas the assay will allow inhibitor screening of ill-characterized membrane proteases.


Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Descoberta de Drogas , Proteínas de Escherichia coli/antagonistas & inibidores , Polarização de Fluorescência/métodos , Proteínas de Membrana/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Proteínas de Ligação a DNA/metabolismo , Endopeptidases/metabolismo , Proteínas de Escherichia coli/metabolismo , Concentração Inibidora 50 , Proteínas de Membrana/metabolismo , Sondas Moleculares , Inibidores de Proteases/química , Especificidade por Substrato
7.
Curr Opin Chem Biol ; 17(1): 102-9, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23273613

RESUMO

Covalent chemical probes enable investigation of a desired fraction of the proteome. It is possible to adjust the selectivity of these probes, so they either react with a certain amino acid in all proteins, a class of proteins or only a single protein species. A combination of specific reactive groups with additional recognition elements can fine tune probes to hit the desired proteins, even in the presence of related family members. Using probes of lower or higher selectivity, screening experiments for inhibitor discovery and imaging experiments for localization studies can be performed, showing only a fraction of the power of covalent small molecule probes.


Assuntos
Aminoácidos/análise , Proteínas/análise , Proteômica/métodos , Aminoácidos/metabolismo , Animais , Descoberta de Drogas/métodos , Humanos , Imagem Molecular/métodos , Sondas Moleculares/análise , Sondas Moleculares/metabolismo , Proteínas/antagonistas & inibidores , Proteínas/metabolismo
8.
Biol Chem ; 388(8): 823-9, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17655501

RESUMO

Anabaena sp. PCC 7120 is a filamentous cyanobacterium that serves as a model to analyze prokaryotic cell differentiation, evolutionary development of plastids, and the regulation of nitrogen fixation. The cell wall is the cellular structure in contact with the surrounding medium. To understand the dynamics of the cell wall proteome during cell differentiation, the cell wall from Anabaena heterocysts was enriched and analyzed. In line with the recently proposed continuity of the outer membrane along the Anabaena filament, most of the proteins identified in the heterocyst cell-wall fraction are also present in the cell wall of vegetative cells, even though the lipid content of both membranes is different.


Assuntos
Anabaena/química , Anabaena/citologia , Parede Celular/química , Proteoma/análise , Anabaena/genética , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA