Cathepsin L Research Articles

Immunodiagnosis of Fasciola gigantica Infection Using Monoclonal Antibody-Based Sandwich ELISA and Immunochromatographic Assay for Detection of Circulating Cathepsin L1 Protease.

BackgroundTropical fasciolosis caused by Fasciola gigantica infection is one of the major diseases infecting ruminants in the tropical regions of Africa and Asia including Thailand. Parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden.MethodsIn this study, we have produced a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1 (rFgCatL1), and developed both sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunochromatographic (IC) test for rapid detection of circulating cathepsin L1 protease (CatL1) in the sera from mice experimentally and cattle naturally infected with Fasciola gigantica. MoAb 4E3 and biotinylated rabbit anti-recombinant CatL1 antibody were selected due to their high reactivities and specificities.ResultsThe lower detection limits of sandwich ELISA and IC test were 3 pg/ml and 0.256 ng/ml, respectively. Sandwich ELISA and IC test could detect F. gigantica infection from day 1 to 35 post infection. In experimental mice, the sensitivity, specificity and accuracy were 95%, 100% and 98.6% (for sandwich ELISA), and 93%, 100% and 98.2% (for IC test), while in natural cattle they were 98.3%, 100% and 99.5% (for sandwich ELISA), and 96.7%, 100% and 99.1% (for IC test).ConclusionsThese two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica.

Cathepsin Lis involved in proliferation and invasion of breast cancer cells.

Cathepsin L(CTSL), alysosomal endopeptidase was found overexpressed in Breast cancer (BC). The purpose of this work was to investigate the possible role of CTSL in the development of BC. RNA interference(RNAi) with aCTSL small hairpin RNAs(CTSL-shRNA) and plasmid with CTSL were used to identify the effects of CTSL on malignant behaviors of BC. MCF-7 and SKBR-3 were selected as cell models in vitro and in vivo. The results showed that down-regulation of CTSL can significantly inhibit the proliferative and invasive ability of MCF-7 cell, while up-regulation of CTSL in SKBR-3 cells had opposite effects. Comparing to parental BC cells, CTSL knockdown cells exhibited attenuated capacities in developing tumor in nude mice, furthermore, the growth of these xenografts were dramatically regressed. In conclusion, our findings suggest that CTSL contributes to the proliferation and metastasis of BC and might be apotent molecular target for BC treatment.

Synthesis and Preclinical Evaluation of a Highly Improved Anticancer Prodrug Activated by Histone Deacetylases and Cathepsin L.

Lack of absolute selectivity against cancer cells is a major limitation for current cancer therapies. In the previous study, we developed a prodrug strategy for selective cancer therapy using a masked cytotoxic agent puromycin [Boc-Lys(Ac)-Puromycin], which can be sequentially activated by histone deacetylases (HDACs) and cathepsin L (CTSL) to kill cancer cells expressing high levels of both enzymes. Despite the promise as a selective cancer therapy, its requirement of relatively high dosage could be a potential issue in the clinical setting. To address this issue, we aimed to further improve the overall efficacy of our prodrug strategy. Since the proteolytic cleavage by CTSL is the rate-limiting step for the drug activation, we sought to improve the substrate structure for CTSL activity by modifying the α-amino protecting group of lysine. Here we show that protection with Fmoc [Fmoc-Lys(Ac)-Puromycin] exhibits a marked improvement in overall anticancer efficacy compared to the original Boc-Lys(Ac)-Puromycin and this is mainly due to the highly efficient cellular uptake besides its improved substrate structure. Furthermore, to address a concern that the improved drug efficacy might direct high toxicity to the normal cells, we confirmed that Fmoc-Lys(Ac)-Puromycin still retains excellent cancer selectivity in vitro and no obvious systemic off-target toxicity in vivo. Thus our preclinical evaluation data presented here demonstrate that the Fmoc-Lys(Ac)-Puromycin exhibits substantially improved anticancer efficacy, further supporting our approach for the selective cancer therapy.

