Search Results (449)

Prostate cancer (PCa) metastasis is reliant on the activity of proteases, such as matrix metalloproteinase-2 (MMP-2). While increased extracellular heat shock protein 90α (eHSP90α) has been linked to increased MMP-2 activity, this has not been examined in the context of cellular stress. We examined stress-induced eHSP90α in human prostate cell lines by immunoblot. Fluorometric gelatin dequenching and zymography assays measured MMP activity. Wound healing and Matrigel drop invasion assays were used to quantify cell motility. HSP90α knockout (KO) cells were established with CRISPR/Cas9. Proteases were profiled with molecular inhibitors and protein arrays and validated by siRNA knockdown, immunoblot, and motility assays. Stress increased eHSP90 in four out of four human prostate cell lines examined. Surprisingly, it concurrently decreased MMP-2 activity. The functional relevance of this was demonstrated when conditioned media from stressed cells decreased the motility of non-stressed cells. Screening for protease inhibitors that would rescue stress-induced decreases in MMP-2 activity identified a single serine protease inhibitor: aprotinin. Yet rescue with aprotinin was lost in HSP90α KO cells. A protease array identified stress-induced increases in kallikrein-related peptidase 6 (KLK6). Knockdown of KLK6 rescued stress-induced MMP-2 activity and cell motility. In conclusion, we identify a novel stress-induced extracellular network that regulates MMP-2 activity and cell motility. We identified KLK6 as a stress-induced extracellular protease leading to decreased MMP-2 activity and cellular invasion, while eHSP90α is required for the rescue of MMP-2 activity once KLK6 is neutralized. Full article

Alpha-1 antitrypsin (AAT) is a serine protease inhibitor with potent anti-inflammatory and immunomodulatory properties, but its role in cancer is context-dependent across tumor types. We integrated transcriptomic analyses of human melanoma cohorts, in vivo studies using AAT-transgenic (hAAT-TG) mice, and in vitro assays in murine and human melanoma cells to define the biological functions of AAT in melanoma. SERPINA1 expression increased progressively from normal skin to nevi and metastatic melanoma, yet higher intratumoral levels correlated with improved overall survival in metastatic disease. In hAAT-TG mice, melanoma growth was markedly inhibited compared with wild-type controls, and the inhibitory effect required CD8⁺ T cells and was enhanced by CD4⁺ T-cell depletion, demonstrating that AAT promotes cytotoxic T-cell activity while attenuating regulatory T-cell suppression. Histologic analysis showed heavily pigmented tumors in hAAT-TG mice. In vitro, hAAT upregulated melanocytic differentiation markers (MITF, TYR, PMEL, MART-1) and increased melanin production in murine and human melanoma lines, suggesting enhanced tumor immunogenicity. In conclusion, hAAT exerts antitumor effects in melanoma indirectly by reprogramming the tumor microenvironment toward differentiation and immune activation. These findings highlight a previously unrecognized role for AAT as a dual immunoregulatory and differentiation-promoting factor and support AAT as a potential immunoregulatory adjuvant in melanoma. Full article

