Cathepsin L Research Articles

GENOMIC, TRANSCRIPTOMIC AND PROTEOMIC TECHNOLOGIES AS A MODERN TOOL FOR HEALTH DISORDERS DIAGNOSTICS, ASSOCIATED WITH THE IMPACT OF ENVIRONMENTAL FACTORS

Introduction. Today, it is relevant to use modern critical technologies for identifying and evaluating the negative effects associated with the effects of chemicals at the stages of pre-nosological changes. This improves the efficiency of the early detection of progress in pre-pathological conditions prior to the onset of pronounced functional changes and the aggravation of the disease. The use of molecular diagnostic methods based on genomic, transcriptomic, and proteomic analysis technologies is one of the most promising approaches. Aim of the work is an analysis of both aspects and practical use of the modern critical technologies capabilities (genomic, transcriptomic and proteomic technologies) in the implementation of biomedical and experimental studies for the tasks of the detection biomarkers of negative effects of chemical risk factors on the example of exposure conditions with aluminum compounds. Material and methods. The proteomic analysis was carried out by the method of two-dimensional electrophoresis, polymorphism of alleles and genotypes of candidate genes by a real-time polymerase chain reaction. The transcriptome state was assessed based on the results of gene expression studies. The expression of membrane and serum proteins was studied by biochemical and immunological methods analysis. Statistical processing of the results was carried out in the systems “Gencalculator,” “Gene Expert” and online program “SNPStats”. Results. The results of using proteomic analysis technologies made it possible to identify proteins annexin-13, SH3-domain protein-RF3, cathepsin L1 and, accordingly, genes CTSL, SH3RF3, THO complex subunit 2 as Ohmic markers of aerogenic exposure of inorganic compounds. The results of the analysis of gene polymorphism in the population exposed to environmental pollution allowed establishing the changed frequency of variant alleles and genotypes of genes: immune control - TLR4 (toll-like receptor); vascular factors - eNOS rs1799983 (endothelial NOsintase); detoxification - coproporphyrinogen oxidase CPOX (rs1131857), cytochrome P450 CYP1A1 (rs 1048943); neuro-humoral regulation of ANKK1 rs1800497 (dopamine receptor gene) and HTR2A rs7997012 (serotonin receptor gene). The results of gene expression analysis made it possible to establish negative transcriptomic effects induced by exposure to amphoteric metals due to the isolation of specific CD4+, CD8+, CD16+ cell phenotypes expressing the proteomic profile gene of blood plasma lipoprotein A (LPA gene). Discussion. The obtained results correspond data of a number of scientific studies, noting the importance of identifying polymorphic deviations of genes determining the individual risk of health problems in a variety of stressful environmental factors affecting humans. Minor genotypes of candidate genes under conditions of excessive contamination with amphoteric metal compounds significantly increase the risk of deviations in immune regulation indices, which modifies apoptosis mechanisms, which are crucial for the formation of atopy and onco-proliferation. Conclusion. The use of genome, transcriptomic and proteomic technologies as a modern tool for the diagnostics of health disorders allowed justifying the set of priority exposition and effect Ohmic-markers, associated with aerogenic effect of amphoteric metals, which have a modifying effect on the pathogenetic mechanisms of the formation of disorders of nervous and immune systems, the 1st and 2nd phase of detoxification, the likelihood of vascular disorders and onco-proliferative processes.

Intracellular complement activation in podocytes aggravates immune kidney injury in trichloroethylene-sensitized mice.

Trichloroethylene (TCE) as a common organic solvent in industrial production can cause occupational medicamentosa-like dermatitis (OMDT) in some exposed workers. In addition to systemic skin damage, OMDT is also accompanied by severe kidney injury. Our previous studies show that complement (C) plays an important role in immune kidney injury caused by TCE. Specifically, C3 is mainly deposited on glomeruli. Recent studies have found that intracellular complement can be activated by cathepsin L (CTSL) and exert a series of biological effects. The purpose of this study was to explore where C3 on glomeruli comes from and what role it plays. A BALB/c mouse model of skin sensitization induced by TCE in the presence or absence of CTSL inhibitor (CTSLi,10 mg/kg). In TCE sensitization-positive mice, C3 was mainly expressed on podocytes and the expression of CTSL significantly increased in podocytes. Kidney function test and related indicators showed abnormal glomerular filtration and transmission electron microscopy revealed ultrastructure damage to podocytes. These lesions were alleviated in TCE/CTSLi positive mice. These results provide the first evidence that in TCE-induced immune kidney injury, intracellular complement in podocytes can be over-activated by CTSL and aggravates podocytes injury, thereby damaging glomerular filtration function. Intracellular complement activation and cathepsin L in podocytes may be a potential target for treating immune kidney injury induced by TCE.

