RESUMO
Human ß-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of allergic inflammatory responses in asthma. Antibodies generally inhibit proteases by blocking substrate access by binding to active sites or exosites or by allosteric modulation. The bivalency of IgG antibodies can increase potency via avidity, but has never been described as essential for activity. Here we report an inhibitory anti-tryptase IgG antibody with a bivalency-driven mechanism of action. Using biochemical and structural data, we determine that four Fabs simultaneously occupy four exosites on the ß-tryptase tetramer, inducing allosteric changes at the small interface. In the presence of heparin, the monovalent Fab shows essentially no inhibition, whereas the bivalent IgG fully inhibits ß-tryptase activity in a hinge-dependent manner. Our results suggest a model where the bivalent IgG acts akin to molecular pliers, pulling the tetramer apart into inactive ß-tryptase monomers, and may provide an alternative strategy for antibody engineering.
Assuntos
Anticorpos Monoclonais/metabolismo , Imunoglobulina G/metabolismo , Triptases/metabolismo , Regulação Alostérica/efeitos dos fármacos , Sequência de Aminoácidos , Heparina/farmacologia , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoglobulina G/química , Modelos Moleculares , Proteínas Mutantes/química , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica , Triptases/químicaRESUMO
Tryptase is a tetrameric serine protease located within the secretory granules of mast cells. In the secretory granules, tryptase is stored in complex with negatively charged heparin proteoglycans and it is known that heparin is essential for stabilizing the enzymatic activity of tryptase. However, recent findings suggest that enzymatically active tryptase also can be found in the nucleus of murine mast cells, but it is not known how the enzmatic activity of tryptase is maintained in the nuclear milieu. Here we hypothesized that tryptase, as well as being stabilized by heparin, can be stabilized by DNA, the rationale being that the anionic charge of DNA could potentially substitute for that of heparin to execute this function. Indeed, we showed that double-stranded DNA preserved the enzymatic activity of human ß-tryptase with a similar efficiency as heparin. In contrast, single-stranded DNA did not have this capacity. We also demonstrated that DNA fragments down to 400 base pairs have tryptase-stabilizing effects equal to that of intact DNA. Further, we showed that DNA-stabilized tryptase was more efficient in degrading nuclear core histones than heparin-stabilized enzyme. Finally, we demonstrated that tryptase, similar to its nuclear localization in murine mast cells, is found within the nucleus of primary human skin mast cells. Altogether, these finding reveal a hitherto unknown mechanism for the stabilization of mast cell tryptase, and these findings can have an important impact on our understanding of how tryptase regulates nuclear events.
Assuntos
DNA/química , Mastócitos/enzimologia , Triptases/química , Células Cultivadas , Estabilidade Enzimática , Humanos , Mastócitos/química , Mastócitos/citologia , Pele/química , Pele/citologia , Pele/enzimologiaRESUMO
ß-Tryptase, a homotetrameric serine protease, has four identical active sites facing a central pore, presenting an optimized setting for the rational design of bivalent inhibitors that bridge two adjacent sites. Using diol, hydroxymethyl phenols or benzoyl methyl hydroxamates, and boronic acid chemistries to reversibly join two [3-(1-acylpiperidin-4-yl)phenyl]methanamine core ligands, we have successfully produced a series of self-assembling heterodimeric inhibitors. These heterodimeric tryptase inhibitors demonstrate superior activity compared to monomeric modes of inhibition. X-ray crystallography validated the dimeric mechanism of inhibition, and compounds demonstrated high selectivity against related proteases, good target engagement, and tryptase inhibition in HMC1 xenograft models. Screening 3872 possible combinations from 44 boronic acid and 88 diol derivatives revealed several combinations that produced nanomolar inhibition, and seven unique pairs produced greater than 100-fold improvement in potency over monomeric inhibition. These heterodimeric tryptase inhibitors demonstrate the power of target-driven combinatorial chemistry to deliver bivalent drugs in a small molecule form.
