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Background: Cathepsin C (CTSC) participates in the development of numerous cancers; however, its function in bladder cancer (BCa) remains largely unknown., Methods: Bioinformatics prediction, quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay, and Western blot assay were used to determine CTSC expression in BCa tissues, paracancer tissues, BCa cells, and normal uroepithelial cells (SV-HUC-1). Colony formation, cell counting kit-8 (CCK-8), and Transwell assays were utilised to ascertain the involvement of CTSC in BCa. The effect of CTSC on BCa was further studied in vivo via animal experiments., Results: CTSC exhibited a heightened expression in BCa cells and tissues; meanwhile, bladder urothelial carcinoma (BLCA) patients with enhanced CTSC expression had a remarkably reduced overall survival than those with low CTSC expression. The overexpression of CTSC substantially enhanced the activity, proliferation, migration, and invasion of BCa cells, whereas its suppression repressed the above biological phenotypes. CTSC could activate the Wnt/β-catenin signalling pathway and upregulate diaphanous-related formin 3 (DIAPH3). CTSC overexpression combined with DIAPH3 knockdown partially reversed the impact of CTSC overexpression on the biological behaviour of BCa cells and the activation of the Wnt/β-catenin signalling pathway., Conclusions: CTSC was upregulated in tissues and BCa cells, and high CTSC expression was associated with poor overall survival. CTSC could enhance the activity, proliferation, migration, and invasion of BCa cells via upregulating DIAPH3 and activating the Wnt/β-catenin pathway., (© 2024 The Author(s). Published by IMR Press.)

Cathepsin C (CTSC) is a lysosomal cysteine protease constitutively expressed at high levels in the lung, kidney, liver, and spleen. It plays a key role in the activation of serine proteases in cytotoxic T cells, natural killer cells (granzymes A and B), mast cells (chymase and tryptase) and neutrophils (cathepsin G, neutrophil elastase, proteinase 3) underscoring its pivotal significance in immune and inflammatory defenses. Here, we comprehensively review the structural attributes, synthesis, and function of CTSC, with a focus on its variants implicated in the etiopathology of several syndromes associated with neutrophil serine proteases, including Papillon-Lefevre syndrome (PLS), Haim-Munk Syndrome (HMS), and aggressive periodontitis (AP). These syndromes are characterized by palmoplantar hyperkeratosis, and early-onset periodontitis (severe gum disease) resulting in premature tooth loss. Due to the critical role played by CTSC in these and several other conditions it is being explored as a potential therapeutic target for autoimmune and inflammatory disorders. The review also discusses in depth the gene variants of CTSC, and in particular their postulated association with chronic obstructive pulmonary disease (COPD), COVID-19, various cancers, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, sudden cardiac death (SCD), atherosclerotic vascular disease, and neuroinflammatory disease. Finally, the therapeutic potential of CTSC across a range of human diseases is discussed., (© 2024. The Author(s).)

Primary graft dysfunction (PGD) is a severe form of acute lung injury resulting from lung ischemia/reperfusion injury (I/R) in lung transplantation (LTx), associated with elevated post-transplant morbidity and mortality rates. Neutrophils infiltrating during reperfusion are identified as pivotal contributors to lung I/R injury by releasing excessive neutrophil extracellular traps (NETs) via NETosis. While alveolar macrophages (AMs) are involved in regulating neutrophil chemotaxis and infiltration, their role in NETosis during lung I/R remains inadequately elucidated. Extracellular histones constitute the main structure of NETs and can activate AMs. In this study, we confirmed the significant involvement of extracellular histone-induced M1 phenotype of AMs (M1-AMs) in driving NETosis during lung I/R. Using secretome analysis, public protein databases, and transwell co-culture models of AMs and neutrophils, we identified Cathepsin C (CTSC) derived from AMs as a major mediator in NETosis. Further elucidating the molecular mechanisms, we found that CTSC induced NETosis through a pathway dependent on NADPH oxidase-mediated production of reactive oxygen species (ROS). CTSC could significantly activate p38 MAPK, resulting in the phosphorylation of the NADPH oxidase subunit p47 phox , thereby facilitating the trafficking of cytoplasmic subunits to the cell membrane and activating NADPH oxidase. Moreover, CTSC up-regulated and activated its substrate membrane proteinase 3 (mPR3), resulting in an increased release of NETosis-related inflammatory factors. Inhibiting CTSC revealed great potential in mitigating NETosis-related injury during lung I/R. These findings suggests that CTSC from AMs may be a crucial factor in mediating NETosis during lung I/R, and targeting CTSC inhition may represent a novel intervention for PGD in LTx., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Cystatin F (CstF) is a protease inhibitor of cysteine cathepsins, including those involved in activating the perforin/granzyme cytotoxic pathways. It is targeted at the endolysosomal pathway but can also be secreted to the extracellular milieu or endocytosed by bystander cells. CstF was shown to be significantly increased in tuberculous pleurisy, and during HIV coinfection, pleural fluids display high viral loads. In human macrophages, our previous results revealed a strong upregulation of CstF in phagocytes activated by interferon γ or after infection with Mycobacterium tuberculosis (Mtb). CstF manipulation using RNA silencing led to increased proteolytic activity of lysosomal cathepsins, improving Mtb intracellular killing. In the present work, we investigate the impact of CstF depletion in macrophages during the coinfection of Mtb-infected phagocytes with lymphocytes infected with HIV. The results indicate that decreasing the CstF released by phagocytes increases the major pro-granzyme convertase cathepsin C of cytotoxic immune cells from peripheral blood-derived lymphocytes. Consequently, an observed augmentation of the granzyme B cytolytic activity leads to a significant reduction in viral replication in HIV-infected CD4 + T-lymphocytes. Ultimately, this knowledge can be crucial for developing new therapeutic approaches to control both pathogens based on manipulating CstF.

Objective: To investigate the molecular mechanisms of chronic rhinosinusitis (CRS), to identify key cell subgroups and genes, to construct effective diagnostic models, and to screen for potential therapeutic drugs. Methods: Key cell subgroups in CRS were identified through single-cell transcriptomic sequencing data. Essential genes associated with CRS were selected and diagnostic models were constructed by hdWGCNA (high dimensional weighted gene co-expression network analysis) and various machine learning algorithms. Causal inference analysis was performed using Mendelian randomization and colocalization analysis. Potential therapeutic drugs were identified using molecular docking technology, and the results of bioinformatics analysis were validated by immunofluorescence staining. Graphpad Prism, R, Python, and Adobe Illustrator software were used for data and image processing. Results: An increased proportion of basal and suprabasal cells was observed in CRS, especially in eosinophilic CRS with nasal polyps (ECRSwNP), with P =0.001. hdWGCNA revealed that the "yellow module" was closely related to basal and suprabasal cells in CRS. Univariate logistic regression and LASSO algorithm selected 13 key genes ( CTSC , LAMB3 , CYP2S1 , TRPV4 , ARHGAP21 , PTHLH , CDH26 , MRPS6 , TENM4 , FAM110C , NCKAP5 , SAMD3 , and PTCHD4 ). Based on these 13 genes, an effective CRS diagnostic model was developed using various machine learning algorithms (AUC=0.958). Mendelian randomization analysis indicated a causal relationship between CTSC and CRS (inverse variance weighted: OR=1.06, P =0.006), and colocalization analysis confirmed shared genetic variants between CTSC and CRS (PPH4/PPH3>2). Molecular docking results showed that acetaminophen binded well with CTSC (binding energy:-5.638 kcal/mol). Immunofluorescence staining experiments indicated an increase in CTSC + cells in CRS. Conclusion: This study integrates various bioinformatics methods to identify key cell types and genes in CRS, constructs an effective diagnostic model, underscores the critical role of the CTSC gene in CRS pathogenesis, and provides new targets for the treatment of CRS.