2'-Hydroxychalcones and their Cyclized Derivatives as Cathepsin L Inhibitors

2'-Hydroxychalcones and their Cyclized Derivatives as Cathepsin L Inhibitors

Cathepsin D in pancreatic acinar cells is implicated in cathepsin B and L degradation, but not in autophagic activity

Cathepsin D (CD) is the major lysosomal aspartic protease and is widely distributed in the cells of various mammalian tissues. CD participates in various physiological events such as regulation of programmed cell death, activation of enzymatic precursors, and metabolic degradation of intracellular proteins through macroautophagy.To investigate the role of CD in pancreatic acinar cells, which constitute the exocrine pancreas, we generated and examined mice specifically deficient for CD in pancreatic acinar cells. CD deficient mice showed normal pancreatic development and autophagic activity, although LC3-II, which is a marker of the autophagosome, accumulates in both physiological and pancreatitis conditions. Moreover, CD deficiency leads to accumulation of matured cathepsin B (CB) and cathepsin L (CL) which are members of the cysteine protease family. We therefore conclude that CD in pancreatic acinar cells is implicated in CB and CL degradation but not in autophagic activity.

Development of a New Antileishmanial Aziridine-2,3-Dicarboxylate-Based Inhibitor with High Selectivity for Parasite Cysteine Proteases.

Leishmaniasis is one of the major neglected tropical diseases of the world. Druggable targets are the parasite cysteine proteases (CPs) of clan CA, family C1 (CAC1). In previous studies, we identified two peptidomimetic compounds, the aziridine-2,3-dicarboxylate compounds 13b and 13e, in a series of inhibitors of the cathepsin L (CL) subfamily of the papain clan CAC1. Both displayed antileishmanial activity in vitro while not showing cytotoxicity against host cells. In further investigations, the mode of action was characterized in Leishmania major. It was demonstrated that aziridines 13b and 13e mainly inhibited the parasitic cathepsin B (CB)-like CPC enzyme and, additionally, mammalian CL. Although these compounds induced cell death of Leishmania promastigotes and amastigotes in vitro, the induction of a proleishmanial T helper type 2 (Th2) response caused by host CL inhibition was observed in vivo. Therefore, we describe here the synthesis of a new library of more selective peptidomimetic aziridine-2,3-dicarboxylates discriminating between host and parasite CPs. The new compounds are based on 13b and 13e as lead structures. One of the most promising compounds of this series is compound s9, showing selective inhibition of the parasite CPs LmaCatB (a CB-like enzyme of L. major; also named L. major CPC) and LmCPB2.8 (a CL-like enzyme of Leishmania mexicana) while not affecting mammalian CL and CB. It displayed excellent leishmanicidal activities against L. major promastigotes (50% inhibitory concentration [IC50] = 37.4 μM) and amastigotes (IC50 = 2.3 μM). In summary, we demonstrate a new selective aziridine-2,3-dicarboxylate, compound s9, which might be a good candidate for future in vivo studies.

T26: Tumour secreted factors cathepsins D and L induce pro-angiogenic changes in human omental microvascular endothelial cells (HOMECs) in ovarian cancer metastasis

Background Epithelial ovarian cancer frequently metastasizes to the omentum, a process that requires pro-angiogenic activation of HOMECs by tumour -secreted factors in their microenvironment. We have previously shown that ovarian cancer cells secrete a range of factors with possible roles in metastatic angiogenesis including the lysosomal proteases cathepsin D (CD) and cathepsin L (CL). However, the role of these proteases in ovarian cancer metastasis to the omentum is not fully understood. Aim To investigate whether proliferative effects of CD and CL are dependent on their catalytic activity in HOMECs. To investigate the intracellular signalling kinases activated by CD and CL. Method HOMEC proliferation was assessed by using a colorimetric (WST1) assay. Potential signalling pathways were examined by phosphokinase array and ELISAs. pH experiments were carried out examine whether the observed effects were due to the catalytic activity of CD and CL. Result CD and CL (50 ng/ml) significantly increased HOMEC proliferation to 141 ± 27% ( p = 0.001, n = 50) and 151% ± 34% ( p = 0.001, n = 45) respectively vs. control (100%) 72 h post-treatment. Inhibitors of CD and CL enzyme activity had no effect on HOMEC proliferation and subsequent pH data suggest a nonproteolytic mitogenic activity of these cathepsins. Both proteins induced phosphorylation of ERK1/2, AKT and p38 α to ∼2, ∼1.5 and ∼1.5 folds respectively relative to total levels (compared to control). Conclusion CD and CL induced proliferation in HOMECs. CD and CL may non- proteolytically contribute to pro-angiogenic responses of the omental microvasculature. Induction of phosphorylation of proliferative kinases ERK1/2, AKT and p38 α suggest possible downstream signalling cascades of these proteins.

