INTRODUCTION: The aging process is intricately linked to alterations in cellular and tissue structures, with the respiratory system being particularly susceptible to age-related changes. Therefore, this study aimed to profile the activity of proteases using activity-based probes in lung tissues of old and young rats, focusing on the expression levels of different, in particular cathepsins G and X and matrix Metalloproteinases (MMPs). Additionally, the impact on extracellular matrix (ECM) components, particularly fibronectin, in relation to age-related histological and ultrastructural changes in lung tissues was investigated. MATERIALS AND METHODS: Lung tissues from old and young rats were subjected to activity-based probe profiling to assess the activity of different proteases. Expression levels of cathepsins G and X were quantified, and zymography was performed to evaluate matrix metalloproteinases activity. Furthermore, ECM components, specifically fibronectin, were examined for signs of degradation in the old lung tissues compared to the young ones. Moreover, histological, immunohistochemical and ultrastructural assessments of old and young lung tissue were also conducted. RESULTS: Our results showed that the expression levels of cathepsins G and X were notably higher in old rat lung tissues in contrast to those in young rat lung tissues. Zymography analysis revealed elevated MMP activity in the old lung tissues compared to the young ones. Particularly, significant degradation of fibronectin, an essential ECM component, was observed in the old lung tissues. Numerous histological and ultrastructural alterations were observed in old lung tissues compared to young lung tissues. DISCUSSION AND CONCLUSION: The findings indicate an age-related upregulation of cathepsins G and X along with heightened MMP activity in old rat lung tissues, potentially contributing to the degradation of fibronectin within the ECM. These alterations highlight potential mechanisms underlying age-associated changes in lung tissue integrity and provide insights into protease-mediated ECM remodeling in the context of aging lungs.
- MeSH
- Extracellular Matrix metabolism ultrastructure MeSH
- Fibronectins * metabolism MeSH
- Cathepsin G metabolism MeSH
- Rats MeSH
- Lysosomes ultrastructure metabolism MeSH
- Matrix Metalloproteinases metabolism MeSH
- Lung * ultrastructure metabolism MeSH
- Peptide Hydrolases metabolism MeSH
- Aging * metabolism MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Schistosomiasis, caused by a parasitic blood fluke of the genus Schistosoma, is a global health problem for which new chemotherapeutic options are needed. We explored the scaffold of gallinamide A, a natural peptidic metabolite of marine cyanobacteria that has previously been shown to inhibit cathepsin L-type proteases. We screened a library of 19 synthetic gallinamide A analogs and identified nanomolar inhibitors of the cathepsin B-type protease SmCB1, which is a drug target for the treatment of schistosomiasis mansoni. Against cultured S. mansoni schistosomula and adult worms, many of the gallinamides generated a range of deleterious phenotypic responses. Imaging with a fluorescent-activity-based probe derived from gallinamide A demonstrated that SmCB1 is the primary target for gallinamides in the parasite. Furthermore, we solved the high-resolution crystal structures of SmCB1 in complex with gallinamide A and its two analogs and describe the acrylamide covalent warhead and binding mode in the active site. Quantum chemical calculations evaluated the contribution of individual positions in the peptidomimetic scaffold to the inhibition of the target and demonstrated the importance of the P1' and P2 positions. Our study introduces gallinamides as a powerful chemotype that can be exploited for the development of novel antischistosomal chemotherapeutics.