Background/Objectives: Oral potentially malignant disorders (OPMDs), including oral leukoplakia (OLK), oral lichen planus (OLP), and actinic cheilitis (AC), have varying risks of progression to oral squamous cell carcinoma (OSCC). Biomarkers such as aldehyde dehydrogenase 1 (ALDH1) and mammary serine protease inhibitor (Maspin) have shown potential for diagnostic and prognostic use in oral cancer. The present study aimed to evaluate the immunoexpression of aldehyde dehydrogenase 1, a cancer stem cell marker associated with aggressiveness, and the mammary serine protease inhibitor, a potential tumor suppressor, in OPMD and OSCC tissues. Methods: A retrospective analysis was performed on 145 biopsy specimens collected from January 2015 to January 2023, including normal epithelium, OPMDs (OLK, OLP, AC), and OSCC. ALDH1 and Maspin expression levels were evaluated using immunohistochemistry, considering both the percentage of positive cells and staining intensity. Statistical analyses were carried out using the Statistical Package for the Social Sciences (SPSS, version 29.0; IBM Corp., Chicago, IL, USA). Results: Normal oral epithelium showed no expression of ALDH1, whereas 40.6% of OPMDs and 44.4% of OSCC samples exhibited high cytoplasmic ALDH1 expression. Nuclear ALDH1 expression was elevated in 29.7% of OPMDs and 38.9% of OSCCs (p < 0.001). Nuclear Maspin expression was high in 95.2% of normal tissues, in 67.2% of OPMDs and in 55.6% of OSCCs (p < 0.001). Maspin showed strong nuclear and cytoplasmic expression in normal tissue, but its expression decreased in OPMDs and OSCCs, with statistically significant reductions in both compartments (p < 0.001). Conclusions: The results indicate that ALDH1 upregulation and Maspin downregulation are hallmark events in oral carcinogenesis. Their combined evaluation provides a powerful tool for assessing dysplastic severity and malignant transformation risk in OPMDs. Future studies on larger cohorts are needed to confirm the prognostic utility of this dual-marker model. Full article

Background: In this study, we report a novel series of proline- and pipecolic acid-based small molecules designed as allosteric inhibitors of the NS2B/NS3 serine proteases from dengue and Zika viruses, key targets in antiviral drug discovery. Results: Enzymatic studies revealed that S-proline derivatives bearing electron-withdrawing substituents on the aromatic ring, particularly that with a trifluoromethyl group in meta position (i.e., compound 3, IC₅₀ = 5.0 µM), were the most potent against DENV NS2B/NS3, while nitro-substituted inhibitors were mostly effective only against the ZIKV protease. R-configured pipecolic acid-based derivatives were the only ones active against DENV NS2B/NS3, even if the mid-micromolar range; however, they demonstrated improved cellular efficacy since inhibitors 24 and 27 exhibiting strong activity in a DENV2 protease reporter gene assay (EC₅₀ = 5.2 and 5.1 µM, respectively). All compounds showed no cytotoxicity (CC₅₀ > 100 µM) and were selective for the viral protease over off-target serine proteases. Structure-based approaches were exploited to map the druggable allosteric site close to Asn152. Conclusions: Our findings led us to identify proline and pipecolic acid-based inhibitors as promising leads for the development of selective flaviviral NS2B/NS3 allosteric inhibitors. Full article

Impaired intestinal proteolytic activity can lead to increased intestinal permeability. Differences in dietary protein intake may influence proteolytic activity in the digestive system. This study investigated whether intestinal proteolytic activity can be influenced by diet. Fecal samples from representative species of each different dietary group—carnivore, herbivore, and omnivore—were analyzed using fluorescence resonance energy transfer (FRET) peptide substrates to measure enzyme activity. Specific protease inhibitors were applied to identify the enzyme classes responsible for substrate degradation. Results showed that total proteolytic activity was significantly higher in feces from carnivores and omnivores than in those of herbivores. The addition of a serine protease inhibitor substantially reduced substrate degradation, indicating that serine proteases accounted for most of the observed activity. These findings demonstrate that proteolytic activity in feces is closely related to dietary protein intake and suggest that the regulation of proteases in the digestive tract may be influenced by feeding behavior and nutritional requirements. Full article

