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Cysteine cathepsins are a fascinating group of proteolytic enzymes that play diverse and crucial roles in numerous biological processes, both in health and disease. Understanding these proteases is essential for uncovering novel insights into the underlying mechanisms of a wide range of disorders, such as cancer. Cysteine cathepsins influence cancer biology by participating in processes such as extracellular matrix degradation, angiogenesis, immune evasion, and apoptosis. In this comprehensive review, we explore foundational research that illuminates the diverse and intricate roles of cysteine cathepsins as diagnostic markers and therapeutic targets for cancer. This review aims to provide valuable insights into the clinical relevance of cysteine cathepsins and explore their capacity to advance personalised and targeted medical interventions in oncology. • Cysteine cathepsins are pivotal in many cancer processes, making them desirable therapeutic targets. • Irregular cysteine cathepsin levels can serve as diagnostic and prognostic biomarkers in various cancers. • Development of molecular probes for cysteine cathepsins enhances diagnostic imaging and image-guided surgery. • Selective inhibitors and novel drug delivery systems targeting cysteine cathepsins hold promise for innovative cancer treatments. • Advances in antibody-drug conjugates utilizing cysteine cathepsins offer major potential in cancer treatment. [ABSTRACT FROM AUTHOR]

Proteolytic activity in the tumour microenvironment is an important factor in cancer development since it can also affect intracellular signalling pathways via positive feedback loops that result in either increased tumour growth or resistance to anticancer mechanisms. In this study, we demonstrated extracellular cathepsin L-mediated cleavage of epidermal growth factor receptor (EGFR) and identified the cleavage site in the extracellular domain after R224. To further evaluate the relevance of this cleavage, we cloned and expressed a truncated version of EGFR, starting at G225, in HeLa cells. We confirmed the constitutive activation of the truncated protein in the absence of ligand binding and determined possible changes in intracellular signalling. Furthermore, we determined the effect of truncated EGFR protein expression on HeLa cell viability and response to the EGFR inhibitors, tyrosine kinase inhibitor (TKI) erlotinib and monoclonal antibody (mAb) cetuximab. Our data reveal the nuclear localization and phosphorylation of EGFR and signal trancducer and activator of transcription 3 (STAT3) in cells that express the truncated EGFR protein and suggest that these phenomena cause resistance to EGFR inhibitors. [ABSTRACT FROM AUTHOR]

Introduction: We previously identified that Cathepsin V (CTSV) expression is associated with poor prognosis in ER+ breast cancer, particularly within the Luminal A subtype. Examination of themolecular role of the proteasewithin Luminal A tumours, revealed that CTSV promotes tumour cell invasion and proliferation, in addition to degradation of the luminal transcription factor, GATA3, via the proteasome. Methods: Cell line models expressing CTSV shRNA or transfected to overexpress CTSV were used to examine the impact of CTSV on cell proliferation byMTT assay and flow cytometry. Western blotting analysis was used to identify the impact of CTSV on histone and chaperone protein expression. Cell fractionation and confocal microscopy was used to illustrate the presence of CTSV in the nuclear compartment. Results: In this work we have identified that CTSV has an impact on breast cancer cell proliferation, with CTSV depleted cells exhibiting delayed progression through the G2/M phase of the cell cycle. Further investigation has revealed that CTSV can control nuclear expression levels of histones H3 and H4 via regulating protein expression of their chaperone sNASP. We have discovered that CTSV is localised to the nuclear compartment in breast tumour cells, mediated by a bipartite nuclear localisation signal (NLS) within the CTSV sequence and that nuclear CTSV is required for cell cycle progression and histone stability in breast tumour cells. Discussion: Collectively these findings support the hypothesis that targeting CTSV may have utility as a novel therapeutic target in ER+ breast cancer by impairing cell cycle progression via manipulating histone stabilisation. [ABSTRACT FROM AUTHOR]

Introduction: We previously identified that Cathepsin V (CTSV) expression is associated with poor prognosis in ER+ breast cancer, particularly within the Luminal A subtype. Examination of the molecular role of the protease within Luminal A tumours, revealed that CTSV promotes tumour cell invasion and proliferation, in addition to degradation of the luminal transcription factor, GATA3, via the proteasome.Methods: Cell line models expressing CTSV shRNA or transfected to overexpress CTSV were used to examine the impact of CTSV on cell proliferation by MTT assay and flow cytometry. Western blotting analysis was used to identify the impact of CTSV on histone and chaperone protein expression. Cell fractionation and confocal microscopy was used to illustrate the presence of CTSV in the nuclear compartment.Results: In this work we have identified that CTSV has an impact on breast cancer cell proliferation, with CTSV depleted cells exhibiting delayed progression through the G2/M phase of the cell cycle. Further investigation has revealed that CTSV can control nuclear expression levels of histones H3 and H4 via regulating protein expression of their chaperone sNASP. We have discovered that CTSV is localised to the nuclear compartment in breast tumour cells, mediated by a bipartite nuclear localisation signal (NLS) within the CTSV sequence and that nuclear CTSV is required for cell cycle progression and histone stability in breast tumour cells.Discussion: Collectively these findings support the hypothesis that targeting CTSV may have utility as a novel therapeutic target in ER+ breast cancer by impairing cell cycle progression via manipulating histone stabilisation.