- MeSH
- Cathepsin B * antagonists & inhibitors metabolism MeSH
- Crystallography, X-Ray MeSH
- Models, Molecular MeSH
- Schistosoma mansoni * enzymology drug effects MeSH
- Schistosomicides pharmacology chemistry MeSH
- Protein Binding MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
Tick saliva injected into the vertebrate host contains bioactive anti-proteolytic proteins from the cystatin family; however, the molecular basis of their unusual biochemical and physiological properties, distinct from those of host homologs, is unknown. Here, we present Ricistatin, a novel secreted cystatin identified in the salivary gland transcriptome of Ixodes ricinus ticks. Recombinant Ricistatin inhibited host-derived cysteine cathepsins and preferentially targeted endopeptidases, while having only limited impact on proteolysis driven by exopeptidases. Determination of the crystal structure of Ricistatin in complex with a cysteine cathepsin together with characterization of structural determinants in the Ricistatin binding site explained its restricted specificity. Furthermore, Ricistatin was potently immunosuppressive and anti-inflammatory, reducing levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α and nitric oxide in macrophages; IL-2 and IL-9 levels in Th9 cells; and OVA antigen-induced CD4+ T cell proliferation and neutrophil migration. This work highlights the immunotherapeutic potential of Ricistatin and, for the first time, provides structural insights into the unique narrow selectivity of tick salivary cystatins determining their bioactivity.
- MeSH
- Cystatins * pharmacology MeSH
- Cysteine metabolism MeSH
- Endopeptidases metabolism MeSH
- Cathepsins metabolism MeSH
- Ixodes * chemistry MeSH
- Vertebrates MeSH
- Peptide Hydrolases metabolism MeSH
- Salivary Cystatins chemistry MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
Renální epiteloidní angiomyolipom (ReAML) je vzácným podtypem angiomyolipomu. Jedná se o benigní onemocnění ledvin, u kterého byly hlášeny případy agresivního chování a malignity. Tyto tumory představují 8 % operovaných angiomyolipomů ledviny. Diagnostika se opírá o histologický nález. Cílem práce je podat přehled informací o této problematice, histologickém obrazu a kazuistice 42leté ženy operované na našem pracovišti, u které bylo prokázáno multicentrické či metastatické postižení lymfatické uzliny retroperitonea.
Renal epithelioid angiomyolipoma (ReAML) is a rare subtype of angiomyolipoma. It is a benign kidney disease, in which cases of aggressive behavior and malignancy have been reported. These tumors represent 8% of operated kidney angiomyolipomas. Diagnosis is based on histological findings. The aim of this text is to review the information on this issue, the histological picture and the case report of a 42-year-old woman operated at our department who was found to have multicentric or metastatic involvement of the retroperitoneal lymph node.
- MeSH
- Angiomyolipoma * surgery diagnostic imaging MeSH
- Adult MeSH
- Epithelioid Cells pathology MeSH
- Cathepsin K analysis MeSH
- Humans MeSH
- Neoplasm Metastasis MeSH
- Kidney Neoplasms * surgery diagnostic imaging MeSH
- Nephrectomy MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Female MeSH
- Publication type
- Case Reports MeSH
Fractured bones can regenerate and restore their biological and mechanical properties to the state prior to the damage. In some cases, however, the treatment of fractures requires the use of supportive implants. For bone healing, three processes are essential: the inflammatory phase, the repair phase and the remodelling phase. A proper course of the first - inflammatory - stage is important to ensure a successful fracture healing process. In our study, we evaluated tissue samples immunohistochemically from the area surrounding the fractures of upper and lower limbs (bone tissue, soft tissue, and the implant-adhering tissue) for markers: CD11b, CD15, CD34, CD44, CD68, Cathepsin K, and TRAcP that are linked to the aforementioned phases. In soft tissue, higher expressions of CD68, CD34, CD15 and CD11b markers were observed than in other locations. TRAcP and Cathepsin K markers were more expressed in the bone tissue, while pigmentation, necrosis and calcification were more observed in the implant-adhering tissue. Since even the implant materials commonly perceived as inert elicit the observed inflammatory responses, new surface treatments and materials need to be developed.
