Reduced expression of cathepsin F predicts poor prognosis in patients with clear cell renal cell carcinoma.

Abnormalities in the extracellular matrix (ECM) play important roles in the regulation and progression of clear cell renal cell carcinoma (ccRCC). The cysteine cathepsin is one of the major proteases involved in ECM remodeling and has been shown to be aberrantly expressed in multiple cancer types. However, the clinical significance and biological function of distinct cysteine cathepsins in ccRCC remain poorly understood. In this study, several bioinformatics databases, including UALCAN, TIMER, GEPIA and the Human Protein Atlas datasets, were used to analyze the expression and prognostic value of different cysteine cathepsin family members in ccRCC. We found that the expression level of CTSF was downregulated in tumor tissues and closely related to the poor survival of ccRCC patients. Further in vitro experiments suggested that CTSF overexpression suppressed the proliferation and migration of ccRCC cells. Moreover, the expression of CTSF was shown to be associated with several immune-infiltrating cells and immunomodulators in ccRCC. These results indicated that CTSF might be a promising diagnostic and prognostic marker in ccRCC.

Cathepsin F genetic mutation is associated with familial papillary thyroid cancer.

BACKGROUND: Thyroid cancer is one of the most common cancers in the world. Genetic factors are important in the occurrence and development of thyroid cancer, and genetic diagnosis has become an important basis for the prognosis of benign and malignant nodules. We identify a family of six siblings with inherited thyroid cancer susceptibility. All six members of this generation have been definitely diagnosed with papillary thyroid carcinoma. This work aims at confirming the relevant causative genes for thyroid cancer in this pedigree. METHODS: We extract DNA from the peripheral blood of six individuals and perform whole genome sequencing. Sanger sequencing and immunohistochemistry further testify the cathepsin F (CTSF) mutation and expression. RESULTS: We identify 57 single nucleotide variations (SNVs) out of at least 4 affected family members via certain filter criteria. The CTSF gene found in five of the six family members is here considered the most promising candidate gene mutation for familial thyroid cancer. Besides, our research also proves several known genes including CTSB, TEKT4, ESR1, MSH6, DIRC3, GNAS, and BANCR that act as probable oncogenic drivers in this family. The Sanger sequencing identifies the existence and veracity of CTSF somatic mutations. The CTSF immunohistochemistry of thyroid cancer tissue specimens displays that higher CTSF expression in mutated patients than that in wild-type patient as well as pericarcinomatous tissue. CONCLUSIONS: We conclude that the evaluation of CTSF gene mutations of patients in thyroid cancer families may be predictive and valuable for the familial heredity of thyroid cancer.

Expression signature, prognosis value and immune characteristics of cathepsin F in non-small cell lung cancer identified by bioinformatics assessment.

BACKGROUND: In recent years, immunotherapies and targeted therapies contribute to population-level improvement in NSCLC cancer-specific survival, however, the two novel therapeutic options have mainly benefit patients containing mutated driven genes. Thus, to explore other potential genes related with immunity or targeted therapies may provide novel options to improve survival of lung cancer patients without mutated driven genes. CTSF is unique in human cysteine proteinases. Presently, CTSF has been detected in several cell lines of lung cancer, but its role in progression and prognosis of lung cancer remains unclear. METHODS: CTSF expression and clinical datasets of lung cancer patients were obtained from GTEx, TIMER, CCLE, THPA, and TCGA, respectively. Association of CTSF expression with clinicopathological parameters and prognosis of lung cancer patients was analyzed using UALCAN and Kaplan-Meier Plotter, respectively. LinkedOmics were used to analyze correlation between CTSF and CTSF co-expressed genes. Protein-protein interaction and gene-gene interaction were analyzed using STRING and GeneMANIA, respectively. Association of CTSF with molecular markers of immune cells and immunomodulators was analyzed with Immunedeconv and TISIDB, respectively. RESULTS: CTSF expression was currently only available for patients with NSCLC. Compared to normal tissues, CTSF was downregulated in NSCLC samples and high expressed CTSF was correlated with favorable prognosis of NSCLC. Additionally, CTSF expression was correlated with that of immune cell molecular markers and immunomodulators both in LUAD and LUSC. Noticeably, high expression of CTSF-related CTLA-4 was found to be associated with better OS of LUAD patients. Increased expression of CTSF-related LAG-3 was related with poor prognosis of LUAD patients while there was no association between CTSF-related PD-1/PD-L1 and prognosis of LUAD patients. Moreover, increased expression of CTSF-related CD27 was related with poor prognosis of LUAD patients while favorable prognosis of LUSC patients. CONCLUSIONS: CTSF might play an anti-tumor effect via regulating immune response of NSCLC.