Assuntos
Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Triptases/antagonistas & inibidores , Animais , Ácidos Borônicos/química , Ácidos Borônicos/farmacologia , Cristalografia por Raios X , Feminino , Humanos , Camundongos , Simulação de Acoplamento Molecular , Conformação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Triptases/química , Triptases/metabolismoRESUMO
Mast cells are tissue-resident cells, which have been proposed to participate in various inflammatory diseases, among them the cardiovascular diseases (CVDs). For mast cells to be able to contribute to an inflammatory process, they need to be activated to exocytose their cytoplasmic secretory granules. The granules contain a vast array of highly bioactive effector molecules, the neutral protease tryptase being the most abundant protein among them. The released tryptase may act locally in the inflamed cardiac or vascular tissue, so contributing directly to the pathogenesis of CVDs. Moreover, a fraction of the released tryptase reaches the systemic circulation, thereby serving as a biomarker of mast cell activation. Actually, increased levels of circulating tryptase have been found to associate with CVDs. Here we review the biological relevance of the circulating tryptase as a biomarker of mast cell activity in CVDs, with special emphasis on the relationship between activation of mast cells in their tissue microenvironments and the pathophysiological pathways of CVDs. Based on the available in vitro and in vivo studies, we highlight the potential molecular mechanisms by which tryptase may contribute to the pathogenesis of CVDs. Finally, the synthetic and natural inhibitors of tryptase are reviewed for their potential utility as therapeutic agents in CVDs.
Assuntos
Doenças Cardiovasculares/metabolismo , Mastócitos/enzimologia , Triptases/metabolismo , Animais , Biomarcadores/metabolismo , Doenças Cardiovasculares/tratamento farmacológico , Humanos , Mastócitos/fisiologia , Triptases/antagonistas & inibidores , Triptases/químicaRESUMO
Psoriasis (PsO) is an autoimmune disease characterized by keratinocyte proliferation, chronic inflammation and mast cell activation. Up to 42% of patients with PsO may present psoriatic arthritis (PsA). PsO and PsA share common pathophysiological mechanisms: keratinocytes and fibroblast-like synoviocytes are resistant to apoptosis: this is one of the mechanism facilitating their hyperplasic growth, and at joint level, the destruction of articular cartilage, and bone erosion and/or proliferation. Several clinical studies regarding diseases characterized by impairment of cell death, either due to apoptosis or necrosis, reported cytochrome c release from the mitochondria into the extracellular space and finally into the circulation. The presence of elevated cytochrome c levels in serum has been demonstrated in diseases as inflammatory arthritis, myocardial infarction and stroke, and liver diseases. Cytochrome c is a signaling molecule essential for apoptotic cell death released from mitochondria to the cytosol allowing the interaction with protease, as the apoptosis protease activation factor, which lead to the activation of factor-1 and procaspase 9. It has been demonstrated that this efflux from the mitochondria is crucial to start the intracellular signaling responsible for apoptosis, then to the activation of the inflammatory process. Another inflammatory marker, the tryptase, a trypsin-like serine protease produced by mast cells, is released during inflammation, leading to the activation of several immune cells through proteinase-activated receptor-2. In this review, we aimed at discussing the role played by cytochrome c and tryptase in PsO and PsA pathogenesis. To this purpose, we searched pathogenetic mechanisms in PUBMED database and review on oxidative stress, cytochrome c and tryptase and their potential role during inflammation in PsO and PsA. To this regard, the cytochrome c release into the extracellular space and tryptase may have a role in skin and joint inflammation.
Assuntos
Artrite Psoriásica/etiologia , Artrite Psoriásica/metabolismo , Citocromos c/metabolismo , Psoríase/etiologia , Psoríase/metabolismo , Triptases/metabolismo , Animais , Apoptose , Artrite Psoriásica/patologia , Autoimunidade , Citocromos c/química , Humanos , Estresse Oxidativo , Psoríase/patologia , Triptases/químicaRESUMO
Human mast cell tryptase has been shown as an activating enzyme in matrix degradation process. The previous study suggest that tryptase either alone or in joining with activation of metalloproteinases, can associate in extra cellular matrix damage and the possible destruction of the basement membrane resulting in photoaging. Therefore the inhibition of tryptase activity is one of the most important therapeutic strategies against the photoaging. Curcumin has been shown to be a potential agent for preventing and/or treating the photoaging induced by UV radiation. However, the protective effect of curcumin against the photoaging through the tryptase inhibition is still inadequately understood. In this work, computational methods to characterize the structural framework and define the atomistic details of the determinants for the tryptase inhibition mechanism by curcuminoids were performed. By molecular docking, three putative binding models able to efficiently bind all curcuminoids were identified. Analysis of molecular dynamics simulations revealed that cyclocurcumin, curcumin glucuronide, and curcumin, the most effective inhibitors from the three models, modified significant tryptase monomer rigidity by binding in all the possible sites. The result of these binding events is the suppression of the functional enzymatic motions involving the binding of substrates to the catalytic site. On the basis of this finding may thus be beneficial for the development of new natural inhibitors for the therapeutic remedy of photoaging, targeting and modulating the activity of tryptase.
