Cathepsin L Research Articles

Moderate protection is induced by a chimeric protein composed of leucine aminopeptidase and cathepsin L1 against Fasciola hepatica challenge in sheep

Leucine aminopeptidase (FhLAP) and cathepsin L1 (FhCL1) of Fasciola hepatica play a critical role in parasite feeding, migration through host tissue, and immune evasion. These antigens have been tested for immune protection as single components with variable degrees of success. The chimeric-protein approach could improve protection levels against fasciolosis. Previously, we reported the design and construction of a chimeric protein composed of antigenic sequences of FhLAP and FhCL1 of F. hepatica. The goal of the present study was to express and evaluate the immune-protective capacity of this chimeric protein (rFhLAP-CL1) in sheep. Animals were randomly allocated into five groups with five animals in each group. Groups 1, 2 and 3 were immunized twice with 100 μg, 200 μg and 400 μg of rFhLAP-CL1 emulsified with Quil A adjuvant, whereas groups 4 and 5 were the adjuvant control and infection control groups, respectively. The animals were then challenged with 200 metacercariae two weeks after the rFhLAP-CL1 booster. The fluke burden was reduced by 25.5%, 30.7% (p < 0.05) and 46.5% (p < 0.01) in sheep immunized with 100 μg, 200 μg and 400 μg of chimeric protein, respectively, in comparison to the infection control group. There was a reduction of 22.7% (p < 0.05) and 24.4% (p < 0.01) in fecal egg count in groups 2 and 3, respectively, compared to the infection control group. Sheep immunized with chimeric protein produced F. hepatica excretion-secretion product-specific total IgG antibody, which were increased after challenge. Moreover, the levels of rFhLAP-CL1-specific IgG1 and IgG2 isotypes in immunized sheep increased rapidly two weeks after the first immunization and were significantly more elevated than those of the control groups, indicating a mixed Th1/Th2 response. This is a preliminary evaluation of the chimeric protein rFhLAP-CL1 as a possible immunogen against F. hepatica infection in sheep.

Cathepsin L promotes secretory IgA response by participating in antigen presentation pathways during Mycoplasma Hyopneumoniae infection.

Cathepsin L (CTSL) has been proved to help contain leishmaniasis and mycoplasma infection in mice by supporting cellular immune responses, but the regulatory functions of CTSL on mucosal immune responses haven’t been tested and remain undefined. Here, we investigated the effects of CTSL on SIgA responses and invariant chain (Ii) degradations in the co-cultured swine dendritic cells (DCs) and B cells system in vitro. When the cells system were transfected with vector CTSL-GFP or incubated with recombinant CTSL (rCTSL) before they were infected with Mycoplasma hyopneumoniae (M.hp), SIgA significantly increased and Ii chain was degraded into smaller intermediates, while SIgA decreased when CTSL was knockdown or inhibited with E64. To confirm the SIgA responses promoted by CTSL contribute to the resistance to mycoplasma pneumonia, pigs injected with rCTSL before they were challenged with M.hp, showed milder clinical symptoms and histopathological damage of lungs, less mycoplasma burden together with higher secretion of SIgA, percentages of CD4+ T cells and level of MHC II molecules comparing with the group without rCTSL. Collectively, these results suggested that rCTSL could provide effective protection for piglets against mycoplasma pneumonia by enhancing M.hp-specific mucosal immune responses through its role in antigen presentation by processing the invariant chain.

