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1.
Cell Mol Life Sci ; 76(10): 2003-2013, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30747251

RESUMO

To successfully feed, ticks inject pharmacoactive molecules into the vertebrate host including cystatin cysteine protease inhibitors. However, the molecular and cellular events modulated by tick saliva remain largely unknown. Here, we describe and characterize a novel immunomodulatory cystatin, Iristatin, which is upregulated in the salivary glands of feeding Ixodes ricinus ticks. We present the crystal structure of Iristatin at 1.76 Å resolution. Purified recombinant Iristatin inhibited the proteolytic activity of cathepsins L and C and diminished IL-2, IL-4, IL-9, and IFN-γ production by different T-cell populations, IL-6 and IL-9 production by mast cells, and nitric oxide production by macrophages. Furthermore, Iristatin inhibited OVA antigen-induced CD4+ T-cell proliferation and leukocyte recruitment in vivo and in vitro. Our results indicate that Iristatin affects wide range of anti-tick immune responses in the vertebrate host and may be exploitable as an immunotherapeutic.


Assuntos
Proteínas de Artrópodes/farmacologia , Cistatinas/farmacologia , Imunossupressores/farmacologia , Cistatinas Salivares/farmacologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Cristalografia por Raios X , Cistatinas/classificação , Cistatinas/genética , Citocinas/metabolismo , Compostos de Epóxi/metabolismo , Feminino , Imunossupressores/química , Imunossupressores/metabolismo , Ixodes/química , Ixodes/genética , Ixodes/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Óxido Nítrico/metabolismo , Filogenia , Proteólise/efeitos dos fármacos , Cistatinas Salivares/química , Cistatinas Salivares/genética , Homologia de Sequência de Aminoácidos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo
2.
J Allergy Clin Immunol ; 143(3): 1153-1162.e12, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30012514

RESUMO

BACKGROUND: Protease allergens disrupt epithelial barriers to exert their allergenicity. Cystatin SN (encoded by CST1) is an endogenous cysteine protease inhibitor upregulated in nasal epithelia in patients with allergic rhinitis (AR). OBJECTIVE: We sought to investigate the protective effect of human cystatin SN on AR symptoms using pollen-induced AR mouse models. METHODS: We performed an in vitro protease activity assay to evaluate the effect of recombinant human cystatin SN (rhCystatin SN) on Japanese cedar (JC) or ragweed proteases. A human nasal epithelial cell line, RPMI 2650, was used to examine tight junction (TJ) disruption in vitro. Mice were sensitized and nasally challenged with JC or ragweed pollens with or without rhCystatin SN to examine the effect of rhCystatin SN on AR symptoms and the epithelial barrier in vivo. Because mice lack CST1, we generated transgenic (Tg) mice expressing human CST1 under control of its genomic control region (hCST1-Tg mice) to examine the role of cystatin SN in physiologically expressed conditions. RESULTS: rhCystatin SN inhibited JC but not ragweed protease activities and prevented JC-induced but not ragweed-induced TJ disruption in vitro. Exogenous administration of rhCystatin SN ameliorated JC-induced but not ragweed-induced sneezing and nasal TJ disruption in vivo. Furthermore, hCST1-Tg mice showed decreased JC-induced but not ragweed-induced sneezing symptoms and nasal TJ disruption compared with wild-type mice. CONCLUSION: Human cystatin SN suppresses AR symptoms through inhibiting allergen protease activities and protecting the nasal TJ barrier in an allergen-specific manner. We propose that upregulation of nasal endogenous protease inhibitors, including cystatin SN, is a novel therapeutic strategy for protease allergen-induced AR.


Assuntos
Rinite Alérgica/imunologia , Cistatinas Salivares/imunologia , Alérgenos/imunologia , Ambrosia/enzimologia , Ambrosia/imunologia , Animais , Antígenos de Plantas/imunologia , Linhagem Celular , Cryptomeria/enzimologia , Cryptomeria/imunologia , Modelos Animais de Doenças , Humanos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Mucosa Nasal/imunologia , Peptídeo Hidrolases/metabolismo , Extratos Vegetais/imunologia , Pólen/imunologia , Inibidores de Proteases/farmacologia , Proteínas Recombinantes/farmacologia , Rinite Alérgica/genética , Cistatinas Salivares/genética , Cistatinas Salivares/farmacologia , Junções Íntimas/metabolismo
3.
Cell Death Dis ; 8(3): e2682, 2017 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-28300829

