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1.
J Immunol Res ; 2021: 6234836, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34869783

RESUMO

Immunomodulation of airway hyperreactivity by excretory-secretory (ES) products of the first larval stage (L1) of the gastrointestinal nematode Trichuris suis is reported by us and others. Here, we aimed to identify the proteins accounting for the modulatory effects of the T. suis L1 ES proteins and studied six selected T. suis L1 proteins for their immunomodulatory efficacy in a murine OVA-induced allergic airway disease model. In particular, an enzymatically active T. suis chitinase mediated amelioration of clinical signs of airway hyperreactivity, primarily associated with suppression of eosinophil recruitment into the lung, the associated chemokines, and increased numbers of RELMα + interstitial lung macrophages. While there is no indication of T. suis chitinase directly interfering with dendritic cell activation or antigen presentation to CD4 T cells, treatment of allergic mice with the worm chitinase influenced the hosts' own chitinase activity in the inflamed lung. The three-dimensional structure of the T. suis chitinase as determined by high-resolution X-ray crystallography revealed high similarities to mouse acidic mammalian chitinase (AMCase) but a unique ability of T. suis chitinase to form dimers. Our data indicate that the structural similarities between the parasite and host chitinase contribute to the disease-ameliorating effect of the helminth-derived chitinase on allergic lung inflammation.

2.
Cells ; 9(11)2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33153033

RESUMO

Spinal Muscular Atrophy (SMA) is a neuromuscular disease caused by decreased levels of the survival of motoneuron (SMN) protein. Post-translational mechanisms for regulation of its stability are still elusive. Thus, we aimed to identify regulatory phosphorylation sites that modulate function and stability. Our results show that SMN residues S290 and S292 are phosphorylated, of which SMN pS290 has a detrimental effect on protein stability and nuclear localization. Furthermore, we propose that phosphatase and tensin homolog (PTEN), a novel phosphatase for SMN, counteracts this effect. In light of recent advancements in SMA therapies, a significant need for additional approaches has become apparent. Our study demonstrates S290 as a novel molecular target site to increase the stability of SMN. Characterization of relevant kinases and phosphatases provides not only a new understanding of SMN function, but also constitutes a novel strategy for combinatorial therapeutic approaches to increase the level of SMN in SMA.


Assuntos
Aminoácidos/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/química , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fosforilação , Fosfosserina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Estabilidade Proteica , Proteólise , Relação Estrutura-Atividade
3.
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
4.
Artigo em Inglês | MEDLINE | ID: mdl-30392576

RESUMO

Lipoxygenases are lipid peroxidizing enzymes, which frequently occur in higher plants and animals. In bacteria, these enzymes are rare and have been introduced via horizontal gene transfer. Since viruses function as horizontal gene transfer vectors and since lipoxygenases may be helpful for releasing assembled virus particles from host cells we explored whether these enzymes may actually occur in viruses. For this purpose we developed a four-step in silico screening strategy and searching the publically available viral genomes for lipoxygenase-like sequences we detected a single functional gene in the genome of a mimivirus infecting Acantamoeba polyphaga. The primary structure of this protein involved two putative metal ligand clusters but the recombinant enzyme did neither contain iron nor manganese. Most importantly, it did not exhibit lipoxygenase activity. These data suggests that this viral lipoxygenase-like sequence does not encode a functional lipoxygenase and that these enzymes do not occur in viruses.


Assuntos
Expressão Gênica , Lipoxigenase , Mimiviridae , Proteínas Virais , Acanthamoeba/virologia , Lipoxigenase/química , Lipoxigenase/genética , Lipoxigenase/isolamento & purificação , Mimiviridae/enzimologia , Mimiviridae/genética , Oxirredução , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação
6.
Artigo em Inglês | MEDLINE | ID: mdl-30131945