Acetaminophen (APAP) overdosing is a major cause of acute liver failure worldwide and an established model for drug-induced acute liver injury (ALI). While studying gene expression during murine APAP-induced ALI by 3'mRNA sequencing (massive analysis of cDNA ends, MACE), we observed splenic mRNA accumulation encoding for the neutrophil serine proteases cathepsin G, neutrophil elastase, and proteinase-3 - all are hierarchically activated by cathepsin C (CtsC). This, along with increased serum levels of these proteases in diseased mice, concurs with the established phenomenon of myeloid cell mobilization during APAP intoxication. Objective: In order to functionally characterize CtsC in murine APAP-induced ALI, effects of its genetic or pharmacological inhibition were investigated. Methods and Results: We report on substantially reduced APAP toxicity in CtsC deficient mice. Alleviation of disease was likewise observed by treating mice with the CtsC inhibitor AZD7986, both in short-term prophylactic and therapeutic protocols. This latter observation indicates a mode of action beyond inhibition of granule-associated serine proteases. Protection in CtsC knockout or AZD7986-treated wildtype mice was unrelated to APAP metabolization but, as revealed by MACE, realtime PCR, or ELISA, associated with impaired expression of inflammatory genes with proven pathogenic roles in ALI. Genes consistently downregulated in protocols tested herein included cxcl2 , mmp9 , and angpt2 . Moreover, ptpn22 , a positive regulator of the toll-like receptor/interferon-axis, was reduced by targeting CtsC. Conclusions: This work suggests CtsC as promising therapeutic target for the treatment of ALI, among others paradigmatic APAP-induced ALI. Being also currently evaluated in phase III clinical trials for bronchiectasis, successful application of AZD7986 in experimental APAP intoxication emphasizes the translational potential of this latter therapeutic approach., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Cathepsin C is a cysteine protease widely found in invertebrates and vertebrates, and has the important physiological role participating in proteolysis in vivo and activating various functional proteases in immune/inflammatory cells in the animals. In order to study the role of cathepsin C in the disease resistance of shrimp, we cloned cathepsin C gene (MjcathC) from Marsupenaeus japonicus, analyzed its expression patterns in various tissues, performed MjcathC-knockdown, and finally challenged experimental shrimps with Vibrio alginolyticus and WSSV. The results have shown the full length of MjcathC is 1782 bp, containing an open reading frame of 1350 bp encoding 449 amino acids. Homology analysis revealed that the predicted amino acid sequence of MjcathC shared respectively 88.42 %, 87.36 % and 87.58 % similarity with Penaeus monodon, Fenneropenaeus penicillatus and Litopenaeus vannamei. The expression levels of MjcathC in various tissues of healthy M. japonicus are the highest in the liver, followed by the gills and heart, and the lowest in the stomach. The expression levels of MjcathC were significantly up-regulated in all examined tissues of shrimp challenged with WSSV or V. alginolyticus. After knockdown-MjcathC using RNAi technology in M. japonicus, the expression levels of lectin and heat shock protein 70 in MjcathC-knockdown shrimp were significantly down-regulated, and the mortality of MjcathC-knockdown shrimp challenged by WSSV and V. alginolyticus significantly increased. Knockdown of the MjcathC reduced the resistance of M. japonicus to WSSV and V. alginolyticus. The above results have indicated that cathepsin C may play an important role in the antibacterial and antiviral innate immunity of M. japonicus., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Objectives: describing the clinical features of twelve Egyptian patients with Papillon-Lefever syndrome (PLS). Five novel mutations in the cathepsin C (CTSC) gene are introduced and the phenotype of the syndrome is expanded by the identification of new clinical features., Design: the clinical, oro-dental data of twelve Egyptian patients from seven unrelated families are described. Sequence analysis of the CTSC gene was performed to identify the causative mutaions., Results: Typical PLS features were presented in all patints but with variable severity. One patient showed atypical dental features including dental structural defect, minimal periodontitis, severe gingivitis, and delayed closure of root apices. Another patient presented with arachnodactyly, dystrophic nails, and buphthalmos in the right eye secondary to uncontrolled congenital glaucoma. Mutational analysis of CTSC gene revealed seven distinct homozygous variants including five novel ones: c.285_286delGT (p.Leu96GlufsTer2), c .302 G>C (p.Trp101Ser), c.622_628delCACAGTC (p.H208Efs*11), c.1331delinsAAAAA (p.G444Efs*4) and c .1343 G>A (p.Cys448Tyr). The previously reported missense variant c .757 G>A (p.Ala253Thr) was found in one patient. This variant is very close to the splice region and by functional studies, we proved that it results in exon skipping and early protein truncation (p.R214Sfs*46)., Conclusion: We report five novel CTSC variants and describe rare and unusual associated clinical and dental findings such as dental structural defects, delayed closure of root apices, and congenital glaucoma. Therefore, our results expand both the phenotypic and mutational spectrum of PLS., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Cathepsin C (CatC, syn. Dipeptidyl peptidase I) is a lysosomal cysteine proteinase expressed in several tissues including inflammatory cells. This enzyme is important for maintaining multiple cellular functions and for processing immune cell-derived proteases. While mutations in the CatC gene were reported in Papillon-Lefèvre syndrome, a rare autosomal recessive disorder featuring hyperkeratosis and periodontitis, evidence from clinical and preclinical studies points toward pro-inflammatory effects of CatC in various disease processes that are mainly mediated by the activation of neutrophil serine proteinases. Moreover, tumor-promoting effects were ascribed to CatC. The aim of this review is to highlight current knowledge of the CatC as a potential therapeutic target in inflammatory disorders., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The research aimed to describe a new Trichinella spiralis dipeptidyl peptidase 1 (TsDPP1) and investigate its functions in the larval invasion of intestinal epithelial cells (IECs). The gene TsDPP1 was successfully replicated and produced in Escherichia coli BL21 (DE3), showing a strong immune response. TsDPP1 was detected in diverse stages of T. spiralis and showed significant expression in the intestine infective larvae (IIL) and adult worms at 6 days post infection, as confirmed by qPCR and Western blot analysis. The primary localization of TsDPP1 in this parasite was observed in cuticles, stichosomes, and embryos by using the indirect immunofluorescence assay (IIFA). rTsDPP1 exhibited the enzymatic function of natural dipeptidyl peptidase and showed specific binding to IECs, and the binding site was found to be localized on cell membrane. Following transfection with dsRNA-TsDPP1, the expression of TsDPP1 mRNA and protein in muscle larvae (ML) were decreased by approximately 63.52 % and 58.68 %, correspondingly. The activity of TsDPP1 in the ML and IIL treated with dsRNA-TsDPP1 was reduced by 42.98 % and 45.07 %, respectively. The acceleration of larval invasion of IECs was observed with rTsDPP1, while the invasion was suppressed by anti-rTsDPP1 serum. The ability of the larvae treated with dsRNA-TsDPP1 to invade IECs was hindered by 31.23 %. In mice infected with dsRNA-treated ML, the intestinal IIL, and adults experienced a significant decrease in worm burdens and a noticeable reduction in adult female length and fecundity compared to the PBS group. These findings indicated that TsDPP1 significantly impedes the invasion, growth, and reproductive capacity of T. spiralis in intestines, suggesting its potential as a target for anti-Trichinella vaccines., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Competing Interests: Conflicts of interest R.L.McC. has held a position funded by Palvella Therapeutics to work on a clinical trial (all unrelated to this work with all funding going to the university. D.G. has been a speaker for Lilly and La Roche-Posay (none applicable to this work). M.T. has been a speaker for AbbVie, Almirall, Eucerin, Janssen and Sanofi (none applicable to this work). E.O’T. has received research funding from Kamari Pharma and Unilever; consultancy fees from Azitra, Palvella Therapeutics and Kamari Pharma; and speaker’s fees from Almirall (all unrelated to this work with all funding going to the university). M.S. declares no conflicts of interest.

