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1.
Sci Adv ; 7(27)2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34215578

RESUMO

The neocortex is stereotypically organized into layers of excitatory neurons arranged in a precise parallel orientation. Here we show that dynamic adhesion both preceding and following radial migration is essential for this organization. Neuronal adhesion is regulated by the Mowat-Wilson syndrome-associated transcription factor Zeb2 (Sip1/Zfhx1b) through direct repression of independent adhesion pathways controlled by Neuropilin-1 (Nrp1) and Cadherin-6 (Cdh6). We reveal that to initiate radial migration, neurons must first suppress adhesion to the extracellular matrix. Zeb2 regulates the multipolar stage by transcriptional repression of Nrp1 and thereby downstream inhibition of integrin signaling. Upon completion of migration, neurons undergo an orientation process that is independent of migration. The parallel organization of neurons within the neocortex is controlled by Cdh6 through atypical regulation of integrin signaling via its RGD motif. Our data shed light on the mechanisms that regulate initiation of radial migration and the postmigratory orientation of neurons during neocortical development.

2.
Cell Syst ; 11(1): 11-24.e4, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32619549

RESUMO

The COVID-19 pandemic is an unprecedented global challenge, and point-of-care diagnostic classifiers are urgently required. Here, we present a platform for ultra-high-throughput serum and plasma proteomics that builds on ISO13485 standardization to facilitate simple implementation in regulated clinical laboratories. Our low-cost workflow handles up to 180 samples per day, enables high precision quantification, and reduces batch effects for large-scale and longitudinal studies. We use our platform on samples collected from a cohort of early hospitalized cases of the SARS-CoV-2 pandemic and identify 27 potential biomarkers that are differentially expressed depending on the WHO severity grade of COVID-19. They include complement factors, the coagulation system, inflammation modulators, and pro-inflammatory factors upstream and downstream of interleukin 6. All protocols and software for implementing our approach are freely available. In total, this work supports the development of routine proteomic assays to aid clinical decision making and generate hypotheses about potential COVID-19 therapeutic targets.


Assuntos
Proteínas Sanguíneas/metabolismo , Infecções por Coronavirus/sangue , Pneumonia Viral/sangue , Proteômica/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus/isolamento & purificação , Biomarcadores/sangue , Proteínas Sanguíneas/análise , COVID-19 , Infecções por Coronavirus/classificação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias/classificação , Pneumonia Viral/classificação , Pneumonia Viral/patologia , Pneumonia Viral/virologia , SARS-CoV-2 , Adulto Jovem
3.
Elife ; 92020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32364493

RESUMO

Mechanisms regulating the turnover of synaptic vesicle (SV) proteins are not well understood. They are thought to require poly-ubiquitination and degradation through proteasome, endo-lysosomal or autophagy-related pathways. Bassoon was shown to negatively regulate presynaptic autophagy in part by scaffolding Atg5. Here, we show that increased autophagy in Bassoon knockout neurons depends on poly-ubiquitination and that the loss of Bassoon leads to elevated levels of ubiquitinated synaptic proteins per se. Our data show that Bassoon knockout neurons have a smaller SV pool size and a higher turnover rate as indicated by a younger pool of SV2. The E3 ligase Parkin is required for increased autophagy in Bassoon-deficient neurons as the knockdown of Parkin normalized autophagy and SV protein levels and rescued impaired SV recycling. These data indicate that Bassoon is a key regulator of SV proteostasis and that Parkin is a key E3 ligase in the autophagy-mediated clearance of SV proteins.


Assuntos
Autofagia , Hipocampo/enzimologia , Proteínas do Tecido Nervoso/deficiência , Terminações Pré-Sinápticas/enzimologia , Vesículas Sinápticas/enzimologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células Cultivadas , Feminino , Hipocampo/ultraestrutura , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Proteólise , Proteostase , Transdução de Sinais , Vesículas Sinápticas/genética , Vesículas Sinápticas/ultraestrutura , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Proteína 2 Associada à Membrana da Vesícula/metabolismo
4.
Sci Data ; 7(1): 146, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415162