SARS-CoV-2 Related Protein Expression may Predict Vulnerability for Developing COVID-19 in Cancer Patients

Dear Editor, Ongoing epidemic of Coronavirus disease (COVID-19) has presented new challenges to the oncologists. Recent studies have suggested higher COVID-19 related mortality in cancer patients (1), reasons for which are currently little understood. Although, COVID-19 primarily causes acute respiratory distress syndrome (ARDS), widespread inflammation and multi-organ dysfunction are common in progressed disease. Increased fragility, age associated factors, and organ specific or systemic dysfunctions in cancer patients may add to the COVID-19 related pathology increasing the chances of mortality (2). Apart from these obvious risk factors, whether there is a cancer specific vulnerability to COVID-19 is scarcely reported. COVID-19 is caused by a new coronavirus strain ‘Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)’ from the family of betacoronaviruses. SARS-CoV-2 binds to angiotensin converting enzyme 2 (ACE2) protein present on the surface of human epithelial cells which facilitates its entry inside the cell (3). For ACE2 mediated cell entry of the SARS-CoV-2 co-expression of proteases like—Transmembrane protease, serine 2 (TMPRSS2), Cathepsin-L (CTSL) and Furin are essential (3). An analysis of tissue specific expression of SARS-CoV-2 cell entry factors ACE2 and TMPRSS2/CTSL/Furin in human cancer patients can inform on their vulnerability for developing COVID-19. We performed in silico analysis of open access data for SARS-CoV-2 related proteins available on Human Protein Atlas (https://www.proteinatlas.org/humanproteome/sars-cov-2). Human Protein Atlas is a standard database for protein expression in normal and pathological human tissues including 20 different types of cancer (4).The details of the laboratory and statistical methods used to generate expression data used for this study is available on https://www.proteinatlas.org/about/assays+annotation. We noted high tissue expression of the viral entry factors in certain specific cancers (Fig. 1). ACE2 expression was detected in many but was specifically enhanced in renal cancer (detected in 100% of cases) (Fig. 1a&b), and had significant expression in colorectal, gastric, and pancreatic cancers (Fig. 1b). All these cancers also had significant expression of TMPRSS2, CTSL, and Furin (Fig. 1b). Higher expression of ACE2 is regarded favourable for the renal cancer, however it may also make patients vulnerable to SARS-CoV-2 infection (4) with an increased chance of renal dysfunction in COVID-19 (5). Uniquely, prostate cancer patients had highest TMPRSS2 RNA expression. TMPRSS2 is an androgen regulated gene and inhibition of TMPRSS2 using androgen-deprivation therapies (ADTs) has been shown to block or decrease the severity of SARS-CoV2 infections (6). A SARS-CoV-2 cell entry factors based vulnerability for developing COVID-19 appears higher in patients of renal and prostate cancers, however this needs to be investigated further. A conclusive knowledge in this regard can help in prevention and therapeutic management of COVID-19 in these patients. Conflict of Interest Authors declare no conflict of interest. "Data accessibility" statement Data related to this manuscript can be accessed from the corresponding author on a reasonable request.

Synthesis and evaluation of Nα,Nε-diacetyl-l-lysine-inositol conjugates as cancer-selective probes for metabolic engineering of GPIs and GPI-anchored proteins.

Two myo-inositol derivatives having an Nα,Nε-diacetyl-l-lysine (Ac2Lys) moiety linked to the inositol 1-O-position through a self-cleavable linker and a metabolically stable 2-azidoethyl group linked to the inositol 3-O- and 4-O-positions, respectively, were designed and synthesized. The Ac2Lys moiety blocking the inositol 1-O-position required for GPI biosynthesis was expected to be removable by a combination of two enzymes, histone deacetylase (HDAC) and cathepsin L (CTSL), abundantly expressed in cancer cells, but not in normal cells, to transform these inositol derivatives into biosynthetically useful products with a free 1-O-position. As a result, it was found that these inositol derivatives could be incorporated into the glycosylphosphatidylinositol (GPI) biosynthetic pathway by cancer cells, but not by normal cells, to express azide-labeled GPIs and GPI-anchored proteins on cell surfaces. Consequently, this study has established a novel strategy and new molecular tools for selective metabolic labeling of cancer cells, which should be useful for various biological studies and applications.

Evasion of Host Immunity During Fasciola hepatica Infection.