Long noncoding RNA LINC00982 upregulates CTSF expression to inhibit gastric cancer progression via the transcription factor HEY1.

Upregulating the expression of long noncoding RNA LINC00982 controlled cell proliferation in gastric cancer, but the regulatory molecular mechanisms are yet to be expounded. We here aimed to elaborate how LINC00982 regulated the malignancy of gastric cancer cells. RT-qPCR and Western blot analysis were used to detect the expression of LINC00982 and cathepsin F (CTSF) in gastric cancer tissues and cells. Modulatory effect of LINC00982 on gastric cancer cells was assessed by CCK-8, colony formation, Transwell migration, and invasion assays. The relationship between LINC00982, YRPW motif 1 (HEY1), and CTSF was examined by RNA-binding protein immunoprecipitation, luciferase assay, and chromatin immunoprecipitation, and their interaction in the regulation of gastric cancer cellular functions was analyzed by performing gain-of-function and rescue assays. The nude mouse model of tumor formation was developed to examine the effects of LINC00982 on tumorigenesis. LINC00982 was lowly expressed in gastric cancer tissues, whereas its overexpression impaired the proliferative, migratory, and invasive properties of gastric cancer cells. Furthermore, LINC00982 could bind to transcription factor HEY1 and inhibited its expression. Through blocking the binding of HEY1 to CTSF promoter, LINC00982 promoted the expression of CTSF. Overexpression of HEY1 or inhibition of CTSF could reverse the antitumor effects of LINC00982 on gastric cancer, which were further demonstrated in vivo. All these taken together, LINC00982 acted as a tumor suppressor in gastric cancer, which is therefore suggested to be a potential antitumor target for gastric cancer.NEW & NOTEWORTHY We identified LINC00982 as a promising antitumor target for the treatment of patients with gastric cancer. We also determined a regulatory network involved in the pathophysiology of gastric cancer wherein LINC00982 could bind to HEY1 to impair its binding to cathepsin F (CTSF) promoter and hence promote CTSF expression, which aids in better understanding of molecular mechanisms related to gastric tumorigenesis.

Expression of Cathepsin F in response to bacterial challenges in Yesso scallop Patinopecten yessoensis.

Cathepsin F is a unique papain cysteine proteinase with highly conserved structures: catalytic triad and a cystatin domain contained in the elongated N-terminal pro-region. It has been reported that cathepsin F is associated with the establishment of innate immune in several vertebrate including fish in aquaculture, but not known in bivalves. In this study, we firstly identified and characterized cathepsin F in the Yesso scallop (Patinopecten yessoensis). The protein structural and phylogenetic analyses were then conducted to determine its identity and evolutionary position. We've also investigated the expression levels of cathepsin F gene at different embryonic developmental stages, in healthy adult tissues and especially in the hemocytes and hepatopancreas after Gram-positive (Micrococcus luteus) and negative (Vibrio anguillarum) challenges using quantitative real-time PCR (qPCR). Cathepsin F was significantly up-regulated 3 h after infection of V. anguillarum in hemocytes, suggesting its participation in immune response. Our findings have provided strong evidence that cathepsin F may be a good target for enhancing the immune activity in Yesso scallop.

Cathepsin F Knockdown Induces Proliferation and Inhibits Apoptosis in Gastric Cancer Cells.