RESUMO

Proteasomes are the main producers of antigenic peptides presented to CD8+ T cells. They can cut proteins and release their fragments or recombine non-contiguous fragments thereby generating novel sequences, i.e. spliced peptides. Understanding which are the driving forces and the sequence preferences of both reactions can streamline target discovery in immunotherapies against cancer, infection and autoimmunity. Here, we present a large database of spliced and non-spliced peptides generated by proteasomes in vitro, which is available as simple CSV file and as a MySQL database. To generate the database, we performed in vitro digestions of 55 unique synthetic polypeptide substrates with different proteasome isoforms and experimental conditions. We measured the samples using three mass spectrometers, filtered and validated putative peptides, identified 22,333 peptide product sequences (15,028 spliced and 7,305 non-spliced product sequences). Our database and datasets have been deposited to the Mendeley (doi:10.17632/nr7cs764rc.1) and PRIDE (PXD016782) repositories. We anticipate that this unique database can be a valuable source for predictors of proteasome-catalyzed peptide hydrolysis and splicing, with various future translational applications.


Assuntos
Bases de Dados de Proteínas , Complexo de Endopeptidases do Proteassoma/fisiologia , Isoformas de Proteínas/química , Antígenos/química , Linfócitos T CD8-Positivos , Humanos , Peptídeos/química
5.
Eur J Immunol ; 50(2): 270-283, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31729751

RESUMO

Dissecting the different steps of the processing and presentation of tumor-associated antigens is a key aspect of immunotherapies enabling to tackle the immune response evasion attempts of cancer cells. The immunodominant glycoprotein gp100209-217 epitope, which is liberated from the melanoma differentiation antigen gp100PMEL17 , is part of immunotherapy trials. By analyzing different human melanoma cell lines, we here demonstrate that a pool of N-terminal extended peptides sharing the common minimal epitope is generated by melanoma proteasome subtypes. In vitro and in cellulo experiments indicate that ER-resident aminopeptidase 1 (ERAP1)-but not ERAP2-defines the processing of this peptide pool thereby modulating the T-cell recognition of melanoma cells. By combining the outcomes of our studies and others, we can sketch the complex processing and endogenous presentation pathway of the gp100209-217 -containing epitope/peptides, which are produced by proteasomes and are translocated to the vesicular compartment through different pathways, where the precursor peptides that reach the endoplasmic reticulum are further processed by ERAP1. The latter step enhances the activation of epitope-specific T lymphocytes, which might be a target to improve the efficiency of anti-melanoma immunotherapy.


Assuntos
Aminopeptidases/imunologia , Apresentação do Antígeno/imunologia , Retículo Endoplasmático/imunologia , Epitopos de Linfócito T/imunologia , Melanoma/imunologia , Melanoma/terapia , Antígenos de Histocompatibilidade Menor/imunologia , Antígenos de Neoplasias , Linhagem Celular Tumoral , Células HeLa , Humanos , Fatores Imunológicos/imunologia , Imunoterapia/métodos , Peptídeos/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Linfócitos T/imunologia
6.
Food Res Int ; 123: 503-515, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31284999

RESUMO

Essential precursors of the cocoa-specific roasting-flavor notes were formed during proteolysis of the cocoa vicilin-class(7S) globulin by a mixture of cocoa aspartic protease and carboxypeptidase. These could be partially purified by ligand-exchange chromatography. Many constituents of this peptide fraction were destroyed by post-treatment with pepsin, but the cocoa-specific flavor-precursor peptides were largely resistant against pepsin treatment. However, these peptides were not generated when the cocoa vicilin-class(7S) globulin was digested with a mixture of pepsin and carboxypeptidase. By nano-liquid chromatography mass spectrometry, the peptide composition of these peptide fractions were compared in order to identify the putative precursors of the cocoa-specific flavor components. These peptides were assigned to five regions of the cocoa vicilin-class(7S) globulin. Analyzing the roasting products of the different protein fractions by headspace solid-phase microextraction, followed by gas chromatography mass spectrometry, eight volatile compounds were detected, whose occurrence correlated with the sensory detection of cocoa-specific flavor notes.