Objective: This in situ study aimed to evaluate the effects of the inhibitors of matrix metalloproteinases (MMPs) and cysteine cathepsins on dentin erosion. Materials and methods: Ten volunteers participated in this study. Each volunteer wore an intraoral appliance containing 4 dentin specimens subjected to different treatments: deionized water as a control, 1 mM 1,10-phenanthroline (an MMP inhibitor), 50 µM E-64 (a cysteine cathepsin inhibitor), and 1 mM 1,10-phenanthroline + 50 µM E-64. The specimens were dipped in 5 ml of the respective solutions for 30 min at room temperature and then exposed to in vivo erosive challenges by rinsing with 150 ml of a cola drink (4 × 5 min/day) for 7 days. The substance loss of the specimens was measured by profilometry. The transverse sections of the specimens were examined using scanning electron microscopy. Thereafter, the demineralized organic matrix (DOM) of the specimens was removed using type I collagen enzyme and assessed by performing profilometry. The differences in substance loss and DOM thickness among the groups were analyzed by one-way repeated-measures analysis of variance (ANOVA) and Bonferroni's test at a level of P < 0.05. Results: Protease inhibitors significantly reduced substance loss in comparison to that of the control group (all P < 0.05). A significantly thicker DOM was observed for the specimens treated with protease inhibitors than for the control specimens (all P < 0.05). No significant differences in substance loss or DOM thickness were found among the MMP inhibitor, cysteine cathepsin inhibitor, and MMP + cysteine cathepsin inhibitor groups. Conclusions: The use of MMP and cysteine cathepsin inhibitors was shown to increase the acid resistance of human dentin, which may be due to the preservation of the DOM. Clinical relevance: The application of protease inhibitors could be considered an appropriate preventive strategy for dentin erosion. [ABSTRACT FROM AUTHOR]

Papain-like cysteine proteases are widely expressed enzymes that mostly regulate protein turnover in the acidic conditions of lysosomes. However, in the last twenty years, these proteases have been evidenced to exert specific functions within different organelles as well as outside the cell. The most studied proteases of this family are human cysteine cathepsins involved both in physiological and pathological processes. The specificity of each protease to its substrates is mostly defined by the structure of the binding cleft. Different patterns of amino acid motif in this area determine the interaction between the protease and the ligands. Moreover, this specificity can be altered under the specific media conditions and in case other proteins are present. Understanding how this network works would allow researchers to design the diagnostic selective probes and therapeutic inhibitors. Moreover, this knowledge might serve as a key for redesigning and de novo engineering of the proteases for a wide range of applications.

[Display omitted] An uncontrolled activity of neutrophil serine proteases (NSPs) contributes to inflammatory diseases. Cathepsin C (CatC) is known to activate NSPs during neutrophilic differentiation and represents a promising pharmacological target in NSP-mediated diseases. In humans, Papillon-Lefèvre syndrome (PLS) patients have mutations in their CTSC gene, resulting in the complete absence of CatC activity. Despite this, low residual NSP activities are detected in PLS neutrophils (<10% vs healthy individuals) , suggesting the involvement of CatC-independent proteolytic pathway(s) in the activation of proNSPs. This prompted us to characterize CatC-independent NSP activation pathways by blocking proCatC maturation. In this study, we show that inhibition of intracellular CatS almost completely blocked CatC maturation in human promyeloid HL-60 cells. Despite this, NSP activation was not significantly reduced, confirming the presence of a CatC-independent activation pathway involving a CatC-like protease that we termed NSPs-AAP-1. Similarly, when human CD34+ progenitor cells were treated with CatS inhibitors during neutrophilic differentiation in vitro , CatC activity was nearly abrogated but ∼30% NSP activities remained, further supporting the existence of NSPs-AAP-1. Our data indicate that NSPs-AAP-1 is a cysteine protease that is inhibited by reversible nitrile compounds designed for CatC inhibition. We further established a proof of concept for the indirect, although incomplete, inhibition of NSPs by pharmacological targeting of CatC maturation using CatS inhibitors. This emphasizes the potential of CatS as a therapeutic target for inflammatory diseases. Thus, preventing proNSP maturation using a CatS inhibitor, alone or in combination with a CatC/NSPs-AAP-1 inhibitor, represents a promising approach to efficiently control the extent of tissue injury in neutrophil-mediated inflammatory diseases. [ABSTRACT FROM AUTHOR]

Papain-like cysteine proteases (PLCPs) are widely expressed enzymes, the main function of which is low-specific-protein turnover in the acidic conditions of lysosomes. Additionally, these proteases provide specific functions in other compartments such as cytosol, nucleus, and extracellular space. The specificity of each protease to its substrates mainly depends on the patterns of the amino acids in the binding cleft. This specificity is highly regulated by media conditions and the presence of accessory proteins. In this study, we examined structural aspects, ensuring the pH-dependent substrate specificity of PLCPs. Experiments employing fluorogenic peptide substrates demonstrated that plant PLCPs and human cathepsins possess a pH-dependent specificity for the residue in the P1 position. X-ray crystallographic studies and molecular simulations allowed the overall structure determination of the enzymes to predict residues in the S1 binding pocket, which can form electrostatic contacts with the substrates. Sequence analysis established the variability of these residues among PLCPs. Based on the obtained data, we designed a peptide inhibitor for human cathepsin L and described its inhibitory potential. As a conclusion, we stated that the S1 binding pocket defines specific pH-dependent recognition of substrates by PLCPs, ensuring multiple physiological functions of these proteases. This work was supported by the Russian Science Foundation (grant No. 22-25-00648).