Field and experimental symptomless infections support wandering donkeys as healthy carriers of Trypanosoma vivax in the Brazilian Semiarid, a region of outbreaks of high mortality in cattle and sheep.

BackgroundThe Brazilian Semiarid is the home of the largest herd of donkeys in South America and of outbreaks of Trypanosoma vivax infection of high mortality in dairy cattle and sheep. For a comprehensive understanding of the underlying mechanisms of these outbreaks and epidemiological role of donkeys, we surveyed for T. vivax in wandering donkeys and follow the experimental infection of donkeys and sheep with a highly virulent isolate from the Semiarid.MethodsBlood samples from 180 randomly selected wandering donkeys from the Brazilian Semiarid region were employed for PCV and parasitemia assessments and tested using the T. vivax-specific TviCATL-PCR assay. PCR-amplifed Cathepsin L (CATL) sequences were employed for genotyping and phylogenetic analysis. Four wandering donkeys were experimentally infected with a T. vivax isolate obtained during an outbreak of high mortality in the Semiarid; the control group consisted of two non-inoculated donkeys.ResultsWe detected T. vivax in 30 of 180 wandering donkeys (16.6 %) using TviCATL-PCR. The prevalence was higher during the dry (15.5 %) than the wet season (1.1 %) and more females (23.1 %) than males (8.9 %) were infected. All the PCR-positive donkeys lacked patent parasitemia and showed normal values of body condition score (BCS) and packed cell volume (PCV). To evaluate the probable tolerance of donkeys to T. vivax, we inoculated five donkeys with a highly virulent isolate (TviBrRp) from the Semiarid. All inoculated donkeys became PCR-positive, but their parasitemia was always subpatent. A control goat inoculated with TviBrRp showed increasing parasitemia concurrently with fever, declining PCV, tachycardia, mucous membrane pallor, enlarged lymph nodes and anorexia. None of these signs were observed in donkeys. However, T. vivax from wandering donkeys shared identical or highly similar genotypes (identified by Cathepsin L sequences) with isolates from cattle and sheep outbreaks of acute disease in the Semiarid.ConclusionsThis is the first report of T. vivax in donkeys in Brazil and, to our knowledge, the first experimental infection of donkeys with T. vivax. The symptomless field and experimental infections corroborated that donkeys are more tolerant to T. vivax than other livestock species as shown in African countries. Therefore, farmers, veterinaries and control programmes should be aware of healthy carrier donkeys as a possible source of T. vivax for susceptible livestock species in the Brazilian Semiarid.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1169-7) contains supplementary material, which is available to authorized users.

Overexpression of Cathepsin L is associated with gefitinib resistance in non-small cell lung cancer.

Lung cancer, the most common malignancy, is still the leading cause of cancer-related death worldwide. Non-small-cell lung cancer (NSCLC) accounts for 80% of all lung cancers. Recent studies showed Cathepsin L (CTSL) is overexpressed in various cancerous tissues; however, the association between CTSL expression and EGFR-TKI resistance remains unknown. In this study, we investigated the expression of CTSL in lung cancer specimens and matched normal tissues by quantitative real-time PCR and IHC. The functional role of CTSL in resistant PC-9/GR cell line was investigated by proliferation and apoptosis analysis compared with control PC-9 cells. Our results found that the level of CTSL expression was higher in NSCLC tissues compared with matched normal adjacent tissue samples, and CTSL was more highly expressed in PC-9/GR cells compared to PC-9 cells. Knocking-down of CTSL in PC-9/GR cells could decrease cell proliferation and potentiate apoptosis induced by gefitinib, suggesting CTSL may contribute to gefitinib resistance in NSCLC. CTSL might be explored as a candidate of therapeutic target for modulating EGFR-TKI sensitivity in NSCLC.

