RESUMO
Serine protease inhibitors of Kazal-type (SPINKs) were widely identified in vertebrates and invertebrates, and played regulatory roles in digestion, coagulation, and fibrinolysis. In this study, we reported the important role of SPINK7 in regulating immune defense of silkworm, Bombyx mori. SPINK7 contains three Kazal domains and has 6 conserved cysteine residues in each domain. Quantitative real-time PCR analyses revealed that SPINK7 was exclusively expressed in hemocytes and was upregulated after infection with two fungi, Saccharomyces cerevisiae and Candida albicans. Enzyme activity inhibition test showed that SPINK7 significantly inhibited the activity of proteinase K from C. albicans. Additionally, SPINK7 inhibited the growth of three fungal spores, including S. cerevisiae, C. albicans, and Beauveria bassiana. The pathogen-associated molecular patterns (PAMP) binding assays suggested that SPINK7 could bind to ß-D-glucan and agglutinate B. bassiana and C. albicans. In vitro assays were performed using SPINK7-coated agarose beads, and indicated that SPINK7 promoted encapsulation and melanization of agarose beads by B. mori hemocytes. Furthermore, co-localization studies using immunofluorescence revealed that SPINK7 induced hemocytes to aggregate and entrap the fungi spores of B. bassiana and C. albicans. Our study revealed that SPINK7 could recognize fungal PAMP and induce the aggregation, melanization, and encapsulation of hemocytes, and provided valuable clues for understanding the innate immunity and cellular immunity in insects.
Assuntos
Beauveria/imunologia , Bombyx/imunologia , Candida albicans/imunologia , Hemócitos/imunologia , Proteínas de Insetos/metabolismo , Micoses/imunologia , Saccharomyces cerevisiae/imunologia , Inibidor da Tripsina Pancreática de Kazal/metabolismo , Animais , Beauveria/metabolismo , Beauveria/patogenicidade , Bombyx/genética , Bombyx/metabolismo , Bombyx/microbiologia , Candida albicans/metabolismo , Candida albicans/patogenicidade , Hemócitos/metabolismo , Hemócitos/microbiologia , Interações entre Hospedeiro e Microrganismos , Imunidade Celular , Imunidade Inata , Proteínas de Insetos/genética , Micoses/genética , Micoses/metabolismo , Moléculas com Motivos Associados a Patógenos/metabolismo , Saccharomyces cerevisiae/patogenicidade , Transdução de Sinais , Inibidor da Tripsina Pancreática de Kazal/genéticaRESUMO
Serpins generally serve as inhibitors that utilize a mobile reactive center loop (RCL) as bait to trap protease targets. Here, we present the crystal structure of serpin18 from Bombyx mori at 1.65 Å resolution, which has a very short and stable RCL. Activity analysis showed that the inhibitory target of serpin18 is a cysteine protease rather than a serine protease. Notably, this inhibitiory reaction results from the formation of an intermediate complex, which then follows for the digestion of protease and inhibitor into small fragments. This activity differs from previously reported modes of inhibition for serpins. Our findings have thus provided novel structural insights into the unique inhibitory mechanism of serpin18. Furthermore, one physiological target of serpin18, fibroinase, was identified, which enables us to better define the potential role for serpin18 in regulating fibroinase activity during B. mori development.
Assuntos
Bombyx/metabolismo , Proteínas de Insetos/química , Inibidores de Proteases/química , Serpinas/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Humanos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Cinética , Dados de Sequência Molecular , Inibidores de Proteases/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Serina Endopeptidases/química , Serpinas/genética , Serpinas/metabolismoRESUMO
Serpins (serine proteinase inhibitors) are widely distributed in different species and are well known for their inhibitory activities towards serine proteinases. Here, we report the functional characterization of Bombyx mori serpin16. Expression analysis showed that serpin16 was specifically expressed at high levels in the silk gland at both the transcriptional and translational levels. Moreover, homology modeling and multi-sequence alignment suggested that serpin16 had a canonical serpin fold, but it contained a unique reactive center loop, which was obviously shorter than that of typical serpins. Inhibitory activity analyses revealed that the target proteinase of serpin18 is a cysteine proteinase, rather than a serine proteinase. Furthermore, a Michaelis complex model of serpin16 with its target proteinase was constructed to explain the structural basis of how serpin16 recognizes the cysteine proteinase and its target specificity.
Assuntos
Estruturas Animais/metabolismo , Bombyx/metabolismo , Cisteína Proteases/metabolismo , Especificidade de Órgãos , Inibidores de Proteases/metabolismo , Seda/metabolismo , Sequência de Aminoácidos , Animais , Regulação da Expressão Gênica , Proteínas de Insetos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Inibidores de Proteases/química , Análise de Sequência de Proteína , Especificidade por SubstratoRESUMO
As a distinct type of aggressive mature large B-cell lymphoma, plasmablastic lymphoma (PBL) poses diagnostic and treatment challenges. PBL is distinguished from other B-cell lymphomas by the presence of plasmacytic differentiation markers such as CD38, CD138, and MUM1. Clinically, PBLs from oral and extra-oral sites are rapidly progressive tumors with frequent relapse after treatment with standard diffuse large B-cell lymphoma regimens. Here, we report a near-complete response of one patient with relapsed PBL following treatment with a non-cytotoxic regimen containing bortezomib, rituximab, and dexamethasone.