RESUMO

Ascariasis is a widespread soil-transmitted helminth infection caused by the intestinal roundworm Ascaris lumbricoides in humans, and the closely related Ascaris suum in pigs. Progress has been made in understanding interactions between helminths and host immune cells, but less is known concerning the interactions of parasitic nematodes and the host microbiota. As the host microbiota represents the direct environment for intestinal helminths and thus a considerable challenge, we studied nematode products, including excretory-secretory products (ESP) and body fluid (BF), of A. suum to determine their antimicrobial activities. Antimicrobial activities against gram-positive and gram-negative bacterial strains were assessed by the radial diffusion assay, while effects on biofilm formation were assessed using the crystal violet static biofilm and macrocolony assays. In addition, bacterial neutralizing activity was studied by an agglutination assay. ESP from different A. suum life stages (in vitro-hatched L3, lung-stage L3, L4, and adult) as well as BF from adult males were analyzed by mass spectrometry. Several proteins and peptides with known and predicted roles in nematode immune defense were detected in ESP and BF samples, including members of A. suum antibacterial factors (ASABF) and cecropin antimicrobial peptide families, glycosyl hydrolase enzymes such as lysozyme, as well as c-type lectin domain-containing proteins. Native, unconcentrated nematode products from intestine-dwelling L4-stage larvae and adults displayed broad-spectrum antibacterial activity. Additionally, adult A. suum ESP interfered with biofilm formation by Escherichia coli, and caused bacterial agglutination. These results indicate that A. suum uses a variety of factors with broad-spectrum antibacterial activity to affirm itself within its microbe-rich environment in the gut.


Assuntos
Antibacterianos/metabolismo , Antibiose , Ascaris suum/metabolismo , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Testes de Aglutinação , Animais , Antibacterianos/análise , Ascaris suum/química , Violeta Genciana/análise , Proteínas de Helminto/análise , Proteínas de Helminto/metabolismo , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Coloração e Rotulagem , Suínos
7.
Biochim Biophys Acta Gen Subj ; 1862(4): 946-957, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29288125

RESUMO

The Guanine-rich RNA sequence binding factor 1 (GRSF1) is a member of the heterogeneous nuclear ribonucleoprotein F/H family and has been implicated in RNA processing, RNA transport and translational regulation. Amino acid alignments and homology modeling suggested the existence of three distinct RNA-binding domains and two auxiliary domains. Unfortunately, little is known about the molecular details of GRSF1/RNA interactions. To explore the RNA-binding mechanisms we first expressed full-length human GRSF1 and several truncation mutants, which include the three separated qRRM domains in E. coli, purified the recombinant proteins and quantified their RNA-binding affinity by RNA electrophoretic mobility shift assays. The expression levels varied between 1 and 10mg purified protein per L bacterial liquid culture and for full-length human GRSF1 a binding constant (KD-value) of 0.5µM was determined. In addition, our mechanistic experiments with different truncation mutants allowed the following conclusions: i) Deletion of either of the three RNA-binding domains impaired the RNA-binding affinity suggesting that the simultaneous presence of the three domains is essential for high-affinity RNA-binding. ii) Deletion of the Ala-rich auxiliary domain did hardly affect RNA-binding. Thus, this structural subunit may not be involved in RNA interaction. iii) Deletion of the acidic auxiliary domain improved the RNA-binding suggesting a regulatory role for this structural motif. iv) The isolated RNA-binding domains did not exhibit sizeable RNA-binding affinities. Taken together these data suggest that a cooperative interaction of the three qRRMs is required for high affinity RNA-binding.


Assuntos
Mutação , Proteínas de Ligação a Poli(A)/genética , Motivos de Ligação ao RNA/genética , RNA/genética , Sequência de Aminoácidos , Sítios de Ligação/genética , Ligação Competitiva , Células HEK293 , Humanos , Cinética , Proteínas de Ligação a Poli(A)/metabolismo , Ligação Proteica , RNA/metabolismo
8.
Sci Rep ; 7: 43718, 2017 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-28276434

RESUMO

Osteopontin is a pleiotropic cytokine that is involved in several diseases including multiple sclerosis. Secreted osteopontin is cleaved by few known proteases, modulating its pro-inflammatory activities. Here we show by in vitro experiments that secreted osteopontin can be processed by extracellular proteasomes, thereby producing fragments with novel chemotactic activity. Furthermore, osteopontin reduces the release of proteasomes in the extracellular space. The latter phenomenon seems to occur in vivo in multiple sclerosis, where it reflects the remission/relapse alternation. The extracellular proteasome-mediated inflammatory pathway may represent a general mechanism to control inflammation in inflammatory diseases.