HLA-DR peptide occupancy can be regulated with a wide variety of small molecules

abstractHLA-DR is the most commonly expressed and likely the most medically important human MHC class II, antigen presenting protein. In a normal immune response, HLA-DR binds to antigenic peptide and the HLA-DR/peptide complex binds to a T-cell receptor, thus contributing to T-cell activation and stimulation of an immune response against the antigen. When foreign antigen is not present, HLA-DR binds endogenous peptide which, under normal conditions does not stimulate an immune response. In most cases, the human peptide is CLIP, but a certain percentage of HLA-DR molecules will be present at the cell surface with other human peptides. We have recently shown that cell surface, CLIP/HLA-DR ratios are a measure of peptide heterogeneity, and in particular, changes in CLIP/HLA-DR ratios represent changes in the occupancy of HLA-DR by other, endogenous peptides. For example, treatment of cells with the HDAC inhibitor, Entinostat, leads to an upregulation of Cathepsin L1 and replacement of Cathepsin L1 senstitive peptides with HLA-DR binding, Cathepsin L1 resistant peptides, an alteration that can be at least partially assessed via assessment of CLIP/HLA-DR cell surface ratios. Here we assay for CLIP/HLA-DR ratios following treatment of immortalized B-cells with a variety of common drugs, almost all of which indicate significant changes in the CLIP/HLA-DR ratios. Furthermore, the CLIP/HLA-DR ratio changes parallel the impact of the drug panoply on cell viability, suggesting that alterations in the HLA-DR peptidome are governed by a variety of mechanisms, rather than exclusively dependent on a dedicated peptide loading process. These results raise questions about how FDA approved drugs may affect the immune response, and whether any of these drugs could be useful as vaccine adjuvants?

Abstract IA19: Regulation of 53BP1 in BRCA1-deficient cells: Why vitamin D matters and how disparities in vitamin D levels could promote aggressive TNBC in women of color

Abstract Breast cancer is the leading cause of cancer death in women worldwide. BRCA1-deficient and triple-negative breast cancers (TNBC) are particularly aggressive, strike younger, Hispanic and African American women, and are most difficult to treat. These tumors exhibit DNA repair defects, making them vulnerable to DNA damaging therapies. Currently, the most promising therapeutic agents are poly(ADP-ribose) polymerase inhibitors (PARPi), which render cells already deficient in DNA repair, with an unbearable load of DNA damage. Excitement about PARPi has been fueled by clinical trials showing that these compounds, either as single agents or in combination with chemotherapy, have very encouraging outcomes in BRCA1-related and TNBC and with minimal toxicity. However, a significant fraction of these cancers acquire resistance to PARPi. Understanding the molecular mechanisms that confer PARPi resistance will have a big therapeutic impact. Elegant studies demonstrated that loss of the DNA repair factor 53BP1 contributes to progression of BRCA1-deficient and TNBC and to their resistance to PARPi. Thus, raising the levels of 53BP1 in these tumors could represent a new therapeutic strategy. Our team identified a new pathway modulating the levels of 53BP1, which could be exploited to stabilize protein levels. We found that BRCA1-deficient cells activate the degradation of 53BP1 by the protease cathepsin-L (CTSL), and that CTSL inhibition by vitamin D stabilizes 53BP1, reduces proliferation, and increases PARPi and radiation sensitivity. Our studies in human breast tumors also suggest that deficiencies in vitamin D/vitamin D receptor (VDR) axis, more prevalent in women of color, could underlie the activation of CTSL-mediated degradation of 53BP1 in BRCA1-deficient and TNBC. Our current studies are testing the ability of vitamin D treatment to reduce the growth of BRCA1-deficient and TNBC cells, as well as their resistance to PARPi, providing a novel modality of treatment for these cancer patients. Citation Format: Susana Gonzalo. Regulation of 53BP1 in BRCA1-deficient cells: Why vitamin D matters and how disparities in vitamin D levels could promote aggressive TNBC in women of color. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr IA19.