Fetal-maternal interactions during the establishment of pregnancy in ruminants

This review integrates established information with new insights into molecular and physiological mechanisms responsible for events leading to pregnancy recognition, endometrial receptivity, and implantation with emphasis on sheep. After formation of the corpus luteum, progesterone acts on the endometrium and stimulates blastocyst growth and elongation to form a filamentous conceptus (embryo/fetus and associated extraembryonic membranes). Recurrent early pregnancy loss in the uterine gland knockout ewe model indicates that endometrial epithelial secretions are essential for peri-implantation blastocyst survival and growth. The elongating sheep conceptus secretes interferon tau (IFNT) that acts on the endometrium to inhibit development of the luteolytic mechanism by inhibiting transcription of the estrogen receptor alpha (ESR1) gene in the luminal (LE) and superficial ductal glandular (sGE) epithelia, which prevents estrogen-induction of oxytocin receptors (OXTR) and production of luteolytic prostaglandin F2-alpha pulses. Progesterone downregulates its receptors (PGR) in LE and then GE, correlating with a reduction of anti-adhesive MUC1 (mucin glycoprotein one) and induction of secreted LGALS15 (galectin 15) and SPP1 (secreted phosphoprotein one), that are proposed to regulate trophectoderm growth and adhesion. IFNT acts on the LE to induce WNT7A (wingless-type MMTV integration site family member 7A) and to stimulate LGALS15, CTSL (cathepsin L), and CST3 (cystatin C), which may regulate conceptus development and implantation. During the peri-implantation period, trophoblast giant binucleate cells (BNC) begin to differentiate from mononuclear trophectoderm cells, migrate and then fuse with the uterine LE as well as each other to form multinucleated syncytial plaques. Trophoblast giant BNC secrete chorionic somatomammotropin (CSH1 or placental lactogen) that acts on the endometrial glands to stimulate their morphogenesis and differentiated function. The interactive, coordinated and stage-specific effects of ovarian and placental hormones regulate endometrial events necessary for fetal-maternal interactions and successful establishment of pregnancy.

Cathepsin L3 From Fasciola hepatica Induces NLRP3 Inflammasome Alternative Activation in Murine Dendritic Cells.

The production of IL-1-family cytokines such as IL-1β and IL-18 is finely regulated by inflammasome activation after the recognition of pathogens associated molecular pattern (PAMPs) and danger associated molecular patterns (DAMPs). However, little is known about the helminth-derived molecules capable of activating the inflammasome. In the case of the helminth trematode Fasciola hepatica, the secretion of different cathepsin L cysteine peptidases (FhCL) is crucial for the parasite survival. Among these enzymes, cathepsin L3 (FhCL3) is expressed mainly in the juvenile or invasive stage. The ability of FhCL3 to digest collagen has demonstrated to be critical for intestinal tissue invasion during juvenile larvae migration. However, there is no information about the interaction of FhCL3 with the immune system. It has been shown here that FhCL3 induces a non-canonical inflammasome activation in dendritic cells (DCs), leading to IL-1β and IL-18 production without a previous microbial priming. Interestingly, this activation was depending on the cysteine protease activity of FhCL3 and the NLRP3 receptor, but independent of caspase activation. We also show that FhCL3 is internalized by DCs, promoting pro-IL-1β cleavage to its mature and biologically active form IL-1β, which is released to the extracellular environment. The FhCL3-induced NLRP3 inflammasome activation conditions DCs to promote a singular adaptive immune response, characterized by increased production of IFN-γ and IL-13. These data reveal an unexpected ability of FhCL3, a helminth-derived molecule, to activate the NLRP3 inflammasome, which is independent of the classical mechanism involving caspase activation.

Precision Proteomics as Protein Biomarker Discovery in Detecting Chronic Lung Allograft Dysfunction

Purpose Chronic lung allograft dysfunction (CLAD) remains the major barrier to long-term success after lung transplantation. A biomarker in blood that can diagnose CLAD would be of great clinical value. In the current study we have conducted broad proteomics analyzes to detect biomarkers for CLAD Methods Plasma from a cohort of 46 lung transplant recipients (CLAD grade 0: 27 samples, CLAD grade 1 - 3: 19 samples were collected. A panel of 639 proteins were analyzed using a high-component, multiplex immunoassay that enables analysis of protein biomarkers. This high multiplexing is achieved with Proximity Extension Assay (PEA) technology. Each biomarker is addressed by a matched pair of antibodies, coupled to unique, partially complementary oligonucleotides, and measured by quantitative real-time PCR. Results Regression analysis comparing 3 groups CLAD grade 0, CLAD grade 1, and CLAD grade 2-3 showed significant differences in plasma proteins expression levels of 12 different proteins: Low affinity immunoglobulin epsilon Fc receptor (FCER2). Corticoliberin (CRH). Interleukin-20 receptor subunit alpha (IL-20RA). TNF-beta (TNFB). Immunoglobulin superfamily member 3 (IGSF3). Cathepsin L1 (CTSL1). Anterior gradient protein 2 homolog (AGR2). Matrix metalloproteinase-9 (MMP-9). Thymic stromal lymphopoietin (TSLP). Importin subunit alpha-5 (KPNA1). Signaling threshold-regulating transmembrane adapter 1 (SIT1). Ras GTPase-activating protein 1 (RASA1). Conclusion Interestingly a significant decrease was observed in TNFB, KPNA1, IGSF3, CRH, FCER2 levels in recipients with CLAD grade 1, but also in recipients with CLAD grade 2 and 3. IL-20RA was significantly decreased in recipents with CLAD grade 1, but not in recipients with CLAD grade 2 and 3. Proteomic patterns may be a viable way in the search of early biomarkers for BOS

