Cathepsin D (CatD, EC 3.4.23.5) is a member belonging to the subfamily of aspartic endopeptidases, which are classified into the MEROPS clan AA, family A1. Helminth parasites express a large set of different peptidases that play pivotal roles in parasite biology and pathophysiology. However, CatD is less well known than the other classes of peptidases in terms of biochemical properties and biological functions. In this study, we identified 2 novel CatDs (CsCatD1 and CsCatD2) of Clonorchis sinensis and partially characterized their properties. Both CsCatDs represent typical enzymes sharing amino acid residues and motifs that are tightly conserved in the CatD superfamily of proteins. Both CsCatDs showed similar patterns of expression in different developmental stages of C. sinensis, but CsCatD2 was also expressed in metacercariae. CsCatD2 was mainly expressed in the intestines and eggs of C. sinensis. Sera obtained from rats experimentally infected with C. sinensis reacted with recombinant CsCatD2 beginning 2 weeks after infection and the antibody titers were gradually increased by maturation of the parasite. Structural analysis of CsCatD2 revealed a bilobed enzyme structure consisting of 2 antiparallel β-sheet domains packed against each other forming a homodimeric structure. These results suggested a plausible biological role of CsCatD2 in the nutrition and reproduction of parasite and its potential utility as a serodiagnostic antigen in clonorchiasis.
Animals ; Aspartic Acid Endopeptidases ; Biology ; Cathepsin D ; Cathepsins ; Clonorchiasis ; Clonorchis sinensis ; Eggs ; Helminths ; Humans ; Intestines ; Metacercariae ; Ovum ; Parasites ; Peptide Hydrolases ; Rats ; Reproduction

OBJECTIVES: Root resorption is an unexpected complication after replantation procedures. Combining anti-osteoclastic medicaments with retrograde root filling materials may avert this resorptive activity. The purpose of this study was to assess effects of a cathepsin K inhibitor with calcium silicate-based cements on osteoclastic activity. METHODS: MC3T3-E1 cells were cultured for biocompatibility analyses. RAW 264.7 cells were cultured in the presence of the receptor activator of nuclear factor-kappa B and lipopolysaccharide, followed by treatment with Biodentine (BIOD) or ProRoot MTA with or without medicaments (Odanacatib [ODN], a cathepsin inhibitor and alendronate, a bisphosphonate). After drug treatment, the cell counting kit-8 assay and Alizarin red staining were performed to evaluate biocompatibility in MC3T3-E1 cells. Reverse-transcription polymerase chain reaction, tartrate-resistant acid phosphatase (TRAP) staining and enzyme-linked immunosorbent assays were performed in RAW 264.7 cells to determine the expression levels of inflammatory cytokines, interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2). Data were analyzed by one-way analysis of variance and Tukey's post hoc test (p < 0.05). RESULTS: Biocompatibility results showed that there were no significant differences among any of the groups. RAW 264.7 cells treated with BIOD and ODN showed the lowest levels of TNF-α and PGE2. Treatments with BIOD + ODN were more potent suppressors of inflammatory cytokine expression (p < 0.05). CONCLUSION: The cathepsin K inhibitor with calcium silicate-based cement inhibits osteoclastic activity. This may have clinical application in preventing inflammatory root resorption in replanted teeth.
Acid Phosphatase ; Alendronate ; Calcium ; Cathepsin K ; Cathepsins ; Cell Count ; Cytokines ; Dinoprostone ; Enzyme-Linked Immunosorbent Assay ; Interleukin-6 ; Interleukins ; Miners ; Necrosis ; Osteoblasts ; Osteoclasts ; Pemetrexed ; Polymerase Chain Reaction ; RAW 264.7 Cells ; Receptor Activator of Nuclear Factor-kappa B ; Replantation ; Root Resorption ; Tooth

Cathepsin S is a cysteine protease which is closely related to autoimmune diseases,psoriasis and other diseases. In this study,we used virtual screening method to screen compounds,which from the natural product library of traditional Chinese medicine,with potential inhibitory effect on cathepsin S. The work involved in study on inhibitory mechanism of representative compounds,then analysis of the distribution of these compounds in traditional Chinese medicine and the correlation with disease,so as to provide a new drug research and data-base for cathepsin S. The complex crystal structure of cathepsin S,2FQ9,was used to establish the pharmacophore model of cathepsin S inhibitor,and the best pharmacophore model was selected. As a result,fifty compounds were selected from TCMD database. After molecular docking,65 potential inhibitors were identified. Potential inhibitors can produce multiple intermolecular interactions with targets,resulting in inhibition. There are 58 kinds of traditional Chinese medicines which include 65 natural inhibitors. Data collection and analysis of the nature,flavor xing,channel entry and modern pharmacological effects of these traditional Chinese medicines showed that most of them were related to the biological activity of cathepsin S,which supported the validity of the screening results. Cathepsin S has a certain correlation with autoimmune diseases and can be used as a target for further study of traditional Chinese medicine.
Cathepsins ; Drugs, Chinese Herbal ; Medicine, Chinese Traditional ; Molecular Docking Simulation