Fasciola hepatica, the common liver fluke, causes infection of livestock throughout temperate regions of the globe. This helminth parasite has an indirect lifecycle, relying on the presence of the mud snail to complete its transition from egg to definitive host (Beesley et al., Transbound Emerg Dis 65:199-216, 2017). Within the definitive host, the parasite excysts in the intestine forming a newly excysted juvenile (NEJ) and migrates via the peritoneal cavity to the liver. Disease resulting from infection can be acute or chronic depending on the host and the number of parasites present. Sheep may succumb to a fatal acute infection if the challenge of metacercariae is great enough. However, in cattle chronic disease is the most likely outcome with parasites surviving for long periods of time. Annual losses are estimated to be in the region of US$ 2000 million to the agricultural industry (Beesley et al., Transbound Emerg Dis 65:199-216, 2017). Management of the disease depends heavily on chemotherapy with triclabendazole being the drug of choice, consistent use for over 20years has resulted in drug-resistant strains emerging worldwide (Beesley et al., Int J Parasitol 47:11-20, 2017). A more sustainable approach to control would be through vaccination and indeed a lead candidate has been identified, cathepsin L1. Despite these promising results the parasite continues to confound our own and host efforts to generate long-lasting and effective immunity. In this brief review we focus our attention on those mechanisms that the parasite utilises to circumvent the innate based defense mechanisms within the host.

Computer-Aided Drug Design for the Identification of Novel Antischistosomal Compounds.

Schistosomiasis is a chronic neglected tropical disease, highlighted by the presence of Schistosoma worms, which presents in advanced cases in approximately 80 countries, affecting almost 300 million people. The treatment is based on only one drug, praziquantel, a drug discovered in the 1970s that shows moderate efficacy against the adult parasite, but low efficacy against the larval stages of the parasite. Therefore, the use of only one drug has brought concerns and losses on drug-resistance cases, necessitating the development of new effective chemotherapeutic agents against Schistosoma species. One of the strategies that have been implemented in drug development is the computer-aided drug design (CADD), investigating the structural characteristics of the compounds and targets in order to understand their actions and biological activities through 3D virtual manipulation, as the QSAR applied to ligands and molecular docking applied to a respective biological target. These studies help to extract information and characteristics relevant to the activity, as well as to predict potential applications and activity. Therefore, this chapter will present the main validated biological targets of the genus Schistosoma, as thioredoxin glutathione reductase (TGR), histone deacetylases (HDAC 1, HDAC 8), dihydroorotate dehydrogenase, sirtuin protein and cathepsin L1, as well as reports of CADD in literature applied to the development of drugs against schistosomiasis, providing compounds with high pharmacological potential and high specificity.

Evaluation of Gelatinolytic and Collagenolytic Activity of Fasciola hepatica Recombinant Cathepsin-L1.

Background: Cysteine proteases of the liver fluke, Fasciola hepatica, participate in catabolism of proteins, migration of the fluke through host tissues and combat host immune systemObjectives: In this study, we evaluated proteolytic activity of F. hepatica recombinant cathepsin L1 (rCL1) against gelatin and collagen as common substratesMaterial and Methods: The coding sequences of F. hepatica CL1 were cloned and expressed in E. coli, in our previous study. The rCL1 was purified by nickel affinity chromatography with a HisTrap Column. The protein concentrations of the purified fractions were determined by Bradford assay. Rat collagen type-1 was treated with distinct amounts of rCL1 at 37 °C, overnight, and the byproduct was analyzed by SDS-PAGE. Furthermore, we used bovine skin gelatin as zymography substrate to evaluate the gelatinolytic activity of the purified rCL1.Results: Recombinant CL1 was capable to digest intact type-1 collagen within 24 h and the gelatinlytic activity of rCL1 was visible at approximately 37 kDa region, with optimal activity at acidified conditions (pH 4)Conclusion: Findings provide a possible mechanism by which a major secretory molecule of F. hepatica could be involved in parasite survival as well as its pathogenesis.

Changes in Proteomic Profiles are Related to Changes in BMI and Fat Distribution During 10 Years of Aging.