Disturbance in the proteolytic process is one of the malignant signs of tumors. Proteolysis is highly orchestrated by cysteine cathepsin and its inhibitors. Cystatin-B (CSTB) is a general cysteine cathepsin inhibitor that prevents cysteine cathepsin from leaking from lysosomes and causing inappropriate proteolysis. Our study found that CSTB was downregulated in both oral squamous cell carcinoma (OSCC) tissues and cells compared with normal controls. Immunohistochemical analysis showed that CSTB was mainly distributed in the epithelial structure of OSCC tissues, and its expression intensity was related to the grade classification. A correlation analysis between CSTB and clinical prognosis was performed using gene expression data and clinical information acquired from The Cancer Genome Atlas (TCGA) database. Patients with lower expression levels of CSTB had shorter disease-free survival times and poorer clinicopathological features (e.g., lymph node metastases, perineural invasion, low degree of differentiation, and advanced tumor stage). OSCC cell models overexpressing CSTB were constructed to assess the effects of CSTB on malignant biological behaviors and upregulation of CSTB inhibited cell proliferation, migration, and invasion in vitro. Weighted gene correlation network analysis (WGCNA) and gene set enrichment analysis (GSEA) were performed based on the TCGA data to explore potential mechanisms, and CSTB appeared to correlate with squamous epithelial proliferation-differentiation processes, such as epidermal cell differentiation and keratinization. Moreover, in WGCNA, the gene module most associated with CSTB expression (i.e., the brown module) was also the one most associated with grade classification. Upregulation of CSTB promoted the expression levels of markers (LOR, IVL, KRT5/14, and KRT1/10), reflecting a tendency for differentiation and keratinization in vitro. Gene expression profile data of the overexpressed CSTB cell line were obtained by RNA sequencing (RNA-seq) technology. By comparing the GSEA enrichment results of RNA-seq data (from the OSCC models overexpressing CSTB) and existing public database data, three gene sets (i.e., apical junction, G2/M checkpoint, etc.) and six pathways (e.g., NOTCH signaling pathway, glycosaminoglycan degradation, mismatch repair, etc.) were enriched in the data from both sources. Overall, our study shows that CSTB is downregulated in OSCC and might regulate the malignant characteristics of OSCC via the epithelial proliferation/differentiation program.

Disturbance in the proteolytic process is one of the malignant signs of tumors. Proteolysis is highly orchestrated by cysteine cathepsin and its inhibitors. Cystatin-B (CSTB) is a general cysteine cathepsin inhibitor that prevents cysteine cathepsin from leaking from lysosomes and causing inappropriate proteolysis. Our study found that CSTB was downregulated in both oral squamous cell carcinoma (OSCC) tissues and cells compared with normal controls. Immunohistochemical analysis showed that CSTB was mainly distributed in the epithelial structure of OSCC tissues, and its expression intensity was related to the grade classification. A correlation analysis between CSTB and clinical prognosis was performed using gene expression data and clinical information acquired from The Cancer Genome Atlas (TCGA) database. Patients with lower expression levels of CSTB had shorter disease-free survival times and poorer clinicopathological features (e.g., lymph node metastases, perineural invasion, low degree of differentiation, and advanced tumor stage). OSCC cell models overexpressing CSTB were constructed to assess the effects of CSTB on malignant biological behaviors and upregulation of CSTB inhibited cell proliferation, migration, and invasion in vitro. Weighted gene correlation network analysis (WGCNA) and gene set enrichment analysis (GSEA) were performed based on the TCGA data to explore potential mechanisms, and CSTB appeared to correlate with squamous epithelial proliferation-differentiation processes, such as epidermal cell differentiation and keratinization. Moreover, in WGCNA, the gene module most associated with CSTB expression (i.e., the brown module) was also the one most associated with grade classification. Upregulation of CSTB promoted the expression levels of markers (LOR, IVL, KRT5/14, and KRT1/10), reflecting a tendency for differentiation and keratinization in vitro. Gene expression profile data of the overexpressed CSTB cell line were obtained by RNA sequencing (RNA-seq) technology. By comparing the GSEA enrichment results of RNA-seq data (from the OSCC models overexpressing CSTB) and existing public database data, three gene sets (i.e., apical junction, G2/M checkpoint, etc.) and six pathways (e.g., NOTCH signaling pathway, glycosaminoglycan degradation, mismatch repair, etc.) were enriched in the data from both sources. Overall, our study shows that CSTB is downregulated in OSCC and might regulate the malignant characteristics of OSCC via the epithelial proliferation/differentiation program. [ABSTRACT FROM AUTHOR]

Purpose: To analyze the effects of different processes during bonding on endogenous cysteine cathepsin activity in dentin. Materials and Methods: Dentin powder, prepared from extracted human third molars, was divided into 10 groups. Two lots of dentin powder were used to detect the effects of the procedure of protein extraction on endogenous cathepsin activity. The others were used to study effects of different acid-etching or adhesive treatments on enzyme activity. Concentrations of 37% phosphoric acid or 10% phosphoric acid, two etch-and-rinse adhesive systems, and two self-etching adhesive systems were used as dentin powder treatments. The untreated mineralized dentin powder was set as the control. After treatment, the proteins of each group were extracted. The total cathepsin activity in the extracts of each group was monitored with a fluorescence reader. Results: In the control group, there were no significant differences in cathepsin activity between the protein extract before EDTA treatment and the protein extract after EDTA treatment (p > 0.05). The cathepsin activities of the three different extracts in the 37% phosphoric acid-treated group were different from each other (p < 0.05). The two acid-etching groups and two etch-and-rinse groups showed significant enzyme activity reduction vs the control group (p < 0.05). There were no significant differences between those four groups (p > 0.05). Treating the dentin powder with any of the two self-etching adhesives resulted in an increase in cathepsin activity (p < 0.05). Conclusions: The activity of cysteine cathepsins can be detected in dentin powder. Treatment with EDTA during protein extraction exerted an influence on cathepsin activity. Acid etching or etch-and-rinse adhesive systems may reduce the activity of endogenous cathepsins in dentin. Self-etching adhesive systems may increase the enzyme activity. [ABSTRACT FROM AUTHOR]

