RESUMO
Aspartic proteases are important biomarkers of human disease and interesting targets for modulation of immune response via MHC class II antigen processing inhibition. The lack of inhibitors with sufficient selectivity hampers precise analysis of the role of cathepsin E and napsin A in samples containing the ubiquitous and highly abundant homolog cathepsin D. Grassystatins from marine cyanobacteria show promising selectivity for cathepsin E but contain several ester bonds that make their synthesis cumbersome and thus limit availability of the inhibitors. Herewith, we present grassystatin-derived cathepsin E inhibitors with greatly facilitated synthesis but retained selectivity profile. We demonstrate their affinity and selectivity with both enzyme kinetic assays and streptavidin-based pull-down from cells and mouse organs. Our findings suggest that grassystatin-like inhibitors are useful tools for targeted inhibition of cathepsin E and thus provide a novel approach for cancer and immunology research.
Assuntos
Catepsina D/antagonistas & inibidores , Catepsina E/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Peptídeos/farmacologia , Catepsina D/metabolismo , Catepsina E/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Células HEK293 , Humanos , Estrutura Molecular , Peptídeos/síntese química , Peptídeos/química , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Three new modified peptides named grassystatins D-F (1-3) were discovered from a marine cyanobacterium from Guam. Their structures were elucidated using NMR spectroscopy and mass spectrometry. The hallmark structural feature in the peptides is a statine unit, which contributes to their aspartic protease inhibitory activity preferentially targeting cathepsins D and E. Grassystatin F (3) was the most potent analogue, with IC50 values of 50 and 0.5 nM against cathepsins D and E, respectively. The acidic tumor microenvironment is known to increase the activation of some of the lysosomal proteases associated with tumor metastasis such as cathepsins. Because cathepsin D is a biomarker in aggressive forms of breast cancer and linked to poor prognosis, the effects of cathepsin D inhibition by 1 and 3 on the downstream cellular substrates cystatin C and PAI-1 were investigated. Furthermore, the functional relevance of targeting cathepsin D substrates was evaluated by examining the effect of 1 and 3 on the migration of MDA-MD-231 cells. Grassystatin F (3) inhibited the cleavage of cystatin C and PAI-1, the activities of their downstream targets cysteine cathepsins and tPA, and the migration of the highly aggressive triple negative breast cancer cells, phenocopying the effect of siRNA-mediated knockdown of cathepsin D.
Assuntos
Ácido Aspártico Proteases/efeitos dos fármacos , Peptídeos/isolamento & purificação , Aminoácidos , Catepsina D/antagonistas & inibidores , Catepsina E/antagonistas & inibidores , Catepsina L/metabolismo , Cianobactérias/química , Relação Dose-Resposta a Droga , Endopeptidases/metabolismo , Feminino , Guam , Humanos , Concentração Inibidora 50 , Lisossomos/metabolismo , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Peptídeos/farmacologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Inibidores de Proteases/farmacologiaRESUMO
The cathepsin family of lysosomal proteases is increasingly being recognized for their altered expression in cancer and role in facilitating tumor progression. The aspartyl protease cathepsin E is overexpressed in several cancers and has been investigated as a biomarker for pancreatic ductal adenocarcinoma (PDAC). Here we show that cathepsin E expression in mouse PDAC tumors is increased by more than 400-fold when compared to healthy pancreatic tissue. Cathepsin E accumulates over the course of disease progression and accounts for more than 3% of the tumor protein in mice with end-stage disease. Through immunoblot analysis we determined that only procathepsin E exists in mouse PDAC tumors and cell lines derived from these tumors. By decreasing the pH, this procathepsion E is converted to the mature form, resulting in an increase in proteolytic activity. Although active site inhibitors can bind procathepsin E, treatment of PDAC mice with the aspartyl protease inhibitor ritonavir did not decrease tumor burden. Lastly, we used multiplex substrate profiling by mass spectrometry to identify two synthetic peptides that are hydrolyzed by procathepsin E near neutral pH. This work represents a comprehensive analysis of procathepsin E in PDAC and could facilitate the development of improved biomarkers for disease detection.