This study investigated howchanges in 84 proteins over a 10-year period of aging were related to changes in measures of body fat and distribution over the same period. Cardiovascular candidate proteins were measured using the proximal extension assay technique, along with BMI and waist-hip ratio (WHR), at ages 70, 75, and 80 in 1,016 participants of the Prospective Investigation of the Vasculature in Uppsala Seniors(PIVUS) cohort. Associations of changes in plasma protein levels, BMI, and WHR over time were analyzed using linear mixed models. Changes in 19 and 16 proteins were significantly associated with changes in BMI and WHR, respectively (P < 0.00059), over the investigated 10-year period. Leptin and fatty acid-binding protein 4 were among the proteins most strongly associated with changes in both BMI and WHR. Four of the proteins significantly tracked with change in BMI (P < 0.00059) but not WHR (P > 0.05): endothelial cell-specific molecule 1, pentraxin-related protein PTX3, ST2protein(also known as interleukin-1 receptor-like 1), and spondin-1. Five proteins tracked with change in WHR (P < 0.00059) but not BMI (P > 0.05): caspase-8, cathepsin L1, oxidized low-density lipoprotein receptor 1, interleukin-6 receptor subunit alpha, and C-C motif chemokine 20. This is the first large longitudinal study of how changes in plasma protein signatures are associated with changes in measures of body fat and distribution over 10 years of aging.

Characterization of the immune roles of cathepsin L in turbot (Scophthalmus maximus L.) mucosal immunity

Cathepsin L (CTSL) is one of the crucial enzymes in cathepsin family, which has been widely known for its involvement in the innate immunity. However, it still remains poorly understood how CTSL modulates the immune system of teleosts. In this study, we captured three cathepsin L genes (SmCTSL, SmCTSL.1 and SmCTSL1) from turbot (Scophthalmus maximus). The coding sequences of SmCTSL, SmCTSL.1 and SmCTSL1 are 1,026 bp, 1,005 bp and 1,017 bp in length and encode 341, 334 and 338 amino acids, respectively. In details, transcripts of CTSL genes share same domains as other CTSL genes, one signal peptide, one propeptide and one papain family cysteine protease domain. Protein interaction network analysis indicated that turbot CTSL genes may play important roles in apoptotic signaling and involve in innate immune response. Evidence from subcellular localization demonstrated that the three Cathepsin L proteins were ubiquitous in nucleus and cytoplasm. The cathepsin L genes were widely expressed in all the tested tissues with the highest expression level of SmCTSL in spleen, and SmCTSL.1 and SmCTSL1 in intestine. Following Vibrio anguillarum, Edwardsiella tarda and Streptococcus iniae challenge, these cathepsin L genes were significantly regulated in mucosal tissues in all the challenges, especially significant down-regulation occurred rapidly in intestine in all the three challenges. In addition, the three cathepsin L genes showed strong binding ability to all the examined microbial ligands (LPS, PGN and LTA). Further studies should be used to analyze the specific function of these three cathepsin L genes. By then, we can use their function to maintain the integrity of the mucosal barrier, thereby promoting the disease resistance line and family selection in turbot.

Expression and Clinical Implications of Cysteine Cathepsins in Gallbladder Carcinoma.

Background: Gallbladder carcinoma (GBC) exhibits poor prognosis due to its detection at an advanced stage. Upregulation of lysosomal cysteine proteases cathepsin L (CTSL) and cathepsin B (CTSB) has been implicated in several tumorigenic processes. However, no such information in GBC was available. Therefore, the present study was planned to investigate the expression and clinical significance of these cathepsins in GBC.Methods: Activities of CTSL and CTSB were assayed in the gallbladder (GB) tissues obtained from GBC patients (n = 43) and control subjects (n = 69). Protein and mRNA levels were quantified using immunohistochemistry and real-time PCR (qPCR), respectively. Finally, serum levels of CTSL and CTSB were estimated by ELISA. Receiver operating characteristic (ROC) curve analysis was used for the assessment of sensitivity, specificity, and diagnostic accuracy of these cysteine cathepsins in GBC. The association of combined CTSL and CTSB activity with overall survival was assessed using Kaplan Meier survival analysis.Results: The expression and activity of both CTSL and CTSB were significantly increased (p < 0.050) in tumors of GBC patients as compared to controls. Enzymatic activity of CTSL+B and CTSB exhibited a strong positive association with tumor stage and lymph node involvement in GBC (p < 0.050). Interestingly, the elevated activity of combined CTSL+B was also associated with increased mortality in these patients. Furthermore, significantly enhanced levels of serum CTSL and CTSB were also observed in GBC (p < 0.050) as compared to controls. ROC analysis revealed high diagnostic significance of serum CTSB and CTSL for distinguishing GBC patients from controls with an area under the curve (AUC) of 82 and 77%, respectively.Conclusion: This study, for the first time, demonstrates the clinical significance of CTSL and CTSB overexpression in GBC. Our findings may help improve the clinical management of this carcinoma.

Cathepsin L interacts with CDK2-AP1 as a potential predictor of prognosis in patients with breast cancer.