Assuntos
Cacau/química , Chocolate/análise , Peptídeos/análise , Sequência de Aminoácidos , Comportamento do Consumidor , Manipulação de Alimentos , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Espectrometria de Massas , Odorantes/análise , Proteínas de Plantas/metabolismo , Proteínas de Armazenamento de Sementes/metabolismo , Sementes/química , Sensibilidade e Especificidade , Microextração em Fase Sólida , Espectrometria de Massas em Tandem , Paladar , Compostos Orgânicos Voláteis/análise
7.
Methods Mol Biol ; 1988: 15-29, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31147929

RESUMO

Mass spectrometry (MS) is today one of the most important analytical techniques in biosciences. The development of electro spray ionization (ESI) as a gentle method, in which molecules are not destroyed, has revolutionized the analytic of peptides. MS is an ideal technique for detection and analysis of peptides generated by purified 20S proteasomes in in vitro experiments. This approach also provides a convenient and sensitive way to monitor the different processing characteristics of proteasome isoforms. The combination of high performance liquid chromatography (HPLC) with ESI-MS allows for the analysis of complex samples with separation in their specific constituents by LC and their subsequent detection by MS.


Assuntos
Antígenos/análise , Espectrometria de Massas/métodos , Peptídeos/análise , Complexo de Endopeptidases do Proteassoma/química , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Análise de Dados , Humanos , Peptídeos/química , Espectrometria de Massas por Ionização por Electrospray
8.
J Biol Chem ; 294(19): 7740-7754, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-30914481

RESUMO

An efficient immunosurveillance of CD8+ T cells in the periphery depends on positive/negative selection of thymocytes and thus on the dynamics of antigen degradation and epitope production by thymoproteasome and immunoproteasome in the thymus. Although studies in mouse systems have shown how thymoproteasome activity differs from that of immunoproteasome and strongly impacts the T cell repertoire, the proteolytic dynamics and the regulation of human thymoproteasome are unknown. By combining biochemical and computational modeling approaches, we show here that human 20S thymoproteasome and immunoproteasome differ not only in the proteolytic activity of the catalytic sites but also in the peptide transport. These differences impinge upon the quantity of peptide products rather than where the substrates are cleaved. The comparison of the two human 20S proteasome isoforms depicts different processing of antigens that are associated to tumors and autoimmune diseases.


Assuntos
Apresentação do Antígeno , Linfócitos T CD8-Positivos/enzimologia , Simulação por Computador , Complexo de Endopeptidases do Proteassoma/química , Células A549 , Animais , Linfócitos T CD8-Positivos/imunologia , Catálise , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/imunologia , Células THP-1
9.
Mol Cell ; 70(5): 881-893.e3, 2018 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-29883607

RESUMO

The assembly of ribosomal subunits is an essential prerequisite for protein biosynthesis in all domains of life. Although biochemical and biophysical approaches have advanced our understanding of ribosome assembly, our mechanistic comprehension of this process is still limited. Here, we perform an in vitro reconstitution of the Escherichia coli 50S ribosomal subunit. Late reconstitution products were subjected to high-resolution cryo-electron microscopy and multiparticle refinement analysis to reconstruct five distinct precursors of the 50S subunit with 4.3-3.8 Å resolution. These assembly intermediates define a progressive maturation pathway culminating in a late assembly particle, whose structure is more than 96% identical to a mature 50S subunit. Our structures monitor the formation and stabilization of structural elements in a nascent particle in unprecedented detail and identify the maturation of the rRNA-based peptidyl transferase center as the final critical step along the 50S assembly pathway.


Assuntos
Escherichia coli/metabolismo , RNA Bacteriano/metabolismo , RNA Ribossômico 23S/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/ultraestrutura , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , RNA Bacteriano/genética , RNA Bacteriano/ultraestrutura , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/ultraestrutura , Subunidades Ribossômicas Maiores de Bactérias/genética , Subunidades Ribossômicas Maiores de Bactérias/ultraestrutura , Relação Estrutura-Atividade
10.
Food Chem ; 255: 209-215, 2018 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-29571468

