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It is estimated that approximately 90% of patients with advanced prostate cancer develop bone metastases; an occurrence that results in a substantial reduction in the quality of life and a drastic worsening of prognosis. The development of novel therapeutic strategies that impair the metastatic process and associated skeletal adversities is therefore critical to improving prostate cancer patient survival. Recognition of the importance of Cathepsin L (CTSL) to metastatic dissemination of cancer cells has led to the development of several CTSL inhibition strategies. The present investigation employed intra-cardiac injection of human PC-3ML prostate cancer cells into nude mice to examine tumor cell dissemination in a preclinical bone metastasis model. CTSL knockdown confirmed the validity of targeting this protease and subsequent intervention studies with the small molecule CTSL inhibitor KGP94 resulted in a significant reduction in metastatic tumor burden in the bone and an improvement in overall survival. CTSL inhibition by KGP94 also led to a significant impairment of tumor initiated angiogenesis. Furthermore, KGP94 treatment decreased osteoclast formation and bone resorptive function, thus, perturbing the reciprocal interactions between tumor cells and osteoclasts within the bone microenvironment which typically result in bone loss and aggressive growth of metastases. These functional effects were accompanied by a significant downregulation of NFκB signaling activity and expression of osteoclastogenesis related NFκB target genes. Collectively, these data indicate that the CTSL inhibitor KGP94 has the potential to alleviate metastatic disease progression and associated skeletal morbidities and hence may have utility in the treatment of advanced prostate cancer patients., (© 2016 UICC.)

A significant proportion of breast cancer patients harbor clinically undetectable micrometastases at the time of diagnosis. If left untreated, these micro-metastases may lead to disease relapse and possibly death. Hence, there is significant interest in the development of novel anti-metastatic agents that could also curb the growth of pre-established micrometastases. Like primary tumor, the growth of metastases also is driven by angiogenesis. Although the role of cysteine protease Cathepsin L (CTSL) in metastasis associated tumor cell functions such as migration and invasion is well recognized, its role in tumor angiogenesis remains less explored. The present study examines the contribution of CTSL to breast cancer angiogenesis and evaluates the anti-angiogenic efficacy of CTSL inhibitor KGP94. CTSL semi-quantitative RT-PCR analysis on breast tissue panels revealed significant upregulation of CTSL in breast cancer patients which strongly correlated with increased relapse and metastatic incidence and poor overall survival. Preclinically, CTSL ablation using shRNA or KGP94 treatment led to a significant reduction in MDA-MB-231 tumor cell induced angiogenesis in vivo. In-vitro assessments demonstrated a significant decrease in various angiogenic properties such as endothelial cell sprouting, migration, invasion, tube formation and proliferation in the presence of KGP94. Microarray analyses revealed a significant upregulation of cell cycle related genes by CTSL. Western blot analyses further confirmed upregulation of members of the cyclin family by CTSL. Collectively, these data indicate that CTSL is an important contributor to tumor angiogenesis and that the CTSL inhibition may have therapeutic utility in the treatment of breast cancer patients.

A critical role for intracellular TLR9 has been described in recognition and host resistance to Leishmania parasites. As TLR9 requires endolysosomal proteolytic cleavage to achieve signaling functionality, we investigated the contribution of different proteases like asparagine endopeptidase (AEP) or cysteine protease cathepsins B (CatB), L (CatL) and S (CatS) to host resistance during Leishmania major (L. major) infection in C57BL/6 (WT) mice and whether they would impact on TLR9 signaling. Unlike TLR9-/-, which are more susceptible to infection, AEP-/-, CatL-/- and CatS-/- mice are as resistant to L. major infection as WT mice, suggesting that these proteases are not individually involved in TLR9 processing. Interestingly, we observed that CatB-/- mice resolve L. major lesions significantly faster than WT mice, however we did not find evidence for an involvement of CatB on either TLR9-dependent or independent cytokine responses of dendritic cells and macrophages or in the innate immune response to L. major infection. We also found no difference in antigen presenting capacity. We observed a more precocious development of T helper 1 responses accompanied by a faster decline of inflammation, resulting in resolution of footpad inflammation, reduced IFNγ levels and decreased parasite burden. Adoptive transfer experiments into alymphoid RAG2-/-γc-/- mice allowed us to identify CD3+ T cells as responsible for the immune advantage of CatB-/- mice towards L. major. In vitro data confirmed the T cell intrinsic differences between CatB-/- mice and WT. Our study brings forth a yet unappreciated role for CatB in regulating T cell responses during L. major infection.

In parasitic flatworms, acid endopeptidases are involved in crucial processes, including digestion, invasion, interactions with the host immune system, etc. In haematophagous monogeneans, however, no solid information has been available about the occurrence of these enzymes. Here we aimed to identify major cysteine and aspartic endopeptidase activities in Eudiplozoon nipponicum, an invasive haematophagous parasite of common carp. Employing biochemical, proteomic and molecular tools, we found that cysteine peptidase activities prevailed in soluble protein extracts and excretory/secretory products (ESP) of E. nipponicum; the major part was cathepsin L-like in nature supplemented with cathepsin B-like activity. Significant activity of the aspartic cathepsin D also occurred in soluble protein extracts. The degradation of haemoglobin in the presence of ESP and worm protein extracts was completely inhibited by a combination of cysteine and aspartic peptidase inhibitors, and diminished by particular cathepsin L, B and D inhibitors. Mass spectrometry revealed several tryptic peptides in ESP matching to two translated sequences of cathepsin L genes, which were amplified from cDNA of E. nipponicum and bioinformatically annotated. The dominance of cysteine peptidases of cathepsin L type in E. nipponicum resembles the situation in, e.g. fasciolid trematodes.

Sunn pest, Eurygaster integriceps, is a serious pest of cereals in the wide area of the globe from Near and Middle East to East and South Europe and North Africa. This study described for the first time, identification of E. integriceps trypsin serine protease and cathepsin-L cysteine, transcripts involved in digestion, which might serve as targets for pest control management. A total of 478 and 500 base pair long putative trypsin and cysteine gene sequences were characterized and named Tryp and Cys, respectively. In addition, the tissue-specific relative gene expression levels of these genes as well as gluten hydrolase (Gl) were determined under different host kernels feeding conditions. Result showed that mRNA expression of Cys, Tryp, and Gl was significantly affected after feeding on various host plant species. Transcript levels of these genes were most abundant in the wheat-fed E. integriceps larvae compared to other hosts. The Cys transcript was detected exclusively in the gut, whereas the Gl and Tryp transcripts were detectable in both salivary glands and gut. Also possibility of Sunn pest gene silencing was studied by topical application of cysteine double-stranded RNA (dsRNA). The results indicated that topically applied dsRNA on fifth nymphal stage can penetrate the cuticle of the insect and induce RNA interference. The Cys gene mRNA transcript in the gut was reduced to 83.8% 2 days posttreatment. Also, it was found that dsRNA of Cys gene affected fifth nymphal stage development suggesting the involvement of this protease in the insect growth, development, and molting., (© 2015 Wiley Periodicals, Inc.)

Rationale: ABCA3 is a lipid transporter in the limiting membrane of lamellar bodies in alveolar type II cells. Mutations in the ABCA3 gene cause respiratory distress syndrome in new-borns and childhood interstitial lung disease. ABCA3 is N-terminally cleaved by an as yet unknown protease, a process believed to regulate ABCA3 activity., Methods: The exact site where ABCA3 is cleaved was localized using mass spectrometry (MS). Proteases involved in ABCA3 processing were identified using small molecule inhibitors and siRNA mediated gene knockdown. Results were verified by in vitro digestion of a synthetic peptide substrate mimicking ABCA3's cleavage region, followed by MS analysis., Results: We found that cleavage of ABCA3 occurs after Lys174 which is located in the proteins' first luminal loop. Inhibition of cathepsin L and, to a lesser extent, cathepsin B resulted in attenuation of ABCA3 cleavage. Both enzymes showed activity against the ABCA3 peptide in vitro with cathepsin L being more active., Conclusion: We show here that, like some other proteins of the lysosomal membrane, ABCA3 is a substrate of cathepsin L. Therefore, cathepsin L may represent a potential target to therapeutically influence ABCA3 activity in ABCA3-associated lung disease.