Microbial pathogens use proteases for their infections, such as digestion of proteins for nutrients and activation of their virulence factors. As an obligate intracellular parasite, Toxoplasma gondii must invade host cells to establish its intracellular propagation. To facilitate invasion, the parasites secrete invasion effectors from microneme and rhoptry, two unique organelles in apicomplexans. Previous work has shown that some micronemal invasion effectors experience a series of proteolytic cleavages within the parasite's secretion pathway for maturation, such as the aspartyl protease (TgASP3) and the cathepsin L-like protease (TgCPL), localized within the post-Golgi compartment and the endolysosomal system, respectively. Furthermore, it has been shown that the precise maturation of micronemal effectors is critical for Toxoplasma invasion and egress. Here, we show that an endosome-like compartment (ELC)-residing cathepsin C-like protease (TgCPC1) mediates the final trimming of some micronemal effectors, and its loss further results in defects in the steps of invasion, egress, and migration throughout the parasite's lytic cycle. Notably, the deletion of TgCPC1 completely blocks the activation of subtilisin-like protease 1 (TgSUB1) in the parasites, which globally impairs the surface-trimming of many key micronemal invasion and egress effectors. Additionally, we found that Toxoplasma is not efficiently inhibited by the chemical inhibitor targeting the malarial CPC ortholog, suggesting that these cathepsin C-like orthologs are structurally different within the apicomplexan phylum. Collectively, our findings identify a novel function of TgCPC1 in processing micronemal proteins within the Toxoplasma parasite's secretory pathway and expand the understanding of the roles of cathepsin C protease. IMPORTANCE Toxoplasma gondii is a microbial pathogen that is well adapted for disseminating infections. It can infect virtually all warm-blooded animals. Approximately one-third of the human population carries toxoplasmosis. During infection, the parasites sequentially secrete protein effectors from the microneme, rhoptry, and dense granule, three organelles exclusively found in apicomplexan parasites, to help establish their lytic cycle. Proteolytic cleavage of these secretory proteins is required for the parasite's optimal function. Previous work has revealed that two proteases residing within the parasite's secretory pathway cleave micronemal and rhoptry proteins, which mediate parasite invasion and egress. Here, we demonstrate that a cathepsin C-like protease (TgCPC1) is involved in processing several invasion and egress effectors. The genetic deletion of TgCPC1 prevented the complete maturation of some effectors in the parasites. Strikingly, the deletion led to a full inactivation of one surface-anchored protease, which globally impaired the trimming of some key micronemal proteins before secretion. Therefore, this finding represents a novel post-translational mechanism for the processing of virulence factors within microbial pathogens., Competing Interests: The authors declare no conflict of interest.

Hypertrophic cardiomyopathy (HCM) is a primary cardiomyopathy characterized by hypertrophic cardiomyocytes. It is one of the leading causes of sudden death in adolescents. However, the molecular mechanism of HCM is not clear. In our study, ribonucleic acid (RNA) sequence data of myocardial tissue in HCM patients were extracted from the Gene Expression Omnibus (GEO) database (GSE130036) and analyzed by weighted gene coexpression network analysis (WGCNA). A total of 31 coexpression modules were identified. The coexpression black module significantly correlated with maximum left ventricular wall thickness (Maxi LVWT). We screened the differentially expressed mRNAs between normal tissues and HCM tissues using the dplyr and tidyr packages in R3.6.2. The genes in the black module and differentially expressed genes were further intersected. We found that the expression of carboxylesterase 1 (CES1) and cathepsin C (CTSC) was downregulated in HCM tissues and negatively correlated with Maxi LVWT. We further verified the expression of CES1 and CTSC was downregulated in HCM clinical blood and negatively correlated with Maxi LVWT. Finally, we demonstrated that overexpression of CTSC and CES1 could alleviate HCM in an HCM cell model. In summary, the study suggests that CES1 and CTSC negatively regulate the development of HCM and have potential as therapeutic and diagnostic targets for HCM.

Background: Papillon Lefevre syndrome (PLS) is an autosomal recessive disorder that results from a mutated gene that encodes a lysosomal peptidase known as cathepsin C (CTSC). The clinical presentation of PLS involves mainly palmoplantar keratosis and periodontitis with a variable degree of severity., Subjects: and methods: Our study included ten patients with a broad spectrum of palmoplantar keratosis and periodontitis severity. CTSC variants were detected by Sanger sequencing. CTSC protein secreted in urine was detected by western blotting., Results: Five patients have missense variants, Four have nonsense variants, and one has splice variants in CTSC. The activation products of cathepsin C protein (Heavy and light chains) were absent in all patients' urine samples except one with a significantly reduced level compared to the controls. The dimeric form of CTSC protein was found in all the studied cases. The monomeric form was found in five cases. The products of proteolytic activation of CTSC by other cathepsins (L and S) were found in the urine samples of five of the patients. Each patient had a characteristic pattern of accumulated CTSC protein maturation/activation substrates, intermediates, and products. 40% of the patients had the activation products of other lysosomal cathepsins., Conclusion: Urinary CTSC in PLS patients could be used as a diagnostic biomarker for the biochemical screening of the disease. Different variants in CTSC result in different profiles of CTSC secreted in the urine of PLS patients. The profiles of secreted CTSC in urine could be correlated to the severity of palmoplantar keratosis., Competing Interests: Declaration of competing interest The authors declare that they have no relevant financial or non-financial interests to disclose., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Human dipeptidyl peptidase I (DPPI) belongs to the family of papain-like cysteine peptidases. Its distinctive features are the unique exclusion domain which enables the eponymous activity and homotetramerization of DPPI, and its dependence on chloride ions for enzymatic activity. The oligomeric state of DPPI is unique in this family of predominantly monomeric peptidases. However, a distant DPPI ortholog from Plasmodium falciparum has been shown to be monomeric, indicating that the oligomeric state of DPPI varies between lineages. The aim of this work was to study the evolution of DPPI, with particular attention to the structural features that determine its characteristic enzymatic activity and preferences, and to reconstruct the evolution of its oligomerization. We analyzed fifty-seven selected sequences of DPPI and confirmed its presence in three lineages, namely, Amorphea (including animals and Amoebozoa), Alveolates and the metamonad Giardia . The amino acid residues that bind the chloride ion are highly conserved in all species, indicating that the dependence on chloride ions for activity is an evolutionarily conserved feature of DPPI. The number of N-glycosylation sites is significantly increased in animals, particularly vertebrates. Analysis of homology models and subunit contacts suggests that oligomerization is likely restricted to DPPIs in the Amorphea group.

Endothelial cell dysfunction is an essential pathophysiological feature of preeclampsia (PE). It has been reported that cathepsin C is upregulated in the maternal vascular endothelium of PE patients. The excessive activation of p38 MAPK leads to various diseases, including PE. NF-κB pathway can promote uteroplacental dysfunction, endothelial stress and development of PE. Moreover, it has been verified that cathepsin C can activate p38 MAPK/NF-κB pathway. In the present work, hypoxia/reoxygenation (H/R) injury model of HUVECs was established to discuss the biological functions of cathepsin C in endothelial cell dysfunction and to elucidate the underlying molecular mechanism. The correlation between cathepsin C and p38 MAPK/NF-κB pathway in H/R-stimulated HUVECs as well as the effects of cathepsin C and p38 MAPK/NF-κB pathway on viability, apoptosis, invasion, in vitro angiogenesis of HUVECs and oxidative stress were assessed. The results revealed that H/R injury elevated cathepsin C expression and activated p38 MAPK/NF-κB pathway in HUVECs and cathepsin C knockdown inhibited the activity of p38 MAPK/NF-κB pathway in H/R-stimulated HUVECs. Downregulation of cathepsin C improved viability, inhibited apoptosis and enhanced invasion of H/R-stimulated HUVECs. In addition, downregulation of cathepsin C alleviated oxidative stress and induced stronger HUVEC angiogenesis in vitro. Furthermore, the protective effects of cathepsin C knockdown against endothelial cell dysfunction were reversed by p38 MAPK activator anisomycin. In other words, downregulation of cathepsin C could improve HUVEC viability and enhance anti-apoptotic capacity, anti-oxidative capability, invasive ability, as well as angiogenic potential of H/R-stimulated HUVECs by repressing p38 MAPK/NF-κB pathway.

Periodontitis is characterized by alveolar bone loss leading to tooth loss. A small proportion of patients develop severe periodontitis at the juvenile or adolescent age without exposure to the main risk factors of the disease. It is considered that these cases carry rare variants with large causal effects, but the specific variants are largely unknown. In this study, we performed exome sequencing of 5 families with children who developed stage IV, grade C, periodontitis between 3 and 18 y of age. In 1 family, we found compound heterozygous variants in the gene CTSC (p.R272H, p.G139R), 1 of which was previously identified in a family with prepubertal periodontitis. Subsequent targeted resequencing of the CTSC gene in 24 patients <25 y of age (stage IV, grade C) identified the known mutation p.I453V (odds ratio = 4.06, 95% CI = 1.6 to 10.3, P = 0.001), which was previously reported to increase the risk for adolescent periodontitis. An affected sibling of another family carried a homozygous deleterious mutation in the gene TUT7 (p.R560Q, CADD score >30 [Combined Annotation Dependent Depletion]), which is implicated in regulation of interleukin 6 expression. Two other affected siblings shared heterozygous deleterious mutations in the interacting genes PADI1 and FLG (both CADD = 36), which contribute to the integrity of the environment-tissue barrier interface. Additionally, we found predicted deleterious mutations in the periodontitis risk genes ABCA1 , GLT6D1 , and SIGLEC5 . We conclude that the CTSC variants p.R272H and p.I453V have different expressivity and diagnostic relevance for prepubertal and adolescent periodontitis, respectively. We propose additional causal variants for early-onset periodontitis, which also locate within genes that carry known susceptibility variants for common forms. However, the genetic architecture of juvenile periodontitis is complex and differs among the affected siblings of the sequenced families.