RESUMO

Sensory evaluation of roasted cocoa not only revealed cocoa-specific but also more or less pronounced nutty-specific aroma notes. Essential precursors of the corresponding volatile compounds could be generated in vitro by proteolysis of the cocoa vicilin-class(7S) globulin using a mixture of cocoa aspartic protease and cocoa carboxypeptidase. Since both proteases have rather different pH optima (pH 3.5 and 5.5-6.0, respectively), we have investigated the pH-dependency of proteolysis of this protein substrate in the presence of both proteases, the liberation of free amino acids and the generated aroma potential. Our findings revealed that the precursors of the nutty aroma notes were generated at higher pH-values (pH 4.8-5.6) than the cocoa-specific precursor peptides (pH 4.4-5.2). Longer peptide fragments of the cocoa vicilin were formed by proteolysis at pH 5.2 than at pH 4.8. Furthermore, our findings indicated that cocoa-vicilin derived peptides are essential precursors of both the cocoa- and the nutty-specific aroma components.


Assuntos
Cacau/química , Odorantes , Proteínas de Plantas/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Ácido Aspártico Proteases/metabolismo , Carboxipeptidases/metabolismo , Manipulação de Alimentos/métodos , Concentração de Íons de Hidrogênio , Nozes , Peptídeos , Proteólise , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/metabolismo , Compostos Orgânicos Voláteis/metabolismo
11.
Cell Rep ; 20(5): 1242-1253, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28768206

RESUMO

Proteasome-catalyzed peptide splicing (PCPS) generates peptides that are presented by MHC class I molecules, but because their identification is challenging, the immunological relevance of spliced peptides remains unclear. Here, we developed a reverse immunology-based multi-level approach to identify proteasome-generated spliced epitopes. Applying this strategy to a murine Listeria monocytogenes infection model, we identified two spliced epitopes within the secreted bacterial phospholipase PlcB that primed antigen-specific CD8+ T cells in L. monocytogenes-infected mice. While reacting to the spliced epitopes, these CD8+ T cells failed to recognize the non-spliced peptide parts in the context of their natural flanking sequences. Thus, we here show that PCPS expands the CD8+ T cell response against L. monocytogenes by exposing spliced epitopes on the cell surface. Moreover, our multi-level strategy opens up opportunities to systematically investigate proteins for spliced epitope candidates and thus strategies for immunotherapies or vaccine design.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Animais , Linfócitos T CD8-Positivos/patologia , Epitopos de Linfócito T/genética , Listeriose/genética , Listeriose/patologia , Camundongos , Complexo de Endopeptidases do Proteassoma/genética
12.
Exp Mol Med ; 48(11): e270, 2016 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-27833096

RESUMO

By changing the relative abundance of generated antigenic peptides through alterations in the proteolytic activity, interferon (IFN)-γ-induced immunoproteasomes influence the outcome of CD8+ cytotoxic T lymphocyte responses. In the present study, we investigated the effects of hepatitis C virus (HCV) infection on IFN-γ-induced immunoproteasome expression using a HCV infection cell culture system. We found that, although IFN-γ induced the transcriptional expression of mRNAs encoding the ß1i/LMP2, ß2i/MECL-1 and ß5i/LMP7 immunoproteasome subunits, the formation of immunoproteasomes was significantly suppressed in HCV-infected cells. This finding indicated that immunoproteasome induction was impaired at the translational or posttranslational level by HCV infection. Gene silencing studies showed that the suppression of immunoproteasome induction is essentially dependent on protein kinase R (PKR). Indeed, the generation of a strictly immunoproteasome-dependent cytotoxic T lymphocyte epitope was impaired in in vitro processing experiments using isolated 20S proteasomes from HCV-infected cells and was restored by the silencing of PKR expression. In conclusion, our data point to a novel mechanism of immune regulation by HCV that affects the antigen-processing machinery through the PKR-mediated suppression of immunoproteasome induction in infected cells.