Assuntos
Esclerose Múltipla/metabolismo , Osteopontina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Sequência de Aminoácidos , Quimiotaxia/imunologia , Células Endoteliais/metabolismo , Espaço Extracelular/metabolismo , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/metabolismo , Humanos , Linfócitos/imunologia , Linfócitos/metabolismo , Modelos Moleculares , Esclerose Múltipla/imunologia , Osteopontina/química , Osteopontina/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Conformação Proteica , Relação Estrutura-Atividade
9.
Data Brief ; 8: 700-8, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27508221

RESUMO

The data provide information in support of the research article, "The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors" (Janek et al., 2016) [1]. Three different protein substrates were partially digested with the aspartic protease isolated from cocoa beans and commercial pepsin, respectively. The obtained peptide fragments were analyzed by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS/MS) and identified using the MASCOT server. The N- and C-terminal ends of the peptide fragments were used to identify the corresponding in-vitro cleavage sites by comparison with the amino acid sequences of the substrate proteins. The same procedure was applied to identify the cleavage sites used by the cocoa aspartic protease during cocoa fermentation starting from the published amino acid sequences of oligopeptides isolated from fermented cocoa beans.

10.
Food Chem ; 211: 320-8, 2016 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-27283639

RESUMO

Particular peptides generated from the vicilin-class(7S) globulin of the cocoa beans by acid-induced proteolysis during cocoa fermentation are essential precursors of the cocoa-specific aroma notes. As revealed by in vitro studies, the formation of the cocoa-specific aroma precursors depends on the particular cleavage specificity of the cocoa aspartic protease, which cannot be substituted by pepsin. Therefore, we have investigated the effects of aspartic protease inhibitors on both enzymes and comparatively studied their cleavage specificities using different protein substrates and MALDI-TOF mass spectrometric analyses of the generated oligopeptides. Three classes of cleavage sites have been identified and characterized: (I) sequences exclusively cleaved by the cocoa enzyme, (II) sequences cleaved by both pepsin and the cocoa enzyme, and (III) those cleaved exclusively by pepsin. In contrast to most aspartic proteases from other origins, basic amino acid residues, particularly lysine, were found to be abundant in the specific cleavage sites of the cocoa enzyme.


Assuntos
Ácido Aspártico Proteases/metabolismo , Cacau/química , Cacau/enzimologia , Sementes/enzimologia , Olfato , Sequência de Aminoácidos , Animais , Ácido Aspártico Endopeptidases/análise , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Ácido Aspártico Proteases/análise , Ácido Aspártico Proteases/genética , Cacau/genética , Chocolate , Fermentação , Sementes/química , Sementes/genética , Suínos
11.
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
12.
J Pept Sci ; 19(9): 588-97, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23893543

RESUMO

Proteasomes are cellular proteases involved in the degradation of numerous cellular proteins. The 20S proteasome is a cylindrical 28-mer protein complex composed of two outer heptameric α-rings forming the entrance for the protein substrate and two inner heptameric ß-rings carrying the catalytic sites. Numerous in vitro studies have provided evidence that the 20S proteasome may degrade peptides of various lengths and even unfolded full-length polypeptide chains. However, a direct demonstration that the 20S proteasome may also cleave surface-attached immobilized peptides is lacking so far. To this end, we used a model system by coupling peptides from different source proteins covalently to the surface of glass beads and applied nanoLC/MS analysis to monitor the generation of proteolytic fragments in the presence of the 20S proteasome. Detectable amounts of cleavage products occurred within a few minutes indicating a much higher cleavage rate than observed with the same substrates in solution. Our finding lends support to the idea that proteasomes may directly degrade segments of membrane-bound proteins protruding into the aqueous phase.


Assuntos
Proteínas Imobilizadas/química , Complexo de Endopeptidases do Proteassoma/química , Sequência de Aminoácidos , Toxinas Bacterianas/química , Proteínas de Choque Térmico/química , Proteínas Hemolisinas/química , Humanos , Proteínas Imediatamente Precoces/química , Dados de Sequência Molecular , Ovalbumina/química , Fragmentos de Peptídeos/química , Proteólise , Técnicas de Síntese em Fase Sólida
13.
Mol Cell Proteomics ; 11(10): 1008-23, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22822185