Resident tissue macrophages are organ-specialized phagocytes responsible for the maintenance and protection of tissue homeostasis. It is well established that tissue diversity is reflected by the heterogeneity of resident tissue macrophage origin and phenotype. However, much less is known about tissue-specific phagocytic and proteolytic macrophage functions. Here, using a quantitative proteomics approach, we identify cathepsins as key determinants of phagosome maturation in primary peritoneum-, lung-, and brain-resident macrophages. The data further uncover cathepsin K (CtsK) as a molecular marker for lung phagosomes required for intracellular protein and collagen degradation. Pharmacological blockade of CtsK activity diminished phagosomal proteolysis and collagenolysis in lung-resident macrophages. Furthermore, profibrotic TGF-β negatively regulated CtsK-mediated phagosomal collagen degradation independently from classical endocytic-proteolytic pathways. In humans, phagosomal CtsK activity was reduced in COPD lung macrophages and non-COPD lung macrophages exposed to cigarette smoke extract. Taken together, this study provides a comprehensive map of how peritoneal, lung, and brain tissue environment shapes phagosomal composition, revealing CtsK as a key molecular determinant of lung phagosomes contributing to phagocytic collagen clearance in lungs.
- MeSH
- Phagosomes * metabolism MeSH
- Cathepsin K * metabolism MeSH
- Collagen metabolism MeSH
- Humans MeSH
- Macrophages * metabolism MeSH
- Lung MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Cathepsin L is a key host cysteine protease utilized by coronaviruses for cell entry and is a promising drug target for novel antivirals against SARS-CoV-2. The marine natural product gallinamide A and several synthetic analogues were identified as potent inhibitors of cathepsin L with IC50 values in the picomolar range. Lead molecules possessed selectivity over other cathepsins and alternative host proteases involved in viral entry. Gallinamide A directly interacted with cathepsin L in cells and, together with two lead analogues, potently inhibited SARS-CoV-2 infection in vitro, with EC50 values in the nanomolar range. Reduced antiviral activity was observed in cells overexpressing transmembrane protease, serine 2 (TMPRSS2); however, a synergistic improvement in antiviral activity was achieved when combined with a TMPRSS2 inhibitor. These data highlight the potential of cathepsin L as a COVID-19 drug target as well as the likely need to inhibit multiple routes of viral entry to achieve efficacy.
- MeSH
- Antiviral Agents chemical synthesis chemistry pharmacology MeSH
- Biological Products chemical synthesis chemistry pharmacology MeSH
- A549 Cells MeSH
- Chlorocebus aethiops MeSH
- COVID-19 metabolism MeSH
- COVID-19 Drug Treatment MeSH
- Cysteine Proteinase Inhibitors chemical synthesis chemistry pharmacology MeSH
- Cathepsin L antagonists & inhibitors metabolism MeSH
- Antimicrobial Cationic Peptides chemical synthesis chemistry pharmacology MeSH
- Humans MeSH
- Microbial Sensitivity Tests MeSH
- Molecular Conformation MeSH
- Proteomics MeSH
- SARS-CoV-2 drug effects MeSH
- Vero Cells MeSH
- Dose-Response Relationship, Drug MeSH
- Structure-Activity Relationship MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
Patients infected with SARS-CoV-2 risk co-infection with Gram-positive bacteria, which severely affects their prognosis. Antimicrobial drugs with dual antiviral and antibacterial activity would be very useful in this setting. Although glycopeptide antibiotics are well-known as strong antibacterial drugs, some of them are also active against RNA viruses like SARS-CoV-2. It has been shown that the antiviral and antibacterial efficacy can be enhanced by synthetic modifications. We here report the synthesis and biological evaluation of seven derivatives of teicoplanin bearing hydrophobic or superbasic side chain. All but one teicoplanin derivatives were effective in inhibiting SARS-CoV-2 replication in VeroE6 cells. One lipophilic and three perfluoroalkyl conjugates showed activity against SARS-CoV-2 in human Calu-3 cells and against HCoV-229E, an endemic human coronavirus, in HEL cells. Pseudovirus entry and enzyme inhibition assays established that the teicoplanin derivatives efficiently prevent the cathepsin-mediated endosomal entry of SARS-CoV-2, with some compounds inhibiting also the TMPRSS2-mediated surface entry route. The teicoplanin derivatives showed good to excellent activity against Gram-positive bacteria resistant to all approved glycopeptide antibiotics, due to their ability to dually bind to the bacterial membrane and cell-wall. To conclude, we identified three perfluoralkyl and one monoguanidine analog of teicoplanin as dual inhibitors of Gram-positive bacteria and SARS-CoV-2.