Plant protease inhibitors (PPI) play a significant role against microbes, insects, and, to a considerable extent, human pathogens. PPIs inactivate hydrolase enzymes or depolarize the plasma membrane of the pathogens, thereby inhibiting their growth, replication, and invasion. Here, an active serine protease inhibitor was isolated and purified from the seeds of Cleome viscosa. The purified inhibitor was homogenous and exhibited a molecular weight of around 12 kDa as a monomer. The secondary structure analysis indicated that the inhibitor was predominantly composed of α-helical content. The kinetics experiments demonstrated a noncompetitive mode of inhibition towards serine protease when casein was used as the enzyme substrate. The inhibitor formed a stable complex with serine protease, having a likely 1:1 stoichiometry, as inferred from ITC, and the dissociation constant was examined to be K_d = 1.9 × 10⁻⁶ M with a Gibbs free energy of ΔG = −8.079 (kcal/mol). The inhibitor exhibits stable protease inhibition up to 90 °C. Further, in vitro preliminary studies revealed its inhibitory effects against HSV-2 function, evidence that it may have a role in the treatment of viral infections. Full article

The premetastatic niche (PMN) represents a specialized microenvironment established in distant organs before the arrival of metastatic cells. This concept has fundamentally altered our understanding of cancer progression, shifting it from a random event-driven process to an orchestrated one. This review examines the critical role of extracellular proteases in PMN formation, focusing on matrix metalloproteinases (MMPs), serine proteases, and cysteine cathepsins that collectively orchestrate extracellular matrix remodeling, immune modulation, and vascular permeability changes essential for metastatic colonization. Key findings demonstrate that MMP9 and MMP2 facilitate basement membrane degradation and the recruitment of bone marrow-derived cells. At the same time, tissue inhibitor of metalloproteinase-1 (TIMP-1) promotes organ-specific hepatic PMN recruitment through neutrophil recruitment mechanisms. The plasminogen–plasmin system emerges as a master regulator through its broad-spectrum proteolytic activity and ability to activate downstream proteases, with S100A10-mediated plasmin generation providing mechanistic pathways for remote PMN conditioning. Neutrophil elastase and cathepsin G contribute to the degradation of anti-angiogenic proteins, thereby creating pro-metastatic microenvironments. These protease-mediated mechanisms represent the earliest interventional window in metastatic progression, offering therapeutic potential to prevent niche formation rather than treat established metastases. However, significant methodological challenges remain, including the need for organ-specific biomarkers, improved in vivo methods for measuring protease activity, and a better understanding of temporal PMN dynamics across different target organs. Full article

Dengue virus (DENV) and Zika virus (ZIKV) are flaviviruses transmitted by Aedes spp. mosquitoes, causing a spectrum of symptoms ranging from mild fevers and joint pain to severe damage to vital organs, including the kidneys, brain, and liver. Unfortunately, there are currently no specific treatments for these viruses. The NS2B/NS3 serine protease has been recognized as a crucial therapeutic target due to its pivotal role in viral replication. Herein, several molecular modeling techniques were employed to search for novel allosteric inhibitors against DENV and ZIKV NS2B/NS3 proteases from a set of 545 in-house compounds. Virtual screening based on molecular docking and MM/GBSA-based free energy calculations indicated that, among 545 derivatives, four compounds demonstrated high binding affinity against both targets, including two sulfonamide-vinyl sulfone hybrids (cpd48_e and cpd50_e), one sulfonamide-chalcone analog (cpd48), and one berberine-cinnamic acid derivative (DN071_f). Their molecular complexation was driven mainly by van der Waals forces rather than electrostatic attraction. Several residues at the enzyme allosteric site, particularly K74, L149, and N152 (DENV) and L76, I123, N152, and V155 (ZIKV), were identified as binding hotspots for the screened compounds. Drug-likeness predictions based on Lipinski’s rule of five further supported their potential as drug candidates. Overall, these findings provide valuable insights for the future design and development of novel antiviral drugs targeting the DENV and ZIKV NS2B/NS3 proteases. Full article