RESUMO

Proteasome-catalyzed peptide splicing (PCPS) represents an additional activity of mammalian 20S proteasomes recently identified in connection with antigen presentation. We show here that PCPS is not restricted to mammalians but that it is also a feature of yeast 20S proteasomes catalyzed by all three active site ß subunits. No major differences in splicing efficiency exist between human 20S standard- and immuno-proteasome or yeast 20S proteasome. Using H(2)(18)O to monitor the splicing reaction we also demonstrate that PCPS occurs via direct transpeptidation that slightly favors the generation of peptides spliced in cis over peptides spliced in trans. Splicing efficiency itself is shown to be controlled by proteasomal cleavage site preference as well as by the sequence characteristics of the spliced peptides. By use of kinetic data and quantitative analyses of PCPS obtained by mass spectrometry we developed a structural model with two PCPS binding sites in the neighborhood of the active Thr1.


Assuntos
Linfócitos B/metabolismo , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Linfócitos B/citologia , Biocatálise , Linhagem Celular Transformada , Cromatografia Líquida , Humanos , Dados de Sequência Molecular , Peptídeos/síntese química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
14.
J Immunol ; 189(2): 529-38, 2012 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-22706083

RESUMO

Control of human CMV (HCMV) infection depends on the cytotoxic activity of CD8(+) CTLs. The HCMV phosphoprotein (pp)65 is a major CTL target Ag and pp65(495-503) is an immunodominant CTL epitope in infected HLA-A*0201 individuals. As immunodominance is strongly determined by the surface abundance of the specific epitope, we asked for the components of the cellular Ag processing machinery determining the efficacy of pp65(495-503) generation, in particular, for the proteasome, cytosolic peptidases, and endoplasmic reticulum (ER)-resident peptidases. In vitro Ag processing experiments revealed that standard proteasomes and immunoproteasomes generate the minimal 9-mer peptide epitope as well as N-terminal elongated epitope precursors of different lengths. These peptides are largely degraded by the cytosolic peptidases leucine aminopeptidase and tripeptidyl peptidase II, as evidenced by increased pp65(495-503) epitope presentation after leucine aminopeptidase and tripeptidyl peptidase II knockdown. Additionally, with prolyl oligopeptidase and aminopeptidase B we identified two new Ag processing machinery components, which by destroying the pp65(495-503) epitope limit the availability of the specific peptide pool. In contrast to cytosolic peptidases, silencing of ER aminopeptidases 1 and 2 strongly impaired pp65(495-503)-specific T cell activation, indicating the importance of ER aminopeptidases in pp65(495-503) generation. Thus, cytosolic peptidases primarily interfere with the generation of the pp65(495-503) epitope, whereas ER-resident aminopeptidases enhance such generation. As a consequence, our experiments reveal that the combination of cytosolic and ER-resident peptidase activities strongly shape the pool of specific antigenic peptides and thus modulate MHC class I epitope presentation efficiency.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por Citomegalovirus/imunologia , Citosol/imunologia , Retículo Endoplasmático/imunologia , Epitopos de Linfócito T/metabolismo , Peptídeo Hidrolases/metabolismo , Apresentação do Antígeno/imunologia , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/virologia , Linhagem Celular , Infecções por Citomegalovirus/enzimologia , Infecções por Citomegalovirus/patologia , Citosol/enzimologia , Citosol/virologia , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/virologia , Epitopos de Linfócito T/biossíntese , Epitopos de Linfócito T/toxicidade , Células HeLa , Humanos , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Peptídeo Hidrolases/biossíntese , Peptídeo Hidrolases/toxicidade
15.
Mol Cell Proteomics ; 11(8): 467-77, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22556278