Assuntos
Curcumina/análogos & derivados , Glucuronídeos/química , Simulação de Acoplamento Molecular , Triptases/química , Curcumina/química , Humanos , Domínios Proteicos , Relação Estrutura-AtividadeRESUMO
Human ß-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of the allergic inflammatory responses in asthma. During acute hypersensitivity reactions, mast cells degranulate, releasing active tetramer as a complex with proteoglycans. Extensive efforts have focused on developing therapeutic ß-tryptase inhibitors, but its unique activation mechanism is less well-explored. Tryptase is active only after proteolytic removal of the pro-domain followed by tetramer formation via two distinct symmetry-related interfaces. We show that the cleaved I16G mutant cannot tetramerize, likely due to impaired insertion of its N terminus into its "activation pocket," indicating allosteric linkage at multiple sites on each protomer. We engineered cysteines into each of the two distinct interfaces (Y75C for small or I99C for large) to assess the activity of each tetramer and disulfide-locked dimer. Using size-exclusion chromatography and enzymatic assays, we demonstrate that the two large tetramer interfaces regulate enzymatic activity, elucidating the importance of this protein-protein interaction for allosteric regulation. Notably, the I99C large interface dimer is active, even in the absence of heparin. We show that a monomeric ß-tryptase mutant (I99C*/Y75A/Y37bA, where C* is cysteinylated Cys-99) cannot form a dimer or tetramer, yet it is active but only in the presence of heparin. Thus heparin both stabilizes the tetramer and allosterically conditions the active site. We hypothesize that each ß-tryptase protomer in the tetramer has two distinct roles, acting both as a protease and as a cofactor for its neighboring protomer, to allosterically regulate enzymatic activity, providing a rationale for direct correlation of tetramer stability with proteolytic activity.
Assuntos
Heparina/metabolismo , Peptídeo Hidrolases/metabolismo , Regiões Promotoras Genéticas , Multimerização Proteica , Triptases/genética , Triptases/metabolismo , Regulação Alostérica , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Subunidades Proteicas , Triptases/químicaRESUMO
Human ß-tryptase, an enzyme with trypsin-like activity in mast cells, is an important target for the treatment of inflammatory and allergy related diseases. Heparin has been inferred to play a vital role in the stabilization of the tryptase structure and the maintenance of its active form. Up to now, the structure-function relationship between heparin and the ßII-tryptase monomer has not been studied with atomic resolution due to the lack of a complex structure of tryptase and heparin. To this end, the exact effect of heparin bonding to the ßII-tryptase monomer structure has been investigated using molecular docking and molecular dynamics (MD) simulation. The MD simulation results combined with MM-GB/SA calculations showed that heparin stabilized the ß-tryptase structure mainly through salt bridge interaction. The averaged noncovalent interaction (aNCI) method was employed for the visualization of nonbonding interactions. A crucial loop, which is located in the core region of ßII-tryptase monomer structure, has been found. Arg188 and Asp189 from this loop act as a salt bridge intermediary between 4-mer heparin and 0GX. The observation of a salt bridge between Asp189 and P1 groups of 0GX confirms the supposed interaction between these two groups. These two residues have been proved to be responsible for the direction of the P1 group of 0GX. Our study revealed that how heparin affected the activity of the human ßII-tryptase monomer (hBTM) through salt bridge interactions. The knowledge of heparin binding characteristics and the key residue contributions in this study may enlighten further the inhibitor design of this enzyme and may also improve our understanding of inflammatory and allergy related diseases.