Lipids play a pivotal role in embryogenesis as structural components of cellular membranes, as a source of energy, and as signaling molecules. On the basis of a collection of temperature-sensitive embryonic lethal mutants, a systematic database search, and a subsequent microscopic analysis of >300 interference RNA (RNAi)-treated/mutant worms, we identified a couple of evolutionary conserved genes associated with lipid storage in Caenorhabditis elegans embryos. The genes include cpl-1 (cathepsin L-like cysteine protease), ccz-1 (guanine nucleotide exchange factor subunit), and asm-3 (acid sphingomyelinase), which is closely related to the human Niemann-Pick disease-causing gene SMPD1. The respective mutant embryos accumulate enlarged droplets of neutral lipids (cpl-1) and yolk-containing lipid droplets (ccz-1) or have larger genuine lipid droplets (asm-3). The asm-3 mutant embryos additionally showed an enhanced resistance against C band ultraviolet (UV-C) light. Herein we propose that cpl-1, ccz-1, and asm-3 are genes required for the processing of lipid-containing droplets in C. elegans embryos. Owing to the high levels of conservation, the identified genes are also useful in studies of embryonic lipid storage in other organisms., (Copyright © 2016 by the Genetics Society of America.)

The presence of glioma stem cells (GSCs) in tumor is relevant for glioma treatment resistance. This study assessed whether knockdown of Cathepsin L can influence GSC growth, tumor radiosensitivity, and clinical outcome. Protein levels of Cathepsin L and stem cell markers (CD133 and Nestin) were analyzed in samples from 90 gliomas of different WHO grades and 6 normal brain tissues by immunohistochemistry. Two glioma stem cell lines with overexpressed Cathepsin L were stably transfected with Cathepsin L short hairpin RNA expression vectors. The effects of Cathepsin L inhibition on radiosensitivity, self-renewal, stemness, DNA damage, and apoptosis were evaluated. In addition, an intracranial animal model and subcutaneous tumor xenografts in nude mice were used to assess tumor response to Cathepsin L inhibition in vivo. Our results proved that expressions of Cathepsin L and CD133, but not of Nestin, correlated with malignant grades of glioma tissues. GSCs with high Cathepsin L and CD133 co-expression were extraordinarily radioresistant. Cathepsin L inhibition with radiotherapy significantly reduced GSC growth, promoted apoptosis, and improved radiosensitivity. Knockdown of Cathepsin L resulted in a dramatic reduction of CD133 expression, as well as the decreased phosphorylation of DNA repair checkpoint proteins (ATM and DNA-PKcs). Furthermore, combination of Cathepsin L inhibition and radiotherapy potently blocked tumor growth and decreased blood vessel formation in vivo. Taken together, these findings suggest Cathepsin L as a promising therapeutic target for clinical therapy in GBM patients., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Cathepsin L, a lysosomal endopeptidase, has been noted for its involvement in the innate immune response in invertebrates. Here, the cathepsin L cDNA of the sea cucumber Apostichopus japonicus (AjCatL) is identified from an EST library and then cloned by the rapid amplification of the cDNA ends (RACE) PCR. The full-length cDNA is 1678 bp long containing an open reading frame (ORF) of 1002 bp, an 80 bp 5' UTR and a 599 bp 3' UTR. The cDNA encodes 333 amino acid residues with a predicted molecular mass of 37.07 kDa and a theoretical isoelectric point (pI) of 5.01. The full-length AjCatL contains three active sites of eukaryotic thiol (cysteine) protease at positions 133-144, 278-288 and 295-314. Analysis of the predicted tertiary structure of prepro-CatL (17-333 aa) and mature-CatL (116-333 aa) reveals that the propeptide region (17-115 aa) blocks access to the substrate-binding cleft. Phylogenetic analysis shows that the AjCatL is clustered together with two other CatLs from Strongylocentrotus purpuratus. The enzymatic activity of AjCatL was verified using a substrate hydrolyzing assay with recombinant mAjCatL. Further analysis of real time-PCR demonstrates that the expression of AjCatL mRNA is significantly up-regulated in the coelomocytes in cases of infection with the common bacterial pathogen, Vibrio splendidus. This suggests that the AjCatL is likely to be involved in the immune response., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Leucine aminopeptidase (LAP) and cathepsin L1 (CL1) are important enzymes for the pathogenesis and physiology of Fasciola hepatica. These enzymes were analysed in silico to design a chimeric protein containing the most antigenic sequences of LAP (GenBank; AAV59016.1; amino acids 192-281) and CL1 (GenBank CAC12806.1; amino acids 173-309). The cloned 681-bp chimeric fragment (rFhLAP-CL1) contains 270 bp from LAP and 411 bp from CL1, comprising three epitopes, DGRVVHLKY (amino acids 54-62) from LAP, VTGYYTVHSGSEVELKNLV (amino acids 119-137) and YQSQTCLPF (amino acids 161-169) from CL1. The ~25 kDa rFhLAP-CL1 chimeric protein was expressed from the pET15b plasmid in the Rosetta (DE3) Escherichia coli strain. The chimeric protein rFhLAP-CL1, which showed antigenic and immunogenic properties, was recognized in Western blot assays using F. hepatica-positive bovine sera, and induced strong, specific antibody responses following immunization in rabbits. The newly generated chimeric protein may be used as a diagnostic tool for detection of antibodies against F. hepatica in bovine sera and as an immunogen to induce protection against bovine fasciolosis.

Cathepsin L, an immune-related protein, was purified from the hepatopancreas of Pacific abalone (Haliotis discus hannai) by ammonium sulfate precipitation and column chromatographies of SP-Sepharose and Sephacryl S-200 HR. Purified cathepsin L appeared as two bands with molecular masses of 28.0 and 28.5 kDa (namely cathepsin La and Lb) on SDS-PAGE under reducing conditions, suggesting that it is a glycoprotein. Peptide mass fingerprinting (PMF) analysis revealed that peptide fragments of 95 amino acid residues was high similarity to cathepsin L of pearl oyster (Pinctada fucata). The optimal temperature and pH of cathepsin L were 35 °C and pH 5.5. Cathepsin L was particularly inhibited by cysteine proteinase inhibitors of E-64 and leupeptin, while it was activated by metalloproteinase inhibitors EDTA and EGTA. The full-length cathepsin L cDNA was further cloned from the hepatopancreas by rapid PCR amplification of cDNA ends (RACE). The open reading frame of the enzyme was 981 bp, encoding 327 amino acid residues, with a conserved catalytic triad (Cys134, His273 and Asn293), a potential N-glycosylation site and conserved ERFNIN, GNYD, and GCGG motifs, which are characteristics of cathepsin L. Western blot and proteinase activity analysis revealed that the expression and enzyme activity of cathepsin L were significantly up-regulated in hepatopancreas at 8 h following Vibrio parahaemolyticus infection, demonstrating that cathepsin L is involved in the innate immune system of abalone. Our present study for the first time reported the purification, characterization, molecular cloning, and tissue expression of cathepsin L in abalone., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Cathepsin L is a cysteine protease with a papain-like structure. It is known to be implicated in multiple processes of mammalian immune response to pathogen infection. In teleost fish, the functionality of cathepsin L is less understood. In this work, we characterized a cathepsin L homologue (designated as SoCatL) from red drum Sciaenops ocellatus, an important farmed fish species in China. SoCatL possesses a typical domain arrangement characteristic of cathepsin L, which comprises a proregion and a protease domain with four catalytically essential residues (Gln137, Cys143, His282 and Asn302) conserved in various organisms. SoCatL shares moderate sequence identities with mammalian cathepsin L and relatively high sequence identities with teleost cathepsin L. Phylogenetic analysis revealed that SoCatL is evolutionally close to fish cathepsin L, especially those belonging to the Perciformes order. The homology model of SoCatL was discovered to exhibit a structure resembling human cathepsin L. Transcriptional expression of SoCatL was found ubiquitous in tissues and enhanced after experimental infection with a bacterial pathogen. Recombinant SoCatL purified from Escherichia coli (designated as rSoCatL) displayed apparent proteolytic activity, which was optimal at 50 °C and pH 7.0. The activity of rSoCatL required the catalytic residue Cys143 and was severely reduced by cathepsin inhibitor. These results suggest that SoCatL is a teleost cathepsin L homologue which functions as a cysteine protease and is likely to participate in the host immune response against bacterial infection.