Assuntos
Catepsina E/metabolismo , Precursores Enzimáticos/metabolismo , Neoplasias Pancreáticas/patologia , Sequência de Aminoácidos , Animais , Catepsina E/antagonistas & inibidores , Catepsina E/química , Linhagem Celular Tumoral , Progressão da Doença , Precursores Enzimáticos/antagonistas & inibidores , Precursores Enzimáticos/química , Regulação Neoplásica da Expressão Gênica , Concentração de Íons de Hidrogênio , Camundongos , Inibidores de Proteases/farmacologia , Neoplasias PancreáticasRESUMO
In search of novel protease inhibitors with therapeutic potential, our efforts exploring the marine cyanobacterium Lyngbya sp. have led to the discovery of tasiamide F (1), which is an analogue of tasiamide B (2). The structure was elucidated using a combination of NMR spectroscopy and mass spectrometry. The key structural feature in 1 is the presence of the Phe-derived statine core, which contributes to its aspartic protease inhibitory activity. The antiproteolytic activity of 1 and 2 was evaluated in vitro against cathepsins D and E, and BACE1. Tasiamide F (1) displayed IC50 values of 57nM, 23nM, and 0.69µM, respectively, indicating greater selectivity for cathepsins over BACE1 compared with tasiamide B (2). Molecular docking experiments were carried out for compounds 1 and 2 against cathepsins D and E to rationalize their activity towards these proteases. The dysregulated activities of cathepsins D and E have been implicated in cancer and modulation of immune responses, respectively, and these proteases represent potential therapeutic targets.
Assuntos
Catepsina D/antagonistas & inibidores , Catepsina D/química , Catepsina E/antagonistas & inibidores , Catepsina E/química , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Animais , Cianobactérias/química , Simulação de Acoplamento Molecular , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Alimentos Marinhos/análiseRESUMO
The synthesis, evaluation, and structure-activity relationships of a class of acyl guanidines which inhibit the BACE-1 enzyme are presented. The prolinyl acyl guanidine chemotype (7c), unlike compounds of the parent isothiazole chemotype (1), yielded compounds with good agreement between their enzymatic and cellular potency as well as a reduced susceptibility to P-gp efflux. Further improvements in potency and P-gp ratio were realized via a macrocyclization strategy. The in vivo profile in wild-type mice and P-gp effects for the macrocyclic analog 21c is presented.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Guanidinas/farmacologia , Compostos Macrocíclicos/farmacologia , Prolina/análogos & derivados , Prolina/farmacologia , Inibidores de Proteases/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Peptídeos beta-Amiloides/biossíntese , Animais , Células CACO-2 , Catepsina D/antagonistas & inibidores , Catepsina E/antagonistas & inibidores , Cães , Guanidinas/síntese química , Humanos , Compostos Macrocíclicos/síntese química , Células Madin Darby de Rim Canino , Masculino , Camundongos , Simulação de Acoplamento Molecular , Pepsina A/antagonistas & inibidores , Prolina/síntese química , Inibidores de Proteases/síntese químicaRESUMO
Stefin B is the major general cytosolic protein inhibitor of cysteine cathepsins. Its main function is to protect the organism against the activity of endogenous potentially hazardous proteases accidentally released from lysosomes. In this study, we investigated the possible effect of endosomal/lysosomal aspartic cathepsins D and E on stefin B after membrane permeabilization. Loss of membrane integrity of lysosomes and endosomes was induced by a lysosomotropic agent L-Leucyl-L-leucine methyl ester (Leu-Leu-OMe). The rat thyroid cell line FRTL-5 was selected as a model cell line owing to its high levels of proteases, including cathepsin D and E. Permeabilization of acid vesicles from FRTL-5 cells induced degradation of stefin B. The process was inhibited by pepstatin A, a potent inhibitor of aspartic proteases. However, degradation of stefin B was prevented by siRNA-mediated silencing of cathepsin D expression. In contrast, cathepsin E silencing had no effect on stefin B degradation. These results showed that cathepsin D and not cathepsin E degrades stefin B. It can be concluded that the presence of cathepsin D in the cytosol affects the inhibitory potency of stefin B, thus preventing the regulation of cysteine cathepsin activities in various biological processes.