Assuntos
Heparina/química , Simulação de Dinâmica Molecular , Triptases/química , Análise por Conglomerados , Heparina/metabolismo , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Triptases/metabolismoAssuntos
Quimiocina CCL17/sangue , Dermatite Atópica/sangue , Interleucinas/sangue , Corticosteroides/química , Corticosteroides/uso terapêutico , Adulto , Biomarcadores/sangue , Quimiocinas/metabolismo , Feminino , Antagonistas dos Receptores Histamínicos/uso terapêutico , Humanos , L-Lactato Desidrogenase/metabolismo , Masculino , Pessoa de Meia-Idade , Prurido/imunologia , Triptases/química , Adulto JovemRESUMO
Tetramer-forming tryptase (hTryptase-ß) was recently discovered to have a prominent role in preventing the internal accumulation of life-threatening fibrin deposits and fibrin-platelet clots. The anticoagulant activity of hTryptase-ß is an explanation for the presence of hemorrhagic disorders in some patients with anaphylaxis or mastocytosis. The fragments of hFibrinogen formed by the proteolysis of this prominent protein by hTryptase-ß could be used as biomarkers in the blood and/or urine for the identification and monitoring of patients with mast cell-dependent disorders. Recombinant hTryptase-ß has potential to be used in clinical settings where it is desirable to inhibit blood coagulation.
Assuntos
Anafilaxia/genética , Coagulação Sanguínea/genética , Regulação Neoplásica da Expressão Gênica , Mastócitos/enzimologia , Mastocitose/genética , Triptases/genética , Anafilaxia/sangue , Fibrina/genética , Fibrina/metabolismo , Fibrinogênio/genética , Fibrinogênio/metabolismo , Histamina/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Mastócitos/patologia , Mastocitose/sangue , Multimerização Proteica , Transdução de Sinais , Pele/enzimologia , Pele/patologia , Trombina/genética , Trombina/metabolismo , Triptases/química , Triptases/metabolismoRESUMO
Fluorescence lifetime (FLT)-based assays have developed to become highly attractive tools in drug discovery. All recently published examples of FLT-based assays essentially describe their use for monitoring enzyme-mediated peptide modifications, such as proteolytic cleavage or phosphorylation/dephosphorylation. Here we report the development of competitive binding assays as novel, inhibitor-centric assays, principally employing the FLT of the acridone dye Puretime 14 (PT14) as the readout parameter. Exemplified with two case studies on human serine proteases, the details of the rationale for both the design and synthesis of probes (i.e., active site-directed low-molecular-weight inhibitors conjugated to PT14) are provided. Data obtained from testing inhibitors with the novel assay format match those obtained with alternative formats such as FLT-based protease activity and time-resolved fluorescence resonance energy transfer-based competitive binding assays.
Assuntos
Descoberta de Drogas/métodos , Inibidores de Proteases/química , Espectrometria de Fluorescência/métodos , Acridonas/química , Ligação Competitiva , Soluções Tampão , Domínio Catalítico , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Humanos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Cinética , Pulmão/enzimologia , Conformação Molecular , Peso Molecular , Peptídeos/química , Ligação Proteica , Proteínas Recombinantes/química , Serina Proteases/química , Triptases/químicaRESUMO
A hallmark feature of mast cells is their high content of cytoplasmic secretory granules filled with various preformed compounds, including proteases of tryptase-, chymase-, and carboxypeptidase A3 type that are electrostatically bound to serglycin proteoglycan. Apart from participating in extracellular processes, serglycin proteoglycan and one of its associated proteases, tryptase, are known to regulate cell death by promoting apoptosis over necrosis. Here we sought to outline the underlying mechanism and identify core histones as primary proteolytic targets for the serglycin-tryptase axis. During the cell death process, tryptase was found to relocalize from granules into the cytosol and nucleus, and it was found that the absence of tryptase was associated with a pronounced accumulation of core histones both in the cytosol and in the nucleus. Intriguingly, tryptase deficiency resulted in defective proteolytic modification of core histones even at baseline conditions, i.e. in the absence of cytotoxic agent, suggesting that tryptase has a homeostatic impact on nuclear events. Indeed, tryptase was found in the nucleus of viable cells and was shown to cleave core histones in their N-terminal tail. Moreover, it was shown that the absence of the serglycin-tryptase axis resulted in altered chromatin composition. Together, these findings implicate histone proteolysis through a secretory granule-derived serglycin-tryptase axis as a novel principle for histone modification, during both cell homeostasis and cell death.