Two cysteine proteases, cathepsin S (CatS) and cathepsin L (CatL), have been identified as the key enzymes involved in the processing of invariant chain (Ii chain) in mammals. However, little is known about the roles of fish cathepsins in the Ii chain processing. In this study, large yellow croaker cathepsin S (LycCatS) and L (LycCatL) were identified and characterized. Based on the sequence comparison and phylogenetic analysis, both LycCatS and LycCatL are highly conserved to their counterparts in teleost. These two cathepsins were constitutively expressed in all tissues and immune-related cells tested, although at different levels. Both recombinant LycCatS (rLycCatS) and LycCatL (rLycCatL) possess the typical cysteine protease activity. Like other mammalian endopeptidase cathepsins, rLycCatS and rLycCatL could be autocatalytically activated to remove propeptides and release active mature peptides. On the other hand, the autocatalytic activation of rLycCatL could be inhibited by recombinant large yellow croaker Ii chain (rLyc-TR-Ii), but the autocatalytic activation of rLycCatS was not affected by rLyc-TR-Ii. Furthermore, the activated rLycCatS can efficiently process rLyc-TR-Ii in a stepwise manner in vitro, while the activated rLycCatL can not. These data indicate that cathepsin S may be the main cathepsin involved in the Ii chain processing in bony fish., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancies and has a poor prognosis due to relatively unspecific early symptoms, and thus often advanced stage, metastasized cancer at presentation. Metastasis of EOC occurs primarily through the transcoelomic route whereby exfoliated tumor cells disseminate within the abdominal cavity, particularly to the omentum. Primary and metastatic tumor growth requires a pool of proangiogenic factors in the microenvironment which propagate new vasculature in the growing cancer. Recent evidence suggests that proangiogenic factors other than the widely known, potent angiogenic factor vascular endothelial growth factor may mediate growth and metastasis of ovarian cancer. In this review we examine the role of some of these alternative factors, specifically cathepsin D and cathepsin L.

During focal cerebral ischemia, the degradation of microvessel basal lamina matrix occurs acutely and is associated with edema formation and microhemorrhage. These events have been attributed to matrix metalloproteinases (MMPs). However, both known protease generation and ligand specificities suggest other participants. Using cerebral tissues from a non-human primate focal ischemia model and primary murine brain endothelial cells, astrocytes, and microglia in culture, the effects of active cathepsin L have been defined. Within 2 hours of ischemia onset cathepsin L, but not cathepsin B, activity appears in the ischemic core, around microvessels, within regions of neuron injury and cathepsin L expression. In in vitro studies, cathepsin L activity is generated during experimental ischemia in microglia, but not astrocytes or endothelial cells. In the acidic ischemic core, cathepsin L release is significantly increased with time. A novel ex vivo assay showed that cathepsin L released from microglia during ischemia degrades microvessel matrix, and interacts with MMP activity. Hence, the loss of microvessel matrix during ischemia is explained by microglial cathepsin L release in the acidic core during injury evolution. The roles of cathepsin L and its interactions with specific MMP activities during ischemia are relevant to strategies to reduce microvessel injury and hemorrhage.

The mucosal surfaces of fish are the first line of host defense against various pathogens. The mucosal immune responses are the most critical events to prevent pathogen attachment and invasion. Cathepsins are a group of peptidases that involved in different levels of immune responses, but the knowledge of the roles of Cathepsin in mucosal immune responses against bacterial infection are still lacking. Therefore, in the present study we characterized the Cathepsin L gene family in channel catfish, and profiled their expression levels after challenging with two different Gram-negative bacterial pathogens. Here, two Cathepsin L genes were identified from channel catfish and were designated CTSL1a and CTSL.1. Comparing to other fish species, the catfish CTSL genes are highly conserved in their structural features. Phylogenetic analysis was conducted to confirm the identification of CTSL genes. Expression analysis revealed that the CTSL genes were ubiquitously expressed in all tested tissues. Following infection, the CTSL genes were significantly induced at most timepoints in mucosal tissues. But the expression patterns varied depending on both pathogen and tissue types, suggesting that CTSL genes may exert disparate functions or exhibit distinct tissue-selective roles in mucosal immune responses. Our findings here, clearly revealed the key roles of CTSL in catfish mucosal immunity; however, further studies are needed to expand functional characterization and examine whether CTSL may also play additional physiological roles in catfish mucosal tissues., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Several members of the papain-like peptidase family have the ability to degrade collagen molecules by cleaving within the triple helix region of this difficult substrate. A common denominator of these peptidases is their ability to cleave substrates with Pro in the P2 position. In humans, cathepsin K is the best-known papain-like collagenase. Here, we investigate the collagenolytic activity of human cathepsin L, which is closely related to cathepsin K. We show that, despite lacking proline specificity, cathepsin L efficiently cleaves type I collagen within the triple helix region and produces a cleavage pattern similar to that of cathepsin K. We demonstrate that both enzymes have similar affinities for type I collagen and are able to release proteolytic fragments from insoluble collagen. Moreover, cathepsin K is only approximately fourfold more potent than cathepsin L in releasing fragments from reconstituted fibrils of FITC-labeled collagen. Replacing active site residues of cathepsin L with those from cathepsin K introduces cathepsin K-like specificity towards synthetic substrates and increases the collagenolytic activity of cathepsin L. Replacing three residues in the S2 subsite is sufficient to produce a mutant with collagenolytic activity on par with human cathepsin K. These results provide a basis for engineering collagenolytic activity into non-collagenolytic papain-like scaffolds., (© 2015 FEBS.)

Cysteine proteinases (cathepsins) from Leishmania spp. are promising molecular targets against leishmaniasis. Leishmania mexicana cathepsin L is essential in the parasite life cycle and a pivotal in virulence factor in mammals. Natural products that have been shown to display antileishmanial activity were screened as part of our ongoing efforts to design inhibitors against the L. mexicana cathepsin L-like rCPB2.8. Among them, agathisflavone (1), tetrahydrorobustaflavone (2), 3-oxo-urs-12-en-28-oic acid (3), and quercetin (4) showed significant inhibitory activity on rCPB2.8 with IC50 values ranging from 0.43 to 18.03 µM. The mechanisms of inhibition for compounds 1-3, which showed Ki values in the low micromolar range (Ki = 0.14-1.26 µM), were determined. The biflavone 1 and the triterpene 3 are partially noncompetitive inhibitors, whereas biflavanone 2 is an uncompetitive inhibitor. The mechanism of action established for these leishmanicidal natural products provides a new outlook in the search for drugs against Leishmania., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Aim: Cathepsin L is a lysosomal cysteine protease that plays important roles in cancer tumorigenesis, proliferation and chemotherapy resistance. The aim of this study was to determine how cathepsin L regulated the radiosensitivity of human glioma cells in vitro., Methods: Human glioma U251 cells (harboring the mutant type p53 gene) and U87 cells (harboring the wide type p53 gene) were irradiated with X-rays. The expression of cathepsin L was analyzed using Western blot and immunofluorescence assays. Cell survival and DNA damage were evaluated using clonogenic and comet assays, respectively. Flow cytometry was used to detect the cell cycle distribution. Apoptotic cells were observed using Hoechst 33258 staining and fluorescence microscopy., Results: Irradiation significantly increased the cytoplasmic and nuclear levels of cathepsin L in U251 cells but not in U87 cells. Treatment with the specific cathepsin L inhibitor Z-FY-CHO (10 μmol/L) or transfection with cathepsin L shRNA significantly increased the radiosensitivity of U251 cells. Both suppression and knockdown of cathepsin L in U251 cells increased irradiation-induced DNA damage and G2/M phase cell cycle arrest. Both suppression and knockdown of cathepsin L in U251 cells also increased irradiation-induced apoptosis, as shown by the increased levels of Bax and decreased levels of Bcl-2., Conclusion: Cathepsin L is involved in modulation of radiosensitivity in human glioma U251 cells (harboring the mutant type p53 gene) in vitro.