Assuntos
Catepsina D/metabolismo , Cistatina B/metabolismo , Citosol/enzimologia , Células Matadoras Naturais/enzimologia , Linfócitos/enzimologia , Macrófagos/enzimologia , Animais , Catepsina D/antagonistas & inibidores , Catepsina D/genética , Catepsina E/antagonistas & inibidores , Catepsina E/genética , Catepsina E/metabolismo , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cistatina B/farmacologia , Citosol/efeitos dos fármacos , Dipeptídeos/farmacologia , Endossomos/efeitos dos fármacos , Endossomos/enzimologia , Expressão Gênica , Células HEK293 , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Pepstatinas/farmacologia , Proteólise/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Glândula Tireoide/citologia , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/enzimologiaRESUMO
When resectable, invasive pancreatic ductal adenocarcinoma (PDAC) is most commonly treated with surgery and radiochemotherapy. Given the intricate local anatomy and locoregional mode of dissemination, achieving clean surgical margins can be a significant challenge. On the basis of observations that cathepsin E (CTSE) is overexpressed in PDAC and that an United States Food and Drug Administration (FDA)-approved protease inhibitor has high affinity for CTSE, we have developed a CTSE optical imaging agent [ritonavir tetramethyl-BODIPY (RIT-TMB)] for potential intraoperative use. We show nanomolar affinity [half maximal inhibitory concentration (IC50) of 39.9 ± 1.2 nM] against CTSE of the RIT-TMB in biochemical assays and intracellular accumulation and target-to-background ratios that allow specific delineation of individual cancer cells. This approach should be useful for more refined surgical staging, planning, and resection with curative intent.
Assuntos
Compostos de Boro , Carcinoma Ductal Pancreático/metabolismo , Catepsina E/metabolismo , Corantes Fluorescentes , Inibidores da Protease de HIV , Neoplasias Pancreáticas/metabolismo , Ritonavir , Animais , Biópsia , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/patologia , Catepsina E/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores da Protease de HIV/farmacologia , Humanos , Concentração Inibidora 50 , Camundongos , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/patologia , Ritonavir/análogos & derivados , Ritonavir/farmacologiaRESUMO
Improving a particular function of molecules is often more difficult than identifying such molecules ab initio. Here, a method to acquire higher affinity and/or more functional peptides was developed as a progressive library selection method. The primary library selection products were utilized to build a secondary library composed of blocks of 4 amino acids, of which selection led to peptides with increased activity. These peptides were further converted to randomly generate paired peptides. Cathepsin E-inhibitors thus obtained exhibited the highest activities and affinities (pM order). This was also the case with cathepsin E-activating peptides, proving the methodological effectiveness. The primary, secondary, and tertiary library selections can be regarded as module-finding, module-shuffling, and module-pairing, respectively, which resembles the progression of the natural evolution of proteins. The mode of peptide binding to their target proteins is discussed in analogy to antibodies and epitopes of an antigen.
Assuntos
Aptâmeros de Peptídeos/química , Catepsina E/antagonistas & inibidores , Inibidores de Proteases/química , Sequência de Aminoácidos , Evolução Molecular Direcionada , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Biblioteca de Peptídeos , Ligação ProteicaRESUMO
The linear depsipeptide grassystatin A, a valuable probe for the study of cathepsin E function, has been synthesized by a [4+6] strategy. It exhibited specific inhibitory activity against cathepsin E with an IC(50) value of 0.8 nM. Our studies indicated that inhibition of cathepsin E did not have an impact on ovalbumin antigen processing and peptide presentation, unique from studies of other aspartic protease inhibitors.