Gastric cancer (GC) is one of the most common cancers in the world. The cathepsin F (CTSF) gene has recently been found to participate in the progression of several types of cancer. However, the clinical characteristics and function of CTSF in GC as well as its molecular mechanisms are not clear. Six GC cell lines and 44 paired adjacent noncancerous and GC tissue samples were used to assess CTSF expression by quantitative polymerase chain reaction (qPCR). We used lentivirus-mediated small hairpin RNA (Lenti-shRNA) against CTSF to knock down the expression of CTSF in GC cells. Western blot and qPCR were used to analyze the mRNA and related protein expression. The biological phenotypes of gastric cells were examined by cell proliferation and apoptosis assays. Microarray-based mRNA expression profile screening was also performed to evaluate the potential molecular pathways in which CTSF may be involved. The CTSF mRNA level was associated with tumor differentiation, depth of tumor invasion, and lymph node metastasis. Downregulation of CTSF expression efficiently inhibited apoptosis and promoted the proliferation of GC cells. Moreover, a total of 1,117 upregulated mRNAs and 1,143 downregulated mRNAs were identified as differentially expressed genes (DEGs). Further analysis identified the involvement of these mRNAs in cancer-related pathways and various other biological processes. Nine DEGs in cancer-related pathways and three downstream genes in the apoptosis pathway were validated by Western blot, which was mainly in agreement with the microarray data. To our knowledge, this is the first report investigating the effect of CTSF on the growth and apoptosis in GC cells and its clinical significance. The CTSF gene may function as a tumor suppressor in GC and may be a potential therapeutic target in the treatment of GC.

Novel compound heterozygous mutations causing Kufs disease type B.

BACKGROUND: Kufs disease type B (also termed CLN13), an adult-onset form of neuronal ceroid lipofuscinosis (NCL), is genetically heterogeneous and challenging to diagnose. Recently, mutations in cathepsin-F have been identified as the causative gene for autosomal recessive Kufs disease type B. RESULTS: Here, we report a sporadic case of Kufs disease type B with novel compound heterozygous mutations, a novel missense mutation c.977G>T (p.C326F) and a novel nonsense mutation c.416C>A (p.S139X), in the cathepsin-F gene. The magnetic resonance imaging findings were consistent with those demonstrated in adult neuronal ceroid lipofuscinosis: diffuse cortical atrophy, mild hyperintensity and reduction of the deep white matter on T2-weighted images. A skin biopsy was negative for abnormalities. CONCLUSIONS: Altogether, our findings broaden the mutation database in relation to the neuronal ceroid lipofuscinosis, and the clinical diagnosis of Kufs disease type B was confirmed.

The involvement of cathepsin F gene (CTSF) in turbot (Scophthalmus maximus L.) mucosal immunity.

Cathepsin F (CTSF) is a recently described papain-like cysteine protease and unique among cathepsins due to an elongated N-terminal pro-region, which contains a cystatin domain. CTSF likely plays a regulatory role in processing the invariant chain which is associated with the major histocompatibility complex (MHC) class II. In this regard, we identified the CTSF gene of turbot as well as its protein structure, phylogenetic relationships, and expression patterns in mucosal tissues following Vibrio anguillarum and Streptococcus iniae challenge. We also determined the expression patterns of CTSF in mucosal tissues after vaccinated with the formalin-inactivated V. vulnificus whole-cell vaccine. Briefly, turbot CTSF gene showed the closest relationship with that of Paralichthys olivaceus in phylogenetic analysis. And CTSF was ubiquitously expressed in all tested tissues with the highest expression level in gill. In addition, CTSF gene showed different expression patterns following different bacterial challenge. The significant quick regulation of CTSF in mucosal surfaces against infection indicated its roles in mucosal immunity. Functional studies should further characterize avail utilization of CTSF function to increase the disease resistance of turbot in maintaining the integrity of the mucosal barriers against infection and to facilitate selection of the disease resistant family/strain in turbot.

Alu-Derived Alternative Splicing Events Specific to Macaca Lineages in CTSF Gene.

Cathepsin F, which is encoded by CTSF, is a cysteine proteinase ubiquitously expressed in several tissues. In a previous study, novel transcripts of the CTSF gene were identified in the crab-eating monkey deriving from the integration of an Alu element-AluYRa1. The occurrence of AluYRa1-derived alternative transcripts and the mechanism of exonization events in the CTSF gene of human, rhesus monkey, and crab-eating monkey were investigated using PCR and reverse transcription PCR on the genomic DNA and cDNA isolated from several tissues. Results demonstrated that AluYRa1 was only integrated into the genome of Macaca species and this lineage-specific integration led to exonization events by producing a conserved 3' splice site. Six transcript variants (V1-V6) were generated by alternative splicing (AS) events, including intron retention and alternative 5' splice sites in the 5' and 3' flanking regions of CTSF_AluYRa1. Among them, V3-V5 transcripts were ubiquitously expressed in all tissues of rhesus monkey and crab-eating monkey, whereas AluYRa1-exonized V1 was dominantly expressed in the testis of the crab-eating monkey, and V2 was only expressed in the testis of the two monkeys. These five transcript variants also had different amino acid sequences in the C-terminal region of CTSF, as compared to reference sequences. Thus, species-specific Alu-derived exonization by lineage-specific integration of Alu elements and AS events seems to have played an important role during primate evolution by producing transcript variants and gene diversification.

