Maturation process and characterization of a novel thermostable and halotolerant subtilisin-like protease with high collagenolytic but low gelatinolytic activity

Enzymatic degradation of collagen is of great industrial and environmental significance; however, little is known about thermophile-derived collagenolytic proteases. Here, we report a novel collagenolytic protease (TSS) from thermophilic Brevibacillus sp. WF146. The TSS precursor comprises a signal peptide, an N-terminal propeptide, a subtilisin-like catalytic domain, a β-jelly roll (βJR) domain, and a prepeptidase C-terminal (PPC) domain. The maturation of TSS involves a stepwise autoprocessing of the N-terminal propeptide and the PPC domain, and the βJR rather than the PPC domain is necessary for correct folding of the enzyme. Purified mature TSS displayed optimal activity at 70°C and pH 9.0, a half-life of 1.5 h at 75°C, and an increased thermostability with rising salinity up to 4 M. TSS possesses an increased number of surface acidic residues and ion pairs, as well as four Ca 2+ -binding sites, which contribute to its high thermostability and halotolerance. At high temperatures, TSS exhibited high activity toward insoluble type I collagen and azocoll, but showed a low gelatinolytic activity, with a strong preference for Arg and Gly at the P1 and P1’ positions, respectively. Both the βJR and PPC domains could bind but not swell collagen, and thus facilitate TSS-mediated collagenolysis via improving the accessibility of the enzyme to the substrate. Additionally, TSS has the ability to efficiently degrade fish scale collagen at high temperatures. IMPORTANCE Proteolytic degradation of collagen at high temperatures has the advantages of increasing degradation efficiency and minimizing the risk of microbial contamination. Reports on thermostable collagenolytic proteases are limited, and their maturation and catalytic mechanisms remain to be elucidated. Our results demonstrate that the thermophile-derived TSS matures in an autocatalytic manner, and represents one of the most thermostable collagenolytic proteases reported so far. At elevated temperatures, TSS prefers hydrolyzing insoluble heat-denatured collagen rather than gelatin, providing new insight into the mechanism of collagen degradation by thermostable collagenolytic proteases. Moreover, TSS has the potential to be used in recycling collagen-rich wastes such as fish scales.

Proteolytic and Structural Changes in Rye and Triticale Roots under Aluminum Stress

Proteolysis and structural adjustments are significant for defense against heavy metals. The purpose of this study was to evaluate whether the Al3+ stress alters protease activity and the anatomy of cereale roots. Azocaseinolytic and gelatinolytic measurements, transcript-level analysis of phytocystatins, and observations under microscopes were performed on the roots of Al3+-tolerant rye and tolerant and sensitive triticales exposed to Al3+. In rye and triticales, the azocaseinolytic activity was higher in treated roots. The gelatinolytic activity in the roots of rye was enhanced between 12 and 24 h in treated roots, and decreased at 48 h. The gelatinolytic activity in treated roots of tolerant triticale was the highest at 24 h and the lowest at 12 h, whereas in treated roots of sensitive triticale it was lowest at 12 h but was enhanced at 24 and 48 h. These changes were accompanied by increased transcript levels of phytocystatins in rye and triticale-treated roots. Light microscope analysis of rye roots revealed disintegration of rhizodermis in treated roots at 48 h and indicated the involvement of root border cells in rye defense against Al3+. The ultrastructural analysis showed vacuoles containing electron-dense precipitates. We postulate that proteolytic-antiproteolytic balance and structural acclimation reinforce the fine-tuning to Al3+.

Endogenous Enzymatic Activity of Primary and Permanent Dentine

Matrix metalloproteinases (MMPs) play an important role in tooth development and influence caries development and hybrid layer degradation. Literature is scant on the differences in the activity of MMPs between primary and permanent dentine. Accordingly, the aim of the present study was to investigate endogenous gelatinolytic activity in primary and permanent dentine. Separate batches of dentine powder were obtained from intact human primary and permanent molars (n = 6). Each batch was divided in two subgroups: (1) mineralised; and (2) demineralised with 10% H3PO4. After protein extraction, gelatine zymography was performed. Furthermore, in situ zymography was performed on dentine sections of the same groups (n = 3). The slices were polished, covered with fluorescein-conjugated gelatine and evaluated using a confocal microscope. In situ zymography data were analysed using two-way analysis of variance and post hoc Holm–Šidák statistics (α = 0.05). Primary dentine showed poorly defined bands in the zymograms that vaguely corresponded to the pro-form and active form of MMP-2 and the pro-form of MMP-9. In permanent dentine, demineralised powder demonstrated stronger gelatinolytic activity than mineralised powder. In situ zymography identified stronger enzymatic activity in primary etched dentine (p < 0.05). Stronger enzymatic activity recorded in primary dentine may be related to the differences in morphology and composition between primary and permanent dentine.

Cyclooxygenase Inhibition Alters Proliferative, Migratory, and Invasive Properties of Human Glioblastoma Cells In Vitro

Prostaglandin E2 (PGE2) is known to increase glioblastoma (GBM) cell proliferation and migration while cyclooxygenase (COX) inhibition decreases proliferation and migration. The present study investigated the effects of COX inhibitors and PGE2 receptor antagonists on GBM cell biology. Cells were grown with inhibitors and dose response, viable cell counting, flow cytometry, cell migration, gene expression, Western blotting, and gelatin zymography studies were performed. The stimulatory effects of PGE2 and the inhibitory effects of ibuprofen (IBP) were confirmed in GBM cells. The EP2 and EP4 receptors were identified as important mediators of the actions of PGE2 in GBM cells. The concomitant inhibition of EP2 and EP4 caused a significant decrease in cell migration which was not reverted by exogenous PGE2. In T98G cells exogenous PGE2 increased latent MMP2 gelatinolytic activity. The inhibition of COX1 or COX2 caused significant alterations in MMP2 expression and gelatinolytic activity in GBM cells. These findings provide further evidence for the importance of PGE2 signalling through the EP2 and the EP4 receptor in the control of GBM cell biology. They also support the hypothesis that a relationship exists between COX1 and MMP2 in GBM cells which merits further investigation as a novel therapeutic target for drug development.