Assuntos
Catepsina E/antagonistas & inibidores , Peptídeos/farmacologia , Inibidores de Proteases/farmacologia , Animais , Catepsina E/metabolismo , Relação Dose-Resposta a Droga , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Estrutura Molecular , Peptídeos/síntese química , Peptídeos/química , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Relação Estrutura-AtividadeRESUMO
The absolute stereostructures of the components of symplocin A (3), a new N,N-dimethyl-terminated peptide from the Bahamian cyanobacterium Symploca sp., were assigned from spectroscopic analysis, including MS, 2D NMR, and Marfey's analysis. The complete absolute configuration of symplocin A, including the unexpected D-configurations of the terminal N,N-dimethylisoleucine and valic acid residues, was assigned by chiral-phase HPLC of the corresponding 2-naphthacyl esters, a highly sensitive, complementary strategy for assignment of N-blocked peptide residues where Marfey's method is ineffectual or other methods fall short. Symplocin A exhibited potent activity as an inhibitor of cathepsin E (IC(50) 300 pM).
Assuntos
Catepsina E/antagonistas & inibidores , Cianobactérias/química , Naftalenos/química , Peptídeos/isolamento & purificação , Bahamas , Cromatografia Líquida de Alta Pressão , Ésteres , Concentração Inibidora 50 , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Peptídeos/farmacologiaRESUMO
BACKGROUND: Aspartic proteases Cathepsin (Cath) E and D are two different proteases, but they share many common characteristics, including molecular weight, catalytic mechanism, substrate preferences, proteolytic conditions and inhibition susceptibility. To define the biological roles of these proteases, it is necessary to elucidate their substrate specificity. In the present study, we report a new peptide-substrate that is only sensitive to Cath E but not Cath D. METHODS: Substrate e, Mca-Ala-Gly-Phe-Ser-Leu-Pro-Ala-Lys(Dnp)-DArg-CONH2, designed in such a way that due to the close proximity of a Mca-donor and a Dnp-acceptor, near complete intramolecular quenching effect was achieved in its intact state. After the proteolytic cleavage of the hydrophobic motif of peptide substrate, both Mca and Dnp would be further apart, resulting in bright fluorescence. RESULTS: Substrate e showed a 265 fold difference in the net fluorescence signals between Cath E and D. This Cath E selectivity was established by having -Leu**Pro- residues at the scissile peptide bond. The confined cleavage site of substrate e was confirmed by LC-MS. The catalytic efficiency (K(cat)/K(M)) of Cath E for substrate e was 16.7 µM⻹S⻹. No measurable catalytic efficiency was observed using Cath D and no detectable fluorescent changes when incubated with Cath S and Cath B. CONCLUSIONS: This study demonstrated the promise of using the developed fluorogenic substrate e as a selective probe for Cath E proteolytic activity measurement. GENERAL SIGNIFICANCE: This study forms the foundation of Cath E specific inhibitor development in further studies.
Assuntos
Catepsina E/química , Oligopeptídeos/química , Catálise , Catepsina D/química , Catepsina D/metabolismo , Catepsina E/antagonistas & inibidores , Catepsina E/metabolismo , Desenho de Fármacos , Fluorescência , Humanos , Oligopeptídeos/metabolismo , Inibidores de Proteases/química , Especificidade por SubstratoRESUMO
In our efforts to explore marine cyanobacteria as a source of novel bioactive compounds, we discovered a statine unit-containing linear decadepsipeptide, grassystatin A (1), which we screened against a diverse set of 59 proteases. We describe the structure determination of 1 and two natural analogues, grassystatins B (2) and C (3), using NMR, MS, and chiral HPLC techniques. Compound 1 selectively inhibited cathepsins D and E with IC(50)s of 26.5 nM and 886 pM, respectively. Compound 2 showed similar potency and selectivity against cathepsins D and E (IC(50)s of 7.27 nM and 354 pM, respectively), whereas the truncated peptide analogue grassystatin C (3), which consists of two fewer residues than 1 and 2, was less potent against both but still selective for cathepsin E. The selectivity of compounds 1-3 for cathepsin E over D (20-38-fold) suggests that these natural products may be useful tools to probe cathepsin E function. We investigated the structural basis of this selectivity using molecular docking. We also show that 1 can reduce antigen presentation by dendritic cells, a process thought to rely on cathepsin E.