Assuntos
Hepacivirus/imunologia , Hepatite C/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , eIF-2 Quinase/imunologia , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Hepacivirus/genética , Humanos , Interferon gama/imunologia , Complexo de Endopeptidases do Proteassoma/genética , RNA Mensageiro/genética
13.
Eur J Immunol ; 46(5): 1109-18, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26909514

RESUMO

CD8(+) T cells responding to infection recognize pathogen-derived epitopes presented by MHC class-I molecules. While most of such epitopes are generated by proteasome-mediated antigen cleavage, analysis of tumor antigen processing has revealed that epitopes may also derive from proteasome-catalyzed peptide splicing (PCPS). To determine whether PCPS contributes to epitope processing during infection, we analyzed the fragments produced by purified proteasomes from a Listeria monocytogenes polypeptide. Mass spectrometry identified a known H-2K(b) -presented linear epitope (LLO296-304 ) in the digests, as well as four spliced peptides that were trimmed by ERAP into peptides with in silico predicted H-2K(b) binding affinity. These spliced peptides, which displayed sequence similarity with LLO296-304 , bound to H-2K(b) molecules in cellular assays and one of the peptides was recognized by CD8(+) T cells of infected mice. This spliced epitope differed by one amino acid from LLO296-304 and double staining with LLO296-304 - and spliced peptide-folded MHC multimers showed that LLO296-304 and its spliced variant were recognized by the same CD8(+) T cells. Thus, PCPS multiplies the variety of peptides that is processed from an antigen and leads to the production of epitope variants that can be recognized by cross-reacting pathogen-specific CD8(+) T cells. Such mechanism may reduce the chances for pathogen immune evasion.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína , Animais , Apresentação do Antígeno/imunologia , Simulação por Computador , Epitopos de Linfócito T/química , Antígenos de Histocompatibilidade Classe I/imunologia , Evasão da Resposta Imune , Listeria monocytogenes/química , Espectrometria de Massas , Camundongos , Peptídeos/química , Peptídeos/imunologia , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/química
14.
Food Chem ; 192: 706-13, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26304401

RESUMO

Essential precursors of the cocoa-specific aroma notes are formed during fermentation of the cocoa beans by acid-induced proteolysis. It has been shown that, in addition to free amino acids, hydrophilic peptides derived from the vicilin-class(7S) globular storage protein are required for the generation of the cocoa-specific aroma notes during the roasting process. To identify those peptides responsible for the generation of the cocoa-specific aroma components, we have developed a procedure for the fractionation of the aroma precursor extract from well-fermented cocoa beans by ligand-exchange and subsequent Sephadex-LH20 chromatography. The cocoa-specific aroma precursor fractions were characterised by matrix-assisted laser-desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) and the determination of their amino acid sequences by electrospray ionisation mass spectrometry (ESI-MS/MS).


Assuntos
Cacau/química , Fermentação , Odorantes/análise , Peptídeos/análise , Aminoácidos/análise , Cacau/metabolismo , Humanos , Peptídeos/metabolismo , Extratos Vegetais/química , Sementes/química , Olfato , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem
15.
J Biol Chem ; 290(51): 30417-28, 2015 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-26507656

RESUMO

MHC class I-restricted epitopes, which carry a tumor-specific mutation resulting in improved MHC binding affinity, are preferred T cell receptor targets in innovative adoptive T cell therapies. However, T cell therapy requires efficient generation of the selected epitope. How such mutations may affect proteasome-mediated antigen processing has so far not been studied. Therefore, we analyzed by in vitro experiments the effect on antigen processing and recognition of a T210M exchange, which previously had been introduced into the melanoma gp100209-217 tumor epitope to improve the HLA-A*02:01 binding and its immunogenicity. A quantitative analysis of the main steps of antigen processing shows that the T210M exchange affects proteasomal cleavage site usage within the mutgp100201-230 polypeptide, leading to the generation of an unique set of cleavage products. The T210M substitution qualitatively affects the proteasome-catalyzed generation of spliced and non-spliced peptides predicted to bind HLA-A or -B complexes. The T210M substitution also induces an enhanced production of the mutgp100209-217 epitope and its N-terminally extended peptides. The T210M exchange revealed no effect on ERAP1-mediated N-terminal trimming of the precursor peptides. However, mutant N-terminally extended peptides exhibited significantly increased HLA-A*02:01 binding affinity and elicited CD8(+) T cell stimulation in vitro similar to the wtgp100209-217 epitope. Thus, our experiments demonstrate that amino acid exchanges within an epitope can result in the generation of an altered peptide pool with new antigenic peptides and in a wider CD8(+) T cell response also towards N-terminally extended versions of the minimal epitope.