Construction of a novel phage display antibody library against Fasciola hepatica, and generation of a single-chain variable fragment specific for F. hepatica cathepsin L1

Phage display technology to produce recombinant monoclonal antibodies or antibody fragments permits the identification of sought after antibodies in short time frames at low cost along with direct and rapid selection for antibody characteristics. Monoclonal antibodies can facilitate the identification and characterisation of parasite molecules that function at the host-parasite interface to help understand at the molecular level the biology of the parasite and disease progression, which often leads to new drug targets, diagnostic antigens or vaccine candidates.The trematode Fasciola hepatica is an important veterinary and human parasite. In this work, we infected rats with F. hepatica and amplified the generated antibody repertoire to produce a single-chain variable fragment (scFv) phage display library. The library was used to identify a scFv that recognises cathepsin L1, a major component of the adult parasites excretory/secretory material and an important vaccine candidate.This is the first report of the construction of a phage display antibody library from a F. hepatica infected host, and also the first instance such a library has been used to identify an affinity-matured monoclonal antibody fragment that binds to a F. hepatica antigen. The scFv library and methods detailed should facilitate future research characterising F. hepatica antigens.

Cathepsin L activated by mutant p53 and Egr-1 promotes ionizing radiation-induced EMT in human NSCLC

BackgroundIonizing radiation (IR) is one of the major clinical therapies of cancer, although it increases the epithelial-mesenchymal transition (EMT) of non-small cell lung cancer (NSCLC), unexpectedly. The cellular and molecular mechanisms underlying this role are not completely understood.MethodsWe used NSCLC cell lines as well as tumor specimens from 78 patients with NSCLC to evaluate p53, Cathepsin L (CTSL) and EMT phenotypic changes. Xenograft models was also utilized to examine the roles of mutant p53 (mut-p53) and CTSL in regulating IR-induced EMT of NSCLC.ResultsExpression of CTSL was markedly increased in human NSCLC tissues with mutant p53 (mut-p53), and p53 mutation positively correlated with metastasis of NSCLC patients. In human non-small cell lung cancer cell line, H1299 cells transfected with various p53 lentivirus vectors, mut-p53 could promote the invasion and motility of cells under IR, mainly through the EMT. This EMT process was induced by elevating intranuclear CTSL which was regulated by mut-p53 depending on Early growth response protein-1 (Egr-1) activation. In the subcutaneous tumor xenograft model, IR promoted the EMT of the cancer cells in the presence of mut-p53, owing to increase expression and nuclear transition of its downstream protein CTSL.ConclusionTaken together, these data reveal the role of the mut-p53/Egr-1/CTSL axis in regulating the signaling pathway responsible for IR-induced EMT.

Identification of reference genes for real-time PCR cytokine gene expression studies in sheep experimentally infected with Fasciola hepatica

The aim of this study was to validate reference genes for gene normalisation using qRT-PCR in hepatic lymph nodes (HLN) and livers from sheep infected with Fasciola hepatica during early and late stages of infection. To this end, a comprehensive statistical approach (RefFinder) encompassing four different methods of analysis (geNorm, BestKeeper, ΔCt method and NormFinder) was used to validate ten candidate reference genes. Stability analysis of gene expression followed by pairwise variation (Vn/Vn + 1) analysis revealed that PGK1, HSP90AA1 and GYPC were the most stable reference genes and suitable for qRT-PCR normalisation in both HLN and liver tissues. These three genes were validated against FoxP3, IL-10, TGF-β, TNF-α and IL-1β genes in the HLN tissue of sheep vaccinated with Cathepsin L1 from F. hepatica and unvaccinated infected and uninfected controls during early stages of infection. In the liver, the three reference genes were validated against TNF-α and IL-1β during chronic stages of infection with F. hepatica and in uninfected controls. Our study is the first to evaluate and validate sheep reference genes in order to provide tools for monitoring cytokines in Fasciola hepatica infected sheep target organs. Our results present an approach to elucidate the role of different cytokines in F. hepatica vaccinated and infected sheep.