Papillon-Lefèvre Syndrome (PLS) is an autosomal recessive monogenic disease caused by loss-of-function mutations in the CTSC gene, thus preventing the synthesis of the protease Cathepsin C (CTSC) in a proteolytically active form. CTSC is responsible for the activation of the pro-forms of the neutrophil serine proteases (NSPs; Elastase, Proteinase 3 and Cathepsin G), suggesting its involvement in a variety of neutrophil functions. In PLS neutrophils, the lack of CTSC protease activity leads to inactivity of the NSPs. Clinically, PLS is characterized by an early, typically pre-pubertal, onset of severe periodontal pathology and palmoplantar hyperkeratosis. However, PLS is not considered an immune deficiency as patients do not typically suffer from recurrent and severe (bacterial and fungal) infections. In this study we investigated an unusual CTSC mutation in two siblings with PLS, a 503A>G substitution in exon 4 of the CTSC gene, expected to result in an amino acid replacement from tyrosine to cysteine at position 168 of the CTSC protein. Both patients bearing this mutation presented with pronounced periodontal pathology. The characteristics and functions of neutrophils from patients homozygous for the 503A>G CTSC mutation were compared to another previously described PLS mutation (755A>T), and a small cohort of healthy volunteers. Neutrophil lysates from patients with the 503A>G substitution lacked CTSC protein and did not display any CTSC or NSP activity, yet neutrophil counts, morphology, priming, chemotaxis, radical production, and regulation of apoptosis were without any overt signs of alteration. However, NET formation upon PMA-stimulation was found to be severely depressed, but not abolished, in PLS neutrophils., Competing Interests: The authors have declared that no competing interests exist.

Cathepsin C, an important lysosomal cysteine protease, mediates the maturation process of neutrophil serine proteases, and participates in the inflammation and immune regulation process associated with polymorphonuclear neutrophils. Therefore, cathepsin C is considered to be an attractive target for treating inflammatory diseases. With INS1007 (trade name: brensocatib) being granted a breakthrough drug designation by FDA for the treatment of Adult Non-cystic Fibrosis Bronchiectasis and Coronavirus Disease 2019, the development of cathepsin C inhibitor will attract attentions from medicinal chemists in the future soon. Here, we summarized the research results of cathepsin C as a therapeutic target, focusing on the development of cathepsin C inhibitor, and provided guidance and reference opinions for the upcoming development boom of cathepsin C inhibitor., Competing Interests: Declaration of competing interest All authors have contributed to the work, have read the manuscript and have agreed to be listed as authors. The submitted manuscript has not been published elsewhere and nor is it currently under review by another publication. No potential conflict of interest relevant to this article., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Emerging evidence suggests that several of the lysosomal cathepsin proteases are genetically associated with type 1 diabetes (T1D) and participate in immune-mediated destruction of the pancreatic β cells. We previously reported that the T1D candidate gene cathepsin H is downregulated by pro-inflammatory cytokines in human pancreatic islets and regulates β-cell function, apoptosis, and disease progression in children with new-onset T1D. In the present study, the objective was to investigate the expression patterns of all 15 known cathepsins in β-cell model systems and examine their role in the regulation of cytokine-induced apoptosis. Real-time qPCR screening of the cathepsins in human islets, 1.1B4 and INS-1E β-cell models identified several cathepsins that were expressed and regulated by pro-inflammatory cytokines. Using small interfering RNAs to knock down (KD) the cytokine-regulated cathepsins, we identified an anti-apoptotic function of cathepsin C as KD increased cytokine-induced apoptosis. KD of cathepsin C correlated with increased phosphorylation of JNK and p38 mitogen-activated protein kinases, and elevated chemokine CXCL10/IP-10 expression. This study suggests that cathepsin C is a modulator of β-cell survival, and that immune modulation of cathepsin expression in islets may contribute to immune-mediated β-cell destruction in T1D.

Objective: Chemokines regulate infiltration of immune cells to brain in inflammation. Cathepsin C (CatC), a lysosomal protease, has been found to participate in neuroinflammation. However, how CatC affects chemokines expression in neuroinflammation triggered by traumatic brain injury (TBI) remains unclear. Here, we investigated the effects of CatC on chemokines and neuroinflammation in TBI., Methods: The present study used CatC knockdown (KD) and overexpression (OE) mice to generate cryogenic brain lesion model and determined effects of CatC on expression of chemokines CCL2, CCL5 and CXCL2 and infiltration of immune cells in acute and chronic phases of the lesion. Further, cellular sources of various chemokines were demonstrated in vitro. Values were compared with wild type (WT) mice., Results: The results found that 6 h after lesion, CatC expression,IL-1β and TNF-α mRNA and protein expression were strongly induced in the lesions; CCL2 and CXCL2 mRNA and protein expression were increased in CatC OE mice, while decreased in CatC KD mice. On the 3rd day after lesion, macrophages and neutrophils were mainly infiltrated to the lesions. Simultaneously, Iba-1+ cells in CatC OE mice were increased, while MPO + cells in CatC KD mice were decreased. In contrast, on the 28th day after lesion, a few lymphocytes were infiltrated surrounding new blood vessels. CatC OE mice showed larger volumes of scar areas, higher expression of CCL2,CXCL2,IL-1β,TNF-α,IL-6 and iNOS, as well as stronger GFAP+ and Iba-1+ signals, while CatC KD mice had reversed effects. No significant differences of CCL5 expression were found in various genotype mice. Further, in vitro study demonstrated CatC-induced expression of CCL2 were mainly derived from microglia and neurons, while CXCL2 derived from microglia and astrocytes., Conclusion: Our data indicate that CatC aggravates neuroinflammation via promoting production of CCL2 and CXCL2 in glial cells and neurons in a cryogenic brain lesion, providing potential cellular and molecular targets for future intervention of TBI and other neuroinflammatory diseases., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objectives: In this study, we analyzed the whole exomes of CTSC gene in a family with history of PLS., Materials and Methods: Genomic DNA was extracted from peripheral blood and genotype analysis was performed. The mutated protein sequence was used to find the best possible tertiary structure for homology modeling. The homology modeling of the novel mutation was then performed using the online Swiss-Prot server. The results were also analyzed for to verify its validity., Results: The analysis of CTSC gene elucidated a novel insertion GAC. The novel mutation was proved by analyzing 50 healthy control volunteers. Modeling of the novel found mutation in CTSC gene revealed structural defects that may have caused the functional abnormalities., Conclusions: The structural analysis of the mutated protein model identifies changes in the stereo-chemical and the energy level of the mutated protein. Since this protein play a role in the activation of granule serine proteases from cytotoxic T lymphocytes, natural killer cells, mast cells, such structural defects may lead to its malfunction causing dysfunctioning of immune defense mechanisms., (© 2021 The Authors. Clinical and Experimental Dental Research published by John Wiley & Sons Ltd.)