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells.

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

Molecular characterization of a cathepsin F-like protease in Trichinella spiralis.

BACKGROUND: Trichinellosis is a re-emerging infectious disease, caused by Trichinella spp. Cathepsin F belongs to cysteine protease that is a major virulence factor for parasitic helminths, and it may be a potential anti-helminth drug target and vaccine candidate. The aim of this study was to clone, express and identify a cathepsin F-like protease in Trichinella spiralis and to investigate its biochemical characteristics. METHODS: The full-length cDNA encoding a putative cathepsin F-like protease in T. spiralis, TsCF1, was cloned and its biochemical characterization and expression profile were analyzed. Transcription of TsCF1 at different developmental stages of T. spiralis was observed by RT-PCR. The recombinant TsCF1 protein was expressed by prokaryotic expression system and recombinant TsCF1 (rTsCF1) was analyzed by western blotting. And expression of TsCF1 at muscle larvae stage was performed by immunofluorescent technique. Molecular modeling of TsCF1 and its binding mode with E-64 and K11777 were analyzed. Enzyme activity and inhibitory test with E-64 as inhibitor were investigated by using Z-Phe-Arg-AMC as specific substrate. RESULTS: Sequence analysis revealed that TsCF1 ORF encodes a protein of 366 aa with a theoretical molecular weight of 41.9 kDa and an isoelectric point of 7.46. The cysteine protease conserved active site of Cys173, His309 and Asn333 were identified and cathepsin F specific motif ERFNAQ like KLFNAQ sequence was revealed in the propeptide of TsCF1. Sequence alignment analysis revealed a higher than 40 % identity with other cathepsin F from parasitic helminth and phylogenetic analysis indicated TsCF1 located at the junction of nematode and trematode. RT-PCR revealed the gene was expressed in muscle larvae, newborn larvae and adult stages. SDS-PAGE revealed the recombinant protein was expressed with the molecular weight of 45 kDa. The purified rTsCF1 was used to immunize rabbit and the immune serum could recognize a band of about 46 kDa in soluble protein of adult, muscle larvae and ES product of muscle larvae. Immunolocalization analysis showed that TsCF1 located on the cuticle and stichosome of the muscle larvae. After renaturation rTsCF1 demonstrated substantial enzyme activity to Z-Phe-Arg-AMC substrate with the optimal pH 5.5 and this activity could be inhibited by cysteine protease inhibitor E-64. Further analysis showed the kinetic parameters of rTsCF1 to be Km = 0.5091 µM and Vmax = 6.12 RFU/s µM at pH 5.5, and the IC50 value of E64 was 135.50 ± 16.90 nM. CONCLUSION: TsCF1 was expressed in all stages of T. spiralis and localized in the cuticle and stichosome. TsCF1 might play a role in the life cycle of T. spiralis and could be used as a potential vaccine candidate and drug target against T. spiralis infection.

Lysosomal integral membrane protein type-2 (LIMP-2/SCARB2) is a substrate of cathepsin-F, a cysteine protease mutated in type-B-Kufs-disease.

The lysosomal integral membrane protein type-2 (LIMP-2/SCARB2) has been identified as a receptor for enterovirus 71 uptake and mannose-6-phosphate-independent lysosomal trafficking of the acid hydrolase ß-glucocerebrosidase. Here we show that LIMP-2 undergoes proteolytic cleavage mediated by lysosomal cysteine proteases. Heterologous expression and in vitro studies suggest that cathepsin-F is mainly responsible for the lysosomal processing of wild-type LIMP-2. Furthermore, examination of purified lysosomes revealed that LIMP-2 undergoes proteolysis in vivo. Mutations in the gene encoding cathepsin-F (CTSF) have recently been associated with type-B-Kufs-disease, an adult form of neuronal ceroid-lipofuscinosis. In this study we show that disease-causing cathepsin-F mutants fail to cleave LIMP-2. Our findings provide evidence that LIMP-2 represents an in vivo substrate of cathepsin-F with relevance for understanding the pathophysiology of type-B-Kufs-disease.