CSN6 Promotes the Migration and Invasion of Cervical Cancer Cells by Inhibiting Autophagic Degradation of Cathepsin L.

CSN6 is one subunit of the highly conserved constitutive photomorphogenesis 9 (COP9) signalosome (CSN), which is overexpressed in many types of cancers, and has received great attention as a regulator of the degradation of cancer-related proteins, suggesting its importance in oncogenic activity. CSN6 has been shown to be overexpressed in cervical cancer (CC) and associated with CC development. CC remains to be one of the most aggressive cancers affecting women. Cathepsin L (CTSL), significantly associated with the autophagy, plays a critical role in degradation of extracellular matrix for metastasis. However, the detailed biological functions of CSN6 on CTSL in CC metastasis have not been well clarified. Our data has shown that CSN6 and CTSL are positively correlated. The overexpression of CSN6 and CTSL might be a strong indicator for CC enhanced aggressiveness. CSN6 could suppress the degradation of CTSL, then facilitated the migration and invasion of CC cells. Interestingly, our results indicated that autophagy is essential for decreasing CTSL, while CSN6 could inhibit the autophagy ability of CC cells. In addition, blocking of the mammalian target of rapamycin (mTOR) pathway reversed CSN6-mediated autophagy inhibition. We further demonstrated that CSN6 positively regulated CTSL expression through an autophagy-lysosomal system. Taken together, we concluded that CSN6 might promote the migration and invasion of cervical cancer cells by inhibiting autophagic degradation of CTSL and serve as a potential gene therapy target for the treatment of CC metastasis.

The Biomarkers NT-proBNP and CA-125 are Elevated in Patients with Idiopathic Atrial Fibrillation.

Blood biomarkers related to AF could be useful to detect silent AF and to develop stratified strategies for AF prevention. Previous studies identified markers that predict incident AF. However, it is difficult to differentiate whether biomarkers relate to underlying cardiovascular diseases, are generated by the atria in response to an AF episode, or both. We therefore measured a panel of blood biomarkers in patients without overt CVD with and without AF to investigate the association between biomarkers and atrial fibrillation (AF) in patients without overt cardiovascular disease (CVD). Blood samples - drawn remote from an AF episode - of 60 patients with AF but without overt forms of CVD (idiopathic AF; iAF) were compared to 120 matched patients with sinus rhythm only. A novel antibody-based method for quantification of blood biomarkers (OlinkProseek Multiplex Cardiovascular) was used to compare 92 biomarkers between the two groups. N-terminal pro-B-type natriuretic peptide (NT-proBNP), Cathepsin L1, Endothelial cell-specific molecule 1, Cancer Antigen-125 (CA-125), Heat shock 27kDa protein, Galanin peptides, Proteinase-activated receptor 1, Stem cell factor, and CD40-ligand were all higher in iAF patients than in SR controls. Both NT-proBNP (OR1.55(1.07-2.25);p=0.022) and CA-125 (OR1.68(1.07-2.64);p=0.026) were independently associated with iAF. This exploratory study, investigating over 90 cardiovascular blood biomarkers in patients without known CVD, identified one established biomarker for paroxysmal AF, NT-proBNP, and a novel marker, CA-125. CA-125 - previously unrelated to paroxysmal AF in an otherwise healthy population - may thus be a potential indicator of remote paroxysms of AF.

Asparaginyl Endopeptidase (Legumain) Supports Human Th1 Induction via Cathepsin L-Mediated Intracellular C3 Activation.

Autocrine activation of the complement receptors C3aR and CD46 by complement activation components C3a and C3b produced through C3 cleavage by the protease cathepsin L (CTSL) during T cell stimulation is a requirement for IFN-γ production and Th1 induction in human CD4+ T cells. Thus, lack of autocrine CD46 activation, such as in CD46-deficient patients, is associated with defective Th1 responses and recurrent infections. We have identified LGMN [the gene coding for legumain, also known as asparaginyl endopeptidase (AEP)] as one of the key genes induced by CD46 co-stimulation during human CD4+ T cell activation. AEP processes and activates a range of proteins, among those α1-thymosin and CTSL, which both drive intrinsically Th1 activity—but has so far not been described to be functionally active in human T cells. Here we found that pharmacological inhibition of AEP during activation of human CD4+ T cells reduced CTSL activation and the CTSL-mediated generation of intracellular C3a. This translated into a specific reduction of IFN-γ production without affecting cell proliferation or survival. In line with these findings, CD4+ T cells isolated from Lgmn−/− mice also displayed a specific defect in IFN-γ secretion and Th1 induction. Furthermore, we did not observe a role for AEP-driven autocrine α1-thymosin activation in T cell-derived IFN-γ production. These data suggest that AEP is an “upstream” activator of the CTSL-C3-IFN-γ axis in human CD4+ T cells and hence an important supporter of human Th1 induction.