Cathepsin L (CTSL) is a lysosomal acid cysteine protease that has been implicated in tumorigenesis and malignant progression. In the present study, the role of CTSL in tumorigenesis and prognosis of breast cancer was evaluated. The prognostic value of CTSL was analyzed using immunohistochemistry in patients with breast cancer, as well as online microarray datasets. CTSL expression was knocked down in the breast cancer cell line T-47D using RNA interference. MTT and colony formation assays were performed to assess the role of CTSL in the proliferation of breast cancer cells. Cell cycle progression and apoptosis were measured using flow cytometry. A physical interaction of CTSL and cyclin dependent kinase 2 associated protein 1 (CDK2-AP1) was determined using a glutathione S-transferase pull-down assay. Endogenous CTSL expression was high in breast cancer cells and exhibited an inverse association with CDK2-AP1 expression; aberrant expression of CTSL in breast cancer tissues predicted an improved clinical outcome and prognosis. In addition, CTSL knockdown decelerated the progression of breast cancer cells by arresting cell cycle progression and increasing apoptosis. Thus, CTSL may be a potential therapeutic target for treating patients with breast cancer.

Cathepsin L induced PC-12 cell apoptosis via activation of B-Myb and regulation of cell cycle proteins

Cathepsin L (CTSL), a cysteine protease, is responsible for the degradation of a variety of proteins. It is known to participate in neuronal apoptosis associated with abnormal cell cycle. However, the mechanisms underlying CTSL-induced cell apoptosis remain largely unclear. We reported here that rotenone caused an activation of CTSL expression in PC-12 cells, while knockdown of CTSL by small interfering RNAs or its inhibitor reduced the rotenone-induced cell cycle arrest and apoptosis. Moreover, elevation of CTSL and increased-apoptosis were accompanied by induction of B-Myb, a crucial cell cycle regulator. We found that B-Myb was increased in rotenone-treated PC-12 cells and knockdown of B-Myb ameliorated rotenone-stimulated cell apoptosis. Further analysis demonstrated that CTSL influenced the expression of B-Myb as suppression of CTSL activity led to a decreased B-Myb expression, whereas overexpression of CTSL resulted in B-Myb induction. Reduction of B-Myb in CTSL-overexpressing cells revealed that regulation of cell cycle-related proteins, including cyclin A and cyclin B1, through CTSL was mediated by the transcription factor B-Myb. In addition, we demonstrated that the B-Myb target, Bim, and its regulator, Egr-1, which was also associated with CTSL closely, were both involved in rotenone-induced apoptosis in PC-12 cells. Our data not only revealed the role of CTSL in rotenone-induced neuronal apoptosis, but also indicated the involvement of B-Myb in CTSL-related cell cycle regulation.

Modulation of Cathepsin L Expression in the Coronary Arteries of Atherosclerotic Swine

IntroductionNeointimal hyperplasia (NIH) and restenosis after percutaneous transluminal coronary angioplasty (PTCA) and intravascular stenting remain a problem on a long-term basis by causing endothelial denudation and damage to the intima and media. Vascular sterile inflammation has been attributed to the formation of NIH. Cathepsin L (CTSL), a lysosome protease, is associated with diet-induced atherogenesis. Vitamin D regulates the actions and regulatory effects of proteases and protease inhibitors in different cell types. Objectives of this study are to evaluate the modulatory effect of vitamin D on CTSL activity in post-PTCA coronary arteries of atherosclerotic swine. MethodsYucatan microswine were fed with high-cholesterol atherosclerotic diets. The swine were stratified to receive three diets: (1) vitamin D–deficient diet, (2) vitamin D–sufficient diet, and (3) vitamin D–supplement diet. After 6 mo, PTCA was performed in the left circumflex coronary artery (LCx). After 1 y, angiography and optical coherence tomography imaging were performed, and swine was euthanized. Coronary arteries were embedded in paraffin. Tissue sections were stained with hematoxylin and eosin. Expression of Ki67 and CTSL were evaluated by immunofluorescence. ResultsIncreased number of Ki67 + cells were observed in the postangioplasty LCx in vitamin D–deficient compared with vitamin D–sufficient or vitamin D–supplemented swine. Notably, the expression of CTSL was significantly increased in postangioplasty LCx of vitamin D–deficient swine compared with the vitamin D–sufficient or vitamin D–supplemented animal groups. ConclusionsIncreased expression of CTSL correlates with the formation of NIH in the PTCA-injured coronary arteries. However, in the presence of sufficient or supplemented levels of vitamin D in the blood, CTSL expression was significantly reduced.