Expression characteristics and specific antibody reactivity of diverse cathepsin F members of Paragonimus westermani.

Paragonimiasis, caused by the lung fluke Paragonimus, is a major food-borne helminthic disease. Differential diagnosis of paragonimiasis from tuberculosis and other infectious granulomas in the lung is a prerequisite to proper management of patients. Cysteine proteases of Paragonimus westermani (PwCPs) invoke specific antibody responses against patient sera, while antibody capturing activity of different PwCPs has not been comparatively analyzed. In this study, we observed the expressional regulation of 11 species of different PwCPs (PwCP1-11). We expressed recombinant PwCPs and assessed diagnostic reliability employing sera from patients with P. westermani (n=138), other trematodiases (n=80), cestodiases (n=60) and pulmonary tuberculosis (n=20), and those of normal controls (n=20). PwCPs formed a monophyletic clade into cathepsin F and showed differential expression patterns along with developmental stages of worm. Bacterially expressed recombinant PwCPs (rPwCPs) exhibited variable sensitivity of 38.4-84.5% and specificity of 87.2-100% in diagnosing homologous infection. rPwCPs recognized specific antibodies of experimental cat sera as early as 3 or 6weeks after infection. Patient sera of fascioliasis, Schistosomiasis japonicum and clonorchiasis demonstrated weak cross-reactions. Our results demonstrate that diverse PwCPs of the cathepsin F family participate in inducing specific antibody responses. Most P. westermani cathepsin F, except for PwCP2 (AAF21461), which showed negligible antibody responses, might be applicable for paragonimiasis serodiagnosis.

Enhanced wound healing, kinase and stem cell marker expression in diabetic organ-cultured human corneas upon MMP-10 and cathepsin F gene silencing.

PURPOSE: Diabetic corneas overexpress proteinases including matrix metalloproteinase-10 (M10) and cathepsin F (CF). Our purpose was to assess if silencing M10 and CF in organ-cultured diabetic corneas using recombinant adenovirus (rAV)-driven small hairpin RNA (rAV-sh) would normalize slow wound healing, and diabetic and stem cell marker expression. METHODS: Sixteen pairs of organ-cultured autopsy human diabetic corneas (four per group) were treated with rAV-sh. Proteinase genes were silenced either separately, together, or both, in combination (Combo) with rAV-driven c-met gene overexpression. Fellow control corneas received rAV-EGFP. Quantitative RT-PCR confirmed small hairpin RNA (shRNA) silencing effect. Ten days after transfection, 5-mm epithelial wounds were made with n-heptanol and healing time recorded. Diabetic, signaling, and putative stem cell markers were studied by immunofluorescence of corneal cryostat sections. RESULTS: Proteinase silencing reduced epithelial wound healing time versus rAV-enhanced green fluorescent protein (EGFP) control (23% for rAV-shM10, 31% for rAV-shCF, and 36% for rAV-shM10 + rAV-shCF). Combo treatment was even more efficient (55% reduction). Staining patterns of diabetic markers (α3ß1 integrin and nidogen-1), and of activated epidermal growth factor receptor and its signaling target activated Akt were normalized upon rAV-sh treatment. Combo treatment also restored normal staining for activated p38. All treatments, especially the combined ones, increased diabetes-altered staining for putative limbal stem cell markers, ΔNp63α, ABCG2, keratins 15 and 17, and laminin γ3 chain. CONCLUSIONS: Small hairpin RNA silencing of proteinases overexpressed in diabetic corneas enhanced corneal epithelial and stem cell marker staining and accelerated wound healing. Combined therapy with c-met overexpression was even more efficient. Specific corneal gene therapy has a potential for treating diabetic keratopathy.

N-terminally truncated forms of human cathepsin F accumulate in aggresome-like inclusions.

