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 is a potential marker for senescent human skin fibroblasts and keratinocytes associated with skin aging.

Cellular senescence is characterized by cell cycle arrest and the senescence-associated secretory phenotype (SASP) and can be triggered by a variety of stimuli, including deoxyribonucleic acid (DNA) damage, oxidative stress, and telomere exhaustion. Cellular senescence is associated with skin aging, and identification of specific markers of senescent cells is essential for development of targeted therapies. Cathepsin F (CTSF) has been implicated in dermatitis and various cancers and participates in cell immortalization through its association with Bcl family proteins. It is a candidate therapeutic target to specifically label and eliminate human skin fibroblasts and keratinocytes immortalized by aging and achieve skin rejuvenation. In this study, we investigated whether CTSF is associated with senescence in human fibroblasts and keratinocytes. In senescence models, created using replicative aging, ionizing radiation exposure, and the anticancer drug doxorubicin, various senescence markers were observed, such as senescence-associated ß-galactosidase (SA-ß-gal) activity, increased SASP gene expression, and decreased uptake of the proliferation marker BrdU. Furthermore, CTSF expression was elevated at the gene and protein levels. In addition, CTSF-positive cells were abundant in aged human epidermis and in some parts of the dermis. In the population of senescent cells with arrested division, the number of CTSF-positive cells was significantly higher than that in the proliferating cell population. These results suggest that CTSF is a candidate for therapeutic modalities targeting aging fibroblasts and keratinocytes.

Cathepsin F and Fibulin-1 as novel diagnostic biomarkers for brain metastasis of non-small cell lung cancer.

BACKGROUND: The lack of non-invasive methods for detection of early micro-metastasis is a major cause of the poor prognosis of non-small cell lung cancer (NSCLC) brain metastasis (BM) patients. Herein, we aimed to identify circulating biomarkers based on proteomics for the early diagnosis and monitoring of patients with NSCLC BM. METHODS: Upregulated proteins were detected by secretory proteomics in the animal-derived high brain metastatic lung cancer cell line. A well-designed study composed of three independent cohorts was then performed to verify these blood-based protein biomarkers: the serum discovery and verification cohorts (n = 80; n = 459), and the tissue verification cohort (n = 76). Logistic regression was used to develop a diagnostic biomarker panel. Model validation cohort (n = 160) was used to verify the stability of the constructed predictive model. Changes in serum Cathepsin F (CTSF) levels of patients were tracked to monitor the treatment response. Progression-free survival (PFS) and overall survival (OS) were analysed to assess their prognostic relevance. RESULTS: CTSF and Fibulin-1 (FBLN1) levels were specifically upregulated in sera and tissues of patients with NSCLC BM compared with NSCLC without BM and primary brain tumour. The combined diagnostic performance of CTSF and FBLN1 was superior to their individual ones. CTSF serum changes were found to reflect the therapeutic response of patients with NSCLC BM and the trends of progression were detected earlier than the magnetic resonance imaging changes. Elevated expression of CTSF in NSCLC BM tissues was associated with poor PFS, and was found to be an independent prognostic factor. CONCLUSIONS: We report a novel blood-based biomarker panel for early diagnosis, monitoring of therapeutic response, and prognostic evaluation of patients with NSCLC BM.

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.

A Human Stem Cell Model of Fabry Disease Implicates LIMP-2 Accumulation in Cardiomyocyte Pathology.

Here, we have used patient-derived induced pluripotent stem cell (iPSC) and gene-editing technology to study the cardiac-related molecular and functional consequences of mutations in GLA causing the lysosomal storage disorder Fabry disease (FD), for which heart dysfunction is a major cause of mortality. Our in vitro model recapitulated clinical data with FD cardiomyocytes accumulating GL-3 and displaying an increased excitability, with altered electrophysiology and calcium handling. Quantitative proteomics enabled the identification of >5,500 proteins in the cardiomyocyte proteome and secretome, and revealed accumulation of the lysosomal protein LIMP-2 and secretion of cathepsin F and HSPA2/HSP70-2 in FD. Genetic correction reversed these changes. Overexpression of LIMP-2 directly induced the secretion of cathepsin F and HSPA2/HSP70-2, implying causative relationship, and led to massive vacuole accumulation. In summary, our study has revealed potential new cardiac biomarkers for FD, and provides valuable mechanistic insight into the earliest pathological events in FD cardiomyocytes.