Association Between Polymorphism in the Human Cathepsin L (CTSL1) Promoter with Hypertension in the Uygur, Kazak and Han Populations in China.

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. Case-control 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. 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. 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). The study results indicate the A-allele of rs3118869 is a protective factor in hypertension.

Cathepsin L targeting in cancer treatment.

Proteolytic enzymes may serve as promising targets for novel therapeutic treatment strategies seeking to impede cancer progression and metastasis. One such enzyme is cathepsin L (CTSL), a lysosomal cysteine protease. CTSL upregulation, a common occurrence in a variety of human cancers, has been widely correlated with metastatic aggressiveness and poor patient prognosis. In addition, CTSL has been implicated to contribute to cancer-associated osteolysis, a debilitating morbidity affecting both life expectancy and the quality of life. In this review, we highlight the mechanisms by which CTSL contributes to tumor progression and dissemination and discuss the therapeutic utility of CTSL intervention strategies aimed at impeding metastatic progression and bone resorption.

Abstract 4185: Role of Cathepsin L in breast cancer angiogenesis

Abstract Recognition of the dependence of angiogenesis on proteolytic enzymes has raised significant interest in evaluation of proteases as potential targets to disrupt tumor angiogenesis. While the role of cysteine proteases in tumor metastasis is well understood, their contribution to tumor angiogenesis remains less explored. Cysteine cathepsins, in particular cathepsin L (CTSL) gets are upregulated in a wide range of human cancers. In transformed cells extracellular CTSL levels can increase up to 200-fold and comprise up to 40% of total secreted proteins. The role of CTSL in promoting tumor metastasis is supported by recent studies in our laboratory which have shown that treatment with the small molecule CTSL inhibitor 3-bromophenyl-3-hydroxyphenyl-ketone thiosemicarbazone (KGP94) significantly impairs the metastatic phenotype of prostate and breast cancer cells. However, the contribution of CTSL to tumor angiogenesis remains less well explored. Thus, the goal of the present studies was to evaluate the role of tumor cell secreted CTSL in tumor cell induced angiogenesis. Upon activation by pro-angiogenic stimuli, endothelial cells migrate and invade through the interstitium to form tubes. The effect of CTSL on the migratory, invasive and tube formation capacity of endothelial cells was therefore investigated. Stimulation of human micro-vascular endothelial cells of the lung (HMVEC-L) with purified human CTSL led to a dose dependent increase in endothelial cell invasion (1.5-fold at 10ng/ml and 2-fold at 100ng/ml). Furthermore, CTSL (100ng/ml) increased the tube forming ability of endothelial cells as demonstrated by an increase in total tube length. Similarly, incubation of HMVEC-L with conditioned media from MDA-MB-231 breast cancer cells, led to a significant increase in their migration, invasion and tube formation capacity (migration 1.3-fold; invasion 2.5-fold). Compared to parental MDA-MB-231 breast cancer cells, CTSL knockdown cells that were inoculated intradermally into ventral skin flaps of female nude mice showed a significant reduction in their ability to induce blood vessel formation (3.5-fold).These proangiogenic endothelial cell functions were accompanied by a significant increase in endothelial cell proliferation upon stimulation with purified CTSL (2-fold). 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. In conclusion, our findings suggest that in addition to its pro-metastatic function, CTSL might further contribute to tumor progression by eliciting various pro-angiogenic endothelial cell functions. Citation Format: Dhivya Sudhan, Belen Rabaglino, Charles Wood, Dietmar Siemann. Role of Cathepsin L in breast cancer angiogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4185. doi:10.1158/1538-7445.AM2015-4185

Vaccine potential of recombinant pro- and mature cathepsinL1 against fasciolosis gigantica in mice

