OBJECTIVE: To explore the interaction between Tubulin beta 4B class IVb (TUBB4B) and Agtpbp1/cytosolic carboxypeptidase- like1 (CCP1) in mouse primary spermatocytes (GC-2 cells) and the role of TUBB4B in regulating the development of GC-2 cells. METHODS: Lentiviral vectors were used to infect GC-2 cells to construct TUBB4B knockdown and negative control (NC-KD) cells. The stable cell lines with TUBB4B overexpression (Tubb4b-OE) and the negative control (NC-OE) cells were screened using purinomycin. RT-qPCR and Western blotting were used to verify successful cell modeling and explore the relationship between TUBB4B and CCP1 expressions in GC-2 cells. The effects of TUBB4B silencing and overexpression on the proliferation and cell cycle of GC-2 cells were evaluated using CCK8 assay and flow cytometry. The signaling pathway proteins showing significant changes in response to TUBB4B silencing or overexpression were identified using Western blotting and immunofluorescence assay and then labeled for verification at the cellular level. RESULTS: Both TUBB4B silencing and overexpression in GC-2 cells caused consistent changes in the mRNA and protein expressions of CCP1 (P < 0.05). Similarly, TUBB4B expression also showed consistent changes at the mRNA and protein after CCP1 knockdown and restoration (P < 0.05). TUBB4B knockdown and overexpression had no significant effect on proliferation rate or cell cycle of GC-2 cells, but caused significant changes in the key proteins of the nuclear factor kappa-B (NF-κB) signaling pathway (p65 and p-p65) and the mitogen-activated protein kinase (MAPK) signaling pathway (ErK1/2 and p-Erk1/2) (P < 0.05); CCP1 knockdown induced significant changes in PolyE expression in GC-2 cells (P < 0.05). CONCLUSIONS TUBB4B and CCP1 interact via a mutual positive regulation mechanism in GC-2 cells. CCP-1 can deglutamize TUBB4B, and the latter is involved in the regulation of NF-κB and MAPK signaling pathways in primary spermatocytes.
Animals ; Male ; Mice ; GTP-Binding Proteins/metabolism* ; Mitogen-Activated Protein Kinases/metabolism* ; NF-kappa B/metabolism* ; RNA, Messenger ; Serine-Type D-Ala-D-Ala Carboxypeptidase/metabolism* ; Signal Transduction ; Spermatocytes ; Tubulin/genetics*

Molting is an important physiological phenomenon of many metamorphosis insects, during which the old and new epidermis are separated by enzymes present in the molting fluid. Various proteomic studies have discovered the presence of Bombyx mori carboxypeptidase A (Bm-CPA) in the molting fluid of silkworm, but its function remains unclear. In order to better understand the role of Bm-CPA in the molting process of silkworm, Bm-CPA was analyzed by bioinformatics analysis, real-time fluorescence quantitative PCR, antibody preparation, immunofluorescence staining, and expression in Pichia pastoris. The results showed that Bm-CPA had a conserved M14 zinc carboxypeptidase domain and glycosylation site. Its expression was regulated by ecdysone 20E, and large expression was observed in the epidermis of the upper cluster stage. Immunofluorescence staining showed that Bm-CPA was enriched in the epidermis during the molting stage, and the inhibitor of Bm-CPA led to the larval death due to the inability to molt. We also successfully obtained a large number of recombinant Bm-CPA proteins by Pichia pastoris expression in vitro. These results may facilitate further understanding the molting development process of silkworm.
Animals ; Molting/genetics* ; Bombyx/genetics* ; Carboxypeptidases A/metabolism* ; Proteomics ; Larva/metabolism* ; Fluorescent Antibody Technique ; Insect Proteins/metabolism*