Objective: To systemically investigate the association between the polymorphism (rs3118869) in cathepsin Lenzyme gene with hypertension in three ethnic groups (Han, Kazak and Uygur) in China., Study Design: Case-control study., Place and Duration of Study: Department of Cardiology, The First Affiliated Hospital, Shihezi Medical College, Shihezi University and Department of Internal Medicine and Genetic Diagnosis Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, from January 2013 to May 2014., Methodology: This case-control study included 1224 patients (422 Uygur, 425 Kazak and 377 Han individuals) with hypertension and 967 healthy unrelated individuals (339 Uygur, 337 Kazak and 291 Han individuals) as controls. The participants came from three ethnic groups (Han, Kazak and Uygur) which were recruited from Xinjiang Province of China. The polymorphism (rs3118869) of the human cathepsin Lgene was genotyped using the TaqMan 5' nuclease assay. Binary logistic regression was built to determine the association of polymorphism with hypertension., Results: The genotype distribution of polymorphism was not significantly different in three ethnic groups. The rs3118869 polymorphism was significantly associated with Essential Hypertension (EH) in co-dominant model (A/C vs. C/C) in total people (OR = 0.697, 95% CI = 0.520 -0.932, p = 0.015), the same result was obtained in recessive model (C/C + A/C vs. A/A) in total people (OR = 0.689, 95% CI = 0.522 -0.910, p = 0.009). Similar finding of rs3118869 in recessive model (C/C + A/C vs. A/A) was also observed after adjusting the variable to the covariates age (OR = 0.629, 95% CI = 0.464 0853, p = 0.003)., Conclusion: The study results indicate the A-allele of rs3118869 is a protective factor in hypertension.

Cathepsin L family, an important cysteine protease found in lysosomes, is categorized into cathepsins B, F, H, K, L, S, and W in vertebrates. This categorization is based on their sequence alignment and traditional functional classification, but the evolutionary relationship of family members is unclear. This study determined the evolutionary relationship of cathepsin L family genes in vertebrates through phylogenetic construction. Results showed that cathepsins F, H, S and K, and L and V were chronologically diverged. Tandem-repeat duplication was found to occur in the evolutionary history of cathepsin L family. Cathepsin L in zebrafish, cathepsins S and K in xenopus, and cathepsin L in mice and rats underwent evident tandem-repeat events. Positive selection was detected in cathepsin L-like members in mice and rats, and amino acid sites under positive selection pressure were calculated. Most of these sites appeared at the connection of secondary structures, suggesting that the sites may slightly change spatial structure. Severe positive selection was also observed in cathepsin V (L2) of primates, indicating that this enzyme had some special functions. Our work provided a brief evolutionary history of cathepsin L family and differentiated cathepsins S and K from cathepsin L based on vertebrate appearance. Positive selection was the specific cause of differentiation of cathepsin L family genes, confirming that gene function variation after expansion events was related to interactions with the environment and adaptability.

Protein N-termini provide useful information for the understanding of posttranslational processing of proteins. The majority of proteins undergo N-terminal processing, such as proteolytic truncation or modifications like acetylation. Multiple methods currently exist for the enrichment of N-terminal peptides for proteomic analyses. Here, we report a novel, simple, and straightforward N-terminomic strategy, based on charge reversal of internal peptides followed by their removal through strong cation exchange chromatography. Our initial proof-of-concept study shows the feasibility of this technique, yielding over 3000 identifications of protein N-termini. We further show the application of this strategy in investigating the N-terminome of mouse embryonic fibroblasts cells deficient for both cathepsin B and L in comparison to wild type) control cells. Finally, we demonstrate that this workflow can be used in combination with a gel-based strategy, allowing preseparation of proteins and thus providing an estimate of the molecular weight of the identified cleavage products., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The cysteine protease cathepsin L (CTSL) is often thought to act as a tumor promoter by enhancing tumor progression and metastasis. This goes along with increased CTSL activity in various tumor entities; however, the mechanisms leading to high CTSL levels are incompletely understood. With the help of the polyoma middle T oncogene driven breast cancer mouse model expressing a human CTSL genomic transgene, we show that CTSL indeed promotes breast cancer metastasis to the lung. During tumor formation and progression high expression levels of CTSL are maintained by enduring translation of CTSL mRNA. Interestingly, human breast cancer specimens expressed the same pattern of 5' untranslated region (UTR) splice variants as the transgenic mice and the human cancer cell line MDA-MB 321. By polyribosome profiling of tumor tissues and human breast cancer cells, we observe an intrinsic resistance of CTSL to stress-induced shutdown of translation. This ability can be attributed to all 5' UTR variants of CTSL and is not dependent on a previously described internal ribosomal entry site motif. In conclusion, we provide in vivo functional evidence for overexpressed CTSL as a promoter of lung metastasis, whereas high CTSL levels are maintained during tumor progression due to stress-resistant mRNA translation., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Purpose: Cathepsin L and ADAMTS-1 are known to play critical roles in follicular rupture, ovulation, and fertility in mice. Similar studies in humans are limited; however, both are known to increase during the periovulatory period. No studies have examined either protease in the follicular fluid of women with unexplained infertility or infertility related to advanced maternal age (AMA). We sought to determine if alterations in cathepsin L and/or ADAMTS-1 existed in these infertile populations., Methods: Patients undergoing in vitro fertilization (IVF) for unexplained infertility or AMA-related infertility were prospectively recruited for the study; patients with tubal or male factor infertility were recruited as controls. Follicular fluid was collected to determine gene expression (via quantitative polymerase chain reaction), enzyme concentrations (via enzyme-linked immunosorbent assays), and enzymatic activities (via fluorogenic enzyme cleavage assay or Western blot analysis) of cathepsin L and ADAMTS-1., Results: The analysis included a total of 42 patients (14 per group). We found no statistically significant difference in gene expression, enzyme concentration, or enzymatic activity of cathepsin L or ADAMTS-1 in unexplained infertility or AMA-related infertility as compared to controls. We also found no statistically significant difference in expression or concentration with advancing age., Conclusions: Cathepsin L and ADAMTS-1 are not altered in women with unexplained infertility or AMA-related infertility undergoing IVF, and they do not decline with advancing age. It is possible that differences exist in natural cycles, contributing to infertility; however, our findings do not support a role for protease alterations as a common cause of infertility.

Lymphatic endothelial cells (LECs) directly express peripheral tissue antigens and induce CD8 T-cell deletional tolerance. LECs express MHC-II molecules, suggesting they might also tolerize CD4 T cells. We demonstrate that when β-galactosidase (β-gal) is expressed in LECs, β-gal-specific CD8 T cells undergo deletion via the PD-1/PD-L1 and LAG-3/MHC-II pathways. In contrast, LECs do not present endogenous β-gal in the context of MHC-II molecules to β-gal-specific CD4 T cells. Lack of presentation is independent of antigen localization, as membrane-bound haemagglutinin and I-Eα are also not presented by MHC-II molecules. LECs express invariant chain and cathepsin L, but not H2-M, suggesting that they cannot load endogenous antigenic peptides onto MHC-II molecules. Importantly, LECs transfer β-gal to dendritic cells, which subsequently present it to induce CD4 T-cell anergy. Therefore, LECs serve as an antigen reservoir for CD4 T-cell tolerance, and MHC-II molecules on LECs are used to induce CD8 T-cell tolerance via LAG-3.

Cathepsin L, a lysosomal acid cysteine protease, was found to be overexpressed in several types of human carcinomas. However, its functional roles in tumor progression and the underlying mechanisms remain largely unclear. In the present study, we investigated a novel functional aspect of cathepsin L in regulating transforming growth factor‑β (TGF‑β)‑induced epithelial‑mesenchymal transition (EMT) in A549 and MCF‑7 cells and examined its possible mechanisms. We found that TGF‑β‑induced cell morphologic changes of EMT were associated with the increased protein level of cathepsin L in A549 and MCF‑7 cells, suggesting that cathepsin L may be involved in the regulation of EMT. Furthermore, we showed that silencing of cathepsin L blocked TGF‑β‑induced cell migration, invasion and actin remodeling and inhibited TGF‑β‑mediated EMT. We also demonstrated that the mechanism of how cathepsin L knockdown regulates EMT may be explained by the suppression of EMT‑inducing molecules, such as Snail, which is associated with the phosphatidylinositol 3‑kinase (PI3K)‑AKT and Wnt signaling pathways. Moreover, we proved that cathepsin L knockdown in A549 cells significantly inhibited xenograft tumor growth and EMT in vivo. The results showed a new mechanism to determine cathepsin L involvement in the regulation of cancer invasion and migration. These results showed that cathepsin L knockdown is important in regulating EMT and suggest that cathepsin L may be utilized as a new target for enhancing the efficacy of chemotherapeutics against epithelial cancer.