Assuntos
Substituição de Aminoácidos , Apresentação do Antígeno/imunologia , Epitopos de Linfócito T/imunologia , Antígeno HLA-A2/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Antígeno gp100 de Melanoma/imunologia , Apresentação do Antígeno/genética , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Epitopos de Linfócito T/genética , Antígeno HLA-A2/genética , Antígenos HLA-B/genética , Antígenos HLA-B/imunologia , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Antígeno gp100 de Melanoma/genética
16.
Eur J Immunol ; 45(12): 3257-68, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26399368

RESUMO

The immunodominant MART-1(26(27)-35) epitope, liberated from the differentiation antigen melanoma antigen recognized by T cells/melanoma antigen A (MART-1/Melan-A), has been frequently targeted in melanoma immunotherapy, but with limited clinical success. Previous studies suggested that this is in part due to an insufficient peptide supply and epitope presentation, since proteasomes containing the immunosubunits ß5i/LMP7 (LMP, low molecular weight protein) or ß1i/LMP2 and ß5i/LMP7 interfere with MART-1(26-35) epitope generation in tumor cells. Here, we demonstrate that in addition the IFN-γ-inducible proteasome subunit ß2i/MECL-1 (multicatalytic endopeptidase complex-like 1), proteasome activator 28 (PA28), and ER-resident aminopeptidase 1 (ERAP1) impair MART-1(26-35) epitope generation. ß2i/MECL-1 and PA28 negatively affect C- and N-terminal cleavage and therefore epitope liberation from the proteasome, whereas ERAP1 destroys the MART-1(26-35) epitope by overtrimming activity. Constitutive expression of PA28 and ERAP1 in melanoma cells indicate that both interfere with MART-1(26-35) epitope generation even in the absence of IFN-γ. In summary, our results provide first evidence that activities of different antigen-processing components contribute to an inefficient MART-1(26-35) epitope presentation, suggesting the tumor cell's proteolytic machinery might have an important impact on the outcome of epitope-specific immunotherapies.


Assuntos
Aminopeptidases/fisiologia , Epitopos/imunologia , Melanoma/imunologia , Proteínas Musculares/fisiologia , Proteínas de Neoplasias/imunologia , Complexo de Endopeptidases do Proteassoma/fisiologia , Linfócitos T/imunologia , Linhagem Celular Tumoral , Cisteína Endopeptidases/fisiologia , Humanos , Antígenos de Histocompatibilidade Menor
17.
Eur J Immunol ; 44(12): 3508-21, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25231383

RESUMO

Immunoproteasomes are considered to be optimised to process Ags and to alter the peptide repertoire by generating a qualitatively different set of MHC class I epitopes. Whether the immunoproteasome at the biochemical level, influence the quality rather than the quantity of the immuno-genic peptide pool is still unclear. Here, we quantified the cleavage-site usage by human standard- and immunoproteasomes, and proteasomes from immuno-subunit-deficient mice, as well as the peptides generated from model polypeptides. We show in this study that the different proteasome isoforms can exert significant quantitative differences in the cleavage-site usage and MHC class I restricted epitope production. However, independent of the proteasome isoform and substrates studied, no evidence was obtained for the abolishment of the specific cleavage-site usage, or for differences in the quality of the peptides generated. Thus, we conclude that the observed differences in MHC class I restricted Ag presentation between standard- and immunoproteasomes are due to quantitative differences in the proteasome-generated antigenic peptides.


Assuntos
Apresentação do Antígeno/fisiologia , Antígenos de Histocompatibilidade Classe I/imunologia , Peptídeos/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Proteólise , Animais , Linhagem Celular Transformada , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Isoenzimas/genética , Isoenzimas/imunologia , Camundongos , Camundongos Mutantes , Peptídeos/genética , Complexo de Endopeptidases do Proteassoma/genética , Especificidade por Substrato/genética , Especificidade por Substrato/imunologia
18.
Ann Hepatol ; 13(4): 429-38, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24927614