Assuntos
Aminoácidos/química , Apresentação de Antígeno/efeitos dos fármacos , Catepsina E/antagonistas & inibidores , Cianobactérias/química , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Animais , Catepsina E/química , Catepsina E/metabolismo , Linhagem Celular Tumoral , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Humanos , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Oceanos e Mares , Oligopeptídeos/isolamento & purificação , Oligopeptídeos/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Especificidade por SubstratoRESUMO
Proteases are involved in various biological functions. Thus, inhibition of their activities is scientifically interesting and medically important. However, there is no systematic method established to date to generate endopeptidase inhibitory peptides. Here, we report a general system to identify endopeptidase inhibitory peptides based on the use of in vitro evolution. Using this system, we generated peptides that inhibit cathepsin E (CE) specifically at a submicromolar IC(50). This system generates protease inhibitor peptides utilizing techniques of cDNA display, selection-by-function, Y-ligation-based block shuffling, and others. We further demonstrated the importance and effectiveness of a secondary library for obtaining small-sized and active peptides. CE inhibitory peptides generated by this method were characterized by a small size (8 to 12 aa) and quite different sequences, suggesting that they bind to different sites on CE. Typical CE inhibitory peptide aptamers obtained here (P(i)101; SCGG IIII SCIA) have half an inhibition activity (K(i); 5 nM) of pepstatin A (potent CE inhibitor) without inhibiting cathepsin D (structurally similar to CE). The general applicability of this system suggests that it may be useful to identify inhibitory peptides for various kinds of proteases and that it may therefore contribute to protein science and drug discovery. The peptide binding to a protein is discussed in comparison with the antibody binding to an antigen.
Assuntos
Aptâmeros de Peptídeos/genética , Aptâmeros de Peptídeos/farmacologia , Catepsina E/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Sequência de Aminoácidos , Aptâmeros de Peptídeos/química , Sequência de Bases , DNA Complementar/genética , Evolução Molecular Direcionada , Descoberta de Drogas , Técnicas In Vitro , Cinética , Dados de Sequência Molecular , Biblioteca de Peptídeos , Inibidores de Proteases/químicaRESUMO
Processing of antigens by proteases in the endocytic compartments of antigen presenting cells (APC) is essential to make them suitable for presentation as antigenic peptides to T lymphocytes. Several proteases of the cysteine, aspartyl and serine classes are involved in this process. It has been speculated, that the aspartyl protease cathepsin E (CatE) is involved in antigen processing in B cell line, monocyte-derived dendritic cells (DC) and murine DC. Here we show the expression of CatE in primary human B cells and DC, which was only elevated in B cells after induction with phorbol 12-myristate 13-acetate (PMA), resulted in enhanced presentation of tetanus toxin C-fragment (TTC) to the respective T cells. Inhibition of aspartyl proteases using pepstatin-A-penetratin (PepA-P), a highly efficient, cell-permeable aspartyl protease inhibitor, reduced significantly T cell activation in PMA activated B cells but not in PMA activated myeloid DC (mDC). Thus we suggest that CatE is important in the processing of TTC in primary human B cells.