Acute kidney injury (AKI) remains a common clinical syndrome associated with high morbidity and mortality. However, effective diagnostic biomarkers and specific therapeutic interventions are still lacking. Secretory leukocyte protease inhibitor (SLPI), a serine protease inhibitor with pleiotropic functions, has emerged as an early diagnostic and prognostic biomarker for AKI. Clinical studies reveal significant elevation of serum SLPI in AKI patients compared to non-AKI patients at the acute phase following post-cardiovascular surgery, supporting its diagnostic potential. Furthermore, evidence also suggests that SLPI showed prognostic value for kidney transplantation and chronic kidney disease progression associated with diverse etiology, including diabetes. In addition, current evidence highlights the biological functions of SLPI in inhibiting NF-κB activities, suppressing neutrophil extracellular trap formation, modulating phagocytosis, regulating cell apoptosis, proliferation, differentiation, and potentially fibrosis across various disease contexts. Preclinical studies demonstrate that administration of recombinant SLPI ameliorates renal dysfunction in multiple AKI models, including ischemia–reperfusion injury and nephrotoxic models induced by gentamicin or cisplatin. Furthermore, the antifibrotic properties of SLPI underscore its therapeutic potential in halting AKI progression to chronic kidney disease. By integrating available evidence, this review aims to elucidate that, as an early acute-phase response molecule, SLPI serves dual roles as not only an early diagnostic and prognostic biomarker for AKI, but also a renoprotective molecule countering kidney injury. Full article

Corneodesmosomes are specialized intercellular junctions that mediate adhesion between corneocytes in the stratum corneum (SC). The degradation of these structures is regulated by kallikrein-related peptidases (KLKs) and their inhibitors. This study aimed to elucidate the effects of Shotokuseki extract (SE), a substance rich in various trace elements, on molecules related to SC adhesion using a three-dimensional cultured human epidermis model. SE was applied to a three-dimensional epidermis model, and analyses were conducted on gene expression, protease activity, protein levels, and tissue structure. SE treatment significantly upregulated the mRNA expression of corneodesmosomal components (desmoglein1, desmocollin1, and corneodesmosin) and the major protease inhibitor serine peptidase inhibitor Kazal type 5. Concurrently, SE increased the mRNA expression of the trypsin-like protease KLK5,while significantly decreasing the mRNA expression and activity of the chymotrypsin-like protease KLK7. Although no significant changes were observed in the protein levels of corneodesmosomal components, histological analysis revealed that SE significantly increased the ratio of SC thickness to total epidermal thickness. These findings suggest that SE contributes to the homeostasis of the SC by simultaneously promoting the expression of genes encoding corneodesmosomal components, and regulating the balance of the protease/inhibitor system involved in their degradation. The selective suppression of KLK7 activity may appropriately regulate the final stage of desquamation, thereby stabilizing barrier function. Full article

Serine protease inhibitor A3 (SERPINA3), also called α-1-antichymotrypsin, is a serine protease involved in placental dysfunction. This study examines SERPINA3 polymorphisms and haplotypes for associations with maternal hypertensive disorders of pregnancy (HDPs) and preeclampsia with severe features (sPE) or Hemolysis, Elevated Liver Enzymes, and Low Platelet (HELLP) syndrome in mother–baby dyads (HDP) and mother–father–baby triads (sPE/HELLP). This retrospective case–control study examined two patient cohorts, HDPs and severe PE/HELLP syndrome. The HDP population included cases (n = 142) and controls (n = 168) of mother–baby dyads recruited from a large, urban, safety-net hospital in Los Angeles. The sPE/HELLP syndrome population included cases (n = 189) and controls (n = 28) of mother–father–baby triads recruited through HELLP syndrome research websites. Cases were verified by medical chart abstraction when possible. Two SERPINA3 SNPs, rs4934 and rs1884082, were genotyped from saliva samples, mouthwash, or buccal swabs. The Haplin package in R was used to perform genetic association analyses. No evidence of increased risk related to individual SERPINA3 SNPs or haplotypes for the developing HDPs or sPE/HELLP was found in individual nor combined cohorts. In the HDP cohort, the g-a haplotype (relative to T-G haplotype) was borderline significant for increased risk of HDPs when carried by the child (double dose: RR = 1.58, 95% CI: (1.00, 2.52), p = 0.05). We observed significant parent-of-origin (PoO) effects in the combined cohort: specifically, an increased risk of HDPs/sPE/HELLP if the mother carries a double copy for both rs4934 (RR = 3.03, 95% CI (1.50, 6.09), p < 0.01) and rs1884082 (RR = 2.38, 95% CI (1.22, 4.71), p = 0.01). A reduced risk of HDPs/sPE/HELLP was observed for rs4934 (RR = 0.54, 95% CI (0.31, 0.98), p = 0.04) and rs1884082 (RR = 0.52, 95% CI (0.30, 0.91), p = 0.02) with child carriage of the maternally inherited allele. In contrast, child carriage of a paternally inherited copy of the variant allele for rs4934 increased risk of HDPs/sPE/HELLP (RR = 1.54, 95% CI (1.09, 2.20), p = 0.02). There was no evidence that SERPINA3 gene polymorphisms and haplotypes were associated with risk of HDPs or sPE/HELLP. However, significant PoO effects were observed in the combined cohort analysis, with child carriage of rs4934 that is maternally inherited decreasing HDPs/sPE/HELLP risk while a paternally inherited copy increases risk, suggesting a role for maternal–fetal genomic incompatibility. Full article