Abstract 3759: Host and neoplastic cell secretions of proteolytic enzymes potentiate metastasis

Abstract The presence of macrophages within breast tumors is associated with metastatic potential. Tumor-associated macrophages are often stimulated by Interleukin-4, resulting in a pro-tumorigenic (M2-like) phenotype and the secretion of growth factors and proteases, including the lysosomal protease cathepsin L (CTSL). CTSL is often secreted by breast cancer cells and stromal cells and contributes to tumor invasion, metastasis, and angiogenesis. We hypothesized that secretion of the proteolytic enzyme cathepsin L by both tumor-associated macrophages and neoplastic cells facilitate tumor invasion and dissemination. Initial studies found that inhibiting CTSL using the novel cathepsin L/K inhibitors KGP94 and KGP207 prevented in vitro M2 macrophage invasion and reduced macrophage-stimulated invasion of 4T1 murine breast cancer cells. Interestingly, KGP94 and KGP207 also reduced the expression of several M2-associated markers, suggesting that CTSL activity may be important for Interleukin-4-driven M0 to M2 differentiation. Furthermore, CTSL shRNA knockdowns revealed that CTSL supplied from both the tumor cell and the macrophage population is important for tumor cell invasion. These data suggest that tumor cells and macrophages may both contribute to the CTSL-driven metastatic phenotype of breast cancer. Taken together, these studies highlight the importance of CTSL in macrophage functions and suggest that cathepsin inhibition strategies may be therapeutically beneficial by impairing the progression of tumors with high infiltration of M2 macrophages. Citation Format: Samantha S. Dykes, Henrietta O. Fasanya, Dietmar W. Siemann. Host and neoplastic cell secretions of proteolytic enzymes potentiate metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3759.

Abstract 988: Synthesis and preclinical evaluation of dual-stimuli responsive doxorubicin prodrug activated by histone deacetylases and cathepsin L

Abstract Doxorubicin (DOX) an anthracycline is a leading anticancer drug with a broad spectrum of activity against numerous solid and hematologic malignancies. However, its clinical application is limited by lower efficacy, severe cardiotoxicity and occurrence of secondary malignancies. There is an urgent need for eliminating the Dox adverse effects while retaining its anticancer efficacy. One of the major goals of cancer therapy is the selective targeting of malignancies over normal tissues. One way to avoid these severe adverse reactions is to develop tumor-targeted prodrugs that are converted to active antitumor drugs at tumor sites in the presence of enzymes that are overexpressed human cancers. Among these, the histone deacetylases (HDACs) and cathepsin L (CTSL) are highly expressed in cancer cells and are considered as potential cancer-specific targets. HDACs are critical enzymes involved in the regulation of histone and non-histone proteins and elevated HDACs in tumor cells are known to be closely associated with tumor initiation, progression, and metastasis. Similarly, the lysosomal cysteine protease CTSL plays key roles at multiple stages of tumor progression and metastasis. In the present study, we developed a new prodrug by coupling an acetylated lysine group to doxorubicin (Lys(Ac)-Dox), a masked cytotoxic agent, which is consecutively activated by HDACs and a CTSL to liberate doxorubicin. We first verified whether Lys(Ac)-Dox could be specifically cleaved by HDACs and CTSL in vitro. The results showed that after incubating with HDACs and CTSL at 37 °C for 20 h, the hydrolysis of Lys(Ac)-Dox reached 99%, suggesting that Lys(Ac)-Dox could be successfully cleaved by the target enzymes. To prove that the Lys(Ac)-Dox would have a much improved growth-inhibitory effect against cancer cells, the cytotoxicity of free DOX and Lys(Ac)-Dox against lung cancer cell lines normal human lung epithelial cell line was determined. The dose-response curves obtained from the cell lines tested indicated that Dox was equally cytotoxic against both cancer and normal cells. In contrast, Lys(Ac)-Dox highly cytotoxic against cancer cells and non-toxic to the normal counterparts. To measure the in vivo anticancer efficacy of Lys(Ac)-Dox vis-vis Dox, we developed subcutaneous xenografts by injecting human lung cancer A549 and H460 cells in nude mice. Lys (Ac)-Dox and Dox were administered daily i.p. at 5 mg/kg. The prodrug showed a significantly higher (&amp;gt;2-fold) tumor regression than Dox. A diminished circulating reticulocyte counts from whole blood after Dox treatment is known to reflect the hematological toxicity caused by the drug. Only Dox caused significant reticulocyte ablation while the prodrug did not, validating our drug design. Biochemical tests involving topo II inhibition and ROS production by the prodrug are in progress (supported by CPRIT grant RP 170207 to KSS). Note: This abstract was not presented at the meeting. Citation Format: Surendra R. Punganuru, Hanumantha Rao Madala, Viswanath Arutla, Kalkunte S. Srivenugopal. Synthesis and preclinical evaluation of dual-stimuli responsive doxorubicin prodrug activated by histone deacetylases and cathepsin L [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 988.