The contribution of individual cysteine cathepsins as positive mediators of programmed cell death is dependent on several factors, such as the type of stimuli, intensity and duration of the stimulus, and cell type involved. Of the eleven human cysteine cathepsins, cathepsin F is the only cathepsin that exhibits an extended N-terminal proregion, which contains a cystatin-like domain. We predicted that the wild-type human cathepsin F contains three natively disordered regions within the enzyme's propeptide and various amino acid stretches with high fibrillation propensity. Wild-type human cathepsin F and its N-terminally truncated forms, Ala(20)-Asp(484) (Δ(19)CatF), Pro(126)-Asp(484) (Δ(125)CatF), and Met(147)-Asp(484) (Δ(146)CatF) were cloned into the pcDNA3 vector and overexpressed in HEK 293T cells. Wild-type human cathepsin F displayed a clear vesicular labeling and colocalized with the LAMP2 protein, a lysosomal marker. However, all three N-terminally truncated forms of human cathepsin F were recovered as insoluble proteins, suggesting that the deletion of at least the signal peptides (Δ(19)CatF), results in protein aggregation. Noteworthy, they concentrated large perinuclear-juxtanuclear aggregates that accumulated within aggresome-like inclusions. These inclusions showed p62-positive immunoreactivity and were colocalized with the autophagy marker LC3B, but not with the LAMP2 protein. In addition, an approximately 2-3 fold increase in DEVDase activity was not sufficient to induce apoptotic cell death. These results suggested the clearance of the N-terminally truncated forms of human cathepsin F via the autophagy pathway, underlying its protective and prosurvival mechanisms.

Cathepsin F mutations cause Type B Kufs disease, an adult-onset neuronal ceroid lipofuscinosis.

Kufs disease, an adult-onset neuronal ceroid lipofuscinosis, is challenging to diagnose and genetically heterogeneous. Mutations in CLN6 were recently identified in recessive Kufs disease presenting as progressive myoclonus epilepsy (Type A), whereas the molecular basis of cases presenting with dementia and motor features (Type B) is unknown. We performed genome-wide linkage mapping of two families with recessive Type B Kufs disease and identified a single region on chromosome 11 to which both families showed linkage. Exome sequencing of five samples from the two families identified homozygous and compound heterozygous missense mutations in CTSF within this linkage region. We subsequently sequenced CTSF in 22 unrelated individuals with suspected recessive Kufs disease, and identified an additional patient with compound heterozygous mutations. CTSF encodes cathepsin F, a lysosomal cysteine protease, dysfunction of which is a highly plausible candidate mechanism for a storage disorder like ceroid lipofuscinosis. In silico modeling suggested the missense mutations would alter protein structure and function. Moreover, re-examination of a previously published mouse knockout of Ctsf shows that it recapitulates the light and electron-microscopic pathological features of Kufs disease. Although CTSF mutations account for a minority of cases of type B Kufs, CTSF screening should be considered in cases with early-onset dementia and may avoid the need for invasive biopsies.

A cathepsin F-like peptidase involved in barley grain protein mobilization, HvPap-1, is modulated by its own propeptide and by cystatins.

Among the C1A cysteine proteases, the plant cathepsin F-like group has been poorly studied. This paper describes the molecular and functional characterization of the HvPap-1 cathepsin F-like protein from barley. This peptidase is N-glycosylated and has to be processed to become active by its own propeptide being an important modulator of the peptidase activity. The expression pattern of its mRNA and protein suggest that it is involved in different proteolytic processes in the barley plant. HvPap-1 peptidase has been purified in Escherichia coli and the recombinant protein is able to degrade different substrates, including barley grain proteins (hordeins, albumins, and globulins) stored in the barley endosperm. It has been localized in protein bodies and vesicles of the embryo and it is induced in aleurones by gibberellin treatment. These three features support the implication of HvPap-1 in storage protein mobilization during grain germination. In addition, a complex regulation exerted by the barley cystatins, which are cysteine protease inhibitors, and by its own propeptide, is also described.

Molecular cloning of a multidomain cysteine protease and protease inhibitor precursor gene from the tobacco hornworm (Manduca sexta) and functional expression of the cathepsin F-like cysteine protease domain.