Assuntos
Mastócitos/citologia , Proteoglicanas/química , Vesículas Secretórias/metabolismo , Triptases/química , Proteínas de Transporte Vesicular/química , Animais , Apoptose , Células da Medula Óssea/citologia , Morte Celular , Núcleo Celular/metabolismo , Citosol/metabolismo , Histonas/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microscopia Eletrônica , Estrutura Terciária de Proteína , Proteoglicanas/genética , Proteínas de Transporte Vesicular/genéticaRESUMO
BACKGROUND: Tryptase, a major secretory product of human mast cells has been implicated as a key mediator of allergic inflammation. Genetic variation in the tryptases is extensive, and α-tryptase, an allelic variant of the more extensively studied ß-tryptase, is absent in substantial numbers of the general population. The degree to which α-tryptase expression may be associated with asthma has not been studied. We have investigated the α-tryptase gene copy number variation and its potential associations with phenotypes of asthma. OBJECTIVES: Caucasian families (n = 341) with at least two asthmatic siblings (n = 1350) were genotyped for the α-tryptase alleles, using high-resolution melting assays. Standards for the possible α-/ß-tryptase ratios were constructed by cloning α-and ß-tryptase PCR products to generate artificial templates. Association analysis of asthma affection status and related phenotypes [total and allergen-specific serum IgE, bronchial hyperresponsiveness to methacholine, forced expiratory volume in 1s (FEV1 ) and atopy and asthma severity scores] was undertaken using family-based association tests (FBAT). RESULTS: Four consistent melting patterns for the α-tryptase genotype were identified with alleles carrying null, one or two copies of the α-tryptase allele. Possessing one copy of α-tryptase was significantly associated with lower serum levels of total and dust mite-specific IgE levels and higher FEV1 measurements, while two copies were related to higher serum concentrations of total and dust mite-specific IgE and greater atopy severity scores. CONCLUSIONS AND CLINICAL RELEVANCE: Associations of α-tryptase copy number with serum IgE levels, atopy scores and bronchial function may reflect roles for tryptases in regulating IgE production and other processes in asthma.
Assuntos
Asma/etiologia , Asma/fisiopatologia , Variação Genética , Imunoglobulina E/imunologia , Triptases/genética , Adolescente , Adulto , Alelos , Alérgenos/imunologia , Animais , Asma/diagnóstico , Sequência de Bases , Criança , Variações do Número de Cópias de DNA , Feminino , Frequência do Gene , Genótipo , Humanos , Imunoglobulina E/sangue , Masculino , Dados de Sequência Molecular , Fenótipo , Pyroglyphidae/imunologia , Testes de Função Respiratória , Alinhamento de Sequência , Triptases/química , Adulto JovemRESUMO
The S1A serine proteases function in many key biological processes such as development, immunity, and blood coagulation. S1A proteases contain a highly conserved disulfide bond (Cys(191)-Cys(220) in chymotrypsin numbering) that links two ß-loop structures that define the rim of the active site pocket. Mast cell ßII-tryptase is a S1A protease that is associated with pathological inflammation. In this study, we have found that the conserved disulfide bond (Cys(220)-Cys(248) in ßII-tryptase) exists in oxidized and reduced states in the enzyme stored and secreted by mast cells. The disulfide bond has a standard redox potential of -301 mV and is stoichiometrically reduced by the inflammatory mediator, thioredoxin, with a rate constant of 350 m(-1) s(-1). The oxidized and reduced enzymes have different substrate specificity and catalytic efficiency for hydrolysis of both small and macromolecular substrates. These observations indicate that ßII-tryptase activity is post-translationally regulated by an allosteric disulfide bond. It is likely that other S1A serine proteases are similarly regulated.
