RESUMO
Neutrophils play major roles against bacteria and fungi infections not only due to their microbicide properties but also because they release mediators like Interleukin-1 beta (IL-1ß) that contribute to orchestrate the inflammatory response. This cytokine is a leaderless protein synthesized in the cytoplasm as a precursor (pro-IL-1ß) that is proteolytically processed to its active isoform and released from human neutrophils by secretory autophagy. In most myeloid cells, pro-IL-1ß is processed by caspase-1 upon inflammasome activation. Here we employed neutrophils from both healthy donors and patients with a gain-of-function (GOF) NLRP3-mutation to dissect IL-1ß processing in these cells. We found that although caspase-1 is required for IL-1ß secretion, it undergoes rapid inactivation, and instead, neutrophil serine proteases play a key role in pro-IL-1ß processing. Our findings bring to light distinctive features of the regulation of caspase-1 activity in human neutrophils and reveal new molecular mechanisms that control human neutrophil IL-1ß secretion.
Assuntos
Autofagia , Caspase 1 , Interleucina-1beta , Neutrófilos , Serina Proteases , Autofagia/genética , Autofagia/imunologia , Caspase 1/genética , Caspase 1/metabolismo , Humanos , Inflamassomos/genética , Inflamassomos/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Neutrófilos/enzimologia , Neutrófilos/imunologia , Serina Endopeptidases/genética , Serina Endopeptidases/imunologia , Serina Proteases/genética , Serina Proteases/imunologiaRESUMO
BACKGROUND: The preparation of broad bean koji is a key process in the production of Pixian broad bean paste (PBP). Protease is essential for the degradation of proteins during PBP fermentation. To obtain broad bean koji with high protease activity using the cocultivated strains of Aspergillus oryzae QM-6 (A. oryzae QM-6) and Aspergillus niger QH-3 (A. niger QH-3), the optimization of acid and neutral protease activities was carried out using BoxBehnken design with response surface methodology (RSM). RESULTS: The optimum conditions were found to be as follows: inoculation proportion (X1), 3:1 (A. oryzae QM-6: A. niger QH-3, w/w); culture temperature (X2), 33°C; inoculum size (X3), 0.5% (w/w); incubation time (X4), 5 d. The acid and neutral protease activities were 605.2 ± 12.4 U/g and 1582.9 ± 23.7 U/g, respectively, which were in good agreement with the predicted values. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles revealed that the broad bean koji extracellular proteins in the case of cocultivation were richer compared to those in the case of A. oryzae QM-6 or A. niger QH-3 strain only. In addition, the free amino acids (FAAs) in the fermentation product were 55% higher in the cocultivation process than in that involving only A. oryzae QM-6, further confirming the diversity of proteases in the fermentation products. CONCLUSIONS: The optimal conditions of koji-making in PBP were obtained using RSM. The cocultivation of A. oryzae and A. niger increases the overall enzyme activities in the culture medium and the FAAs content, which would thus have potential application in the PBP industry.
Assuntos
Peptídeo Hidrolases/metabolismo , Aspergillus niger , Aspergillus oryzae , Fabaceae/enzimologia , Técnicas de Cocultura , Vicia faba , Eletroforese em Gel de Poliacrilamida , Fermentação , AminoácidosRESUMO
Effects of hypothermia and subsequent self-warming on activity of Ca(2+)-dependent neutral proteases were studied in tissues of ground squirrels and rats. Moderate hypothermia did not significantly change activity of Ca(2+)-dependent neutral proteases in the analyzed tissues of ground squirrels, but reduced protease activity in rat heart. Severe hypothermia reduced enzyme activity in the analyzed tissues of rats and ground squirrels. Differences in activity of Ca(2+)-dependent neutral proteases after long-term hypothermia and subsequent self-warming were found only in the heart.