Expression of free radicals by peritoneal cells of sheep during the early stages of Fasciola hepatica infection

BackgroundThe majority of vaccination studies against infection with F. hepatica in a natural host have been conducted at the late stage of the infection when the host's immune response is already immunomodulated by the parasite towards a Th2 non-protective response. This study was aimed at analysing the dynamic of the cell populations present in peritoneal liquid and the production of free radicals by the peritoneal leukocytes in infected and vaccinated sheep with recombinant cathepsin L1 of F. hepatica (rFhCL1) in early stages of the infection.MethodsForty-five sheep were divided into three groups: Group 1 remained as negative control (n = 5), Group 2 (n = 20) was challenged with F. hepatica and Group 3 (n = 20) was vaccinated with rFhCL1 and challenged with F. hepatica. After the slaughtering, peritoneal lavages were carried out at 1, 3, 9 and 18 days post-infection (dpi) to isolate peritoneal cell populations. Flow cytometry was conducted to assess levels of hydrogen peroxide (H2O2) and nitric oxide (NO).ResultsThere was a significant increase in the total number of leukocytes at 9 and 18 dpi in infected and vaccinated groups. Production of H2O2 was significantly increased in peritoneal granulocytes in both infected and vaccinated groups. Production of nitric oxide showed a significant rise in the granulocytes and monocytes/macrophages in infected and vaccinated sheep. The NO production by granulocytes at 3 and 9 dpi was significantly higher in the vaccinated than in the infected animals.ConclusionsExperimental infection induced an increase in the total number of leukocytes within the abdominal cavity at 9 and 18 dpi, being more noticeable in vaccinated animals. Production of H2O2 occurred mainly in granulocytes of vaccinated and infected animals. Production of NO was incremented in vaccinated and non-vaccinated animals in all peritoneal cells. Vaccinated animals produced significant higher level of H2O2 and NO than infected animals.

Molecular characterization of a cathepsin L1 highly expressed in phagocytes of pacific oyster Crassostrea gigas

Cathepsin L1 (CTSL1) is a lysosomal cysteine protease with a papain-like structure. It is known to be implicated in multiple processes of immune response against pathogen infection based on the proteolytic activity. In the present study, a CTSL1 homologue (designated as CgCTSL1) was identified from Crassostrea gigas. It contained a typically single Pept_C1 domain with three conserved catalytically essential residues (Gln25, His135 and Asn178). The mRNA of CgCTSL1 was ubiquitously expressed in oyster tissues with the highest expression level in important immune tissues such as gill and hemocytes. CgCTSL1 proteins were mainly detected in gill and hepatopancreas by immunohistochemistry. Recombinant CgCTSL1 (rCgCTSL1) exhibited proteolytic activity to cleave the substrate Ac-FR-amino-4-trifluoromethyl coumarin (AFC) in a dose-dependent manner, and the inhibitor could reduce its proteolytic activity. After the interference of CgCTSL1 mRNA, the proteolytic activity of oyster hemocytes was significantly down-regulated with the released AFC fluorescence value decreasing from 375.84 to 179.21 (p < 0.05). Flow cytometry analysis revealed that the expression of CgCTSL1 protein was higher in phagocytes with the mean fluorescence intensity (MFI) value of 21,187 (4.13-fold, p < 0.01) compared to the MFI value of 5,130 in non-phagocytic hemocytes. The further confocal analysis demonstrated that the actively phagocytic hemocytes with green bead signals were co-localized with stronger CgCTSL1 positive signals. The mRNA expression levels of CgCTSL1 in phagocyte-like sub-populations of granulocytes and semi-granulocytes were 298.12-fold (p < 0.01) and 2.75-fold (p < 0.01) of that in agranulocytes, respectively. Western blotting analysis of the hemocyte proteins revealed that CgCTSL1 was relatively abundant in granulocytes and semi-granulocytes compared to that in agranulocytes. These results collectively suggested that CgCTSL1, a CTSL1 homologue highly expressed in phagocyte-like hemocytes, was possibly involved in cellular immune response dependent on its conserved proteolytic activity, which might provide clues for the divergence between phagocytes and non-phagocytic hemocytes as well as the identification of promising molecular markers for phagocytes in oyster C. gigas.