Aim: Cathepsin L, a lysosomal cysteine proteinase, is exclusively elevated in a variety of malignancies, including gliomas. In this study we investigated the relationship between cathepsin L and NF-κB, two radiation-responsive elements, in regulating the sensitivity of human glioma cells ionizing radiation (IR) in vitro., Methods: Human glioma U251 cells were exposed to IR (10 Gy), and the expression of cathepsin L and NF-κB was measured using Western blotting. The nuclear translocation of NF-κB p65 and p50 was analyzed with immunofluorescence assays. Cell apoptosis was examined with clonogenic assays. NF-κB transcription and NF-κB-dependent cyclin D1 and ATM transactivation were monitored using luciferase reporter and ChIP assays, respectively. DNA damage repair was investigated using the comet assay., Results: IR significantly increased expression of cathepsin L and NF-κB p65 and p50 in the cells. Furthermore, IR significantly increased the nuclear translocation of NF-κB, and NF-κB-dependent cyclin D1 and ATM transactivation in the cells. Knockdown of p65 did not change the expression of cathepsin L in IR-treated cells. Pretreatment with Z-FY-CHO (a selective cathepsin L inhibitor), or knockdown of cathepsin L significantly attenuated IR-induced nuclear translocation of NF-κB and cyclin D1 and ATM transactivation, and sensitized the cells to IR. Pretreatment with Z-FY-CHO, or knockdown of p65 also decreased IR-induced DNA damage repair and clonogenic cell survival, and sensitized the cells to IR., Conclusion: Cathepsin L acts as an upstream regulator of NF-κB activation in human glioma cells and contributes to their sensitivity to IR in vitro. Inhibition of cathepsin L can sensitize the cells to IR.

Background: Larvae of the tenebrionids Tenebrio molitor and Tribolium castaneum have highly compartmentalized guts, with primarily cysteine peptidases in the acidic anterior midgut that contribute to the early stages of protein digestion., Results: High throughput sequencing was used to quantify and characterize transcripts encoding cysteine peptidases from the C1 papain family in the gut of tenebrionid larvae. For T. castaneum, 25 genes and one questionable pseudogene encoding cysteine peptidases were identified, including 11 cathepsin L or L-like, 11 cathepsin B or B-like, and one each F, K, and O. The majority of transcript expression was from two cathepsin L genes on chromosome 10 (LOC659441 and LOC659502). For cathepsin B, the major expression was from genes on chromosome 3 (LOC663145 and LOC663117). Some transcripts were expressed at lower levels or not at all in the larval gut, including cathepsins F, K, and O. For T. molitor, there were 29 predicted cysteine peptidase genes, including 14 cathepsin L or L-like, 13 cathepsin B or B-like, and one each cathepsin O and F. One cathepsin L and one cathepsin B were also highly expressed, orthologous to those in T. castaneum. Peptidases lacking conservation in active site residues were identified in both insects, and sequence analysis of orthologs indicated that changes in these residues occurred prior to evolutionary divergence. Sequences from both insects have a high degree of variability in the substrate binding regions, consistent with the ability of these enzymes to degrade a variety of cereal seed storage proteins and inhibitors. Predicted cathepsin B peptidases from both insects included some with a shortened occluding loop without active site residues in the middle, apparently lacking exopeptidase activity and unique to tenebrionid insects. Docking of specific substrates with models of T. molitor cysteine peptidases indicated that some insect cathepsins B and L bind substrates with affinities similar to human cathepsin L, while others do not and have presumably different substrate specificity., Conclusions: These studies have refined our model of protein digestion in the larval gut of tenebrionid insects, and suggest genes that may be targeted by inhibitors or RNA interference for the control of cereal pests in storage areas.

Trypanosoma theileri is a hemoprotozoan parasite that infects various ruminant species. We investigated the epidemiology of this parasite among cattle and water buffalo populations bred in Sri Lanka, using a diagnostic PCR assay based on the cathepsin L-like protein (CATL) gene. Blood DNA samples sourced from cattle (n=316) and water buffaloes (n=320) bred in different geographical areas of Sri Lanka were PCR screened for T. theileri. Parasite DNA was detected in cattle and water buffaloes alike in all the sampling locations. The overall T. theileri-positive rate was higher in water buffaloes (15.9%) than in cattle (7.6%). Subsequently, PCR amplicons were sequenced and the partial CATL sequences were phylogenetically analyzed. The identity values for the CATL gene were 89.6-99.7% among the cattle-derived sequences, compared with values of 90.7-100% for the buffalo-derived sequences. However, the cattle-derived sequences shared 88.2-100% identity values with those from buffaloes. In the phylogenetic tree, the Sri Lankan CATL gene sequences fell into two major clades (TthI and TthII), both of which contain CATL sequences from several other countries. Although most of the CATL sequences from Sri Lankan cattle and buffaloes clustered independently, two buffalo-derived sequences were observed to be closely related to those of the Sri Lankan cattle. Furthermore, a Sri Lankan buffalo sequence clustered with CATL gene sequences from Brazilian buffalo and Thai cattle. In addition to reporting the first PCR-based survey of T. theileri among Sri Lankan-bred cattle and water buffaloes, the present study found that some of the CATL gene fragments sourced from water buffaloes shared similarity with those determined from cattle in this country., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Cytotoxic T-lymphocyte antigen-2α (CTLA-2α) is a potent inhibitor of cathepsin L-like cysteine proteases. Recombinant CTLA-2α is known to be a potent, competitive inhibitor of cathepsin L-like cysteine proteases. In this study, cathepsin L, cathepsin C, and tubulointerstitial nephritis antigen-related protein 1 (TINAGL1) were identified as novel interactive proteins of CTLA-2α by the yeast two-hybrid screening system. The direct interactions and co-localization of these proteins with CTLA-2α were confirmed using co-immunoprecipitation and immunofluorescence staining, respectively. The disulfide-bonded CTLA-2α/cathepsin L complex was isolated from mouse tissue. CTLA-2α was found to be specific and consistently expressed on the maternal side of the mouse placenta. Double immunofluorescence analysis showed that CTLA-2α was co-localized with cathepsin L, cathepsin C, and TINAGL1 in placenta. A simple cell-based fluorescence assay revealed that CTLA-2α exhibited inhibitory activity toward cathepsin C in live cells, which indicated that CTLA-2α is a novel endogenous inhibitor of cathepsin C.

Cathepsin L (CTSL) is a lysosomal cysteine protease that has been found to be overexpressed in ovarian cancer (OC). The aim of the present study was to investigate the possible involvement of CTSL in the development of OC. In this study, RNA interference with a CTSL small hairpin RNA (CTSL-shRNA), and a plasmid carrying CTSL were used to identify the effects of this enzyme on the regulation of the malignant behavior of OC cells. OV-90 and SKOV3 human ovarian cancer cell lines were selected as cell models in vitro and in vivo. The results showed that downregulation of CTSL significantly inhibits the proliferative and invasive capability of SKOV3 cells, and that upregulation of CTSL in OV-90 cells leads to opposite effects. Compared with parental OC cells, cells in which CTSL was silenced exhibited a reduced capacity to develop into tumors in nude mice, while the growth of tumor xenografts derived from these cells was markedly constrained. In conclusion, the results suggested that CTSL contributes to the proliferation and metastasis of OC, and that CTSL may be a novel molecular target for OC treatment.