This study aims to investigate the therapeutic effect of icariin(ICA) on thioacetamide(TAA)-induced femoral osteolysis in rats. RAW264.7 cells were treated with TAA and ICA. Cell counting kit-8(CCK-8) assay was used to detect cell proliferation, and tartrate-resistant acid phosphatase(TRAP) staining to examine the formation of osteoclasts. The expression of TRAP, cathepsin K, c-FOS, and NFATc1 in RAW264.7 cells was determined by Western blot and immunofluorescence method. Thirty-two SD rats were randomized into the control group, TAA group(intraperitoneal injection of TAA at 300 mg·kg~(-1)), ICA group(gavage of ICA at 600 mg·kg~(-1)) and TAA + ICA group(intraperitoneal injection of TAA at 300 mg·kg~(-1) and gavage of ICA at 600 mg·kg~(-1)). Administration was performed every other day for 6 weeks. Body weight and length of femur were recorded at execution. Pathological injury and osteoclast differentiation of femur were observed based on hematoxylin-eosin(HE) staining and TRAP staining, and the changes of bone metabolism-related indexes alkaline phosphatase(ALP), calcium(Ca), phosphorus(P), magnesium(Mg), and cross-linked N-telopeptide of type Ⅰ collagen(NTX-Ⅰ) in serum were detected. Three-point bending test and micro-CT were applied to evaluate the quality of femur, and Western blot to detect the levels of osteoclast-related proteins TRAP, cathepsin K, RANK, RANKL, p38, p-p38, ERK, p-ERK, JNK, p-JNK, c-Fos, and NFATc1. The results showed ICA could inhibit TAA-induced production of TRAP-positive cells, the expression of osteoclast-related proteins, and nuclear translocation of NFATc1. ICA alleviated the weight loss, reduction of femur length, and growth inhibition induced by TAA in SD rats. ICA ameliorated the decline of femur elastic modulus caused by TAA and significantly restored trabecular bone mineral density(BMD), trabecular pattern factor(Tb.Pf), trabecular number(Tb.N), trabecular thickness(Tb.Th), and structure model index(SMI), thus improving bone structure. Western blot results showed ICA suppressed femoral osteoclast differentiation induced by TAA through RANKL-p38/ERK-NFATc1 signaling pathway. ICA inhibits osteoclast differentiation and prevents TAA-induced osteolysis by down-regulating RANKL-p38/ERK-NFAT signaling pathway.
Rats ; Animals ; Osteoclasts ; Cathepsin K/pharmacology* ; Thioacetamide/pharmacology* ; Bone Resorption/pathology* ; Osteolysis/pathology* ; Cell Differentiation ; Rats, Sprague-Dawley ; NFATC Transcription Factors/metabolism*

Plant serine carboxypeptidase-like acyltransferases (SCPL-AT) have similar structural characteristics and high homology compared to the serine carboxypeptidase. They can transfer the acyl from acyl glucose esters to many natural products, participate in the acylation modification of plant secondary metabolites, enrich the structural diversity of natural products, and improve the physicochemical properties such as water solubility and stability of compounds. This review summarizes the structural characteristics, catalytic mechanism, functional characterization, and biocatalytic applications of SCPL-AT from plants. This will help to promote the functional characterization of these acyltransferase genes and the biosynthesis of useful plant secondary metabolites by synthetic biotechnology.
Acylation ; Acyltransferases/metabolism* ; Carboxypeptidases/metabolism* ; Plants/enzymology*

Objective To explore the effects of cathepsin B(CTSB)on the activation of nucleotide-binding domain and leucine-rich-repeat-containing family and pyrin domain-containing 3(NLRP3)inflammasome via transient receptor potential mucolipin-1(TRPML1)in cell oxidative stress model and specific gene silencing cell model. Methods BV2 cells cultured in vivo were treated separately or simultaneously with hydrogen peroxide(HO),calcium-sensitive receptor agonist gadolinium trichloride(GdCl),and CTSB inhibitor CA-074Me,and interleukin-1(IL-1)beta and caspase-1 protein were detected by enzyme-linked immunosorbent assay.The growth activity of BV2 cells in each group was measured by MTT.BV2 cells were treated with different concentrations of HO.Cystatin C mRNA and TRPML1 mRNA in BV2 cells were detected by real-time quantitative polymerase chain reaction and the proteins of TRPML1,CTSB,cathepsin D(CTSD),cathepsin L(CTSL)and cathepsin V(CTSV)were detected by Western blot.Specific small interfering RNA was designed for TRPML1 gene target sequence.TRPML1 gene silencing cell lines(named Tr-si-Bv2 cells)were established in BV2 cells and treated with or without HO.TRPML1,CTSB and transcription factor EB(TFEB)proteins in Tr-si-Bv2 cells or control cells were detected by Western blot. Results After treatment with HO,the expression of caspase-1 protein and NLRP3 mRNA in BV2 cells was increased,and IL-1beta protein in BV2 cells was significantly increased after treatment with GdCl(P=0.0036).After treatment with CA-074Me,the doses of NLRP3 mRNA(P=0.037),caspase-1(P=0.021),and IL-1β(P= 0.036)were significantly reduced.Cells in the HO group and HO+GdCl group grew more slowly.The expressions of CTSB mRNA and TRPML1 mRNA,or CTSB and TRPML1 proteins in BV2 cells in the treatment group with 200 μmol/L of HO concentration were similar.HO-induced CTSB protein expression was inhibited after silencing TRPML1 gene.The changes of other cathepsins were not affected for the different concentration of HO.In the BV2 cells treated with TRPML1 gene silencing,the expression of CTSB protein was significantly reduced and the difference was statistically significant(P=0.021)between the HO +siRNA treatment group and the HO treatment group.<b>Conclusionb> CTSB regulates the activation of NLRP3 inflammasome in the oxidative stress model of microglia cells,probably mediated by calcium channel protein TRPML1.
Animals ; Cathepsin B ; antagonists & inhibitors ; metabolism ; Cell Line ; Gene Silencing ; Hydrogen Peroxide ; Inflammasomes ; metabolism ; Interleukin-1beta ; Mice ; Microglia ; NLR Family, Pyrin Domain-Containing 3 Protein ; metabolism ; Oxidative Stress ; Pyrin Domain ; Transient Receptor Potential Channels ; metabolism