Acanthamoeba spp. are free-living protozoa that are opportunistic pathogens for humans. Cysteine proteases of Acanthamoeba have been partially characterized, but their biochemical and functional properties are not clearly understood yet. In this study, we isolated a gene encoding cysteine protease of A. castellanii (AcCP) and its biochemical and functional properties were analyzed. Sequence analysis of AcCP suggests that this enzyme is a typical cathepsin L family cysteine protease, which shares similar structural characteristics with other cathepsin L-like enzymes. The recombinant AcCP showed enzymatic activity in acidic conditions with an optimum at pH 4.0. The recombinant enzyme effectively hydrolyzed human proteins including hemoglobin, albumin, immunoglobuins A and G, and fibronectin at acidic pH. AcCP mainly localized in lysosomal compartment and its expression was observed in both trophozoites and cysts. AcCP was also identified in cultured medium of A. castellanii. Considering to lysosomal localization, secretion or release by trophozoites and continuous expression in trophozoites and cysts, the enzyme could be a multifunctional enzyme that plays important biological functions for nutrition, development and pathogenicity of A. castellanii. These results also imply that AcCP can be a promising target for development of chemotherapeutic drug for Acanthamoeba infections.
Acanthamoeba castellanii ; Acanthamoeba ; Cathepsin L ; Cathepsins ; Cysteine Proteases ; Cysteine ; Fibronectins ; Genes, vif ; Humans ; Hydrogen-Ion Concentration ; Lysosomes ; Sequence Analysis ; Trophozoites ; Virulence

Toxoplasma gondii cathepsin C proteases (TgCPC1, 2, and 3) are important for the growth and survival of T. gondii. In the present study, B-cell and T-cell epitopes of TgCPC1 were predicted using DNAstar and the Immune Epitope Database. A TgCPC1 DNA vaccine was constructed, and its ability to induce protective immune responses against toxoplasmosis in BALB/c mice was evaluated in the presence or absence of the adjuvant α-GalCer. As results, TgCPC1 DNA vaccine with or without adjuvant α-GalCer showed higher levels of IgG and IgG2a in the serum, as well as IL-2 and IFN-γ in the spleen compared to controls (PBS, pEGFP-C1, and α-Galcer). Upon challenge infection with tachyzoites of T. gondii (RH), pCPC1/α-Galcer immunized mice showed the longest survival among all the groups. Mice vaccinated with DNA vaccine without adjuvant (pCPC1) showed better protective immunity compared to other controls (PBS, pEGFP-C1, and α-Galcer). These results indicate that a DNA vaccine encoding TgCPC1 is a potential vaccine candidate against toxoplasmosis.
Animals ; B-Lymphocytes ; Cathepsin C* ; Cathepsins* ; DNA* ; Epitopes, T-Lymphocyte ; Immunoglobulin G ; Interleukin-2 ; Mice* ; Peptide Hydrolases ; Spleen ; Toxoplasma* ; Toxoplasmosis* ; Vaccines, DNA*

To determine the effect of the tumor necrosis factor-α (TNF-α) in odontoclast formation, we administrated a TNF-α inhibitor in rats with diabetes rats with periodontitis. The rats included in the study were divided into three groups: control rats without diabetes or periodontitis (the C group), rats with periodontitis and diabetes (the PD group), and rats with periodontitis and diabetes treated by infliximab, the TNF inhibitor (the PD+infliximab group). The PD and PD+ infliximab groups received intravenous administrations of streptozotocin (STZ, 50 mg/kg) to induce diabetes. After 7 days of STZ injections, the mandibular first molars were ligatured to induce periodontitis. The PD+infliximab group was intrapenitoneally administrated by infliximab (5 mg/kg). On days 3 and 20 after the ligature administration, odontoclast formation along root surfaces was evaluated by tartrate resistant acid phosphatase (TRAP) staining and cathepsin K immunohistochemistry. On day 3, the number of TRAP- and cathepsin K-positive cells increased more so in the PD group than in the C group. The PD+infliximab group showed a lower number of positive cells than the PD group. There was no difference in all the groups on day 20. On day 3, the cathepsin-K positive multinucleated and mononucleated cells were higher in the PD group than in the C group. The number of cathepsin-K positive multinucleated cells was lower in the PD+infliximab group than in the PD group. The PD group showed more cathepsin K-positive cells in the furcation and distal surfaces than the c group. The Cathepsin K-positive cells of the PD+infliximab group were lower than that of the PD group in furcation. These results suggest that TNF-α stimulates odontoclast formation in diabetes with periodontitis.
Acid Phosphatase ; Administration, Intravenous ; Animals ; Cathepsin K ; Cathepsins ; Immunohistochemistry ; Infliximab ; Ligation ; Molar ; Necrosis ; Osteoclasts* ; Periodontitis* ; Rats* ; Streptozocin