In Fasciola gigantica cathepsin L1 (CatL1) is a family of predominant proteases that is expressed in caecal epithelial cells and secreted into the excretory-secretory products (ES). CatL1 isotypes are expressed in both early and late stages of the life cycle and the parasites use them for migration and digestion. Therefore, CatL1 is a plausible target for vaccination against this parasite. Recombinant pro-F.gigantica CatL1 (rproFgCatL1) and recombinant mature F.gigantica CatL1 (rmatFgCatL1) were expressed in Escherichia coli BL21. The vaccination was performed in Imprinting Control Region (ICR) mice (n=10) by subcutaneous injection with 50μg of rproFgCatL1 and rmatFgCatL1 combined with Freund’s adjuvant. Two weeks after the second boost, mice were infected with 15 metacercariae by the oral route. The level of protection of rproFgCatL1 and rmatFgCatL1 vaccines was estimated to be 39.1, 41.7% and 44.9, 47.2% when compared with non vaccinated-infected and adjuvant-infected controls, respectively. Antibodies in the immune sera of vaccinated mice were shown by immuno-blotting to react with the native FgCatL1 in the extract of newly excysted juveniles (NEJ), 4-week-old juveniles and the ES products of 4 week-old juveniles. By determining the levels of IgG1 and IgG2a in the immune sera, which are indicative of Th2 and Th1 immune response, respectively, it was found that both Th1 and Th2 responses were significantly increased in rproFgCatL1- and rmatFgCatL1-immunized groups compared with the control groups, with higher levels of Th2 (IgG1) than Th1 (IgG2a). The levels of serum aspartate aminotransferase (AST) and alanine transaminase (ALT) in rmatFgCatL1-immunized group showed a significant decrease when compared to rproFgCatL1-immunized group, indicating that rmatFgCatL1-vaccinated mice had reduced liver parenchyma damage. The pathological lesions of liver in vaccinated groups were significantly decreased when compared with control groups. This study indicates that rFgCatL1 has a potential as a vaccine candidate against F. gigantica in mice, and this potential will be tested in ruminants.

Stress-resistant Translation of Cathepsin L mRNA in Breast Cancer Progression

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.

OBJECTIVE FUNCTION, LEAN MASS, AND ASSOCIATED GENETIC ADAPTATIONS OF THE OPERATED LEG FOLLOWING TOTAL HIP ARTHROPLASTY

Purpose: To characterize muscle recovery following total hip arthroplasty (THA) combining genetic adaptations in the affected leg with objective function and body composition assessment. Methods: Preoperatively and at six weeks postoperatively, objective function was assessed by: maximal voluntary contraction of the operated leg quadriceps (MVCOLQ) in Newtons (N), 30[Formula: see text]s chair sit-to-stand (ST), and six-minute walk test (6MWT), with lean mass of the operated leg estimated by dual energy X-ray absorptiometry (DEXA). Genetic adaptations were assessed from vastus lateralis (VL) biopsies by quantitative polymerase chain reaction (qPCR) analysis of markers of hypertrophy (FOS, calpain2 (CAPN2)), atrophy (20[Formula: see text]s proteasome alpha subunit 7 (PSMA7), cathepsin L2 (CTSL2), inflammation (Tumour necrosis factor alpha (TNF-[Formula: see text]), Interleukin-6 (IL-6)) and lipid metabolism (lipoprotein lipase, LPL and peroxisome proliferated activated receptor gamma (PPARAG). Results: 14 patients were recruited. At six weeks, no significant differences, relative to preoperative values, were noted in either objective function or leg lean mass. Markers for hypertrophy were increased (FOS [Formula: see text]1463%, [Formula: see text]), with atrophy (PSMA7 [Formula: see text]44.8%, [Formula: see text]; CTSL2 [Formula: see text]42.5%, [Formula: see text]), inflammation (TNF [Formula: see text]29.6%, [Formula: see text]) and lipid metabolism markers showing a decreasing trend (LPL [Formula: see text]42.45%, [Formula: see text]). Conclusion: The initial post-THA intramuscular environment appears supportive of anabolism. However, this is not reflected in objective function or lean mass measures at six weeks, suggesting longer duration may be required for physiological adaptation to occur.