Diaprepes abbreviatus is an important pest that causes extensive damage to citrus in the USA. Analysis of an expressed sequence tag (EST) library from the digestive tract of larvae and adult D. abbreviatus identified cathepsins as major putative digestive enzymes. One class, sharing amino acid sequence identity with cathepsin L's, was the most abundant in the EST dataset representing 14.4% and 3.6% of the total sequences in feeding larvae and adults, respectively. The predominant cathepsin (Da-CTSL1) among this class was further studied. Three dimensional modeling of the protein sequence showed that the mature Da-CTSL1 protein folds into an expected cathepsin L structure producing a substrate binding pocket with appropriate positioning of conserved amino acid residues. A full-length cDNA was obtained and the proCTSL1 encoding sequence was expressed in Rosetta™ Escherichia coli cells engineered to express tRNAs specific for eukaryotic codon usage. The Da-CTSL1 was expressed as a fusion protein with GST and His6 tags and purified in the presence of 1% Triton X-100 by Ni-NTA affinity and size exclusion chromatography. Recombinant mature Da-CTSL1 (23 KDa) exhibits optimal activity at pH 8, rather than at acidic pH that was shown of all previously characterized cathepsins L. Substrate specificity supports the hypothesis that Da-CTSL1 is a unique basic cathepsin L and protease inhibitor studies also suggest unique activity, unlike other characterized acidic cathepsin Ls. This paper describes for the first time a prokaryotic expression system for the production of a functional eukaryotic cathepsin L1 from larval gut of D. abbreviatus., (Published by Elsevier Ltd.)

Cathepsin Ls (CatLs), the major cysteine protease secreted by Fasciola spp., are important for parasite digestion and tissue invasion. Fasciola gigantica cathepsin L1H (FgCatL1H) is the isotype expressed in the early stages for migration and invasion. In the present study, a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1H (rFgCatL1H) was produced by hybridoma technique using spleen cells from BALB/c mice immunized with recombinant proFgCatL1H (rproFgCatL1H). This MoAb is an immunoglobulin (Ig)G1 with κ light chain isotype. The MoAb reacted specifically with rproFgCatL1H, the native FgCatL1H at a molecular weight (MW) 38 to 48 kDa in the extract of whole body (WB) of metacercariae and newly excysted juvenile (NEJ) and cross-reacted with rFgCatL1 and native FgCatLs at MW 25 to 28 kDa in WB of 2- and 4-week-old juveniles, adult, and adult excretory-secretory (ES) fractions by immunoblotting and indirect ELISA. It did not cross-react with antigens in WB fractions from other parasites, including Gigantocotyle explanatum, Paramphistomum cervi, Gastrothylax crumenifer, Eurytrema pancreaticum, Setaria labiato-papillosa, and Fischoederius cobboldi. By immunolocalization, MoAb against rFgCatL1H reacted with the native protein in the gut of metacercariae and NEJ and also cross-reacted with CatL1 in 2- and 4-week-old juveniles and adult F. gigantica. Therefore, FgCatL1H and its MoAb may be used for immunodiagnosis of both early and late fasciolosis in ruminants and humans.

Avian coccidiosis, caused by Eimeria spp., is one of the major parasitic diseases in birds. Cysteine protease is a major virulence factor in parasitic protozoa, and it may be a suitable chemotherapeutic target and vaccine candidate molecule. A 100 amino acid (aa.) partial sequence of cathepsin L, which is a cysteine protease, was reported by Katrib et al. (Ac. No. CDJ41293) (2012). A 219 aa. sequence was reported by Reid et al. (Ac. No. AFV92863) (2013). However, the open reading frame (ORF) was not reported. In this study, a full sequence of a cathepsin-L-like peptidase in Eimeria tenella (EtcatL) was obtained and its biochemical characterizations and expression profiles were analyzed across different stages of the parasite's life cycle. Results showed that the EtcatL gene encodes a protein 470 aa. in length, with 47 and 49% identity to Toxoplasma gondii and Eimeria acervulina. Considering the close phylogenetic relationship, TgcatL (PDB. ID 3F75) was selected for use as a template for homology modeling with quality factors of 90.9. Gelatin SDS-PAGE showed it to exert protease activity at ≈38 and ≈26 kDa. Further analysis showed the kinetic parameters of the recombinant peptidase to be K m  = 8.9 μM and V max = 5.7 RFU/s μM at pH 5.5 containing 10 mM dithiothreitol (DTT) in the reaction matrix, and the IC50 value of E64 was 65.32 ± 3.02 nM. The recombinant protein was active from 25 to 50 °C, with optimal activity at 42 °C. The RT-PCR and Western blot results showed it to be expressed mainly at the endogenous stages and the initial phase of the sporulation. The protective experiment showed that chickens immunized with 100 and 200 μg rEtcatL had reduction of weight loss values 48.7 and 57.9% those of infected controls, respectively. Their reduction of lesion scores (RLS) were 25.0 and 47.2% that of control chickens, and relative oocyst production (ROP) was 39.6 and 15.5% that of control chickens. These results indicate that the EtcatL can be used as an effective immunogen, and further studies are needed to enhance its potential as a vaccine candidate molecule.

Objectives: To investigate the expression and role of Cathepsin L (CTSL) in Hepatocellular carcinoma (HCC) tissue and cell line (MHCC-97H), and to evaluate the clinical and prognostic significance of CTSL protein in patients with HCC., Methods: The expression of CTSL was examined in HCC tissue and MHCC-97H cells by Western-blotting, Real-time PCR and immunohistochemical staining. Cell growth curve assay and colony formation assay were used to verify the effect of CTSL on the proliferation and tumor progression ability of MHCC-97H cells. Tumor formation assay in nude mice was used to analyze the effect of CTSL on the tumorigenicity of MHCC-97H cells., Results: The status of CTSL protein in carcinoma tissues is much higher than that in paracarcinoma tissues. The overall survival of the patients with high CTSL expression was significantly shorter than the low CTSL expression group. high CTSL expression was significantly correlated with advanced clinical staging, histological grade and tumor recurrence. In vitro experiments demonstrated that over-expression of CTSL in MHCC-97H cells promoted cell proliferation and tumor progression ability. Down-regulation of CTSL showed the opposite effects. Over-expression of CTSL increase the tumorigenicity of MHCC-97H cells by in vivo experiments. Moreover, multivariate analysis suggested that CTSL expression might be an independent prognostic indicator for the survival of HCC patients after curative surgery., Conclusions: CTSL might involve in the development and progression of HCC as a oncogene, and thereby may be a valuable prognostic marker for HCC patients.

In mammalian cells, the protozoan pathogen Toxoplasma resides in a nonfusiogenic vacuole that segregates it from host cell resources. How the parasite acquires nutrients and whether it is capable of internalizing host macromolecules have been long-standing mysteries. By exploiting a mutant of Toxoplasma lacking the cathepsin protease L, Dou et al. observed the accumulation of host cytosolic-derived proteins in a multivesicular post-Golgi compartment, which establishes the existence of a functional heterophagic pathway in Toxoplasma., (Copyright © 2014. Published by Elsevier Ltd.)

Cathepsin L is one of the crucial enzyme superfamilies and involved in the immune responses. The Cathepsin L cDNA and genome of Cristaria plicata（CpCL） was cloned from the hemocytes using degenerate primers by the rapid amplification of cDNA ends (RACE) PCR. The genomic DNA was 9353 bp long and had a total of six introns and seven exons. The full-length cDNA of CpCL was 1144 bp, the cDNA contained a 5' untranslated region (UTR) of 34 nucleotides, the 3' UTR of 108 bp with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 1002 bp, encoding 333 amino acid residues with 37.65 kDa predicted molecular weight. The theoretical isoelectric point was 8.61. The prepro-cathepsin L was consisted of a typical signal peptide (Met1-Gly20), a pro-region peptide (Leu21-Glu116) and a mature peptide (Tyr117-Val333). Many members of the papain family possessed of a proline residue at position 2 in the mature enzymem, this was also observed in CpCL. The preproprotein included an oxyanion hole (Gln 135), the active center formed by Cys141, His280 and Asn 300, the potential N-glycosylation site (Asn38, Asn 113 and Asn 272) and the conserved GCXGG motifs, which was characteristic of cathepsin, the conserved ERWNIN and GNFD motifs, which were characteristic for cathepsin L. Homology analysis revealed that the CpCL shared 49-87% identity to other known cathepsin L sequences. The phylogenetic tree showed that the CpCL clustered with the invertebrate cathepsin L cysteine proteases, and was closely related to the cathepsin L of Hyriopsis cumingii. The expression of CpCL mRNA was detected in hepatopancreas, hemocytes, mantle, gills and adductor muscle, and the higher expression level was in hepatopancreas. After A. hydrophila stimulation, the expression of the CpCL mRNA was up-regulated in hemocytes and hepatopancreas, and the expression level was significantly lower in gill than one after PBS challenge group., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The lysosomal protease cathepsin L has been reported to cleave various functionally important cytosolic or nuclear proteins. To explain nucleo-cytosolic localization of cathepsin L, it has been hypothesized that skipping of the first start codon during translation initiation results in an N-terminally truncated protein lacking the endoplasmic reticulum-import signal. Here we demonstrate that out-of-frame AUGs prevent translation of truncated cathepsin L in cell culture as well as in a new knock-in mouse model. We further evaluate potential roles of nuclear cathepsin L during early embryonic development. Our analysis reveals normal epiblast development of cathepsin L-deficient embryos, but uncovers a pronounced lysosomal storage phenotype in the extra-embryonic tissue of the visceral endoderm. In conclusion, the phenotypes of cathepsin L deficiency can be fully assigned to lack of canonically targeted cathepsin L, while the biogenesis and functionality of nucleo-cytosolic cathepsin L remain elusive.