OBJECTIVE: Root mobility due to reciprocating movement of the tooth (jiggling) may exacerbate orthodontic root resorption (ORR). "Jiggling" describes mesiodistal or buccolingual movement of the roots of the teeth during orthodontic treatment. In the present study, buccolingual movement is described as "jiggling." We aimed to investigate the relationship between ORR and jiggling and to test for positive cell expression in odontoclasts in resorbed roots during experimental tooth movement (jiggling) in vivo. METHODS: Male Wistar rats were divided into control, heavy force (HF), optimal force (OF), and jiggling force (JF) groups. The expression levels of cathepsin K, matrix metalloproteinase (MMP)-9 protein, interleukin (IL)-6, cytokine-induced neutrophil chemoattractant 1 (CINC-1; an IL-8-related protein in rodents), receptor activator of nuclear factor κB ligand (RANKL), and osteoprotegerin protein in the dental root were determined using immunohistochemistry. RESULTS: On day 21, a greater number of root resorption lacunae, which contained multinucleated odontoclasts, were observed in the palatal roots of rats in the JF group than in rats from other groups. Furthermore, there was a significant increase in the numbers of cathepsin K-positive and MMP-9-positive odontoclasts in the JF group on day 21. Immunoreactivities for IL-6, CINC-1, and RANKL were stronger in resorbed roots exposed to jiggling than in the other groups on day 21. Negative reactivity was observed in the controls. CONCLUSIONS: These results suggest that jiggling may induce ORR via inflammatory cytokine production during orthodontic tooth movement, and that jiggling may be a risk factor for ORR.
Animals ; Cathepsin K ; Cathepsins ; Humans ; Immunohistochemistry ; Interleukin-6 ; Interleukins ; Male ; Models, Animal* ; Neutrophils ; Osteoclasts ; Osteoprotegerin ; Rats* ; Rats, Wistar ; Risk Factors ; Root Resorption* ; Tooth Movement* ; Tooth*

To investigate the effect of the jianpi-jiedu formula (JPJD) on the expression of angiogenesis-relevant genes in colon cancer.
 Methods: Crude extract was obtained from JPJD by water extract method. The effect of JPJD crude extract on colon cancer cell proliferation capacity was determined by MTT assays. The IC50 value was calculated by GraphPad Prism5 software. Affymetrix gene expression profiling chip was used to detect significant differences in expressions of genes after JPJD intervention, and pathway enrichment analysis was performed to analyze the differentially expressed genes. Ingenuity Pathway Analysis software was applied to analyze differentially expressed genes relevant to tumor angiogenesis based on mammalian target of rapamycin (mTOR) signaling pathway and then the network diagram was built. Western blot was used to verify the protein levels of key genes related to tumor angiogenesis.
 Results: JPJD crud extract inhibited the proliferation capacity in colon cancer cells. The IC50 values in 24, 48, and 72 hours after treatment were 13.060, 9.646 and 8.448 mg/mL, respectively. The results of chip showed that 218 genes significantly upgraded, and 252 genes significantly downgraded after JPJD treatment. Most of the genes were related to the function of biosynthesis, metabolism, cell apoptosis, antigen extraction, angiogenesis and so on. There were 12 differentially expressed angiogenesis genes. IPA software analysis showed that the JPJD downregulated expression of sphingomyelin phosphodiesterase 3 (SMPD3), VEGF, vascular endothelial growth factor A (VEGFA), integrin subunit alpha 1 (ITGA1), cathepsin B (CTSB), and cathepsin S (CTSS) genes, while upregulated expressions of GAB2 and plasminogen activator, urokinase receptor (PLAUR) genes in the colorectal cancer cell. Western blot results demonstrated that JPJD obviously downregulated expressions of phospho-mTOR (P-mTOR), signal transducer and activator of transcription 3 (STAT3), hypoxia inducible factor-1α (HIF-1α), and VEGF proteins, while obviously upregulated the level of phospho-P53 (P-P53) protein.
 Conclusion: JPJD may inhibit colorectal tumor angiogenesis through regulation of the mTOR-HIF-1α-VEGF signal pathway.
Animals ; Blotting, Western ; Cathepsin B ; drug effects ; metabolism ; Cathepsins ; drug effects ; metabolism ; Cell Line, Tumor ; drug effects ; Colorectal Neoplasms ; blood supply ; genetics ; Down-Regulation ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Profiling ; methods ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; drug effects ; metabolism ; Integrin alpha Chains ; drug effects ; metabolism ; Neovascularization, Pathologic ; genetics ; Receptors, Urokinase Plasminogen Activator ; drug effects ; metabolism ; STAT3 Transcription Factor ; drug effects ; metabolism ; Signal Transduction ; Sphingomyelin Phosphodiesterase ; drug effects ; metabolism ; TOR Serine-Threonine Kinases ; drug effects ; metabolism ; Tumor Suppressor Protein p53 ; drug effects ; metabolism ; Up-Regulation ; Vascular Endothelial Growth Factor A ; drug effects ; metabolism