Cysteine cathepsins are described as lysosomal proteases with housekeeping and highly specialised functions in many organisms. In this study, cysteine cathepsins were identified from the transcriptome profile of blunt snout bream (Megalobrama amblycephala). A total of 41 cysteine cathepsin-like sequences were found and divided into groups of cathepsin (Cts): B, C, F, H, K, L, S, and Z. Twenty-nine of these sequences contained coding sequences, encoding 92-473 amino acids, which exhibited the highest (78%-95%) homology to the counterparts from zebrafish (Danio rerio). Multiple sequence alignment of the amino acids showed highly conserved domains among the cysteine cathepsins of M. amblycephala to its homologs. The putative proteins, including maCtsb1, maCtsc1, maCtsf2, maCtsh1, maCtsk5, maCtsl5, maCtss1, and maCtsz5 were characterised and structured using in silico methods. Additionally, codon usage of full length open reading frame sequences of these cathepsins was analysed. This study provided basic information for further studies on the specific functions of cysteine cathepsins of M. amblycephala in the future.

The intracellular accumulation of α-synuclein (α-syn) amyloid fibrils is a hallmark of Parkinson's disease. Because lysosomes are responsible for degrading aggregated species, enhancing lysosomal function could alleviate the overburden of α-syn. Previously, we showed that cysteine cathepsins (Cts) is the main class of lysosomal proteases that degrade α-syn, and in particular, CtsL was found to be capable of digesting α-syn fibrils. Here, we report that CtsK is a more potent protease for degrading α-syn amyloids. Using peptide mapping by liquid chromatography with mass spectrometry, critical cleavage sites involved in destabilizing fibril structure are identified. CtsK is only able to devour the internal regions after the removal of both N- and C-termini, indicating their protective role of the amyloid core from proteolytic attack. Our results suggest that if overexpressed in lysosomes, CtsK has the potential to ameliorate α-syn pathology. Image 1 • Cathepsin K was identified as a potent disaggregase of α-syn fibrils. • Mechanistic insights were revealed by detailed peptide mapping by LC-MS. • N- and C-termini protect the amyloid core from proteolytic attack. • Critical cleavages sites involved in degradation were identified. • Cathepsin K offers therapeutic approach for decreasing fibril load in PD. [ABSTRACT FROM AUTHOR]

We designed a near-infrared fluorescent substrate-based probe (SBP), termed MG101, for monitoring extracellular cathepsin S (CatS) activity. We conceived a fused peptide hairpin loop-structure, combining a CatS recognition domain, an electrostatic zipper (with complementary charges of a polyanionic (D-Glu) 5 segment and a polycationic (D-Arg) 5 motif, as well as a N and C terminal Förster resonance energy transfer pair (donor: AlexaFluor680; quencher: BHQ3) to facilitate activity-dependent imaging. MG101 showed excellent stability since no fluorescence release corresponding to a self-dequenching was observed in the presence of either 2 M NaCl or after incubation at a broad range of pH (2.2–8.2). Cathepsins B, D, G, H, and K, neutrophil elastase and proteinase 3 did not cleave MG101, while CatS, and to a lesser extent CatL, hydrolysed MG101 at pH 5.5. However MG101 was fully selective for CatS at pH 7.4 (k cat /K m = 140,000 M−1 s−1) and sensitive to low concentration of CatS (<1 nM). The selectivity of MG101 was successfully endorsed ex vivo , as it was hydrolysed in cell lysates derived from wild-type but not knockout CatS murine spleen. Furthermore, application of the SBP probe with confocal microscopy confirmed the secretion of active CatS from THP-1 macrophages, which could be abrogated by pharmacological CatS inhibitors. Taken together, present data highlight MG101 as a novel near-infrared fluorescent SBP for the visualization of extracellular active CatS from macrophages and other cell types. Image 1 [ABSTRACT FROM AUTHOR]

Cathepsin S (CatS) is a secreted cysteine protease that cleaves certain extracellular matrix proteins, regulates antigen presentation in antigen-presenting cells (APC), and promotes M2-type macrophage and dendritic cell polarization. CatS is overexpressed in many solid cancers, and overall, it appears to promote an immune-suppressive and tumor-promoting microenvironment. While most data suggest that CatS inhibition or knockdown promotes anti-cancer immunity, cell-specific inhibition, especially in myeloid cells, appears to be important for therapeutic efficacy. This makes the design of CatS selective inhibitors and their targeting to tumor-associated M2-type macrophages (TAM) and DC an attractive therapeutic strategy compared to the use of non-selective immunosuppressive compounds or untargeted approaches. The selective inhibition of CatS can be achieved through optimized small molecule inhibitors that show good pharmacokinetic profiles and are orally bioavailable. The targeting of these inhibitors to TAM is now more feasible using nanocarriers that are functionalized for a directed delivery. This review discusses the role of CatS in the immunological tumor microenvironment and upcoming possibilities for a nanocarrier-mediated delivery of potent and selective CatS inhibitors to TAM and related APC to promote anti-tumor immunity.