217 Altered Regulation of the Inhibitor IκBα Contributes to Changes in NF-κB Regulated Genes in the TILRR ko Mouse

<h3>Introduction</h3> Inflammatory responses are key drivers in the pathogenesis of atherosclerosis. We have identified a co-receptor, TILRR, a prominent activator of NF-κB controlled responses, which we have demonstrated is highly expressed in atherosclerotic lesions. Using <i>Apoe^–/–</i>(Apo-lipoprotein E knockout) mice we have recently shown that administration of a blocking peptide antibody, which selectively targets amplification of inflammatory responses through TILRR, significantly reduces progression of atherosclerosis and promotes plaque stability. This study examines the molecular mechanisms underlying the reduced NF-κB activation in the TILRR knockout mouse. <h3>Methods and results</h3> Microarray analysis of blood and spleen monocytes from TILRR knockout mice and littermate controls, revealed pronounced reductions in NF-κB controlled inflammatory genes relevant to development of cardiovascular disease including Cathepsin L1, CXCL1 and CXCL13. Western analysis demonstrated a 65% decrease in IκBα expression and a 60% reduction in inhibitor degradation in IL-1 stimulated monocytes from TILRR-/- mice compared to responses in littermate controls. Parallel <i>in vitro</i> studies were carried out to determine the underlying mechanisms and consequences of these changes on interaction of the inhibitor with NF-κB. Deletion of the ANK 2 region of IκBα resulted in loss of function while the impact of ANK deletions 3, 4, and 5 was not significantly different from the wild type protein. 3-D modelling of the ANK2 deleted protein revealed that the modified protein maintained its overall structure. The modified protein lost its ability to interact with NF-κB, identifying a role for this region in IkBa/NF-kB complex formation. Ongoing studies are evaluating alanine-scanning mutants of conserved residues within the ANK2 region to determine their impact on amplified NF-kB responses. <h3>Conclusion</h3> Our results are consistent with a role for TILRR in regulation of NF-κB controlled inflammatory gene activation <i>in vivo</i>, and suggest that functional alterations in IκBα regulation in part are responsible for the reductions in the inflammatory response.

Insights into the Interactions of Fasciola hepatica Cathepsin L3 with a Substrate and Potential Novel Inhibitors through In Silico Approaches.

Background Fasciola hepatica is the causative agent of fascioliasis, a disease affecting grazing animals, causing economic losses in global agriculture and currently being an important human zoonosis. Overuse of chemotherapeutics against fascioliasis has increased the populations of drug resistant parasites. F. hepatica cathepsin L3 is a protease that plays important roles during the life cycle of fluke. Due to its particular collagenolytic activity it is considered an attractive target against the infective phase of F. hepatica.Methodology/Principal FindingsStarting with a three dimensional model of FhCL3 we performed a structure-based design of novel inhibitors through a computational study that combined virtual screening, molecular dynamics simulations, and binding free energy (ΔGbind) calculations. Virtual screening was carried out by docking inhibitors obtained from the MYBRIDGE-HitFinder database inside FhCL3 and human cathepsin L substrate-binding sites. On the basis of dock-scores, five compounds were predicted as selective inhibitors of FhCL3. Molecular dynamic simulations were performed and, subsequently, an end-point method was employed to predict ΔGbind values. Two compounds with the best ΔGbind values (-10.68 kcal/mol and -7.16 kcal/mol), comparable to that of the positive control (-10.55 kcal/mol), were identified. A similar approach was followed to structurally and energetically characterize the interface of FhCL3 in complex with a peptidic substrate. Finally, through pair-wise and per-residue free energy decomposition we identified residues that are critical for the substrate/ligand binding and for the enzyme specificity.Conclusions/SignificanceThe present study is the first computer-aided drug design approach against F. hepatica cathepsins. Here we predict the principal determinants of binding of FhCL3 in complex with a natural substrate by detailed energetic characterization of protease interaction surface. We also propose novel compounds as FhCL3 inhibitors. Overall, these results will foster the future rational design of new inhibitors against FhCL3, as well as other F. hepatica cathepsins.

A randomized vehicle-controlled proof-of-concept study of human cathepsin-L injections for the treatment of edematous fibrosclerotic panniculopathy (cellulite)

A randomized vehicle-controlled proof-of-concept study of human cathepsin-L injections for the treatment of edematous fibrosclerotic panniculopathy (cellulite)