PURPOSE: Despite the high success rates of endosseous dental implants, their placement is restricted according to the height and volume of bone available. The use of short or mini dental implants could be one way to overcome this limitation. Thus, this study aimed to compare standard, short, and mini dental implants with regard to associated clinical parameters and peri-implant crevicular fluid (PICF) levels of cathepsin -K (CTSK), RANK ligand (RANKL), and osteoprotegerin (OPG), after prosthodontic loading. METHODS: A total of 78 non-submerged implants (Euroteknika, Aesthetica+2, Sallanches, France) were installed in 30 subjects (13 male, 17 female; range, 26-62 years) who visited the clinic of the Periodontology Department, Faculty of Dentistry, Selcuk University. Sampling and measurements were performed on the loading date (baseline) and 2, 14, and 90 days after loading. Assessment of the peri-implant status for the implant sites was performed using the pocket probing depth (PPD), modified plaque index, modified gingival index, modified sulcular bleeding index, and radiographic signs of bone loss. PICF samples collected from each implant were evaluated for CTSK, RANKL, and OPG levels using the ELISA method. Keratinized tissue and marginal bone loss (MBL) were also noted. RESULTS: Clinical parameters statistically significantly increased in each group but did not show statistical differences between groups without PPD. Although implant groups showed a higher MBL in the upper jaw, only the standard dental group demonstrated a statistically significant difference. At 90 days, the OPG: sRANKL ratio and total amounts of CTSK for each group did not differ from baseline. CONCLUSIONS: Within the limitations of this study, both short and mini dental implants were achieving the same outcomes as the standard dental implants in the early period after loading.
Alveolar Bone Loss ; Cathepsin K ; Cathepsins ; Dental Implants* ; Dentistry ; Enzyme-Linked Immunosorbent Assay ; Female ; Hemorrhage ; Humans ; Jaw ; Male ; Osteoprotegerin ; Periodontal Index ; Prosthodontics* ; RANK Ligand

Silkworm is a holometabolous insect of Lepidoptera. During metamorphosis, significant morphological changes happen including the dissociation of old tissues and remodeling of new tissues. It has been reported that cathepsins are involved in these processes. Cathepsin is a kind of intracellular proteinase that exists in many species. It includes some subfamilies like cathepsin B, H and L. The studies on cathepsin are useful for clarifying the details of silkworm metamorphosis process. In total, 13 cathepsins were identified by screening the silkworm genome database. The basic information and the expression patterns about these genes were analyzed. Interestingly, an ovary-specific cathepsin L gene (Gene ID: BGIBMGAOO4622) was investigated by the data of silkworm microarray and real-time quantitative PCR (qPCR). The full-length cDNA is 1,209 bp, encoding a protein with 402 amino acids. Sequences alignment revealed that it has a high sequence similarity with cathepsin L of other species, and it is highly conserved in the active-site of the enzyme. The phylogenetic analysis showed that ovary-specific cathepsin L is clustered with other lepidopterous insects. Furthermore, this gene was cloned and prokaryotic expressed. Recombinant protein was present in inclusion body. Importantly, the qPCR result showed that the expression level of this gene is increasing during the early stage of pupal development and reaches the highest value at the 3rd day of pupal stage, which suggests that this gene may be involved in the process of development of the ovary and oocyte.
Animals ; Bombyx ; genetics ; Cathepsins ; genetics ; Insect Proteins ; genetics ; Phylogeny