Structural basis of the cystein protease inhibitor Clonorchis sinensis Stefin-1.

Cystein protease plays a critical role as a virulence factor in the development and progression of various diseases. Cystatin is a superfamily of cysteine protease inhibitors that participates in various physiological and pathological processes. The cysteine protease inhibitor CsStein-1 isolated from Clonorchis sinensis belongs to the type 1 stefin of cystatins. This inhibitor regulates the activity and processing of CsCF (Cathepsin F of Clonorchis sienesis), which plays an important role in parasite nutrition and host-parasite interaction. CsStefin-1 has also been proposed as a host immune modulator and a participant in the mechanism associated with anti-inflammatory ability. Here, we report the first crystal structure of CsStefin-1 determined by the multi-wavelength anomalous diffraction (MAD) method to 2.3 Å. There are six molecules of CsStefin-1 per asymmetric unit, with a solvent content of 36.5%. The structure of CsStefin-1 is composed of twisted four-stranded antiparallel ß-sheets, a central α-helix, and a short α-helix. We also demonstrate that CsStefin-1 binds to CsCF-8 cysteine protease and inhibits its activity. In addition, a molecular docking model of CsStefin-1 and CsCF-8 was developed using homology modeling based on their structures. The structural information regarding CsStefin-1 and molecular insight into its interaction with CsCF-8 are important to understanding their biological function and to design of inhibitors that modulate cysteine protease activity.

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.

Chicken IgY-based coproantigen capture ELISA for diagnosis of human opisthorchiasis.

Diagnosis of Opisthorchis viverrini infection by conventional stool examination is increasingly difficult due to the low intensity of the infection after several rounds of control programmes in endemic regions as well as coinfections with intestinal flukes. Therefore sensitive and specific diagnostic test is needed. In this study, a coproantigen sandwich ELISA using recombinant O. viverrini cathepsin F (rOv-CF) was developed. This sandwich ELISA employing chicken IgY raised against rOv-CF in combination with rabbit IgG antibody to the somatic O. viverrini antigens showed a lower detection limit (LLD) of 70ng native O. viverrini somatic antigens by spiking the parasite antigens into control feces. When applied to the diagnosis, the IgY-based sandwich ELISA exhibited sensitivity and specificity of 93.3% and 76.7%, respectively, in an investigation of 90 human cases positive or negative for opisthorchiasis. The positive predictive value (PPV) and negative predictive value (NPV) for this coproantigen detection were 66.7% and 95.2%, respectively. This IgY-based sandwich ELISA using parasite cathepsin F detection shows a promising immunodiagnostic alternative for human opisthorchiasis in endemic regions.

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.

Active Site Mapping of Human Cathepsin†F with Dipeptide Nitrile Inhibitors.

Cleavage of the invariant chain is the key event in the trafficking pathway of major histocompatibility complex class II. Cathepsin S is the major processing enzyme of the invariant chain, but cathepsin F acts in macrophages as its functional synergist which is as potent as cathepsin S in invariant chain cleavage. Dedicated low-molecular-weight inhibitors for cathepsin F have not yet been developed. An active site mapping with 52 dipeptide nitriles, reacting as covalent-reversible inhibitors, was performed to draw structure-activity relationships for the non-primed binding region of human cathepsin F. In a stepwise process, new compounds with optimized fragment combinations were designed and synthesized. These dipeptide nitriles were evaluated on human cysteine cathepsins F, B, L, K and S. Compounds 10 (N-(4-phenylbenzoyl)-leucylglycine nitrile) and 12 (N-(4-phenylbenzoyl)leucylmethionine nitrile) were found to be potent inhibitors of human cathepsin F, with Ki values <10 nM. With all dipeptide nitriles from our study, a 3D activity landscape was generated to visualize structure-activity relationships for this series of cathepsin F inhibitors.

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.

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.

Selective nitrile inhibitors to modulate the proteolytic synergism of cathepsins S and F.

A series of dipeptide nitriles with different P3 substituents was designed to explore the S3 binding pocket of cathepsin S. Racemic 7-16 and the enantiopure derivative (R)-22 proved to be potent inhibitors of human cathepsin S and exhibited notable selectivity over human cathepsins L, K, and B. Inhibition of cathepsin F, the functional synergist of cathepsin S, was not observed. The azadipeptide analogue of 22, compound 26, was highly potent but nonselective.