Background: Papilon-Lefevre syndrome (PLS; OMIM 245000) is a rare autosomal recessive disease characterized by aggressive periodontitis and palmoplantar keratoderma. The prevalence of PLS in the general population is one to four cases per million. Although the etiology and pathogenic mechanisms underlying PLS remain largely unclear, existing evidence shows loss-of-function mutations of the cathepsin C gene (CTSC; OMIM 602365) could cause PLS. Here we found a novel variant of the CTSC gene in a Chinese PLS family and predicted the effect of the variant on the physic-chemical characters and tertiary structure of the protein., Methods: The 1-7 coding exons and exon-intron boundaries of CTSC gene of the proband and her family were amplified and sequenced directly, and Chromas was used to read sequencing files. Furthermore, the PolyPhen-2, PROVEAN, and Mutation Taster were utilized to predict the pathogenicity of the variant. Besides, the physic-chemical and structural characters of the protein were analyzed by ProtParam, ProtScale, and SWISS-MODEL., Results: Our study identified a novel homozygous variant c.763T>C (p.Cys255Arg) in exon 6 of the CTSC gene, and it was a likely pathogenic variant as predicted by PolyPhen-2, PROVEAN, and Mutation Taster. Moreover, ProtParam and Protscale revealed the variant increased the isoelectric point and hydrophilicity of the protein, and the SWISS-MODEL analysis suggested the variant was located in a critical domain for protein activity., Conclusion: Our study analyzed a Chinese family with PLS and identified a novel missense variant in the CTSC gene. Besides, this study retrospectively summarized 113 variants of CTSC in the world and highlighted the features of 27 CTSC variants in Chinese PLS patients. In addition, this study paid much particular attention to the relationship between CTSC variants and different phenotypes., (© 2021 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Papillon-Lefèvre syndrome (PLS) is a rare autosomal recessive genodermatosis characterized by palmoplantar keratoderma and severe periodontitis leading to premature loss of primary and permanent teeth. PLS is caused by loss-of-function mutations in CTSC, lacking functional cathepsin C, which impairs the activation of neutrophil serine proteases. Precise pathogenesis of periodontal damage is unknown. Patient 1 presented with well-demarcated, transgredient, diffuse, palmoplantar keratoderma and psoriasiform lesions from the age of 2 years. Based on severe and recurrent periodontal inflammation, his dentist had diagnosed PLS at the age of 3 years and provided a strict oral hygiene regimen with repeated adjunct antibiotic therapies. Oral acitretin 10 mg/day along with tretinoin ointment at the age of 9 greatly improved palmoplantar keratoderma. Aged 18 years, the patient exhibited an intact permanent dentition and absence of periodontal disease. Patient 2, a 30-year-old man, suffered from transgredient, diffuse, palmoplantar keratoderma with fissuring from the age of 2 months, marked psoriasiform plaques on elbows and knees, and nail dystrophy. Intriguingly, without specific dental treatment, teeth and dental records were unremarkable. He was referred with a suspected diagnosis of psoriasis. Both patients were otherwise healthy, blood tests and sonography of internal organs were within normal limits. Panel sequencing revealed loss-of-function mutations in CTSC, c.322A>T (p.Lys108Ter) and c.504C>G (p.Tyr168Ter) in patient 1 and homozygous c.415G>T (p.Gly139Ter) in patient 2. The final diagnosis of unusual PLS was made. PLS should be considered in palmoplantar keratoderma lacking periodontitis or tooth loss., (© 2021 Japanese Dermatological Association.)

Brain arteriovenous malformations (bAVMs) are congenital anomalies of blood vessels that cause intracranial hemorrhage in children and young adults. Chromosomal rearrangements and fusion genes play an important role in tumor pathogenesis, though the role of fusion genes in bAVM pathophysiological processes is unclear. The aim of this study was to identify fusion transcripts in bAVMs and analyze their effects. To identify fusion transcripts associated with bAVM, RNA sequencing was performed on 73 samples, including 66 bAVM and 7 normal cerebrovascular samples, followed by STAR-Fusion analysis. Reverse transcription polymerase chain reaction and Sanger sequencing were applied to verify fusion transcripts. Functional pathway analysis was performed to identify potential effects of different fusion types. A total of 21 fusion transcripts were detected. Cathepsin C (CTSC)-Ras-Related Protein Rab-38 (RAB38) was the most common fusion and was detected in 10 of 66 (15%) bAVM samples. In CTSC-RAB38 fusion-positive samples, CTSC and RAB38 expression was significantly increased and activated immune/inflammatory signaling. Clinically, CTSC-RAB38 fusion bAVM cases had a higher hemorrhage rate than non-CTSC-RAB38 bAVM cases (p < 0.05). Our study identified recurrent CTSC-RAB38 fusion transcripts in bAVMs, which may be associated with bAVM hemorrhage by promoting immune/inflammatory signaling., (© 2020 American Association of Neuropathologists, Inc. All rights reserved.)

Cathepsins are lysosomal acid hydrolases that make crucial contributions to tumor progression through a variety of signaling mechanisms, including autophagy, cell survival, chemotherapeutic resistance, and metastasis. Herein, we report that cathepsin C (CTSC) silencing upregulates the anticancer potential of curcumin in colorectal cancer cells (CRCs) both in vitro and in athymic mice xenografts. Curcumin treatment enhances CTSC level in CRCs; however, CTSC silencing with subsequent curcumin treatment (sequential treatment) induces ER stress and autophagic dysregulation accompanied by lysosomal permeabilization and ROS generation. This lysosomal permeabilization triggered the cytosolic CTSB mediated BID-dependent mitochondrial membrane permeabilization and thereby caspase-dependent apoptosis. This phenotype can be rescued by CTSB inhibition and NAC, which further supported the involvement of ROS and CTSB in apoptosis following sequential treatment. Indeed, the sequential CTSC silencing and curcumin treatment also significantly curtailed tumor volume as well as ameliorated cytosolic cyt c and tBID protein levels in tumor tissues compared to those in control and individual treatments of CTSC targeting and on curcumin treatment in nude mice xenografts. The results reveal that CTSC can controls the curcumin-induced cytotoxic insult through autophagy maintenance both in vitro and in athymic mice xenografts, thereby providing an insight into the role of CTSC in chemoprevention of CRCs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A growing body of evidence suggests a role of proteolytic enzymes in the development of diabetic nephropathy. Cathepsin C (CatC) is a well-known regulator of inflammatory responses, but its involvement in podocyte and renal injury remains obscure. We used Zucker rats, a genetic model of metabolic syndrome and insulin resistance, to determine the presence, quantity, and activity of CatC in the urine. In addition to the animal study, we used two cellular models, immortalized human podocytes and primary rat podocytes, to determine mRNA and protein expression levels via RT-PCR, Western blot, and confocal microscopy, and to evaluate CatC activity. The role of CatC was analyzed in CatC-depleted podocytes using siRNA and glycolytic flux parameters were obtained from extracellular acidification rate (ECAR) measurements. In functional analyses, podocyte and glomerular permeability to albumin was determined. We found that podocytes express and secrete CatC, and a hyperglycemic environment increases CatC levels and activity. Both high glucose and non-specific activator of CatC phorbol 12-myristate 13-acetate (PMA) diminished nephrin, cofilin, and GLUT4 levels and induced cytoskeletal rearrangements, increasing albumin permeability in podocytes. These negative effects were completely reversed in CatC-depleted podocytes. Moreover, PMA, but not high glucose, increased glycolytic flux in podocytes. Finally, we demonstrated that CatC expression and activity are increased in the urine of diabetic Zucker rats. We propose a novel mechanism of podocyte injury in diabetes, providing deeper insight into the role of CatC in podocyte biology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Papillon Lefevre syndrome (PLS) manifests with palmoplantar keratoderma, combined with a rapidly progressive periodontitis associated with mutations in Cathepsin C (CTSC) gene. This article reports a 15-year old male proband with typical PLS traits having a novel compound heterozygote with p.Q49X mutation in exon 1 and p.Y259C missense mutation in exon 6 of CTSC gene respectively. The exon 1 mutation, p.Q49X, (found in proband's mother) was located in exclusion domain and exon 6 mutation, p.Y259C (found in proband's father), was present in peptidase C1A, papain C-terminal domain. Interestingly, missense mutation p.Y259C identified in this study was found to be not reported so far. Upon computational analysis, this missense mutation was found to be lethal. Moreover, our protein modelling approach using mutant protein revealed the presence of monomeric structure on contrary to the tetrameric structure of the wild type protein. In addition, in vitro functional characterization of mutant p.Y259C expressed in HEK293 cells showed a significant reduction in CTSC activity (0.015 ± 0.009 mU/ml) when compared with wild type protein (0.21 ± 0.008 mU/ml). Thus, in this study, we have demonstrated that the pathogenic missense mutant p.Y259C might cause PLS by impaired CTSC function.