Inhibition of Plasmodium falciparum cysteine protease falcipain-2 by a human cross-class inhibitor serpinB3: A mechanistic insight

Falcipain-2(FP2), a cysteine protease from Plasmodium falciparum, cleaves host erythrocyte hemoglobin and specific membrane skeleton components during the parasite life cycle. Therefore its inhibition has been considered as an attractive approach to combat the disease. SerpinB3 (SPB3) belongs to the ovalbumin-serpin family and is a potent cross-class inhibitor of cysteine cathepsins L, K, S and papain. This study explored the possibility of inhibition of FP2 by SPB3. It turned out that general proteolytic activities as well as specific hemoglobinolytic activity of FP2 have been inhibited by SPB3. Furthermore, studies have been designed to investigate and characterize the mechanism of inhibition in comparison with proteases Cathepsin L (CTSL) and papain. The Ki value of inhibition for FP2, measured against its specific substrate (VLK-pNA), is 338.11 nM and stoichiometry (I/E ratio) of inhibition is 1. These values are comparable to CTSL and papain. Analytical gel filtration profile and CD spectroscopy data confirm FP2-SPB3 complex formation. Our studies revealed that interaction of SPB3 with FP2 is non-covalent type like that of CTSL and papain but unlike other serine protease-inhibiting serpins. An in-silico docking and simulation study have been performed with FP2 as well as CTSL and results suggest different binding mode for FP2 and CTSL, though both the complexes are stable with significant contribution from electrostatic energy of interaction. We further showed a disease state mutant SPB3-Gly351Ala performed better anti-protease activity against FP2. This study, for the first time, has shown a serpin family inhibitor from human could efficiently inhibit activity of FP2.

Cathepsin L is A Potential Marker for Triple-Negative Breast Cancer

Breast cancer is the second cancer-related death among women worldwide. Triple-negative breast cancer (TNBC) is the most aggressive and lethal subtype, which is associated with high metastasis and poor prognosis. Cathepsins, a family of lysosomal cysteine proteases, such as cathepsin L (CTSL), are involved in cancer invasion and metastasis. Thus, CTSL may emerge as a marker for TNBC. So, we characterized the expression of CTSL mRNA in tissue specimen of TNBC (n = 10) and non-TNBC (non-TN) (n = 10) using quantitative real-time PCR. Data were statistically analyzed using Mann-Whitney U-test. Our data demonstrate that CTSL mRNA expression was up-regulated in TN BC vs. non-TN BC patients. In conclusion, the high expression of CTSL may represent a marker for TNBC and its targeting could have therapeutic implications

Porcine deltacoronavirus enters cells via two pathways: A protease-mediated one at the cell surface and another facilitated by cathepsins in the endosome

Porcine deltacoronavirus (PDCoV) is a pathogen belonging to the genus Deltacoronavirus that in 2014 caused outbreaks of piglet diarrhea in the United States. To identify suitable therapeutic targets, a more comprehensive understanding of the viral entry pathway is required, particularly of the role of proteases. Here, we identified the proteases that activate the viral spike (S) glycoprotein to initiate cell entry and also pinpointed the host-cellular pathways that PDCoV uses for entry. Our results revealed that cathepsin L (CTSL) and cathepsin B (CTSB) in lysosomes and extracellular trypsin in cell cultures independently activate the S protein for membrane fusion. Pretreating the cells with the lysosomal acidification inhibitor bafilomycin-A1 (Baf-A1) completely inhibited PDCoV entry, and siRNA-mediated ablation of CTSL or CTSB expression significantly reduced viral infection, indicating that PDCoV uses an endosomal pathway for entry. Of note, trypsin treatment of cell cultures also activated PDCoV entry, even when the endosomal pathway was inhibited. This observation indicated that trypsin-induced S protein cleavage and activation in cell cultures enables viral entry directly from the cell surface. Our results provide critical insights into the PDCoV infection mechanism, uncovering two distinct viral entry pathways: one through cathepsin L and cathepsin B in the endosome and another via a protease at the cell surface. Because PDCoV infection sites represent a proteases-rich environment, these findings suggest that endosome inhibitor treatment alone is insufficient to block PDCoV entry into intestinal epithelial cells in vivo Therefore, approaches that inhibit viral entry from the cell membrane should also be considered.