Aim: The aim of this study was to comparatively assess the gelatinolytic activity of matrix metalloproteinases(MMPs) and Cysteine Cathepsins (CCs) in the adhesive interface using etch and rinse adhesive at different time intervals using zymographic technique. Methodology: Twenty freshly extracted non-carious human third molars were used in this study. Occlusal surfaces were ground flat and 1mm thick horizontal dentin slabs were obtained from each tooth using a diamond disc. The dentin surface was polished with 600-grit silicon-carbide paper. Five out of 20 samples were directly pulverized. In the remaining fifteen samples, the dentin was etched and adhesive was applied and light cured according to the manufacturer's instructions. A 1mm thick flowable composite was build up and light cured. Bonded specimens were cut vertically into 3 to 4 dentin slabs by means of diamond disc to expose the adhesive/dentin interfaces. These were then ground down to 500 µm thick resin-dentin interface using a hard tissue microtome. These sections were then pulverised into powder. Following this, every five samples were subjected to zymographic analysis after 1 day, 7 days and 21 days. Results: Zymograms showed clear, thicker bands on all three isoforms in the etched samples compared to control samples at 1st and 7th day intervals and became inactive at 21st day for all three isoforms. MMP 9 activity was relatively higher when compared to CCs and MMP 2. Conclusion: Etch and rinse adhesive activated MMPs and CCs within the hybrid layer that remained active till 7th day and no gelatinolytic activity was found on 21st day and MMPs are more active compared to CCs and MMP-2.

L-leucyl-L-leucine methyl ester (LLOMe) induces apoptosis, which is thought to be mediated by release of lysosomal cysteine cathepsins from permeabilized lysosomes into the cytosol. Here, we demonstrated in HeLa cells that apoptotic as well as sub-apoptotic concentrations of LLOMe caused rapid and complete lysosomal membrane permeabilization (LMP), as evidenced by loss of the proton gradient and release into the cytosol of internalized lysosomal markers below a relative molecular mass of 10,000. However, there was no evidence for the release of cysteine cathepsins B and L into the cytosol; rather they remained within lysosomes, where they were rapidly inactivated and degraded. LLOMe-induced adverse effects, including LMP, loss of cysteine cathepsin activity, caspase activation and cell death could be reduced by inhibition of cathepsin C, but not by inhibiting cathepsins B and L. When incubated with sub-apoptotic LLOMe concentrations, lysosomes transiently lost protons but annealed and re-acidified within hours. Full lysosomal function required new protein synthesis of cysteine cathepsins and other hydrolyses. Our data argue against the release of lysosomal enzymes into the cytosol and their proposed proteolytic signaling during LLOMe-induced apoptosis. [ABSTRACT FROM AUTHOR]

Lysosomes and lysosomal hydrolases, including the cathepsins, have been shown to change their properties with aging brain a long time ago, although their function was not really understood. The first biochemical and clinical studies were followed by a major expansion in the last 20 years with the development of animal disease models and new approaches leading to a major advancement of understanding of the role of physiological and degenerative processes in the brain at the molecular level. This includes the understanding of the major role of autophagy and the cathepsins in a number of diseases, including its critical role in the neuronal ceroid lipofuscinosis. Similarly, cathepsins and some other lysosomal proteases were shown to have important roles in processing and/or degradation of several important neuronal proteins, thereby having either neuroprotective or harmful roles. In this review, we discuss lysosomal cathepsins and their regulation with the focus on cysteine cathepsins and their endogenous inhibitors, as well as their role in several neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Aim: The aim of this study was to comparatively assess the gelatinolytic activity of matrix metalloproteinases(MMPs) and Cysteine Cathepsins (CCs) in the adhesive interface using etch and rinse adhesive at different time intervals using zymographic technique.Methodology: Twenty freshly extracted non-carious human third molars were used in this study. Occlusal surfaces were ground flat and 1mm thick horizontal dentin slabs were obtained from each tooth using a diamond disc. The dentin surface was polished with 600-grit silicon-carbide paper. Five out of 20 samples were directly pulverized. In the remaining fifteen samples, the dentin was etched and adhesive was applied and light cured according to the manufacturer's instructions. A 1mm thick flowable composite was build up and light cured. Bonded specimens were cut vertically into 3 to 4 dentin slabs by means of diamond disc to expose the adhesive/dentin interfaces. These were then ground down to 500 µm thick resin-dentin interface using a hard tissue microtome. These sections were then pulverised into powder. Following this, every five samples were subjected to zymographic analysis after 1 day, 7 days and 21 days.Results: Zymograms showed clear, thicker bands on all three isoforms in the etched samples compared to control samples at 1st and 7th day intervals and became inactive at 21st day for all three isoforms. MMP 9 activity was relatively higher when compared to CCs and MMP 2.Conclusion: Etch and rinse adhesive activated MMPs and CCs within the hybrid layer that remained active till 7th day and no gelatinolytic activity was found on 21st day and MMPs are more active compared to CCs and MMP-2. [ABSTRACT FROM AUTHOR]

The synthesis and evaluation against various cysteine cathepsins with endopeptidase activity, of two new families of hitherto unknown 1,3,5-triazines, substituted by a nitrile function and either a cyclohexylamine moiety ( 5 -like) or a piperazine moiety ( 9 -like) are described. The structure-activity relationship was discussed; from 16 synthesized novel compounds, 9h was the most active and selectively inhibitor of Cat K (IC 50 = 28 nM) and Cat S (IC 50 = 23 nM). Molecular docking of 9h to X-ray crystal structure of cathepsins K and S confirmed a common binding mode with a crucial covalent bond with Cys25. We observed for 9h that p -trifluorophenyl group is located in S2 pocket and possess hydrophobic interactions with Tyr67 and Met68. Triazine and piperazine moieties are located in S′1 pocket and interact with Gly23, Cys63, Gly64 and Gly65. Altogether, these results indicate that the new analogs can make them effective agents against some viruses for which the glycoprotein cleavage is mediated by an array of proteases. [ABSTRACT FROM AUTHOR]