Cathepsin C (CTSC) mediates neutrophil serine protease (NSP) maturation, contributing to inflammatory cascades, making it a key therapeutic target. In this study, through large-scale screening of a natural product library, marrubiin, a diterpenoid lactone compound, was identified as a potent CTSC inhibitor, which holds potential value in the treatment of inflammatory diseases. It inhibited human recombinant CTSC (IC₅₀ = 57.5 nM) and intracellular CTSC (IC₅₀ = 51.6 nM) with acceptable cytotoxicity, and reduced the activity and protein levels of downstream NSPs in vitro. Functionally, marrubiin inhibited lipopolysaccharide-induced nitric oxide release and regulated the levels of cytokines and chemokines. Docking result predicted marrubiin may achieve CTSC activity inhibition by using lactone structure as a covalent unit to target Cys234. In vivo study indicated that high-dose marrubiin (IC₅₀ = 30 mg/kg) reduced CTSC and NSPs activities in blood and bone marrow in mice without toxicity, and its efficacy was comparable to that of positive compound AZD7986. In the adjuvant-induced arthritis model, high-dose marrubiin (IC₅₀ = 60 mg/kg) exerted a therapeutic effect by reducing the activities of CTSC and NSPs. These findings indicated marrubiin is a promising natural CTSC inhibitor, which can be used for the treatment of neutrophil-related inflammatory diseases. Full article

Background/Objectives: Peritonsillar abscess (PTA) and cellulitis (PTC) often present with similar clinical features, making differentiation challenging despite imaging. This study evaluates the diagnostic performance of serum HMGB1 and kallistatin levels as potential independent biomarkers to distinguish PTA from PTC. Methods: In this single-center prospective cohort study, 97 patients aged 18 to 65 years who met the inclusion criteria and presented with peritonsillar infection (39 PTA; 58 PTC) between February and July 2025 were enrolled. Serum levels of HMGB1, kallistatin, and routine inflammatory markers were measured and compared. Univariate and multivariate logistic regression analyses identified independent predictors for distinguishing PTA from PTC. Receiver operating characteristic (ROC) curve analysis assessed the diagnostic accuracy of biomarkers. Decision curve analysis (DCA) was performed to evaluate the clinical net benefit of individual biomarkers and their combinations across a range of threshold probabilities. Results: Compared to controls, patients with peritonsillar infection had significantly higher WBC, neutrophil, CRP, procalcitonin, and HMGB1 levels and significantly lower kallistatin levels (all p < 0.05). Within the infection group, PTA patients showed significantly higher CRP (p = 0.036) and HMGB1 (p = 0.003) levels and lower kallistatin (p < 0.001) levels compared to PTC patients. In univariate analysis, CRP, HMGB1, and kallistatin were significantly associated with PTA; however, in multivariate analysis, only elevated HMGB1 (OR: 1.21; 95% CI: 1.09–1.35; p < 0.001) and reduced kallistatin (OR: 0.395; 95% CI: 0.24–0.648; p < 0.001) remained independent predictors. ROC analysis showed that both HMGB1 and kallistatin demonstrated good discriminative ability in distinguishing PTA from PTC. DCA revealed that the three-biomarker combination (kallistatin + HMGB1 + CRP) achieved the highest mean net benefit (0.183) across all threshold probabilities, outperforming individual biomarkers (kallistatin: 0.131, HMGB1: 0.111, CRP: 0.099) and the two-biomarker model (0.176). The combined model maintained superior net benefit across threshold probabilities of 25–75%, indicating optimal clinical utility within this decision range. Conclusions: Serum HMGB1 and kallistatin may be effective adjunctive biomarkers for differentiating PTA from PTC. Full article