Assuntos
Cisteína/química , Mastócitos/enzimologia , Oxirredução , Triptases/química , Regulação Alostérica , Animais , Sítios de Ligação , Catálise , Domínio Catalítico , Linhagem Celular Tumoral , Cisteína/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Humanos , Mastócitos/metabolismo , Camundongos , Relação Estrutura-Atividade , Especificidade por Substrato , Triptases/metabolismoRESUMO
BACKGROUND: A salivary proteome-transcriptome project on the hard tick Ixodes scapularis revealed that Kunitz peptides are the most abundant salivary proteins. Ticks use Kunitz peptides (among other salivary proteins) to combat host defense mechanisms and to obtain a blood meal. Most of these Kunitz peptides, however, remain functionally uncharacterized, thus limiting our knowledge about their biochemical interactions. RESULTS: We discovered an unusual cysteine motif in a Kunitz peptide. This peptide inhibits several serine proteases with high affinity and was named tryptogalinin due to its high affinity for ß-tryptase. Compared with other functionally described peptides from the Acari subclass, we showed that tryptogalinin is phylogenetically related to a Kunitz peptide from Rhipicephalus appendiculatus, also reported to have a high affinity for ß-tryptase. Using homology-based modeling (and other protein prediction programs) we were able to model and explain the multifaceted function of tryptogalinin. The N-terminus of the modeled tryptogalinin is detached from the rest of the peptide and exhibits intrinsic disorder allowing an increased flexibility for its high affinity with its inhibiting partners (i.e., serine proteases). CONCLUSIONS: By incorporating experimental and computational methods our data not only describes the function of a Kunitz peptide from Ixodes scapularis, but also allows us to hypothesize about the molecular basis of this function at the atomic level.
Assuntos
Proteínas de Artrópodes/química , Ixodes/metabolismo , Proteínas e Peptídeos Salivares/química , Inibidores de Serina Proteinase/química , Triptases/química , Motivos de Aminoácidos , Animais , Proteínas de Artrópodes/classificação , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Cisteína/química , Cisteína/genética , Humanos , Ixodes/química , Ixodes/genética , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/classificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhipicephalus/química , Rhipicephalus/genética , Rhipicephalus/metabolismo , Proteínas e Peptídeos Salivares/classificação , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/metabolismo , Homologia de Sequência de Aminoácidos , Inibidores de Serina Proteinase/classificação , Inibidores de Serina Proteinase/genética , Inibidores de Serina Proteinase/metabolismo , Triptases/antagonistas & inibidores , Triptases/metabolismoRESUMO
AIMS AND HYPOTHESIS: Mast cells are immune cells known for their role in several inflammatory and fibrotic diseases. Recent works in mice suggest that mast cells could be cellular actors involved in the pathophysiology of obesity, a disease characterized by white adipose tissue (WAT) and systemic inflammation. The aim of the study was to better characterize mast cells in WAT of obese with or without type 2 diabetes and lean subjects as well as to explore the relationship with WAT inflammation and fibrosis. METHODS: Subcutaneous and omental adipose tissue from six lean subjects, 10 obese nondiabetic, and 10 diabetic patients was analyzed by immunohistochemistry and real-time PCR for inflammatory and fibrosis markers. Cytokines secretion of mast cells isolated from WAT and cultured in different conditions was estimated by cytokine array kit. RESULTS: We found that mast cells are activated in human adipose tissue and localized preferentially in fibrosis depots, a local condition that stimulates their inflammatory state. Mast cells with tryptase(+) chymase(+) staining tended to be higher in obese omental adipose tissue. We found positive links between mast cell number and several characteristics of obese WAT including fibrosis, macrophage accumulation, and endothelial cell inflammation. Mast cell number and their inflammatory phenotype are associated with diabetes parameters. CONCLUSION AND INTERPRETATION: Mast cells are cellular actors of WAT inflammation and possibly fibrotic state found in obesity and diabetes. Whether mast cells could be involved in the pathophysiology of diabetes needs additional study as well as the positioning of these cells in driving pathological alterations of WAT in these chronic metabolic diseases.