RESUMO

BACKGROUND: The 20S proteasome is the proteolytic core of the major intracellular protein degradative system, the ubiquitin-proteasome system. Since little is known about proteasomes of human liver, we have investigated the proteasome spectrum in adult human liver. MATERIAL AND METHODS: 20S proteasomes were chromatographically purified from adult human liver and from HuH7 cells. They were divided into subpopulations and subtypes and characterized with regard to their proteolytic activities using short fluorogenic oligo- and long poly-peptide substrates. Their subunit composition was studied by immunoblotting. RESULTS: Proteasomes from adult human liver tissue can be separated into three subpopulations (I, II, III), each of which is composed of several subtypes, which total to a spectrum of 14 different subtypes. Two minor subtypes contain only the immuno-subunits ß1i and ß5i but not their standard counterparts; all others are intermediate subtypes containing ß1 and ß5 standard- and ß1i and ß5i immuno-subunits in various compositions. With regard to the proteolytic activities we observed that a decreasing content of subunit ß1i in the subtypes goes along with a decreasing ratio of chymotrypsin-like/caspase-like activity, whereas the degradation rate of a 30 mer polypeptide substrate increased with decreasing ß1i content. By comparison, 20S proteasomes from HuH7 cells do not contain immuno-subunits but are pure standard proteasomes, which can be separated into three subtypes. CONCLUSION: These findings suggest that adult human liver contains a spectrum of 14 different 20S proteasome subtypes with different enzymatic properties reflecting most probably an adaptive response of liver cell functions to challenging factors during lifetime.


Assuntos
Eritrócitos/enzimologia , Fígado/enzimologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Baço/enzimologia , Linhagem Celular , Eletroforese , Humanos
19.
Age (Dordr) ; 36(1): 57-72, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23690132

RESUMO

Aging induces alterations of tissue protein homoeostasis. To investigate one of the major systems catalysing intracellular protein degradation we have purified 20S proteasomes from rat liver of young (2 months) and aged (23 months) animals and separated them into three subpopulations containing different types of intermediate proteasomes with standard- and immuno-subunits. The smallest subpopulation ΙΙΙ and the major subpopulation Ι comprised proteasomes containing immuno-subunits ß1i and ß5i beside small amounts of standard-subunits, whereas proteasomes of subpopulation ΙΙ contained only ß5i beside standard-subunits. In favour of a relative increase of the major subpopulation Ι, subpopulation ΙΙ and ΙΙΙ were reduced for about 55 % and 80 %, respectively, in aged rats. Furthermore, in all three 20S proteasome subpopulations from aged animals standard-active site subunits were replaced by immuno-subunits. Overall, this transformation resulted in a relative increase of immuno-subunit-containing proteasomes, paralleled by reduced activity towards short fluorogenic peptide substrates. However, depending on the substrate their hydrolysing activity of long polypeptide substrates was significantly higher or unchanged. Furthermore, our data revealed an altered MHC class I antigen-processing efficiency of 20S proteasomes from liver of aged rats. We therefore suggest that the age-related intramolecular alteration of hepatic proteasomes modifies its cleavage preferences without a general decrease of its activity. Such modifications could have implications on protein homeostasis as well as on MHC class I antigen presentation as part of the immunosenescence process.


Assuntos
Envelhecimento/metabolismo , Fígado/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Imuno-Histoquímica , Masculino , Ratos , Ratos Wistar
20.
Methods Mol Biol ; 960: 15-29, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23329475

RESUMO

Mass spectrometry (MS) is today one of the most important analytical techniques in biosciences. The development of electro spray ionization (ESI) as a gentle ionization method, in which molecules are not destroyed, has revolutionized the analytic of peptides. MS is an ideal technique for detection and analysis of peptides generated by in vitro experiments using purified 20S proteasomes. It also provides a convenient and sensitive way to monitor the processing activity of enzymes. The combination of high performance liquid chromatography (HPLC) with ESI-MS allows the analysis of complex samples with separation in their specific constituents by LC and their subsequent detection by MS.


Assuntos
Antígenos/química , Fragmentos de Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Antígenos/metabolismo , Cromatografia Líquida de Alta Pressão , Epitopos/química , Epitopos/metabolismo , Antígenos HLA-A/imunologia , Proteólise
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