Biomarkers of blood cadmium and incidence of cardiovascular events in non-smokers: results from a population-based proteomics study

BackgroundCadmium is a toxic metal with multiple adverse health effects, including risk of cardiovascular disease (CVD). The mechanistic link between cadmium and CVD is unclear. Our aim was to examine the associations between blood cadmium (B-Cd) and 88 potential protein biomarkers of CVD.MethodsB-Cd and 88 plasma proteins were measured in a community-based prospective cohort, the Malmö Diet and Cancer study. The primary analysis was performed in never smokers (n = 1725). Multiple linear regression was used with adjustments for age and sex, and correction for multiple comparisons using the false discovery rate method. Proteins significantly associated with B-Cd were replicated in long-term former smokers (n = 782). Significant proteins were then studied in relation to incidence of CVD (i.e., coronary events or ischemic stroke) in never smokers.ResultsFifteen proteins were associated with B-Cd in never smokers. Eight of them were replicated in long-term former smokers. Kidney injury molecule-1, fibroblast growth factor-23 (FGF23), tumor necrosis factor receptor-2, matrix metalloproteinase-12, cathepsin L1, urokinase plasminogen activator receptor, C-C motif chemokine-3 (CCL3), and chemokine (C-X3-C motif) ligand-1 were associated with B-Cd both in never smokers and long-term former smokers. Except for CCL3 and FGF23, these proteins were also significantly associated with incidence of CVD.ConclusionsB-Cd in non-smokers was associated with eight potential plasma biomarkers of CVD and kidney injury. The results suggest pathways for the associations between B-Cd and CVD and kidney injury.

Cathepsin B Affects the Activation of Nucleotide-binding Domain and Leucine-rich-repeat-containing Family and Pyrin Domain-containing 3 Inflammasome via Transient Receptor Potential Mucolipin-1

Objective To explore the effects of cathepsin B(CTSB)on the activation of nucleotide-binding domain and leucine-rich-repeat-containing family and pyrin domain-containing 3(NLRP3)inflammasome via transient receptor potential mucolipin-1(TRPML1)in cell oxidative stress model and specific gene silencing cell model. Methods BV2 cells cultured in vivo were treated separately or simultaneously with hydrogen peroxide(H2O2),calcium-sensitive receptor agonist gadolinium trichloride(GdCl3),and CTSB inhibitor CA-074Me,and interleukin-1(IL-1)beta and caspase-1 protein were detected by enzyme-linked immunosorbent assay.The growth activity of BV2 cells in each group was measured by MTT.BV2 cells were treated with different concentrations of H2O2.Cystatin C mRNA and TRPML1 mRNA in BV2 cells were detected by real-time quantitative polymerase chain reaction and the proteins of TRPML1,CTSB,cathepsin D(CTSD),cathepsin L(CTSL)and cathepsin V(CTSV)were detected by Western blot.Specific small interfering RNA was designed for TRPML1 gene target sequence.TRPML1 gene silencing cell lines(named Tr-si-Bv2 cells)were established in BV2 cells and treated with or without H2O2.TRPML1,CTSB and transcription factor EB(TFEB)proteins in Tr-si-Bv2 cells or control cells were detected by Western blot. Results After treatment with H2O2,the expression of caspase-1 protein and NLRP3 mRNA in BV2 cells was increased,and IL-1beta protein in BV2 cells was significantly increased after treatment with GdCl3(P=0.0036).After treatment with CA-074Me,the doses of NLRP3 mRNA(P=0.037),caspase-1(P=0.021),and IL-1β(P= 0.036)were significantly reduced.Cells in the H2O2 group and H2O2+GdCl3 group grew more slowly.The expressions of CTSB mRNA and TRPML1 mRNA,or CTSB and TRPML1 proteins in BV2 cells in the treatment group with 200 μmol/L of H2O2 concentration were similar.H2O2-induced CTSB protein expression was inhibited after silencing TRPML1 gene.The changes of other cathepsins were not affected for the different concentration of H2O2.In the BV2 cells treated with TRPML1 gene silencing,the expression of CTSB protein was significantly reduced and the difference was statistically significant(P=0.021)between the H2O2 +siRNA treatment group and the H2O2 treatment group.<b>Conclusion</b> CTSB regulates the activation of NLRP3 inflammasome in the oxidative stress model of microglia cells,probably mediated by calcium channel protein TRPML1.