Fasciola hepatica induces Foxp3 T cell, proinflammatory and regulatory cytokine overexpression in liver from infected sheep during early stages of infection

The expression of T regulatory cells (Foxp3), regulatory (interleukin [IL]-10 and transforming growth factor beta [TGF-β]) and proinflammatory (tumor necrosis factor alpha [TNF-α] and interleukin [IL]-1β) cytokines was quantified using real time polymerase chain reaction (qRT-PCR) in the liver of sheep during early stages of infection with Fasciola hepatica (1, 3, 9, and 18 days post-infection [dpi]). Portal fibrosis was also evaluated by Masson’s trichrome stain as well as the number of Foxp3+ cells by immunohistochemistry. Animals were divided into three groups: (a) group 1 was immunized with recombinant cathepsin L1 from F. hepatica (FhCL1) in Montanide adjuvant and infected; (b) group 2 was uniquely infected with F. hepatica; and (c) group 3 was the control group, unimmunized and uninfected. An overexpression of regulatory cytokines of groups 1 and 2 was found in all time points tested in comparison with group 3, particularly at 18 dpi. A significant increase of the number of Foxp3+ lymphocytes in groups 1 and 2 was found at 9 and 18 dpi relative to group 3. A progressive increase in portal fibrosis was found in groups 1 and 2 in comparison with group 3. In this regard, group 1 showed smaller areas of fibrosis than group 2. There was a significant positive correlation between Foxp3 and IL-10 expression (by immunohistochemistry and qRT-PCR) just as between portal fibrosis and TGF-β gene expression. The expression of proinflammatory cytokines increased gradually during the experience. These findings suggest the induction of a regulatory phenotype by the parasite that would allow its survival at early stages of the disease when it is more vulnerable.

Human brain gene expression profiles of the cathepsin V and cathepsin L cysteine proteases, with the PC1/3 and PC2 serine proteases, involved in neuropeptide production

Proteases are required to generate active peptide neurotransmitters, known as neuropeptides, from pro-neuropeptides. Model animal systems have recently illustrated roles for the cathepsin V (CTSV) and cathepsin L (CTSL) cysteine proteases, combined with the serine proteases PC1/3 (PCSK1) and PC2 (PCSK2), and exopeptidases in the production of neuropeptides. There is notable interest in the human-specific cathepsin V gene which is not present in rodent and other animal models used in prior studies of neuropeptide production. A gap in the field is knowledge of the human brain gene expression patterns of these neuropeptide-producing protease systems. Therefore, the goal of this study was to characterize the expression profiles of these pro-neuropeptide processing proteases in human brain. Quantitative gene expression microarray data for 169 human brain regions was obtained from the Allen Institute Human Brain Atlas resource, analyzed as log2 of gene expression intensity normalized to the mean of human genes (21,245 genes) expressed in human brain. These proteases had log2 values of 2–12, indicating expression levels above the average of all genes in the human brain, with varying expression levels among the 169 brain regions. CTSV and CTSL displayed moderate to high expression values of 1.9–8.6 and 7.1–10.6, respectively. Interestingly, CTSV and CTSL showed high expression in white matter composed of myelinated axons, consistent with the knowledge that neuropeptide production occurs in axons within transported neuropeptide secretory vesicles to nerve terminals. PCSK1 had a broad range of moderate to very high expression with log2 of 2–12. PCSK2 had somewhat lower expression levels than PCSK1. The exopeptidase genes RNPEP, CTSH, and CPE each showed fairly even levels of expression throughout the brain, with CPE displaying high expression. The prevalence of these processing proteases throughout human brain regions, including areas rich in neuropeptides such as hypothalamus, is consistent with their roles for neuropeptide production. Further, proenkephalin and NPY precursors, substrates of CTSV and CTSL shown in prior model animal studies, were co-expressed with CTSV and CTSL. These data demonstrate that the human brain expresses the neuropeptide-producing cysteine and serine proteases, with exopeptidases, throughout a multitude of brain regions.