The role of cellular proteases and endosome maturation in the entry of caliciviruses including porcine enteric calicivirus (PEC), murine norovirus (MNV)-1 and feline calicivirus (FCV) were investigated. Treatment with chloroquine or cathepsin L inhibitors, but not cathepsin B inhibitors, significantly reduced the replication of PEC, MNV and FCV. When concentrated PEC, MNV or FCV were incubated with recombinant cathepsin L, the minor capsid protein VP2 of PEC and the major capsid protein VP1 of MNV and FCV were cleaved by the protease based on the Western blot analysis. Confocal microscopy analysis of PEC and MNV-1 showed that viral capsid proteins were retained in the endosomes in the presence of a cathepsin L inhibitor or chloroquine during virus entry. The results of this study suggest the important role of endosome maturation and cathepsin L in the entry of caliciviruses, and cathepsin L as a potential therapeutic target for calicivirus infection., (Copyright © 2014 Elsevier Inc. All rights reserved.)

RNA interference (RNAi) has been widely and successfully used for gene inactivation in insects, including aphids, where dsRNA administration can be performed either by feeding or microinjection. However, several aspects related to the aphid response to RNAi, as well as the influence of the administration method on tissue response, or the mixed success to observe phenotypes specific to the gene targeted, are still unclear in this insect group. In the present study, we made the first direct comparison of two administration methods (injection or feeding) for delivery of dsRNA targeting the cathepsin-L gene in the pea aphid, Acyrthosiphon pisum. In order to maximize the possibility of discovering specific phenotypes, the effect of the treatment was analyzed in single individual aphids at the level of five body compartments: the bacteriocytes, the gut, the embryonic chains, the head and the remaining body carcass. Our analysis revealed that gene expression knockdown effect in each single body compartment was dependent on the administration method used, and allowed us to discover new functions for the cathepsin-L gene in aphids. Injection of cathepsin-L dsRNA was much more effective on carcass and head, inducing body morphology alterations, and suggesting a novel role of this gene in the molting of these insects. Administration by feeding provoked cathepsin-L knockdown in the gut and specific gut epithelial cell alteration, therefore allowing a better characterization of tissue specific role of this gene in aphids., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cathepsins (CTSs) are peptidases that have biological roles in degrading extracellular matrix, catabolism of intracellular proteins, and processing of pro-hormones. Of these, cathepsin L (CTSL) is closely associated with morphological changes in reproductive organs required for proper function in mammals, including humans and mice, but little is known about CTSL in avian species. In the present study, the expression of CTSL was investigated in the oviduct of hens during regression and recrudescence in response to molting. Our results revealed that expression of CTSL mRNA increased (P<0.001) when the oviduct underwent regression during the molting period in hens. In situ hybridization and immunohistochemial analyses detected CTSL mRNA and protein predominantly in the luminal (LE) and glandular epithelia (GE) during regression of the oviduct, but not during regeneration of the oviduct. Expression of CTSL decreased in the oviduct of chicks treated with diethylstilbestrol (DES, a synthetic estrogen agonist). Furthermore, we discovered four miRNAs including miR-23b, miR-551, miR-1464 and miR-1803 that regulate expression of the CTSL gene at the post-transcriptional level, which suggests that CTSL mRNA can be regulated by specific miRNAs via 3'-UTR in chickens. Results of the present research suggest that estrogen regulates expression of CTSL during regression of the oviduct during molting and that down-regulation of CTSL is likely a prerequisite for the normal regeneration of oviductal tissues following molting in laying hens., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The chemistries within phagosomes of APCs mediate microbial destruction as well as generate peptides for presentation on MHC class II. The antimicrobial effector NADPH oxidase (NOX2), which generates superoxide within maturing phagosomes, has also been shown to regulate activities of cysteine cathepsins through modulation of the lumenal redox potential. Using real-time analyses of lumenal microenvironmental parameters, in conjunction with hydrolysis pattern assessment of phagocytosed proteins, we demonstrated that NOX2 activity not only affects levels of phagosomal proteolysis as previously shown, but also the pattern of proteolytic digestion. Additionally, it was found that NOX2 deficiency adversely affected the ability of bone marrow-derived macrophages, but not dendritic cells, to process and present the I-A(b)-immunodominant peptide of the autoantigen myelin oligodendrocyte glycoprotein (MOG). Computational and experimental analyses indicated that the I-A(b) binding region of the immunodominant peptide of MOG is susceptible to cleavage by the NOX2-controlled cysteine cathepsins L and S in a redox-dependent manner. Consistent with these findings, I-A(b) mice that were deficient in the p47(phox) or gp91(phox) subunits of NOX2 were partially protected from MOG-induced experimental autoimmune encephalomyelitis and displayed compromised reactivation of MOG-specific CD4(+) T cells in the CNS, despite eliciting a normal primary CD4(+) T cell response to the inoculated MOG Ag. Taken together, this study demonstrates that the redox microenvironment within the phagosomes of APCs is a determinant in MHC class II repertoire production in a cell-specific and Ag-specific manner, which can ultimately impact susceptibility to CD4(+) T cell-driven autoimmune disease processes., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Closely related African trypanosomes cause lethal diseases but display distinct host ranges. Specifically, Trypanosoma brucei brucei causes nagana in livestock but fails to infect humans, while Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense cause sleeping sickness in humans. T. b. brucei fails to infect humans because it is sensitive to innate immune complexes found in normal human serum known as trypanolytic factor (TLF) 1 and 2; the lytic component is apolipoprotein-L1 in both TLFs. TLF resistance mechanisms of T. b. gambiense and T. b. rhodesiense are now known to arise through either gain or loss-of-function, but our understanding of factors that render T. b. brucei susceptible to lysis by human serum remains incomplete. We conducted a genome-scale RNA interference (RNAi) library screen for reduced sensitivity to human serum. Among only four high-confidence 'hits' were all three genes previously shown to sensitize T. b. brucei to human serum, the haptoglobin-haemoglobin receptor (HpHbR), inhibitor of cysteine peptidase (ICP) and the lysosomal protein, p67, thereby demonstrating the pivotal roles these factors play. The fourth gene identified encodes a predicted protein with eleven trans-membrane domains. Using chemical and genetic approaches, we show that ICP sensitizes T. b. brucei to human serum by modulating the essential cathepsin, CATL, a lysosomal cysteine peptidase. A second cathepsin, CATB, likely to be dispensable for growth in in vitro culture, has little or no impact on human-serum sensitivity. Our findings reveal major and novel determinants of human-serum sensitivity in T. b. brucei. They also shed light on the lysosomal protein-protein interactions that render T. b. brucei exquisitely sensitive to lytic factors in human serum, and indicate that CATL, an important potential drug target, has the capacity to resist these factors.