Assuntos
Tecido Adiposo/patologia , Diabetes Mellitus Tipo 2/patologia , Inflamação/patologia , Mastócitos/patologia , Obesidade Mórbida/patologia , Tecido Adiposo Branco/patologia , Adulto , Biomarcadores/análise , Glicemia/metabolismo , Contagem de Células , Separação Celular , Quimases/química , Células Endoteliais/patologia , Feminino , Fibrose/patologia , Homeostase/fisiologia , Humanos , Imuno-Histoquímica , Lipídeos/sangue , Testes de Função Hepática , Masculino , Pessoa de Meia-Idade , Obesidade Mórbida/metabolismo , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Triptases/químicaRESUMO
Tick-derived protease inhibitor (TdPI) is a tight-binding Kunitz-related inhibitor of human tryptase ß with a unique structure and disulfide-bond pattern. Here we analyzed its oxidative folding and reductive unfolding by chromatographic and disulfide analyses of acid-trapped intermediates. TdPI folds through a stepwise generation of heterogeneous populations of one-disulfide, two-disulfide, and three-disulfide intermediates, with a major accumulation of the nonnative three-disulfide species IIIa. The rate-limiting step of the process is disulfide reshuffling within the three-disulfide population towards a productive intermediate that oxidizes directly into the native four-disulfide protein. TdPI unfolds through a major accumulation of the native three-disulfide species IIIb and the subsequent formation of two-disulfide and one-disulfide intermediates. NMR characterization of the acid-trapped and further isolated IIIa intermediate revealed a highly disordered conformation that is maintained by the presence of the disulfide bonds. Conversely, the NMR structure of IIIb showed a native-like conformation, with three native disulfide bonds and increased flexibility only around the two free cysteines, thus providing a molecular basis for its role as a productive intermediate. Comparison of TdPI with a shortened variant lacking the flexible prehead and posthead segments revealed that these regions do not contribute to the protein conformational stability or the inhibition of trypsin but are important for both the initial steps of the folding reaction and the inhibition of tryptase ß. Taken together, the results provide insights into the mechanism of oxidative folding of Kunitz inhibitors and pave the way for the design of TdPI variants with improved properties for biomedical applications.
Assuntos
Inibidores de Proteases/química , Animais , Cisteína/química , Dissulfetos/química , Relação Dose-Resposta a Droga , Glutationa/química , Humanos , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Estresse Oxidativo , Oxigênio/química , Dobramento de Proteína , Carrapatos , Triptases/antagonistas & inibidores , Triptases/químicaRESUMO
The saccharin ring system has gained considerable attention in the past decades, especially in the field of medicinal chemistry. The wide applicability of saccharin derivatives remains the driving force behind the constant development of novel routes and methods that provide new access to the construction of saccharin. Since the functionalization of this heterocycle has proved difficult, except for N- and O-alkylation, novel strategic approaches are much sought-after and thus constitute a great value to any medicinal chemist. In addition to the synthetic novelties introduced into the synthesis and functionalization of this particular heterocycle, the numerous newly discovered biological activities of saccharin and its derivatives are also reviewed. Saccharin may be considered to constitute a privileged framework on account of its role as a key structural element in several biologically active compounds ranging from enzyme inhibitors to receptor ligands and beyond.
Assuntos
Sacarina/química , Aldeído Redutase/química , Aldeído Redutase/metabolismo , Ciclização , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacologia , Humanos , Isomerismo , Sacarina/síntese química , Sacarina/farmacologia , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Triptases/química , Triptases/metabolismoRESUMO
Here we report the design, chemical and recombinant synthesis, and functional properties of a series of novel inhibitors of human mast cell tryptase beta, a protease of considerable interest as a therapeutic target for the treatment of allergic asthma and inflammatory disorders. These inhibitors are derived from a linear variant of the cyclic cystine knot miniprotein MCoTI-II, originally isolated from the seeds of Momordica cochinchinensis. A synthetic cyclic miniprotein that bears additional positive charge in the loop connecting the N- and C-termini inhibits all monomers of the tryptase beta tetramer with an overall equilibrium dissociation constant K(i) of 1 nM and thus is one of the most potent proteinaceous inhibitors of tryptase beta described to date. These cystine knot miniproteins may therefore become valuable scaffolds for the design of a new generation of tryptase inhibitors.