Expression and activity of cathepsin L2 and its effect on the degradation of melanosomes in skin lesions of seborrheic keratosis

Objective To determine the expression of cathepsin L2 (CTSL2) and evaluate its activity in skin lesions of seborrheic keratosis (SK) , to observe the ultrastructural changes of melanosomes in the skin lesions of SK, and to estimate the effect of CTSL2 on the degradation of melanosomes. Methods Twenty patients with SK were enrolled from the Department of Dermatology, Renmin Hospital of Wuhan University. The lesional tissue and the perilesional normal skin were biopsied from each patient. Among 15 of the 20 patients, hematoxylin and eosin (HE) staining and Fontana-Masson silver staining were performed to observe the distribution of melanin granules, transmission electron microscopy (TEM) was conducted to observe the ultrastructural changes of melanosomes, and immunohistochemical staining was performed to estimate the cellular proliferative activity. RT-PCR and fluorogenic substrate cleavage assay were performed in the other 5 patients to determine the mRNA expression of CTSL2 and evaluate its activity, respectively. Sucrose density gradient ultracentrifugation was performed to isolate and purify melanosomes from the retinal pigment epithelium (RPE) harvested from a discarded eyeball of a 35-year old male patient with informed consent. The purified melanosomes were incubated with epidermal lysates of SK lesions, and TEM was used to observe the changes in the membrane structure of melanosomes. Statistical analysis was carried out by paired t test, and a P value < 0.05 was considered statistically significant. Results A large number of melanin granules were deposited in SK lesions, while the linear deposition of melanin granules was only seen in the basal layer of the normal skin. TEM showed that the percentage of damaged melanosomes was much higher in the normal skin (49.00% ± 4.00%) than in the SK lesions (24.33% ± 3.06%) (t= 8.49, P < 0.05) . RT-PCR revealed that the mRNA expression and activity of CTSL2 were both significantly lower in the SK lesions than in the normal skin (mRNA: 0.35 ± 0.09 vs. 0.43 ± 0.08, t= 3.17, P < 0.05; activity: 17.46 ± 0.45 vs. 28.78 ± 0.58, t= 34.29, P < 0.05) . Moreover, TEM also showed that the percentage of damaged melanosome was lower in the SK lesion lysate-treated group (32.33% ± 4.93%) than in the normal skin lysate-treated group (43.00% ± 2.65%, t= 3.30, P < 0.05) . Conclusion Decreased expression of CTSL2 in the SK lesions can affect the degradation of melanosomes by keratinocytes. However, whether CTSL2 directly takes part in the pathogenesis of SK or not is still needed to be further confirmed. Key words: Keratosis, seborrheic; Melanosomes; Cathepsin L

Intranasal delivery of a formulation containing stage-specific recombinant proteins of Fasciola hepatica cathepsin L5 and cathepsin B2 triggers an anti-fecundity effect and an adjuvant-mediated reduction in fluke burden in sheep

Fasciola hepatica infection continues to be a major problem in the agriculture sector, particularly in sheep and cattle. Cathepsin L and B proteases are major components of the excretory/secretory material of the parasite, and their roles in several important aspects of parasite invasion and survival has led to their use as targets in rational vaccine design. Previous studies in rats demonstrated that the use of stage-specific antigens, cathepsin B2 and cathepsin L5, as part of a multivalent vaccine, was able to confer significant protection against challenge. In the present study, recombinant versions of cathepsin L5 and cathepsin B2 produced in yeast were used in combination to vaccinate sheep. Intramuscular and intranasal forms of administration were applied, and sheep were subsequently challenged with 150 F. hepatica metacercariae. Intramuscular vaccination was able to induce a strong systemic antibody response against both antigens, but failed to confer significant protection. Conversely, no elevated antibody response was detected against the vaccine antigens following nasal vaccination; however, a reduction in parasite egg viability (>92%) and a statistically significant (p = 0.006), predominantly adjuvant-mediated reduction in worm burdens was observed.