Assuntos
Apresentação de Antígeno , Linfócitos B/imunologia , Catepsina E/biossíntese , Fragmentos de Peptídeos/imunologia , Toxina Tetânica/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Catepsina E/antagonistas & inibidores , Células Dendríticas/enzimologia , Células Dendríticas/imunologia , Humanos , Ativação Linfocitária , Pepstatinas/farmacologia , Fragmentos de Peptídeos/metabolismo , Toxina Tetânica/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
It has been strongly demanded by a number of people to elevate activities of molecules of a particular function. Currently, there is no general guide available for this purpose. Here we present a novel approach for this; a local sequence space map-directed method for exploring molecules of a higher activity. This approach exploits the knowledge of a local sequence space so far established and obtains the shape of sequence space (map) by intra- and extrapolating the known landscape, which was drawn through the principal coordinates analysis. In this method, we successfully obtained 16 DNA aptamers of cathepsin E (CE) inhibitory activity that have comparable or higher activities than the ancestral ones on which the designed molecules were based. Some of them had a 30% higher activity than the previously reported top one (SFR-6-3). This high efficiency in obtaining functional molecules (16 out of 21 newly designed ones) is by no means usual because most of molecules generated at random are known to have no function, showing the effectiveness of the map-based approach. The selected molecules were confirmed to have the i-motif structure, consistent to the fact that they have a C-rich sequence and their CE-inhibitory activities were measured at an acidic pH, both of which are favorable for the i-motif. This structure of CE-inhibitory aptamers was inferred to contribute to the structural stability but not to the function itself directly.
Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/farmacologia , Catepsina E/antagonistas & inibidores , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Sequência de Bases , Dicroísmo Circular , Clonagem Molecular , Primers do DNA , Conformação de Ácido NucleicoRESUMO
Cathepsin E is a major intracellular aspartic protease which is predominantly present in the cells of immune system and is frequently implicated in antigen processing via the MHC class II pathway. In the present review some of the known features of cathepsin E such as tissue distribution, subcellular localization, enzymatic properties, intracellular trafficking, gene regulation and associated physiological conditions are highlighted.
Assuntos
Catepsina E/fisiologia , Animais , Apresentação de Antígeno/fisiologia , Catepsina E/antagonistas & inibidores , Catepsina E/genética , Indução Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Humanos , Especificidade de Órgãos/fisiologia , Processamento de Proteína Pós-Traducional , Especificidade por Substrato/fisiologiaRESUMO
The most abundant autoreactive T cells in patients with Goodpasture's disease are specific for peptides in the autoantigen that have high affinity for the disease-associated HLA class II molecule, DR15. How can such T cells escape self-tolerance mechanisms? This study showed that these peptides are highly susceptible to destruction in the earliest stages of antigen processing, and some must be cleaved for antigen digestion to be possible ("unlocking"). Goodpasture autoantigen [collagen alpha3(IV)NC1; approximately 31 kD] that was incubated with B cell lysosomes was cleaved within a few minutes to form approximately 9- and approximately 22-kD fragments, then increasing quantities of smaller peptides. The processing was completely abrogated by pepstatin A, a specific inhibitor of cathepsin D/E, even though lysosomal extracts contain a rich array of proteases. Purified cathepsin D generated the same major alpha3(IV)NC1 fragments as entire lysosomes, suggesting that cathepsin D cleavages are required to initiate alpha3(IV)NC1 processing. The initial unlocking cleavages destroyed two major self-epitopes, and subsequent preferred cleavages destroyed all of the other T cell epitopes that are recognized by most patients' autoreactive T cells. The responses of T cell clones that are specific for a major disease-associated peptide to antigen-pulsed intact antigen-presenting cells were substantially enhanced by pepstatin A treatment. Therefore, cathepsin D activity significantly diminishes presentation of alpha3(IV)NC1 peptides that are recognized by patients' T cells by destroying the peptides in early processing. These observations can explain why the mature T cell repertoire includes reactivity toward these self-peptides and suggests that a key factor in disease initiation is likely to be a shift in antigen processing.