RESUMO

The post-translational modification of proteins with O-GlcNAc is involved in various cellular processes including signal transduction, transcription, translation, and nuclear transport. This transient protein modification enables cells or tissues to adapt to nutrient conditions or stress. O-Glycosylation of the 26 S proteasome ATPase subunit Rpt2 is known to influence the stability of proteins by reducing their proteasome-dependent degradation. In contrast, knowledge of the sites of O-GlcNAcylation on the subunits of the catalytic core of the 26 S proteasome, the 20 S proteasome, and the impact on proteasome activity is very limited. This is predominantly because O-GlcNAc modifications are often substoichiometric and because 20 S proteasomes represent a complex protein mixture of different subtypes. Therefore, identification of O-GlcNAcylation sites on proteasome subunits essentially requires effective enrichment strategies. Here we describe an adapted ß-elimination-based derivatization method of O-GlcNAc peptides using a novel biotin-cystamine tag. The specificity of the reaction was increased by differential isotopic labeling with either "light" biotin-cystamine or deuterated "heavy" biotin-cystamine. The enriched peptides were analyzed by LC-MALDI-TOF/TOF-MS and relatively quantified. The method was optimized using bovine α-crystallin and then applied to murine 20 S proteasomes isolated from spleen and brain and murine Hsp90 isolated from liver. Using this approach, we identified five novel and one known O-GlcNAc sites within the murine 20 S proteasome core complex that are located on five different subunits and in addition two novel O-GlcNAc sites on murine Hsp90ß, of which one corresponds to a previously described phosphorylation site.


Assuntos
Acetilglucosamina/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Biotina/metabolismo , Western Blotting , Radioisótopos de Carbono/metabolismo , Bovinos , Cromatografia Líquida , Cistamina/metabolismo , Glucose/farmacologia , Glicosilação/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , alfa-Cristalinas/metabolismo
16.
Mol Cell ; 38(6): 879-88, 2010 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-20620957

RESUMO

The proteasome, the central protease of eukaryotic cells, is composed of one core particle (CP) and one or two adjacent regulatory particles (RP), which contain multiple subunits. Several proteasome-dedicated chaperones govern the assembly of CP and RP, respectively. We sought for proteins that regulate final steps of RP-CP assembly in yeast and found Ecm29, a conserved HEAT-like repeat protein. Here, we show that Ecm29 controls the integrity of RP-CP assemblies. Ecm29 recognizes RP-CP species in which CP maturation is stalled due to the lack of distinct beta subunits. Reconstitution assays revealed that Ecm29 functions as scaffold protein during the remodeling of incompletely matured RP-CP assemblies into regular enzymes. Upon the completion of CP maturation, Ecm29 is degraded and RP-CP is dissociated.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/enzimologia , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/fisiologia , Subunidades Proteicas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
17.
PLoS Comput Biol ; 6(6): e1000830, 2010 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-20613855

RESUMO

The identification of proteasome-generated spliced peptides (PSP) revealed a new unpredicted activity of the major cellular protease. However, so far characterization of PSP was entirely dependent on the availability of patient-derived cytotoxic CD8+ T lymphocytes (CTL) thus preventing a systematic investigation of proteasome-catalyzed peptide splicing (PCPS). For an unrestricted PSP identification we here developed SpliceMet, combining the computer-based algorithm ProteaJ with in vitro proteasomal degradation assays and mass spectrometry. By applying SpliceMet for the analysis of proteasomal processing products of four different substrate polypeptides, derived from human tumor as well as viral antigens, we identified fifteen new spliced peptides generated by PCPS either by cis or from two separate substrate molecules, i.e., by trans splicing. Our data suggest that 20S proteasomes represent a molecular machine that, due to its catalytic and structural properties, facilitates the generation of spliced peptides, thereby providing a pool of qualitatively new peptides from which functionally relevant products may be selected.


Assuntos
Algoritmos , Biologia Computacional/métodos , Fragmentos de Peptídeos/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Software , Sequência de Aminoácidos , Antígenos Nucleares/química , Antígenos Nucleares/metabolismo , Autoantígenos/química , Autoantígenos/metabolismo , Linfócitos T CD8-Positivos , Simulação por Computador , Bases de Dados de Proteínas , Epitopos de Linfócito T , Fator 5 de Crescimento de Fibroblastos/química , Fator 5 de Crescimento de Fibroblastos/metabolismo , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem , Antígeno gp100 de Melanoma
18.
J Mol Biol ; 373(1): 1-10, 2007 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-17804016