Pancreatitis is an inflammatory disease of the pancreas characterized by dysregulated activity of digestive enzymes, necrosis, immune infiltration, and pain. Repeated incidence of pancreatitis is an important risk factor for pancreatic cancer. Legumain, a lysosomal cysteine protease, has been linked to inflammatory diseases such as atherosclerosis, stroke, and cancer. Until now, legumain activation has not been studied during pancreatitis. We used a fluorescently quenched activity- based probe to assess legumain activation during caerulein-induced pancreatitis in mice. We detected activated legumain by ex vivo imaging, confocal microscopy, and gel electrophoresis. Compared with healthy controls, legumain activity in the pancreas of caeruleintreated mice was increased in a time-dependent manner. Legumain was localized to CD68+ macrophages and was not active in pancreatic acinar cells. Using a small-molecule inhibitor of legumain, we found that this protease is not essential for the initiation of pancreatitis. However, it may serve as a biomarker of disease, since patients with chronic pancreatitis show strongly increased legumain expression in macrophages. Moreover, the occurrence of legumain-expressing macrophages in regions of acinar-to-ductal metaplasia suggests that this protease may influence reprogramming events that lead to inflammation- induced pancreatic cancer. [ABSTRACT FROM AUTHOR]

Cathepsin L is a ubiquitously expressed papain-like cysteine protease involved in the endosomal degradation of proteins and has numerous roles in physiological and pathological processes, such as arthritis, osteoporosis, and cancer. Insight into the specificity of cathepsin L is important for elucidating its physiological roles and drug discovery. To study interactions with synthetic ligands, we prepared a presumably inactive mutant and crystallized it. Unexpectedly, the crystal structure determined at 1.4 Å revealed that the cathepsin L molecule is cleaved, with the cleaved region trapped in the active site cleft of the neighboring molecule. Hence, the catalytic mutant demonstrated low levels of catalytic activity. [ABSTRACT FROM AUTHOR]

Cysteine peptidases are involved in physiological processes of insect development and have been considered as potential targets for the development of insect control strategies. In this study, we obtained a recombinant cysteine cathepsin L (AsCathL) from leaf-cutting ant (Atta sexdens), a species from the order Hymenoptera who causes enormous damage to crops, natural forests and reforested areas. RT-qPCR showed AsCathL expression throughout insect development and in all body parts of the adult insect analysed, suggesting its role as a lysosomal cathepsin. AsCathL encodes a protein of 320 amino acid residues consisting of a pro-peptide and the mature with amino acids sequence over 67% similarity with lysosomal cathepsin L of species from Lepidoptera and Diptera. Phylogenetic tree revealed that AsCathL is very similar to predicted cathepsins found in other ants. Recombinant AsCathL was expressed in insoluble form by Escherichia coli Arctic Express (DE3) RIL, purified under denaturing conditions and refolded. The enzyme showed hydrolytic activity in vitro towards synthetic substrate Z-Phe-Arg-AMC at acidic pH. Synthetic inhibitor E−64 acted against peptidase activity and a study regarding the interaction between E−64 and AsCathL using nuclear magnetic resonance (NMR) revealed that 83.18% from all E−64 molecules are irreversibly bound to AsCathL. In addition, the proteolytic activity of AsCathL was strongly inhibited by recombinant sugarcane cystatins with K i ranging from 0.6 nM to 2.95 nM. To the best of our knowledge this is the first report characterizing a cysteine peptidase from leaf-cutting ants, which may contribute to future studies of ants' cathepsins. • Cathepsin L from Atta sexdens has been cloned and characterized for the first time. • Transcript has higher expression in worker than pupae and larvae as analysed by RT-qPCR. • NMR experiments showed irreversible interaction between AsCathL and E−64. • Five canecystatins effectively inhibited the activity of the recombinant AsCathL. [ABSTRACT FROM AUTHOR]

Fasciolosis is a worldwide parasitic disease of ruminants and an emerging human disease caused by the liver fluke Fasciola hepatica. The cystatin superfamily of cysteine protease inhibitors is composed of distinct families of intracellular stefins and secreted true cystatins. FhCyLS-2 from F. hepatica is an unusual member of the superfamily, where our sequence and 3D structure analyses in this study revealed that it combines characteristics of both families. The protein architecture demonstrates its relationship to stefins, but FhCyLS-2 also contains the secretion signal peptide and disulfide bridges typical of true cystatins. The secretion status was confirmed by detecting the presence of FhCyLS-2 in excretory/secretory products, supported by immunolocalization. Our high-resolution crystal structure of FhCyLS-2 showed a distinct disulfide bridging pattern and functional reactive center. We determined that FhCyLS-2 is a broad specificity inhibitor of cysteine cathepsins from both the host and F. hepatica, suggesting a dual role in the regulation of exogenous and endogenous proteolysis. Based on phylogenetic analysis that identified several FhCyLS-2 homologues in liver/intestinal foodborne flukes, we propose a new group within the cystatin superfamily called cystatin-like stefins., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Cysteine cathepsins are widely spread on living organisms associated to protein degradation in lysosomes, but some groups of Arthropoda (Heteroptera, Coleoptera, Crustacea and Acari) present these enzymes related to digestion of the meal proteins. Although spiders combine a mechanism of extra-oral with intracellular digestion, the sporadic studies on this subject were mainly concerned with the digestive fluid (DF) analysis. Thus, a more complete scenario of the digestive process in spiders is still lacking in the literature. In this paper we describe the identification and characterization of cysteine cathepsins in the midgut diverticula (MD) and DF of the spider Nephilengys cruentata by using enzymological assays. Furthermore, qualitative and quantitative data from transcriptomic followed by proteomic experiments were used together with biochemical assays for results interpretation. Five cathepsins L, one cathepsin F and one cathepsin B were identified by mass spectrometry, with cathepsins L1 (NcCTSL1) and 2 (NcCTSL2) as the most abundant enzymes. The native cysteine cathepsins presented acidic characteristics such as pH optima of 5.5, pH stability in acidic range and zymogen conversion to the mature form after in vitro acidification. NcCTSL1 seems to be a lysosomal enzyme with its recombinant form displaying acidic characteristics as the native ones and being inhibited by pepstatin. Evolutionarily, arachnid cathepsin L may have acquired different roles but its use for digestion is a common feature to studied taxa. Now a more elucidative picture of the digestive process in spiders can be depicted, with trypsins and astacins acting extra-orally under alkaline conditions whereas cysteine cathepsins will act in an acidic environment, likely in the digestive vacuoles or lysosome-like vesicles. [ABSTRACT FROM AUTHOR]