RESUMO

Cystatin SN (CST1) is a specific inhibitor belonging to the cystatin superfamily that controls the proteolytic activities of cysteine proteases such as cathepsins. Our previous study showed that high CST1 expression enhances tumor metastasis and invasiveness in colorectal cancer. Recently, auranofin (AF), a gold(I)-containing thioredoxin reductase 1 (TrxR1) inhibitor, has been used clinically to treat rheumatoid arthritis. AF is a proteasome-associated deubiquitinase inhibitor and can act as an anti-tumor agent. In this study, we investigated whether CST1 expression induces autophagy and tumor cell survival. We also investigated the therapeutic effects of AF as an anti-tumor agent in colorectal cancer (CRC) cells. We found that CRC cells expressing high levels of CST1 undergo increased autophagy and exhibit chemotherapeutic resistance to AF-induced cell death, while those expressing low levels of CST1 are sensitive to AF. We also observed that knockdown of CST1 in high-CST1 CRC cells using CST1-specific small interfering RNAs attenuated autophagic activation and restored AF-induced cell mortality. Conversely, the overexpression of CST1 increased autophagy and viability in cells expressing low levels of CST1. Interestingly, high expression of CST1 attenuates AF-induced cell death by inhibiting intracellular reactive oxygen species (ROS) generation, as demonstrated by the fact that the blockage of ROS production reversed AF-induced cell death in CRC cells. In addition, upregulation of CST1 expression increased cellular glutathione reductase (GR) activity, reducing the cellular redox state and inducing autophagy in AF-treated CRC cells. These results suggest that high CST1 expression may be involved in autophagic induction and protects from AF-induced cell death by inhibition of ROS generation through the regulation of GR activity.


Assuntos
Auranofina/farmacologia , Autofagia/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Glutationa Redutase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Cistatinas Salivares/farmacologia , Catepsinas/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Células HT29 , Humanos , Oxirredução/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Cistatinas Salivares/metabolismo , Tiorredoxina Redutase 1/metabolismo , Regulação para Cima/efeitos dos fármacos
4.
J Periodontal Res ; 47(5): 661-73, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22582873

RESUMO

BACKGROUND AND OBJECTIVE: Healthy subjects who do not have Aggregatibacter actinomycetemcomitans in their oral cavity may possess factors in saliva that might demonstrate antibacterial activity against the bacterium. The aim of this study was to identify and purify proteins from saliva of healthy subjects that might demonstrate antibacterial activity against A. actinomycetemcomitans and test the same against the bacteria. MATERIAL AND METHODS: Saliva from 10 healthy volunteers was tested individually for its anti-A. actinomycetemcomitans activity. Among the 10 subjects, eight demonstrated anti-A. actinomycetemcomitans activity. Saliva was collected from one healthy volunteer who demonstrated the highest antimicrobial activity against A. actinomycetemcomitans. After clarifying the saliva, it was subjected to an affinity chromatography column with A. actinomycetemcomitans. The proteins bound to A. actinomycetemcomitans were eluted from the column and identified using mass spectrometry (MALDI-TOF/TOF MS). Among other proteins that bound to A. actinomycetemcomitans, which included lactoferrin, immunoglobulin A and kallikrein, cystatin SA was observed in significantly higher concentrations, and this was purified from the eluate. The purified cystatin SA was tested at different concentrations for its ability to kill A. actinomycetemcomitans in a 2 h cell killing assay. The bacteria were also treated with a proteinase inhibitor, leupeptin, to clarify whether the antimicrobial effect of cystatin SA was related to its protease inhibitory function. Cystatin SA was also tested for its ability to prevent binding of A. actinomycetemcomitans to buccal epithelial cells (BECs) in an A. actinomycetemcomitans-BEC binding assay. RESULTS: Cystatin SA (0.1 mg/mL) demonstrated a statistically significant antimicrobial activity against A. actinomycetemcomitans. The effect of cystatin SA decreased with lower concentrations, with 0.01 mg/mL showing no effect. The addition of monoclonal cystatin SA antibodies to the purified sample completely negated the antimicrobial effect. Treatment of A. actinomycetemcomitans with leupeptin resulted in no antimicrobial effect, suggesting that the antimicrobial activity of cystatin SA is independent of its protease inhibitory function. A. actinomycetemcomitans pretreated with cystatin SA showed reduced binding to BECs, suggesting a potential role for cystatin SA in decreasing the colonization of A. actinomycetemcomitans. CONCLUSION: The present study shows that cystatin SA demonstrates antimicrobial activity against the periodontopathogen A. actinomycetemcomitans, and future studies determining the mechanism of action are necessary. The study also shows the ability of cystatin SA to reduce significantly the binding of A. actinomycetemcomitans to BECs.


Assuntos
Aggregatibacter actinomycetemcomitans/efeitos dos fármacos , Antibacterianos/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Cistatinas Salivares/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Catepsinas/antagonistas & inibidores , Cromatografia de Afinidade , Inibidores de Cisteína Proteinase/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Endopeptidase K/farmacologia , Células Epiteliais/microbiologia , Fusobacterium nucleatum/efeitos dos fármacos , Humanos , Imunoglobulina A Secretora/isolamento & purificação , Calicreínas/isolamento & purificação , Lactoferrina/isolamento & purificação , Leupeptinas/farmacologia , Microscopia Confocal , Mucosa Bucal/citologia , Mucosa Bucal/microbiologia , Porphyromonas gingivalis/efeitos dos fármacos , Saliva/efeitos dos fármacos , Cistatinas Salivares/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo
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