Background: Papillon-Lefévre syndrome (PLS; OMIM 245000) and Haim-Munk syndrome (HMS; OMIM 245010), which are both characterized by palmoplantar hyperkeratosis and periodontitis, are phenotypic variants of the same disease caused by mutations of the cathepsin C (CTSC) gene., Aim: To identify putative genetic modifying factors responsible for the differential development of the PLS or HMS phenotypes, we investigated two Hungarian patients with different phenotypic variants (PLS and HMS) but carrying the same homozygous nonsense CTSC mutation (c.748C/T; p.Arg250X)., Methods: To gain insights into phenotype-modifying associations, whole exome sequencing (WES) was performed for both patients, and the results were compared to identify potentially relevant genetic modifying factors., Results: WES revealed two putative phenotype-modifying variants: (i) a missense mutation (rs34608771) of the SH2 domain containing 4A (SH2D4A) gene encoding an adaptor protein involved in intracellular signalling of cystatin F, a known inhibitor of the cathepsin protein, and (ii) a missense variant (rs55695858) of the odorant binding protein 2A (OBP2A) gene, influencing the function of the cathepsin protein through the glycosyltransferase 6 domain containing 1 (GLT6D1) protein., Conclusion: Our study contributes to the accumulating evidence supporting the clinical importance of phenotype-modifying genetic factors, which have high potential to aid the elucidation of genotype-phenotype correlations and disease prognosis., (© 2020 The Authors. Clinical and Experimental Dermatology published by John Wiley & Sons Ltd on behalf of British Association of Dermatologists.)

Renal cell carcinoma (RCC) is the most common adult kidney cancer, and accounts for 85% of all cases of kidney cancers worldwide. Praeruptorin B (Pra-B) is a bioactive constituent of Peucedanum praeruptorum Dunn and exhibits several pharmacological activities, including potent antitumor effects. However, the anti-RCC effects of Pra-B and their underlying mechanisms are unclear; therefore, we explored the effects of Pra-B on RCC cells in this study. We found that Pra-B nonsignificantly influenced the cell viability of human RCC cell lines 786-O and ACHN at a dose of less than 30 μM for 24 h treatment. Further study revealed that Pra-B potently inhibited the migration and invasion of 786-O and ACHN cells, as well as downregulated the mRNA and protein expression of cathepsin C (CTSC) and cathepsin V (CTSV) of 786-O and ACHN cells. Mechanistically, Pra-B also reduced the protein levels of phospho (p)-epidermal growth factor receptor (EGFR), p-mitogen-activated protein kinase kinase (MEK), and p-extracellular signal-regulated kinases (ERK) in RCC cells. In addition, Pra-B treatment inhibited the effect of EGF on the upregulation of EGFR-MEK-ERK, CTSC and CTSV expression, cellular migration, and invasion of 786-O cells. Our findings are the first to demonstrate that Pra-B can reduce the migration and invasion ability of human RCC cells through suppressing the EGFR-MEK-ERK signaling pathway and subsequently downregulating CTSC and CTSV. This evidence suggests that Pra-B can be developed as an effective antimetastatic agent for the treatment of RCC.

Haim-Munk syndrome (HMS) and Papillon-Lefevre syndrome (PLS) are phenotypic variants of palmoplantar keratoderma (PPK) with progressive early-onset periodontitis and dental caries. HMS and PLS have been associated with homozygous or compound heterozygous mutations in the lysosomal protease gene Cathepsin C (CTSC). There have been only a few documented cases of CTSC mutations in patients from South-East Asia. We report the clinical findings of two Cambodian brothers who presented with diffuse, demarcated PPK with transgrediens extending to the elbows and knees, as well as pachyonychia and dental caries. Arachnodactyly and periodontitis were also found in the older brother. Next-generation sequencing unveiled a homozygous missense variant in CTSC (NM&#95;001814.5: c.1337AC: p.(Asp446Ala)) in both brothers. Both parents were heterozygous for the variant, while an unaffected older brother was homozygous for the wild-type allele. Our study adds to the spectrum of mutations and associated clinical presentations for this rare genodermatosis., (© 2019 Wiley Periodicals, Inc.)

Increasing evidence indicates that in response to environmental changes, macrophages can dynamically change into two main functional phenotypes, namely M1 and M2. Depending on these different phenotypes, macrophages can produce either pro-inflammatory or anti-inflammatory factors which may affect the outcome of inflammation. Mastering the switching of M1/M2 phenotypes may provide therapeutic approaches to chronic inflammatory disease, such as atherosclerosis, rheumatoid arthritis, even the metabolic disorders. Cathepsin C (CTSC), as a member of the papain family of cysteine proteases, is a key enzyme in the activation of granule serine proteases thereby involved in modulating the inflammatory responses. Moreover, abundant expression of CTSC has been found in M1 macrophages in plaques of atherosclerosis and related to the progression of disease. However, whether CTSC can regulate macrophage activation status in inflammatory responses has not been fully investigated. In the present study, using peritoneal macrophages (PMs) and mouse macrophage cell line RAW264.7 treated with LPS and active monomer of CTSC, we found that CTSC was not only expressed in macrophages in M1 activation status, but also facilitated macrophages towards M1 phenotype, suggesting a self-activation mechanism involved in this process which may lead to a vicious circle in chronic inflammation. Then we attempted to explore the underlying molecular mechanisms of CTSC resulting in M1 activation. Focal adhesion kinase (FAK) is one of the non-receptor cytoplasmic protein tyrosine kinases, serving as an upstream mediator that leads to transcription of many pro-inflammatory factors. We found FAK expression was up-regulated at both mRNA and protein levels following CTSC stimulation, and FAK phosphorylation level was also significantly increased. The p38MAPK/NF-κB pathway, as the downstream of FAK, were also found activated in CTSC-treated macrophages, suggesting that CTSC may promote macrophage towards M1 activation status through FAK-induced p38MAPK/NF-κB signaling pathway activation. Our study provides a new therapeutic target in the treatment of chronic inflammatory diseases., (Copyright © 2019. Published by Elsevier Inc.)

Cathepsin C (CatC) is a cysteine protease involved in a variety of immune and inflammatory pathways such as activation of cytotoxicity of various immune cells. Homozygous or compound heterozygous variants in the CatC coding gene CTSC cause different conditions that have in common severe periodontitis. Periodontitis may occur as part of Papillon-Lefèvre syndrome (PLS; OMIM#245000) or Haim-Munk syndrome (HMS; OMIM#245010), or may present as an isolated finding named aggressive periodontitis (AP1; OMIM#170650). AP1 generally affects young children and results in destruction of the periodontal support of the primary dentition. In the present study we report exome sequencing of a three generation consanguineous Turkish family with a recessive form of early-onset AP1. We identified a novel homozygous missense variant in exon 2 of CTSC (NM&#95;148170, c.G302C, p.Trp101Ser) predicted to disrupt protein structure and to be disease causing. This is the first described CTSC variant specific to the nonsyndromic AP1 form. Given the broad phenotypic spectrum associated with CTSC variants, reporting this novel variant gives new insights on genotype/phenotype correlations and might improve diagnosis of patients with early-onset AP1.

Objectives: To identify the molecular basis of Papillon-Lefèvre syndrome in two Chinese families., Methods: Peripheral blood and mouth swab samples were obtained, from which genomic DNA and RNA were isolated. Sanger sequencing was employed to identify the mutations. mRNA expression was tested by real-time quantitative PCR. Evolutionary conservation, pathogenicity prediction and impact of protein structures of the mutations were conducted with bioinformatics tools and homology modelling. HEK293 cells were transfected with plasmids expressing wild-type or mutated CTSC. CTSC protein expression level and enzyme activity were explored., Results: Mutation analysis revealed two novel compound heterozygous mutations, the c.190-191insA and c.1211-1212delA in patient 1 and the c.716A>G and c.757+1G>A in patient 2. In both patients, the levels of CTSC mRNA were significantly lower than in their relatives. Homology modelling analysis predicted that the mutations affect the structure and stability of the protein, and in vitro study showed that the CTSC proteins containing the mutations c.190-191insA and c.1211-1212delA, which result in truncated versions of protein, display impaired enzyme activity. The protein containing c.716A>G mutation showed quite similar enzyme activity compared to wild-type CTSC., Conclusion: Our data support the molecular mechanism of PLS and enlarge the scope of CTSC gene mutations related to PLS., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.)