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.
Cathepsin C ; genetics ; DNA Mutational Analysis ; Exons ; Humans ; Male ; Mutation ; Papillon-Lefevre Disease ; genetics ; Pedigree ; Phenotype

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

Humanin (HN) is a mitochondrial peptide that exhibits cytoprotective actions against various stresses and diseases. HN has been shown to induce the phosphorylation of AMP-activated protein kinase (AMPK), which is a negative regulator of receptor activator of nuclear factor-κB ligand (RANKL). However, the role of HN in osteoclastogenesis or other skeletal disorders remains unknown. Here, we examined whether HN regulates osteoclastogenesis via AMPK activation using bone marrow-derived macrophage (BMM) cultures. Our results show that HN inhibited RANKL-induced osteoclast formation and reduced the expression of genes involved in osteoclastogenesis, including nuclear factor of activated T-cells cytoplasmic 1, osteoclast-associated receptor, cathepsin K, and tartrate-resistant acid phosphatase. Moreover, HN increased the levels of phosphorylated AMPK protein; compound C, an AMPK inhibitor, recovered HN-induced osteoclast differentiation. In addition, we found that HN significantly decreased the levels of RANKL-induced reactive oxygen species in BMMs. Therefore, these results indicate that HN plays an important role in osteoclastogenesis and may function as an inhibitor of bone disorders via AMPK activation.
Acid Phosphatase ; AMP-Activated Protein Kinases ; Cathepsin K ; Cytoplasm ; Macrophages ; Osteoclasts ; Phosphorylation ; Reactive Oxygen Species ; T-Lymphocytes

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

BACKGROUND: Tetrabromobisphenol A (TBBPA), one of the most widely used brominated flame-retardants, is a representative persistent organic pollutants group. Studies on TBBPA toxicity have been conducted using various target cells; however, few studies have investigated TBBPA toxicity in bone cells. Therefore, this study investigated the in vitro effects of TBBPA on osteoclasts, a cell type involved in bone metabolism. METHODS: RAW264.7 cells were cultured in medium containing 50 ng/mL receptor activator of nuclear factor kappa B ligand (RANKL) and varying concentrations of TBBPA. To evaluate the effects of TBBPA on the differentiation and function of osteoclasts, osteoclast-specific gene expression, tartrate-resistant acid phosphatase (TRAP) activity, bone resorbing activity, mitochondrial membrane potential (MMP) and mitochondrial superoxide were measured. RESULTS: The presence of 20 μM TBBPA significantly increased TRAP activity in RANKL-stimulated RAW264.7 cells, the bone resorbing activity of osteoclasts, and the gene expression of Akt2, nuclear factor of activated T-cells cytoplasmic 1, and chloride channel voltage-sensitive 7. However, TBBPA treatment caused no change in the expression of carbonic anhydrase II, cathepsin K, osteopetrosis-associated transmembrane protein 1, Src, extracellular signal-related kinase, GAB2, c-Fos, or matrix metalloproteinase 9. Furthermore, 20 μM TBBPA caused a significant decrease in MMP and a significant increase in mitochondrial superoxide production. CONCLUSION: This study suggests that TBBPA promotes osteoclast differentiation and activity. The mechanism of TBBPA-stimulated osteoclastogenesis might include increased expression of several genes involved in osteoclast differentiation and reactive oxygen species production.
Acid Phosphatase ; Carbonic Anhydrase II ; Cathepsin K ; Chloride Channels ; Cytoplasm ; Gene Expression ; In Vitro Techniques ; Matrix Metalloproteinase 9 ; Membrane Potential, Mitochondrial ; Metabolism ; Osteoclasts ; Phosphotransferases ; RANK Ligand ; Reactive Oxygen Species ; Receptor Activator of Nuclear Factor-kappa B ; Superoxides ; T-Lymphocytes