Abundant clinical evidences indicate that up-regulation of several cathepsins in many human cancers is correlated with malignant progression and poor patient prognosis. In addition, a decrease in catalase activity or accumulation of hydrogen peroxide correlates with cancer metastasis. Recent studies indicate that cathepsin activation and expression can be modulated via H2O2 treatment. However, the actual relationship between catalase and cathepsins is not yet fully understood. In the present study, we found that catalase expression (or activity) was higher, while intracellular and extracellular Cat S, Cat L, and Cat K activities were lower in the non-invasive CL1-0 cells compared to the highly invasive CL1-5 cells. After CL1-0 cells were transfected with catalase-shRNA, the corresponding ROS (H2O2) level and Cat S, Cat L, or Cat K expression (or activity) was up-regulated, accompanied by an increase in cell migration and invasion. On the other hand, ROS (H2O2) level, cathepsin S, L, and K activities, cell migration and invasion were decreased in catalase-overexpressed CL1-5 cells. It is suggested that catalase may regulate cathepsin activity by controlling the production of ROS (H2O2), leading to variation in migration and invasion ability of lung cancer cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

RNA interference (RNAi) mediated crop protection against insect pests is a technology that is greatly anticipated by the academic and industrial pest control communities. Prior to commercialization, factors influencing the potential for evolution of insect resistance to RNAi should be evaluated. While mutations in genes encoding the RNAi machinery or the sequences targeted for interference may serve as a prominent mechanism of resistance evolution, differential effects of RNAi on target pests may also facilitate such evolution. However, to date, little is known about how variation of field insect populations could influence the effectiveness of RNAi treatments. To approach this question, we evaluated the effects of RNAi treatments on adults of three western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte) populations exhibiting different levels of gut cysteine protease activity, tolerance of soybean herbivory, and immune gene expression; two populations were collected from crop rotation-resistant (RR) problem areas and one from a location where RR was not observed (wild type; WT). Our results demonstrated that RNAi targeting DvRS5 (a highly expressed cysteine protease gene) reduced gut cysteine protease activity in all three WCR populations. However, the proportion of the cysteine protease activity that was inhibited varied across populations. When WCR adults were treated with double-stranded RNA of an immune gene att1, different changes in survival among WT and RR populations on soybean diets occurred. Notably, for both genes, the sequences targeted for RNAi were the same across all populations examined. These findings indicate that the effectiveness of RNAi treatments could vary among field populations depending on their physiological and genetic backgrounds and that the consistency of an RNAi trait's effectiveness on phenotypically different populations should be considered or tested prior to wide deployment. Also, genes that are potentially subjected to differential selection in the field should be avoided for RNAi-based pest control., (Published by Elsevier Inc.)

Cysteine cathepsins (CTSs) are involved in the degradation and remodeling of the extracellular matrix and are associated with cellular transformation, differentiation, motility and adhesion in cancer development. Previous studies indicate that CTSs may be involved in ovarian cancer invasion and metastasis. However, due to the lack of large sample clinical studies and direct experimental evidence for the relationship between the expression of CTSs and invasion and metastasis, the diagnostic and prognostic value of CTSs in ovarian cancer progression has not been elucidated. In the present study, we observed that expression levels of CTSB, CTSL and CC in malignant ovarian tumors were significantly higher than the expression levels in benign tumors and normal ovarian tissues, yet their associations with clinicopathological features varied. In particular, CTSL was related to lymph node metastasis, CC was related to liver metastasis and omental metastasis, and CTSB and CTSL expression levels were found to be independent prognostic factors in ovarian cancer. Further study indicated that the serum level of CTSL was significantly higher in patients with ovarian malignant tumors than the levels in benign tumors and healthy controls, and the levels were elevated in low grade and advanced stage compared to the levels in high grade and early stage disease, suggesting that the serum level of CTSL may be a useful serum marker for the diagnosis of ovarian cancer. Furthermore, the expression of CTSL in ovarian cancer cells can greatly enhance the ability of cell invasion and metastasis, although no change was observed for cell adhesion. Taken together, we demonstrated that the overexpression of CTSL is involved in tumor invasion and metastasis, and the CTSL level in serum may be a marker for invasion and metastasis in ovarian cancer.

Alveolar macrophages play an important role in chronic obstructive pulmonary disease via production of matrix metalloproteinases (MMPs) and cathepsins as well as their inhibitors, tissue inhibitors of metalloproteinases and cystatin C. We hypothesised that expression levels of these molecules by alveolar macrophages at baseline and after stimulation would be influenced by genotype and associated with chronic obstructive pulmonary disease phenotypes. Quantitative PCR and ELISAs/gelatine zymography were used to investigate expression levels of mRNA and protein, respectively. The relationships of expression with genotype, pulmonary function and emphysema were analysed. The results showed that basal expression level of MMP12 mRNA was inversely related to the diffusing capacity of the lung for carbon monoxide/alveolar volume and to forced expiratory volume in 1 s/forced vital capacity after correction for multiple comparisons. The expression level of MMP12 protein stimulated with lipopolysaccharide was also inversely related to the diffusing capacity of the lung for carbon monoxide/alveolar volume and was positively related to the extent of emphysema. The basal expression of MMP1 mRNA was positively correlated with the extent of emphysema. Cathepsin L protein level was positively associated with forced expiratory volume in 1 s % predicted. We conclude that increased MMP12 and MMP1 expression may play a role in the pathogenesis of emphysema. Cathepsin L and MMP9 may be involved in the development of airflow limitation.

Men with prostate cancer who receive androgen deprivation therapy show profound skeletal muscle loss. We hypothesized that the androgen deficiency activates not only the ubiquitin-proteasome systems but also the autophagy and affects key aspects of the molecular cross talk between protein synthesis and degradation. Here, 2-month-old male mice were castrated and treated with either testosterone (T) propionate or vehicle for 7 days (short term) or 43 days (long term), and with and without hydroxyflutamide. Castrated mice showed rapid and profound atrophy of the levator ani muscle (high androgen responder) at short term and lesser atrophy of the triceps muscle (low androgen responder) at long term. Levator ani and triceps muscles of castrated mice showed increased level of autophagy markers and lysosome enzymatic activity; only the levator ani showed increased proteasomal enzymatic activity. The levator ani muscle of the castrated mice showed increased level and activation of forkhead box protein O3A, the inhibition of mechanistic target of rapamicyn, and the activation of tuberous sclerosis complex protein 2 and 5'-AMP-activated protein kinase. Similar results were obtained in the triceps muscle of castrated mice. T rescued the loss of muscle mass after orchiectomy and inhibited lysosome and proteasome pathways dose dependently and in a seemingly IGF-I-dependent manner. Hydroxyflutamide attenuated the effect of T in the levator ani muscle of castrated mice. In conclusion, androgen deprivation in adult mice induces muscle atrophy associated with proteasomal and lysosomal activity. T optimizes muscle protein balance by modulating the equilibrium between mechanistic target of rapamicyn and 5'-AMP-activated protein kinase pathways.

BRCA1 and 53BP1 play decisive roles in the choice of DNA double-strand break repair mechanisms. BRCA1 promotes DNA end resection and homologous recombination (HR) during S/G 2 phases of the cell cycle, while 53BP1 inhibits end resection and facilitates non-homologous end-joining (NHEJ), primarily during G 1. This competitive relationship is critical for genome integrity during cell division. However, their relationship in the many cells in our body that are not cycling is unknown. We discovered profound differences in 53BP1 and BRCA1 regulation between cycling and non-cycling cells. Cellular growth arrest results in transcriptional downregulation of BRCA1 and activation of cathepsin-L (CTSL)-mediated degradation of 53BP1. Accordingly, growth-arrested cells do not form BRCA1 or 53BP1 ionizing radiation-induced foci (IRIF). Interestingly, cell cycle re-entry reverts this scenario, with upregulation of BRCA1, downregulation of CTSL, stabilization of 53BP1, and 53BP1 IRIF formation throughout the cycle, indicating that BRCA1 and 53BP1 are important in replicating cells and dispensable in non-cycling cells. We show that CTSL-mediated degradation of 53BP1, previously associated with aggressive breast cancers, is an endogenous mechanism of non-cycling cells to balance NHEJ (53BP1) and HR (BRCA1). Breast cancer cells exploit this mechanism to ensure genome stability and viability, providing an opportunity for targeted therapy.