Earlier studies showed that the oxidant menadione (MD) induces apoptosis in certain cells and also has anticancer effects. Most of these studies emphasized the role of the mitochondria in this process. However, the engagement of other organelles is less known. Particularly, the role of lysosomes and their proteolytic system, which participates in apoptotic cell death, is still unclear. The aim of this study was to investigate the role of lysosomal cathepsins on molecular signaling in MD-induced apoptosis in U937 cells. MD treatment induced translocation of cysteine cathepsins B, C, and S, and aspartic cathepsin D. Once in the cytosol, some cathepsins cleaved the proapoptotic molecule, Bid, in a process that was completely prevented by E64d, a general inhibitor of cysteine cathepsins, and partially prevented by the pancaspase inhibitor, z-VAD-fmk. Upon loss of the mitochondrial membrane potential, apoptosome activation led to caspase-9 processing, activation of caspase-3-like caspases, and poly (ADP-ribose) polymerase cleavage. Notably, the endogenous protein inhibitor, stefin B, was degraded by cathepsin D and caspases. This process was prevented by z-VAD-fmk, and partially by pepstatin A-penetratin. These findings suggest that the cleaved Bid protein acts as an amplifier of apoptotic signaling through mitochondria, thus enhancing the activity of cysteine cathepsins following stefin B degradation., (© 2019 Wiley Periodicals, Inc.)

Cathepsin C is a tetrameric lysosomal protease that acts as a dipeptidyl-peptidase due to the presence of the exclusion domain that is unique among papain-like cysteine proteases. Here we describe a recombinant form of cathepsin C lacking its exclusion domain (CatCΔEx) produced in a bacterial expression system (E. coli). CatCΔEx is a monomer with endoprotease activity and affinity for hydrophobic residues such as Phe, Leu or Pro, but not Val, in the P2 position. As opposed to cathepsin C, it does not require chloride ions for its activity. Despite lower turnover rates of hydrolysis of synthetic substrates, CatCΔEx has elastolytic and gelatinolytic activity comparable to other cysteine cathepsins., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The dipeptide glycyl-l-phenylalanine 2-naphthylamide (GPN) is widely used to perturb lysosomes because its cleavage by the lysosomal enzyme cathepsin C is proposed to rupture lysosomal membranes. We show that GPN evokes a sustained increase in lysosomal pH (pH ly ), and transient increases in cytosolic pH (pH cyt ) and Ca 2+ concentration ([Ca 2+ ] c ). None of these effects require cathepsin C, nor are they accompanied by rupture of lysosomes, but they are mimicked by structurally unrelated weak bases. GPN-evoked increases in [Ca 2+ ] c require Ca 2+ within the endoplasmic reticulum (ER), but they are not mediated by ER Ca 2+ channels amplifying Ca 2+ release from lysosomes. GPN increases [Ca 2+ ] c by increasing pH cyt , which then directly stimulates Ca 2+ release from the ER. We conclude that physiologically relevant increases in pH cyt stimulate Ca 2+ release from the ER in a manner that is independent of IP 3 and ryanodine receptors, and that GPN does not selectively target lysosomes., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Objective: This study aimed to investigate the gene mutational characteristics of cathepsin C (CTSC) gene in a Chinese patient with Papillon-Lefèvre syndrome (PLS) and further confirm the genetic basis for the phenotype of PLS., Methods: Peripheral blood samples were obtained from the PLS proband and his family members (his parents and younger brother) for genomic DNA extraction. The coding region and exon boundaries of the CTSC gene were amplified and sequenced by polymerase chain reaction and direct sequencing of DNA., Results: Compound heterozygous mutations of CTSC gene were identified in the patient. A heterozygous missense mutation occurred in the 800th base of exon 6, and the base T in the base pair was replaced by C (c.800T>C). The encoded amino acid leucine changed to proline (p. L267P). A heterozygous missense mutation occurred in the 1015th base of exon 7, and base C in the base pair was replaced by T (c.1015C>T). The encoded amino acid arginine changed to cysteine (p.R339C). Among the mutations, c.800T>C originated from the mother, c.1015C>T was identified from the father. No mutations were detected in the younger brother., Conclusions: Mutations of CTSC gene are responsible for the phenotype of PLS.

Background: Microglia-derived lysosomal cathepsins are important inflammatory mediators to trigger signaling pathways in inflammation-related cascades. Our previous study showed that the expression of cathepsin C (CatC) in the brain is induced predominantly in activated microglia in neuroinflammation. Moreover, CatC can induce chemokine production in brain inflammatory processes. In vitro studies further confirmed that CatC is secreted extracellularly from LPS-treated microglia. However, the mechanisms of CatC affecting neuroinflammatory responses are not known yet., Methods: CatC over-expression (CatCOE) and knock-down (CatCKD) mice were treated with intraperitoneal and intracerebroventricular LPS injection. Morris water maze (MWM) test was used to assess the ability of learning and memory. Cytokine expression in vivo was detected by in situ hybridization, quantitative PCR, and ELISA. In vitro, microglia M1 polarization was determined by quantitative PCR. Intracellular Ca 2+ concentration was determined by flow cytometry, and the expression of NR2B, PKC, p38, IkBα, and p65 was determined by western blotting., Results: The LPS-treated CatCOE mice exhibited significantly increased escape latency compared with similarly treated wild-type or CatCKD mice. The highest levels of TNF-α, IL-1β, and other M1 markers (IL-6, CD86, CD16, and CD32) were found in the brain or serum of LPS-treated CatCOE mice, and the lowest levels were detected in CatCKD mice. Similar results were found in LPS-treated microglia derived from CatC differentially expressing mice or in CatC-treated microglia from wild-type mice. Furthermore, the expression of NR2B mRNA, phosphorylation of NR2B, Ca 2+ concentration, phosphorylation of PKC, p38, IκBα, and p65 were all increased in CatC-treated microglia, while addition of E-64 and MK-801 reversed the phosphorylation of above molecules., Conclusion: The data suggest that CatC promotes microglia M1 polarization and aggravates neuroinflammation via activation of Ca 2+ -dependent PKC/p38MAPK/NF-κB pathway. CatC may be one of key molecular targets for alleviating and controlling neuroinflammation in neurological diseases.

Acute pancreatitis is characterized by premature intracellular protease activation and infiltration of inflammatory cells, mainly neutrophil granulocytes and macrophages, into the organ. The lysosomal proteases cathepsin B, D, and L have been identified as regulators of early zymogen activation and thus modulators of the severity of pancreatitis. Cathepsin C (CTSC, syn. dipeptidly-peptidase I) is a widely expressed, exo-cystein-protease involved in the proteolytic processing of various other lysosomal enzymes. We have studied its role in pancreatitis. We used CTSC-deleted mice and their WT littermates in two experimental models of pancreatitis. The mild model involved eight hourly caerulein injections and the severe model partial duct ligation. Isolated pancreatic acini and spleen-derived leukocytes were used for ex vivo experiments. CTSC is expressed in the pancreas and in inflammatory cells. CTSC deletion reduced the severity of pancreatitis (more prominently in the milder model) without directly affecting intra-acinar cell trypsin activation in vitro The absence of CTSC reduced infiltration of neutrophil granulocytes impaired their capacity for cleaving E-cadherin in adherens junctions between acinar cells and reduced the activity of neutrophil serine proteases polymorphonuclear (neutrophil) elastase, cathepsin G, and proteinase 3, but not neutrophil motility. Macrophage invasion was not dependent on the presence of CTSC. CTSC is a regulator and activator of various lysosomal enzymes such as cathepsin B, D, and L. Its loss mitigates the severity of pancreatitis not by reducing intra-acinar cell zymogen activation but by reducing infiltration of neutrophil granulocytes into the pancreas. In this context one of its key roles is that of an activator of neutrophil elastase., (© 2019 John et al.)