Demineralization in dentinal caries and erosion exposes dentine organic matrix. This exposed matrix, containing type I collagen and non-collagenous proteins, is then degraded by host collagenolytic enzymes, matrix metalloproteinases (MMPs) and cysteine cathepsins. The knowledge of the identities and function of these enzymes in dentine has accumulated only within the last 15 years, but has already formed a field of research called 'dentine degradomics'. This research has demonstrated the role of endogenous collagenolytic enzymes in caries and erosion development. In demineralized dentine, the enzymes degrade triple-helical collagen molecules, leading to the gradual loss of collagen matrix. Even before that, they can cleave off the terminal non-helical ends of collagen molecules called telopeptides, leading to the structural changes at the intramolecular gap areas, which may affect or even prevent intrafibrillar remineralization, which is considered essential in restoring the dentine's mechanical properties. They may also cause the loss of non-collagenous proteins that could serve as nucleation sites for remineralization. Here we review the findings demonstrating that inhibition of salivary or dentine endogenous MMPs and cysteine cathepsins may provide preventive means against the progression of caries or erosion. Furthermore, we also suggest the future directions for the new experimental preventive research to gain more knowledge of the enzymes and their function during and after dentine demineralization, and the pathways to find the clinically acceptable means to prevent the functional activity of these enzymes. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Elevated cathepsin S (Cat S) level is correlated with higher migration ability in tumor cells. This study investigates the inhibitory effect of novel synthetic α-ketoamide compounds on cathepsin activity and cancer cell migration. The effect of several α-ketoamide compounds on the activity of recombinant cathepsins (Cat S, Cat L and Cat K) was examined. Two highly metastatic cancer cell lines were incubated with three Cat S-specific compounds (6n, 6w and 6r) to analyze their effect on cellular Cat S activity and cell migration. At a 100 nM concentration, compounds 6n, 6r and 6w effectively inhibited Cat S activity. Cat S activity and cell migration were significantly reduced in CL1-3 cells after treatment with either 6n or 6w at 5 μM. Similar results were also obtained when A2058 cells were treated with 6n. These results highlight the therapeutic potential of α-ketoamide compounds, especially 6n and 6w, to prevent or delay cancer metastasis. [ABSTRACT FROM AUTHOR]

Abstract: Abundant clinical evidences indicate that up-regulation of several cathepsins in many human cancers is correlated with malignant progression and poor patient prognosis. In addition, a decrease in catalase activity or accumulation of hydrogen peroxide correlates with cancer metastasis. Recent studies indicate that cathepsin activation and expression can be modulated via H2O2 treatment. However, the actual relationship between catalase and cathepsins is not yet fully understood. In the present study, we found that catalase expression (or activity) was higher, while intracellular and extracellular Cat S, Cat L, and Cat K activities were lower in the non-invasive CL1-0 cells compared to the highly invasive CL1-5 cells. After CL1-0 cells were transfected with catalase-shRNA, the corresponding ROS (H2O2) level and Cat S, Cat L, or Cat K expression (or activity) was up-regulated, accompanied by an increase in cell migration and invasion. On the other hand, ROS (H2O2) level, cathepsin S, L, and K activities, cell migration and invasion were decreased in catalase-overexpressed CL1-5 cells. It is suggested that catalase may regulate cathepsin activity by controlling the production of ROS (H2O2), leading to variation in migration and invasion ability of lung cancer cells. [Copyright &y& Elsevier]

Abstract: Objectives: Contemporary adhesives lose their bond strength to dentin regardless of the bonding system used. This loss relates to the hydrolysis of collagen matrix of the hybrid layers. The preservation of the collagen matrix integrity is a key issue in the attempts to improve the dentin bonding durability. Methods: Dentin contains collagenolytic enzymes, matrix metalloproteinases (MMPs) and cysteine cathepsins, which are responsible for the hydrolytic degradation of collagen matrix in the bonded interface. Results: The identities, roles and function of collagenolytic enzymes in mineralized dentin has been gathered only within last 15years, but they have already been demonstrated to have an important role in dental hard tissue pathologies, including the degradation of the hybrid layer. Identifying responsible enzymes facilitates the development of new, more efficient methods to improve the stability of dentin–adhesive bond and durability of bond strength. Significance: Understanding the nature and role of proteolytic degradation of dentin–adhesive interfaces has improved immensely and has practically grown to a scientific field of its own within only 10years, holding excellent promise that stable resin–dentin bonds will be routinely available in a daily clinical setting already in a near future. [Copyright &y& Elsevier]