RESUMO

The 20 S proteasomes are cylinder-shaped heteromeric dimers with a subunit configuration of alpha7, beta7, beta7, alpha7. Replacement of the three active site-containing standard beta-subunits (beta1, beta2, beta5) by immuno-beta-subunits (beta1i, beta2i, beta5i) results in formation of 20 S immuno-proteasomes, while only partial replacement leads to intermediate-type proteasomes. Synthesis of immuno-subunits can be induced by interferon-gamma, which causes a complete transformation of three subtypes of standard proteasomes into three subtypes of intermediate-type proteasomes in HeLa cells, a process that results in a change in the proteolytic activities of the enzymes. HeLa cells producing the proteasome beta1-subunit tagged with the Fc region-binding ZZ domain of protein A were grown in the presence of interferon-gamma. From these cells, we have purified 20 S proteasomes by using IgG-affinity resin and analysed them by 2D PAGE. Our study showed that subunit replacement can be confined to one half of the proteasome cylinder, resulting in the formation of intermediate-type proteasomes with "asymmetric" subunit composition. Analysis of proteasomes purified from the cytoplasm, nucleoplasm, and microsomes of HeLa S3 cells reveals that all three compartments are furnished with intermediate-type proteasomes of different subtype and subunit composition, exhibiting different specific proteolytic activities.


Assuntos
Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Conformação Proteica , Subunidades Proteicas/metabolismo , Animais , Sítios de Ligação , Células HeLa , Humanos , Interferon gama/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Subunidades Proteicas/química , Subunidades Proteicas/genética
19.
Proteomics ; 6(16): 4622-32, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16858736

RESUMO

Quantitative protein profiling is an essential part of proteomics and requires technologies that accurately, reproducibly, and comprehensively identify and quantify proteins. Over the past years, many quantitative proteomic methods have been developed. Here, 20S proteasome subtypes isolated from rat were compared by four approaches based on the combination of isotope-coded affinity tag (ICAT), 2-DE, LC and ESI and MALDI MS: (i) 2-DE, (ii) ICAT/2-DE MALDI-MS, (iii) ICAT/LC-ESI-MS, (iv) ICAT/LC-MALDI-MS. A definite qualitative advantage of 2-DE gels was the separation of all known protein species, the identification of cysteine sulfoxide of alpha-4 (RC6-IS) and N-terminal acetylation of several subunits. Furthermore, quantitative differences between the standard subunits beta-2, and beta-5 and their immunosubunits were only detected by 2-DE image analysis revealing a higher replacement of standard- by immuno-beta-subunits in subtype IV. It was obvious that for relative quantification only protein spot and mass peaks with a certain level of intensity displayed acceptable values of SD. However, ICAT in conjunction with LC/MALDI-MS was the most accurate method for quantification. The experimental data of this investigation are accessible via http://www.mpiib-berlin.mpg.de/2D-PAGE/.


Assuntos
Fígado/química , Complexo de Endopeptidases do Proteassoma/análise , Proteoma/análise , Acetilação , Sequência de Aminoácidos , Animais , Eletroforese em Gel Bidimensional , Marcação por Isótopo , Extratos Hepáticos/química , Dados de Sequência Molecular , Subunidades Proteicas/análise , Ratos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
20.
J Biol Chem ; 279(36): 37751-62, 2004 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-15210724

RESUMO

26 S proteasomes fulfill final steps in the ubiquitin-dependent degradation pathway by recognizing and hydrolyzing ubiquitylated proteins. As the 26 S proteasome mainly localizes to the nucleus in yeast, we addressed the question how this 2-MDa multisubunit complex is imported into the nucleus. 26 S proteasomes consist of a 20 S proteolytically active core and 19 S regulatory particles, the latter composed of two subcomplexes, namely the base and lid complexes. We have shown that 20 S core particles are translocated into the nucleus as inactive precursor complexes via the classic karyopherin alphabeta import pathway. Here, we provide evidence that nuclear import of base and lid complexes also depends on karyopherin alphabeta. Potential classic nuclear localization sequences (NLSs) of base subunits were analyzed. Rpn2 and Rpt2, a non-ATPase subunit and an ATPase subunit of the base complex, harbor functional NLSs. The Rpt2 NLS deletion yielded wild type localization. However, the deletion of the Rpn2 NLS resulted in improper nuclear proteasome localization and impaired proteasome function. Our data support the model by which nuclear 26 S proteasomes are assembled from subcomplexes imported by karyopherin alphabeta.


Assuntos
Núcleo Celular/metabolismo , Sinais de Localização Nuclear , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , alfa Carioferinas/metabolismo , beta Carioferinas/metabolismo , Sequência de Aminoácidos , Hidrólise , Microscopia de Fluorescência , Dados de Sequência Molecular , Proteínas de Saccharomyces cerevisiae/química
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