A Manduca sexta (tobacco hornworm) cysteine protease inhibitor, MsCPI, purified from larval hemolymph has an apparent molecular mass of 11.5 kDa, whereas the size of the mRNA is very large (∼9 kilobases). MsCPI cDNA consists of a 9,273 nucleotides that encode a polypeptide of 2,676 amino acids, which includes nine tandemly repeated MsCPI domains, four cystatin-like domains and one procathepsin F-like domain. The procathepsin F-like domain protein was expressed in Escherichia coli and processed to its active mature form by incubation with pepsin. The mature enzyme hydrolyzed Z-Leu-Arg-MCA, Z-Phe-Arg-MCA and Boc-Val-Leu-Lys-MCA rapidly, whereas hydrolysis of Suc-Leu-Tyr-MCA and Z-Arg-Arg-MCA was very slow. The protease was strongly inhibited by MsCPI, egg-white cystatin and sunflower cystatin with K(i) values in the nanomolar range. When the MsCPI tandem protein linked to two MsCPI domains was treated with proteases, it was degraded by the cathepsin F-like protease. However, tryptic digestion converted the MsCPI tandem protein to an active inhibitory form. These data support the hypothesis that the mature MsCPI protein is produced from the MsCPI precursor protein by trypsin-like proteases. The resulting mature MsCPI protein probably plays a role in the regulation of the activity of endogenous cysteine proteases.

A family of cathepsin F cysteine proteases of Clonorchis sinensis is the major secreted proteins that are expressed in the intestine of the parasite.

Cysteine proteases of helminth parasites play essential roles in parasite physiology as well as in a variety of important pathobiological processes. In this study, we identified a multigene family of cathepsin F cysteine proteases in Clonorchis sinensis (CsCFs). We identified a total of 12 CsCF genes through cDNA cloning using degenerate PCR primers followed by RACE. Sequence and phylogenetic analysis of the genes suggested they belonged to the cathepsin F-like enzyme family and further clustered into three different subfamilies. Enzymatic and proteomic analysis of C. sinensis excretory and secretory products (ESP) revealed that multiple isoforms of CsCF were the major proteins present in the ESP and the proteolytic activity of the ESP is mainly attributable to the enzymes. Comparative analysis of representative enzymes for each subfamily, CsCF-4, CsCF-6, and CsCF-11, showed that they share similar biochemical properties typical for cathepsin F-like enzymes, but significant differences were also identified. The enzymes were expressed throughout various developmental stages of the parasite and the transcripts increased gradually in accordance with the maturation of the parasite. Immunolocalization analysis of CsCFs showed that they were mainly localized in the intestine and intestinal contents of the parasite. These results collectively suggested that CsCFs, which are apparently synthesized in the epithelial cells lining the parasite intestine and secreted into the intestinal lumen of the parasite, might have a cooperative role for nutrient uptake in the parasite. Furthermore, they were eventually secreted into outside of the parasite and may perform additional functions for host-parasite interactions.

Adenovirus-driven overexpression of proteinases in organ-cultured normal human corneas leads to diabetic-like changes.

Our previous data suggested the involvement of matrix metalloproteinase-10 (MMP-10) and cathepsin F (CTSF) in the basement membrane and integrin changes occurring in diabetic corneas. These markers were now examined in normal human organ-cultured corneas upon recombinant adenovirus (rAV)-driven transduction of MMP-10 and CTSF genes. Fifteen pairs of normal autopsy human corneas were used. One cornea of each pair was transduced with rAV expressing either CTSF or MMP-10 genes. 1-2 x 10(8) plaque forming units of rAV per cornea were added to cultures for 48 h with or without sildenafil citrate. The fellow cornea of each pair received control rAV with vector alone. After 6-10 days additional incubation without rAV, corneas were analyzed by Western blot or immunohistochemistry, or tested for healing of 5-mm circular epithelial wounds caused by topical application of n-heptanol. Sildenafil significantly increased epithelial transduction efficiency, apparently by stimulation of rAV endocytosis through caveolae. Corneas transduced with CTSF or MMP-10 genes or their combination had increased epithelial immunostaining of respective proteins compared to fellow control corneas. Staining for diabetic markers integrin alpha(3)beta(1), nidogen-1, nidogen-2, and laminin gamma2 chain became weaker and irregular upon proteinase transduction. Expression of phosphorylated Akt was decreased in proteinase-transduced corneas. Joint overexpression of both proteinases led to significantly slower corneal wound healing that became similar to that observed in diabetic corneas. The data suggest that MMP-10 and CTSF may be responsible for abnormal marker patterns and impaired wound healing in diabetic corneas. Inhibition of these proteinases in diabetic corneas may alleviate diabetic keratopathy symptoms.