Abstract: It is more than 50years since the lysosome was discovered. Since then its hydrolytic machinery, including proteases and other hydrolases, has been fairly well identified and characterized. Among these are the cysteine cathepsins, members of the family of papain-like cysteine proteases. They have unique reactive-site properties and an uneven tissue-specific expression pattern. In living organisms their activity is a delicate balance of expression, targeting, zymogen activation, inhibition by protein inhibitors and degradation. The specificity of their substrate binding sites, small-molecule inhibitor repertoire and crystal structures are providing new tools for research and development. Their unique reactive-site properties have made it possible to confine the targets simply by the use of appropriate reactive groups. The epoxysuccinyls still dominate the field, but now nitriles seem to be the most appropriate “warhead”. The view of cysteine cathepsins as lysosomal proteases is changing as there is now clear evidence of their localization in other cellular compartments. Besides being involved in protein turnover, they build an important part of the endosomal antigen presentation. Together with the growing number of non-endosomal roles of cysteine cathepsins is growing also the knowledge of their involvement in diseases such as cancer and rheumatoid arthritis, among others. Finally, cysteine cathepsins are important regulators and signaling molecules of an unimaginable number of biological processes. The current challenge is to identify their endogenous substrates, in order to gain an insight into the mechanisms of substrate degradation and processing. In this review, some of the remarkable advances that have taken place in the past decade are presented. This article is part of a Special Issue entitled: Proteolysis 50years after the discovery of lysosome. [Copyright &y& Elsevier]

Abstract: Near-infrared fluorophore (NIRF)-labeled imaging probes are becoming increasingly important in bio-molecular imaging applications, that is, in animal models for tumor imaging or inflammation studies. In this study we showed that the previously introduced chemical concept of ‘Reverse Design’ represents an efficient strategy for the generation of selective probes for cysteine proteases from chemically optimized protease inhibitors for investigations in proteomic lysates as well as for in vivo molecular imaging studies. The newly developed activity-based probe AW-091 was demonstrated to be highly selective for cathepsin S in vitro and proved useful in monitoring cysteine cathepsin activity in vivo, that is, in zymosan-induced mouse model of inflammation. AW-091 showed higher signal-to-background ratios at earlier time points than the commercially available polymer-based ProSense680 (VisEn Medical) and thus represents an efficient new tool for studying early proteolytic processes leading to various diseases, including inflammation, cancer, and rheumatoid arthritis. In addition, the fluorescent signal originating from the cleaved AW-091 was shown to be reduced by the administration of an anti-inflammatory drug, dexamethasone and by the cathepsin inhibitor E-64, providing a valuable system for the evaluation of small-molecule inhibitors of cathepsins. [Copyright &y& Elsevier]

Abstract: Proteolytic activity is required for several key processes in cancer development and progression, including tumor growth, invasion and metastasis. Accordingly, high levels of protease expression and activity have been found to correlate with malignant progression and poor patient prognosis in a wide variety of human cancers. Members of the papain family of cysteine cathepsins are among the protease classes that have been functionally implicated in cancer. Therefore, the discovery of effective cathepsin inhibitors has considerable potential for anti-cancer therapy. In this study we describe the identification of a novel, reversible cathepsin inhibitor, VBY-825, which has high potency against cathepsins B, L, S and V. VBY-825 was tested in a pre-clinical model of pancreatic islet cancer and found to significantly decrease tumor burden and tumor number. Thus, the identification of VBY-825 as a new and effective anti-tumor drug encourages the therapeutic application of cathepsin inhibitors in cancer. [ABSTRACT FROM AUTHOR]

Abstract: Lysosomes are membrane-bound organelles, which contain an arsenal of different hydrolases, enabling them to act as the terminal degradative compartment of the endocytotic, phagocytic and autophagic pathways. During the last decade, it was convincingly shown that destabilization of lysosomal membrane and release of lysosomal content into the cytosol can initiate the lysosomal apoptotic pathway, which is dependent on mitochondria destabilization. The cleavage of BID to t-BID and degradation of anti-apoptotic BCL-2 proteins by lysosomal cysteine cathepsins were identified as links to the mitochondrial cytochrome c release, which eventually leads to caspase activation. There have also been reports about the involvement of lysosome destabilization and lysosomal proteases in the extrinsic apoptotic pathway, although the molecular mechanism is still under debate. In the present article, we discuss the cross-talk between lysosomes and mitochondria during apoptosis and its consequences for the fate of the cell. [Copyright &y& Elsevier]

Although cysteine cathepsins, including cathepsin K, are sensitive to oxidation, proteolytically active forms are found at inflammatory sites. Regulation of cathepsin K activity was analyzed in the presence of H2O2 to gain an insight into these puzzling observations. H2O2 impaired processing of procathepsin K and inactivated its mature form in a time- and dose-dependent mode. However, as a result of the formation of a sulfenic acid, as confirmed by trapping in the presence of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol, approximately one-third of its initial activity was restored by dithiothreitol. This incomplete inactivation may partially explain why active cysteine cathepsins are still found during acute lung inflammation. [ABSTRACT FROM AUTHOR]

The article examines the health benefits of ß-carotene particularly in immunity. It was found that supplementing ß-carotene in diet increases glutathione in murine splenocytes. Moreover, the redox effect of ß-carotene was observed in vivo, upregulating the activity of cysteine cathepsin. Results suggest that ß-carotene has a positive effect in redox status of immune cells.