SILAC\u2013based quantitative MS approach for real-time recording protein-mediated cell-cell interactions

In tumor microenvironment, interactions among multiple cell types are critical for cancer progression. To understand the molecular mechanisms of these complex interplays, the secreted protein analysis between malignant cancer cells and the surrounding nonmalignant stroma is a good viewpoint to investigate cell-cell interactions. Here, we developed two stable isotope labeling of amino acids in cell culture (SILAC)-based mass spectrometry (MS)/MS approaches termed spike-in SILAC and triple-SILAC to quantify changes of protein secretion level in a cell co-cultured system. Within the co-culture system of CT26 and Ana-1 cells, the spike-in SILAC and triple-SILAC MS approaches are sensitive to quantitatively measure protein secretion changes. Three representative quantified proteins (Galectin-1, Cathepsin L1 and Thrombospondin-1) by two SILAC-based MS methods were further validated by Western blotting, and the coming result matched well with SILACs’. We further applied these two SILACs to human cell lines, NCM460 and HT29 co-culture system, for evaluating the feasibility, which confirmed the spike-in and triple SILAC were capable of monitoring the changed secreted proteins of human cell lines. Considering these two strategies in time consuming, sample complexity and proteome coverage, the triple-SILAC way shows more efficiency and economy for real-time recording secreted protein levels in tumor microenvironment.

Design and synthesis of a new peptide derived from Fasciola gigantica cathepsin L1 with potential application in serodiagnosis of fascioliasis

Fascioliasis is a global parasitic disease that affects domestic animals and causes considerable economic losses in the process of domestic animal breeding in endemic regions. The cause of the disease involves a liver trematode of the genus Fasciola, which secretes materials into a host's body (mainly proteins) in order to protect it from the host's immune system. These materials can be involved in the migration, growth, and nutrition of the parasite. Among the expressive proteins of Fasciola, proteases have been introduced as the appropriate targets for diagnosis, treatment, and vaccination against parasites. Cathepsin L (CL) is a member of cysteine proteases; it is widely expressed in the Fasciola species. The aim of this study was to evaluate two synthetic peptides from F. gigantica CL1 for improving serological diagnosis of the Fasciola infection. Therefore, the potential diagnostic value of the surface epitopes of CL1 was assessed using ELISA. In the current study, bioinformatics tools were applied to select two appropriate epitopes of Fasciola Cathepsin L1 as synthetic antigens. Their diagnostic values were evaluated by two methods of indirect ELISA and dot blot analysis.The findings revealed that the first peptide at a dilution ratio of 1:400 and the second peptide at a dilution ratio of 1:100 had the best results and the best concentration of antigens was introduced at 4 μg/ml. Moreover, 191 sera samples were analyzed by both peptides by using the ELISA method, including fascioliasis sera, other parasitic sera and negative sera. The sensitivity of the peptides 1-ELISA and peptide 2-ELISA for the diagnosis of the various cases was 100%. The specificity of the first peptide was 87.3% and its efficacy was determined to be 93.65%. The specificity and the efficacy of the second peptide were 79% and 89.5%, respectively. The positive predictive values of the first and second peptides were obtained to be 86.27% and 79.27% respectively, and the negative predictive values of both peptides was calculated as 100%. In conclusion, the results of this study indicated that the peptide 1 from CL1 may be used as an appropriate antigen for the diagnosis of fascioliasis if the findings are backed up by using other serodiagnostic methods for checking serological cross-reactivity linked to other parasites.

Full length histone H3 conjugated electrochemical biosensor for extracellular proteolytic Cathepsin L activity detection

Timely and accurate detection of proteolytic extracellular Cathepsin L (CTSL) activity is vital for assessing the metastatic potential of cancer. In this article, extracellular CTSL activity was detected using biotinylated full length histone H3 (BFLH3) treated extended gate (EG) field effect transistors (FETs) with 104 times the sensitivity of fluorometric CTSL activity assays. Upon active reaction by CTSL, the treated BFLH3 is cleaved after amino acid 21, inducing a significant charge change on the EG surface due to the large size and charge of the cleaved BFLH3. This change in EG surface charge can be detected by FET, enabling the quantification of proteolytic extracellular CTSL activity. Moreover, self-normalized quantification of CTSL activity was achieved in heterogeneous LNCaP and PANC-1 cultured cell media, which accurately represented higher CTSL levels in LNCaP derived from metastatic lesion of human prostatic adenocarcinoma. CTSL activity detection with EG FET allows multiple measurements of CTSL activity with one FET and opens up the possibility of detecting other proteolytic enzymes for cancer diagnosis and prognosis.