Background/aims: Diabetic cardiomyopathy (DCM) is characterized by structural and functional alterations that can lead to heart failure. Several mechanisms are known to be involved in the pathogenesis of DCM, however, the molecular mechanism that links inflammation to DCM is incompletely understood. To learn about this mechanism, we investigated the role of inflammatory serine proteases (ISPs) during the development of DCM., Methods: Eight weeks old mice with deletion of dipeptidyl peptidase I (DPPI), an enzyme involved in the maturation of major ISPs, and wild type (WT) mice controls were injected with streptozotocin (50 mg/kg for 5 days intraperitoneally) and studied after 4, 8, 16, and 20 week after induction of type 1 diabetes mellitus (T1DM). Induction of diabetes was followed by echocardiographic measurements, glycemic and hemoglobulin A1c profiling, immunoblot, qPCR, enzyme activity assays, and immunohistochemistry (IHC) analysis of DPPI, ISPs, and inflammatory markers. Fibrosis was determined from left ventricular heart by Serius Red staining and qPCR. Apoptosis was determined by TUNEL assay and immunoblot analysis., Results: In the diabetic WT mice, DPPI expression increased along with ISP activation, and DPPI accumulated abundantly in the left ventricle mainly from infiltrating neutrophils. In diabetic DPPI-knockout (DPPI-KO) mice, significantly decreased activation of ISPs, myocyte apoptosis, fibrosis, and cardiac function was improved compared to diabetic WT mice. In addition, DPPI-KO mice showed a decrease in overall inflammatory status mediated by diabetes induction which was manifested by decreased production of pro-inflammatory cytokines like TNF-α, IL-1β and IL-6., Conclusion: This study elucidates a novel role of ISPs in potentiating the immunological responses that lead to the pathogenesis of DCM in T1DM. To the best of our knowledge, this is the first study to report that DPPI expression and activation promotes the inflammation that enhances myocyte apoptosis and contributes to the adverse cardiac remodeling that subsequently leads to DCM., Competing Interests: The authors declare no conflict of interests., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

The lysosomal cysteine protease cathepsin C plays a pivotal role in regulation of inflammatory and immune responses. However, the function of fish cathepsin C in virus replication remains largely unknown. In this study, cathepsin C gene (Ec-CC) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-CC cDNA was composed of 2077 bp. It contained an open reading frame (ORF) of 1374 bp and encoded a 458-amino acid protein which shared 89% identity to cathepsin C from bicolor damselfish (Stegastes partitus). Amino acid alignment analysis showed that Ec-CC contained an N-terminal signal peptide, the propeptide region and the mature peptide. RT-PCR analysis showed that Ec-CC transcript was expressed in all the examined tissues which abundant in spleen and head kidney. After challenged with Singapore grouper iridovirus (SGIV) stimulation, the relative expression of EC-CC was significantly increased at 24 h post-infection. Subcellular localization analysis revealed that Ec-CC was distributed mainly in the cytoplasm. Further studies showed that overexpression of Ec-CC in vitro significantly delayed the cytopathic effect (CPE) progression evoked by SGIV and inhibited the viral genes transcription. Moreover, overexpression of Ec-CC significantly increased the expression of proinflammatory cytokines during SGIV infection. Taken together, our results demonstrated that Ec-CC might play a functional role in SGIV infection by regulating the inflammation response., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Rheumatoid arthritis (RA) is an autoimmune disease and characterized by the excessive cell proliferation, abnormal cell cycle of lymphocytes and synovial cells. The therapeutic effects of curcumin in active RA patients were reported, but limited by its insolubility and rapid systemic elimination. Dimethyl curcumin (DiMC) is a metabolically stable analogue of curcum with anti-inflammatory property. In this study, liposomes encapsulated dimethyl curcumin (Lipo-DiMC) was prepared to improve the bioavailability and metabolic-stability; collagen induced arthritis (CIA) rat model was employed to investigate the effects of Lipo-DiMC treatments during CIA progress. Physical assessments and routine-blood-test were performed. Fresh spleen lymphocytes were isolated from normal, CIA and Lipo-DiMC-treated CIA rats; flow-cytometry for cell-cycle analysis, western-blotting for intracellular signal pathway protein expressions, gelatin-zymography for matrix-metalloproteases 2/9 (MMP-2/9) and GF-AFC for dipeptidyl-peptidase I (DPPI) activity assay. Compared with untreated CIA rats, Lipo-DiMC treatments relieved paw-swellings, suppressed the increments of immunocytes numbers and inhibited DPPI and MMP-2/9 over-activity in blood. Lipo-DiMC adjusted CIA-induced cell cycle dysfunction at G0/G1-phase and S-phase of spleen lymphocytes for CIA rats. The intracellular expression-trends of P38, P21, Bcl-2, JNK-1 and DPPI of spleen lymphocytes were observed during CIA progress with and without Lipo-DiMC administrations. Lipo-DiMC exhibited its therapeutic functions by attenuating CIA development in rats, associated with down-regulating CIA-induced lymphocytes numbers, inhibiting over-expressed of DPPI and MMP-2/9, and adjusting cell cycles. These findings provide a new insight into the mechanism of Lipo-DiMC treatment in CIA rat model and suggest that Lipo-DiMC could be considered as a potential drug for RA treatment., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Cathepsin C (CatC) is a lysosomal enzyme involved in activation of serine proteases from immune and inflammatory cells. Several loss-of-function mutations in the CatC gene have been shown to be the genetic mark of Papillon-Lefèvre syndrome (PLS), a rare autosomal recessive disease characterized by severe early-onset periodontitis, palmoplantar hyperkeratosis, and increased susceptibility to infections. Deficiencies or dysfunction in other cathepsin family proteins, such as cathepsin B or D, have been associated with autophagic and lysosomal disorders., Objectives: Here we characterized the basis for autophagic dysfunction in patients with PLS by analyzing skin fibroblasts derived from patients with several mutations in the CatC gene and reduced enzymatic activity., Methods: Skin fibroblasts were isolated from patients with PLS assessed by using genetic analysis. Authophagic flux dysfunction was evaluated by examining accumulation of p62/SQSTM1 and a bafilomycin assay. Ultrastructural analysis further confirmed abnormal accumulation of autophagic vesicles in mutant cells. A recombinant CatC protein was produced by a baculovirus system in insect cell cultures., Results: Mutant fibroblasts from patients with PLS showed alterations in oxidative/antioxidative status, reduced oxygen consumption, and a marked autophagic dysfunction associated with autophagosome accumulation. These alterations were accompanied by lysosomal permeabilization, cathepsin B release, and NLR family pyrin domain containing 3 (NLRP3) inflammasome activation. Treatment of mutant fibroblasts with recombinant CatC improved cell growth and autophagic flux and partially restored lysosomal permeabilization., Conclusions: Our data provide a novel molecular mechanism underlying PLS. Impaired autophagy caused by insufficient lysosomal function might represent a new therapeutic target for PLS., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Major depression has been interpreted as an inflammatory disease characterized by cell-mediated immune activation, which is generally triggered by various stresses. Microglia has been thought to be the cellular link between inflammation and depression-like behavioural alterations. The expression of cathepsin C (Cat C), a lysosomal proteinase, is predominantly induced in microglia in neuroinflammation. However, little is known about the role of Cat C in pathophysiology of depression. In the present study, Cat C transgenic mice and wild type mice were subjected to an intraperitoneal injection of LPS (0.5 mg/kg) and 6-week unpredictable chronic mild stress (UCMS) exposure to establish acute and chronic stress-induced depression model. We examined and compared the behavioural and proinflammatory cytokine alterations in serum and depression-targeted brain areas of Cat C differentially expressed mice in stress, as well as indoleamine 2,3-dioxygenase (IDO) and 5-hydroxytryptamine (5HT) levels in brain. The results showed that Cat C overexpression (Cat C OE) promoted peripheral and central inflammatory response with significantly increased TNFα, IL-1β and IL-6 in serum, hippocampus and prefrontal cortex, and resultant upregulation of IDO and downregulation of 5HT expression in brain, and thereby aggravated depression-like behaviours accessed by open field test, forced swim test and tail suspension test. In contrast, Cat C knockdown (Cat C KD) partially prevented inflammation, which may help alleviate the symptoms of depression in mice. To the best of our knowledge, we are the first to demonstrate that Cat C aggravates neuroinflammation involved in disturbances of behaviour and neurochemistry in acute and chronic stress-induced murine model of depression.

In demyelinating diseases such as multiple sclerosis (MS), an imbalance between the demyelination and remyelination rates underlies the degenerative processes. Microglial activation is observed in demyelinating lesions; however, the molecular mechanism responsible for the homeostatic/environmental change remains elusive. We previously found that cystatin F (CysF), a cysteine protease inhibitor, is selectively expressed in microglia only in actively demyelinating/remyelinating lesions but ceases expression in chronic lesions, suggesting its role in remyelination. Here, we report the effects of manipulating the expression of CysF and cathepsin C (CatC), a key target of CysF, in a murine model of transgenic demyelinating disease, Plp 4e/- . During the active remyelinating phase, both CysF knockdown (CysFKD) and microglial-selective CatC overexpression (CatCOE) showed a worsening of the demyelination in Plp 4e/- transgenic mice. Conversely, during the chronic demyelinating phase, CatC knockdown (CatCKD) ameliorated the demyelination. Our results suggest that the balance between CatC and CysF expression controls the demyelination and remyelination process., (© 2017 Wiley Periodicals, Inc.)