Myeloperoxidase Inhibition Decreases the Expression of Collagen and Metallopeptidase in Mare Endometria under In Vitro Conditions

Neutrophils can originate neutrophil extracellular traps (NETs). Myeloperoxidase (MPO) is a peroxidase found in NETs associated to equine endometrosis and can be inhibited by 4-aminobenzoic acid hydrazide (ABAH). Metallopeptidases (MMPs) participate in extracellular matrix stability and fibrosis development. The objectives of this in vitro work were to investigate, in explants of mare’s endometrium, (i) the ABAH capacity to inhibit MPO-induced collagen type I (COL1) expression; and (ii) the action of MPO and ABAH on the expression and gelatinolytic activity of MMP-2/-9. Explants retrieved from the endometrium of mares in follicular or mid-luteal phases were treated with MPO, ABAH, or their combination, for 24 or 48 h. The qPCR analysis measured the transcription of COL1A2, MMP2, and MMP9. Western blot and zymography were performed to evaluate COL1 protein relative abundance and gelatinolytic activity of MMP-2/-9, respectively. Myeloperoxidase elevated COL1 relative protein abundance at both treatment times in follicular phase (p < 0.05). The capacity of ABAH to inhibit MPO-induced COL1 was detected in follicular phase at 48 h (p < 0.05). The gelatinolytic activity of activated MMP-2 augmented in mid-luteal phase at 24 h after MPO treatment, but it was reduced with MPO+ABAH treatment. The activity of MMP-9 active form augmented in MPO-treated explants. However, this effect was inhibited by ABAH in the follicular phase at 48 h (p < 0.05). By inhibiting the pro-fibrotic effects of MPO, it might be possible to reduce the development of endometrosis. Metallopeptidase-2 might be involved in an acute response to MPO in the mid-luteal phase, while MMP-9 might be implicated in a prolonged exposition to MPO in the follicular phase.

The In Vitro Inhibitory Effect of Sivelestat on Elastase Induced Collagen and Metallopeptidase Expression in Equine Endometrium

Neutrophil extracellular traps (NETs) fight endometritis, and elastase (ELA), a protease found in NETs, might induce collagen type I (COL1) accumulation in equine endometrium. Metallopeptidases (MMPs) are involved in extracellular matrix balance. The aim was to evaluate the effects of ELA and sivelestat (selective elastase inhibitor) on MMP-2 and MMP-9 expression and gelatinolytic activity, as well as the potential inhibitory effect of sivelestat on ELA-induced COL1 in equine endometrium. Endometrial explants from follicular (FP) and mid-luteal (MLP) phases were treated for 24 or 48 h with ELA, sivelestat, and their combination. Transcripts of COL1A2, MMP2, and MMP9 were evaluated by qPCR; COL1 protein relative abundance by Western blot, and MMP-2 and MMP-9 gelatinolytic activity by zymography. In response to ELA treatment, there was an increase in MMP2 mRNA transcription (24 h) in active MMP-2 (48 h), both in FP, and in MMP9 transcripts in FP (48 h) and MLP (24 h) (p < 0.05). Sivelestat inhibited ELA-induced COL1A2 transcripts in FP (24 h) and MLP (24 h, 48 h) (p < 0.05). The sivelestat inhibitory effect was detected in MMP9 transcripts in FP at 48 h (p < 0.05), but proteases activity was unchanged. Thus, MMP-2 and MMP-9 might be implicated in endometrium fibrotic response to ELA. In mare endometrium, sivelestat may decrease ELA-induced COL1 deposition and hinder endometrosis development.

N-acetyl-seryl-aspartyl-lysyl-proline treatment protects heart against excessive myocardial injury and heart failure in mice

Myocardial infarction (MI) in mice results in cardiac rupture at 4–7 days after MI, whereas cardiac fibrosis and dysfunction occur later. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) has anti-inflammatory, anti-fibrotic, and pro-angiogenic properties. We hypothesized that Ac-SDKP reduces cardiac rupture and adverse cardiac remodeling, and improves function by promoting angiogenesis and inhibiting detrimental reactive fibrosis and inflammation after MI. C57BL/6J mice were subjected to MI and treated with Ac-SDKP (1.6 mg/kg per day) for 1 or 5 weeks. We analyzed (1) intercellular adhesion molecule-1 (ICAM-1) expression; (2) inflammatory cell infiltration and angiogenesis; (3) gelatinolytic activity; (4) incidence of cardiac rupture; (5) p53, the endoplasmic reticulum stress marker CCAAT/enhancer binding protein homology protein (CHOP), and cardiomyocyte apoptosis; (6) sarcoplasmic reticulum Ca2+ ATPase (SERCA2) expression; (7) interstitial collagen fraction and capillary density; and (8) cardiac remodeling and function. Acutely, Ac-SDKP reduced cardiac rupture, decreased ICAM-1 expression and the number of infiltrating macrophages, decreased gelatinolytic activity, p53 expression, and myocyte apoptosis, but increased capillary density in the infarction border. Chronically, Ac-SDKP improved cardiac structures and function, reduced CHOP expression and interstitial collagen fraction, and preserved myocardium SERCA2 expression. Thus, Ac-SDKP decreased cardiac rupture, ameliorated adverse cardiac remodeling, and improved cardiac function after MI, likely through preserved SERCA2 expression and inhibition of endoplasmic reticulum stress.