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry is facilitated by transmembrane protease serine 2 (TMPRSS2), which is regulated by the androgen receptor (AR). Androgen deprivation therapy (ADT), widely used in prostate cancer treatment, may potentially modulate TMPRSS2 expression, affecting SARS-CoV-2 infection susceptibility and severity. We evaluated the impact of ADT on pulmonary TMPRSS2 expression in prostate cancer patients and analyzed differences in expression patterns associated with specific ADT regimens. We examined TMPRSS2 immunohistochemical expression in lung tissue from 20 consecutive autopsy cases of men with prostate cancer (6 receiving ADT at time of death), compared with non-ADT prostate cancer patients and age-matched women controls. Histoscores were calculated by assessing the percentage and intensity of pneumocyte TMPRSS2 expression. Prostate cancer patients receiving ADT showed significantly reduced pulmonary TMPRSS2 expression compared to non-ADT patients (mean histoscores: 152.7 vs. 225.0, p = 0.037) and age-matched women controls (mean histoscores: 152.7 vs. 238.0, p = 0.024). Direct AR antagonists (apalutamide, bicalutamide) produced greater TMPRSS2 suppression than Gonadotropin-Releasing Hormone modulators or androgen biosynthesis inhibitors. No significant correlation was observed between the TMPRSS2 expression and Gleason score, PSA levels, or underlying lung pathology. Our findings demonstrate that ADT significantly reduces pulmonary TMPRSS2 expression, with direct AR antagonists showing the strongest effect. This suggests a potential mechanistic explanation for differential COVID-19 susceptibility and provides a rationale for investigating AR-targeted therapies as potential protective interventions against SARS-CoV-2 infection severity. Full article

Bothrops species are responsible for the majority of envenomations in Argentina. In particular, Bothrops diporus is among the main species responsible for the majority of envenomations in Argentina and causes significant injury and coagulopathy. Given the significance of this venom, the authors sought to define the toxin responsible for coagulopathy with specialized spectrophotometric and thromboelastographic methods. Utilizing clotting time, spectrophotometry, and thromboelastography, it was determined that B. diporus venom has potent, procoagulant activity in human plasma and buffer milieu. Calcium-dependent and -independent activities consistent with serine protease activity were identified. The activity included both thrombin-generating and thrombin-like enzymatic activity. The venom cleaved the serine protease-specific chromogenic substrate β-Ala-Gly-Arg-p-nitroanilide diacetate, and its activity was inhibited in plasma by antithrombin after addition of heparin. Further, venom exposed in isolation to RuCl₃, a known inhibitor of serine protease-containing venoms, demonstrated decreased activity in human plasma. In conclusion, the present study contributes to a better understanding of B. diporus venom and may have implications for the rational design of inhibitors, antivenom formulations, or preclinical models to study venom-induced coagulopathies. Full article