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Ácido Aspártico Endopeptidases/metabolismo , Autoantígenos/metabolismo , Catepsina D/farmacologia , Colágeno Tipo IV/metabolismo , Proteínas Serina-Treonina Quinases/imunologia , Linfócitos T/imunologia , Doença Antimembrana Basal Glomerular , Ácido Aspártico Endopeptidases/imunologia , Autoantígenos/efeitos dos fármacos , Catepsina D/antagonistas & inibidores , Catepsina E/antagonistas & inibidores , Colágeno Tipo IV/efeitos dos fármacos , Epitopos/imunologia , Humanos , Lisossomos/enzimologia , Lisossomos/fisiologia , Pepstatinas/farmacologia , Proteínas Serina-Treonina Quinases/efeitos dos fármacosRESUMO
A method for efficient enrichment of protease inhibitors out of a DNA library was developed by introducing SF-link technology. A two-step selection strategy was designed consisting of the initial enrichment of aptamers based on binding function while the second enrichment step was based on the inhibitory activity to a protease, cathepsin E (CE). The latter was constructed by covalently linking of a biotinylated peptide substrate to each of the ssDNA molecule contained in the preliminarily selected DNA library, generating 'SF-link'. Gradual enrichment of inhibitory DNAs was attained in the course of selection. One molecule, SFR-6-3, showed an IC(50) of around 30 nM, a K(d) of around 15 nM and high selectivity for CE. Sequence and structure analysis revealed a C-rich sequence without any guanine and possibly an i-motif structure, which must be novel to be found in in vitro-selected aptamers. SF-link technology, which is novel as the screening technology, provided a remarkable enrichment of specific protease inhibitors and has a potential to be further developed.
Assuntos
Catepsina E/antagonistas & inibidores , Biblioteca Gênica , Inibidores de Proteases/farmacologia , Técnica de Seleção de Aptâmeros/métodos , Sequência de Aminoácidos , Animais , Aptâmeros de Peptídeos/química , Sequência de Bases , Catepsina D/antagonistas & inibidores , DNA/química , DNA/genética , Cinética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ratos , Especificidade por SubstratoRESUMO
The molecular details of antigen processing, including the identity of the enzymes involved, their intracellular location and their substrate specificity, are still incompletely understood. Selective inhibition of proteolytic antigen processing enzymes such as cathepsins D and E, using small molecular inhibitors such as pepstatin, has proven to be a valuable tool in investigating these pathways. However, pepstatin is poorly soluble in water and has limited access to the antigen processing compartment in antigen presenting cells. We have synthesised mannose-pepstatin conjugates, and neomannosylated BSA-pepstatin conjugates, as tools for the in vivo study of the antigen processing pathway. Conjugation to mannose and to neomannosylated BSA substantially improved the solubility of the conjugates relative to pepstatin. The mannose-pepstatin conjugates showed no reduction in inhibition of cathepsin E, whereas the neomannosylated BSA-pepstatin conjugates showed some loss of inhibition, probably due to steric factors. However, a neomannosylated BSA-pepstatin conjugate incorporating a cleavable disulfide linkage between the pepstatin and the BSA showed the best uptake to dendritic cells and the best inhibition of antigen processing.
Assuntos
Apresentação de Antígeno/efeitos dos fármacos , Manose/farmacologia , Pepstatinas/farmacologia , Animais , Catepsina E/antagonistas & inibidores , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Manose/química , Pepstatinas/química , Soroalbumina Bovina/química , SolubilidadeRESUMO
The aspartic proteinase cathepsin E (CatE) has been implicated in Ag processing. In this study we report that CatE expression is negatively regulated by the MHC class II transactivator (CIITA). CIITA-deficient murine and human B cells expressed greater CatE than wild-type B cells, whereas overexpression of CIITA in a human gastric carcinoma cell line, AGS, resulted in decreased CatE mRNA and protein. AGS cells expressing CIITA also exhibited decreased processing of OVA Ag. Inhibition of CatE expression is specific to the type III CIITA isoform and maps to the acidic and proline/serine/threonine-rich (PST) protein domains of CIITA. We found that CatE expression is inducible by PU.1 and p300, and that this induction can be reversed by CIITA. These findings demonstrate a novel phenomenon: regulation of CatE Ag processing by CIITA in an isoform-dependent manner.