- MeSH
- Anti-Bacterial Agents chemistry MeSH
- Antiviral Agents chemistry MeSH
- COVID-19 * MeSH
- Fluorocarbons * pharmacology MeSH
- Glycopeptides chemistry MeSH
- Gram-Positive Bacteria MeSH
- Cathepsins pharmacology MeSH
- Humans MeSH
- SARS-CoV-2 MeSH
- Teicoplanin pharmacology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Parkinson's disease (PD) is generally considered a sporadic disorder, but a strong genetic background is often found. The aim of this study was to identify the underlying genetic cause of PD in two affected siblings and to subsequently assess the role of mutations in Cathepsin B (CTSB) in susceptibility to PD. A typical PD family was identified and whole-exome sequencing was performed in two affected siblings. Variants of interest were validated using Sanger sequencing. CTSB p.Gly284Val was genotyped in 2077 PD patients and 615 unrelated healthy controls from the Czech Republic, Ireland, Poland, Ukraine, and the USA. The gene burden analysis was conducted for the CTSB gene in an additional 769 PD probands from Mayo Clinic Florida familial PD cohort. CTSB expression and activity in patient-derived fibroblasts and controls were evaluated by qRT-PCR, western blot, immunocytochemistry, and enzymatic assay. The CTSB p.Gly284Val candidate variant was only identified in affected family members. Functional analysis of CTSB patient-derived fibroblasts under basal conditions did not reveal overt changes in endogenous expression, subcellular localization, or enzymatic activity in the heterozygous carrier of the CTSB variant. The identification of the CTSB p.Gly284Val may support the hypothesis that the CTSB locus harbors variants with differing penetrance that can determine the disease risk.
- MeSH
- Genotype MeSH
- Heterozygote MeSH
- Cathepsin B genetics metabolism MeSH
- Humans MeSH
- Parkinson Disease * genetics MeSH
- Penetrance MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Cathepsin K (CatK) is a target for the treatment of osteoporosis, arthritis, and bone metastasis. Peptidomimetics with a cyanohydrazide warhead represent a new class of highly potent CatK inhibitors; however, their binding mechanism is unknown. We investigated two model cyanohydrazide inhibitors with differently positioned warheads: an azadipeptide nitrile Gü1303 and a 3-cyano-3-aza-β-amino acid Gü2602. Crystal structures of their covalent complexes were determined with mature CatK as well as a zymogen-like activation intermediate of CatK. Binding mode analysis, together with quantum chemical calculations, revealed that the extraordinary picomolar potency of Gü2602 is entropically favoured by its conformational flexibility at the nonprimed-primed subsites boundary. Furthermore, we demonstrated by live cell imaging that cyanohydrazides effectively target mature CatK in osteosarcoma cells. Cyanohydrazides also suppressed the maturation of CatK by inhibiting the autoactivation of the CatK zymogen. Our results provide structural insights for the rational design of cyanohydrazide inhibitors of CatK as potential drugs.
- MeSH
- Hydrazines chemistry pharmacology MeSH
- Protease Inhibitors chemistry pharmacology MeSH
- Cathepsin K antagonists & inhibitors metabolism MeSH
- Humans MeSH
- Models, Molecular MeSH
- Molecular Structure MeSH
- Tumor Cells, Cultured MeSH
- Nitriles chemistry pharmacology MeSH
- Dose-Response Relationship, Drug MeSH
- Structure-Activity Relationship MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