Secreted cysteine proteases of the carcinogenic liver fluke, Opisthorchis viverrini: regulation of cathepsin F activation by autocatalysis and trans-processing by cathepsin B.

Opisthorchis viverrini is an important helminth pathogen of humans that is endemic in Thailand and Laos. Adult flukes reside within host bile ducts and feed on epithelial tissue and blood cells. Chronic opisthorchiasis is associated with severe hepatobiliary diseases such as cholangiocarcinoma. Here we report that adult O. viverrini secrete two major cysteine proteases: cathepsin F (Ov-CF-1) and cathepsin B1 (Ov-CB-1). Ov-CF-1 is secreted as an inactive zymogen that autocatalytically processes and activates to a mature enzyme at pH 4.5 via an intermolecular cleavage at the prosegment-mature domain junction. Ov-CB-1 is also secreted as a zymogen but, in contrast to Ov-CF-1, is fully active against peptide and macromolecular substrates despite retaining the N-terminal prosegment. The active Ov-CB-1 zymogen was capable of trans-activating Ov-CF-1 by proteolytic removal of its prosegment at pH 5.5, a pH at which the Ov-CF-1 zymogen cannot autocatalytically activate. Both cathepsins hydrolyse human haemoglobin but their combined action more efficiently degrades haemoglobin to smaller peptides than each enzyme alone. Ov-CF-1 degraded extracellular matrix proteins more effectively than Ov-CB-1 at physiological pH. We propose that Ov-CB-1 regulates Ov-CF-1 activity and that both enzymes work together to degrade host tissue contributing to the development of liver fluke-associated cholangiocarcinoma.

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.

Biolistic transformation of Schistosoma mansoni: Studies with modified reporter-gene constructs containing regulatory regions of protease genes.

Biolistics of the flatworm parasite Schistosoma mansoni facilitates the accurate spatial expression of transgenes under the control of gene-specific promoter elements. To improve transgene expression, either in the number of positive worms and/or an increased transgene signal per worm, we tested plasmid constructs incorporating 5' and 3' gene-specific genomic fragments, and parts of the open reading frame for two S. mansoni proteases, cathepsins F and D (SmCF and SmCD). GFP-expression was gut-localized, a novel finding for SmCD and consistent with previous data for SmCF. The mCherry fluorescent protein can also operate as a reporter. Though certain constructs imparted stronger and better distributed signals per positive worm, the low yields throughout (1-5% positive per experiment) precluded further quantifications of improvement. Electroporation of the same constructs was also weakly efficient (1-10% positives per experiment). However, reporter signals were found in tissues other than the gut, which may represent dysregulated transcription.

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.

Molecular cloning, mRNA expression and enzymatic characterization of cathepsin F from olive flounder (Paralichthys olivaceus).

Cathepsin F is a recently described papain-like cysteine protease of unknown function, and unique among cathepsins due to an elongated N-terminal pro-region, which contains a cystatin domain. In the present study, the cDNA of olive flounder (Paralichthys olivaceus) cathepsin F (PoCtF) was cloned by the combination of homology molecular cloning and rapid amplification of cDNA ends (RACE) approaches. The PoCtF gene was determined to consist of the 1844 bp nucleotide sequence which encodes for a 475-amino acid polypeptide. The results of RT-PCR analysis revealed ubiquitous expression throughout the entirety of healthy flounder tissues; however the PoCtF expressions increased significantly in gill at 3h post-injection with lipopolysaccharide (LPS). Also, immunostaining using anti-PoCtF antibody was strongest on the epidermal mucus in the fin. The cDNA encoding mature enzyme of PoCtF was expressed in Escherichia coli using the pGEX-4T-1 expression vector system. Its activity was quantified by cleaving the synthetic peptide Z-Phe-Arg-AMC, a substrate commonly used for functional characterization of cysteine proteinases, and the optimal pH for the protease activity was 7.5. The findings of the present study suggest that PoCtF has a higher optimum pH than mammalian cathepsin F, and PoCtF is an interesting target for future investigations of the role of cathepsin F in the epidermal mucus and fish innate immune system.