scholarly journals Cysteine cathepsins are altered by flow within an engineered in vitro microvascular niche

2020 ◽  
Vol 4 (4) ◽  
pp. 046102
Author(s):  
Simone A. Douglas ◽  
Kristina Haase ◽  
Roger D. Kamm ◽  
Manu O. Platt
Keyword(s):  
Cysteine Cathepsins

Trial of the cysteine cathepsin inhibitor JPM-OEt on early and advanced mammary cancer stages in the MMTV-PyMT-transgenic mouse model

2008 ◽  
Vol 389 (8) ◽  
Author(s):  
Uta Schurigt ◽  
Lisa Sevenich ◽  
Corinne Vannier ◽  
Mieczyslaw Gajda ◽  
Anne Schwinde ◽  
...  
Keyword(s):  
Lung Metastases ◽  
Control Group ◽  
Breast Cancers ◽  
Cancer Trial ◽  
Tissue Extracts ◽  
Cysteine Cathepsins ◽  
Cysteine Cathepsin ◽  
Effective Inhibition ◽  
Pharmacokinetic Properties

Abstract Recent data suggest proteases of the papain-like cysteine cathepsin family as molecular targets for cancer therapy. Here, we report the treatment of polyoma middle T oncogene-induced breast cancers in mice with the cell-permeable broad-spectrum cysteine cathepsin inhibitor JPM-OEt. Up to 100 mg/kg inhibitor was intraperitoneally injected once per day in two trials on early and advanced cancers. In both trials, transient delays in tumour growth were observed. However, at the endpoint of both experiments no significant differences in tumour weights, histopathology and lung metastasis were found between the inhibitor and the control group. The invasive strand formation of collagen I-embedded tumour cell spheroids generated from primary tumours of inhibitor-treated mice in the early cancer trial could be inhibited in vitro by JPM-OEt; a result arguing against induction of resistance to the inhibitor. Measurement of cysteine cathepsin activities in tissue extracts after intraperitoneal injection of JPM-OEt revealed effective inhibition of cysteine cathepsins in pancreas, kidneys and liver, while activities in mammary cancers and in lungs were not significantly affected. We conclude that the pharmacokinetic properties of JPM-OEt, which result in poor bioavailability, may prohibit its use for stand-alone treatment of solid mammary cancers and their lung metastases.

Toward understanding recurrent meningioma: the potential role of lysosomal cysteine proteases and their inhibitors

2010 ◽  
Vol 112 (5) ◽  
pp. 940-950 ◽  
Author(s):  
Tamara T. Lah ◽  
Isabelle Nanni ◽  
Miha Trinkaus ◽  
Philipe Metellus ◽  
Christophe Dussert ◽  
...  
Keyword(s):  
Cystatin C ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Who Grade ◽  
Simpson Grade ◽  
Cysteine Cathepsins ◽  
Chick Heart ◽  
Embryonic Chick Heart ◽  
Meningioma Recurrence

Object The first aim of this study was to diagnose more aggressive and potentially recurrent meningiomas using an in vitro embryonic chick heart invasiveness assay in which lysosomal enzyme cathepsin B was used as the invasiveness marker. The second aim was to confirm if cathepsin B and/or cathepsin L and their endogenous inhibitors were also prognostic parameters in the clinical study of 119 patients with meningioma. Methods Primary meningioma cultured spheroids were “confronted” with embryonic chick heart spheroids in vitro, and cathepsin B was used as molecular marker to immunolabel the invasive tumor cells. In vitro invasion assays of the malignant meningioma cells were used to assess the invasive potential related to the cysteine cathepsins. As to the second aim, the possible association of cathepsin B along with selected molecular markers, cathepsin L, and endogenous cysteine protease inhibitors (stefins A and B and cystatin C) with meningioma malignancy was determined using enzyme-linked immunosorbent assays in tumor homogenates. Univariate and multivariate analyses were used to compare these parameters with established biological markers of meningioma recurrence in 119 patients with meningiomas. Results The more invasive tumors, which characteristically overgrew the normal tissue, were identified even within a group of histologically benign meningiomas. More intensive staining of cathepsin B in these tumors was not only found at the tumor front, but also in the invading pseudopodia of a single migrating tumor cells. Matrigel invasion of malignant meningioma cells was significantly altered by modulating cathepsin B activity and by stefin B silencing. In the clinical samples of meningioma, the levels of cathepsins B and L, stefin B, and cystatin C were highest in the tumors of higher histological grades, whereas stefin A and progesterone receptor were the only markers that were significantly increased and decreased, respectively, in WHO Grade III lesions. With respect to the prognosis of relapse, cathepsin L (p = 0.035), stefin B (p = 0.007), cystatin C (p = 0.008), and progesterone receptor (p = 0.049) levels were significant, whereas cathepsin B was not a prognosticator. As expected, WHO grade, age, and Simpson grade (complete tumor resection) were prognostic, with Simpson grade only relevant in the short term (up to 90 months) but not in longer-term follow-up. Of note, the impact of all these parameters was lost in multivariate analysis, due to overwhelming prognostic impact of stefin B (p = 0.039). Conclusions The data indicate that the cysteine cathepsins and their inhibitors are involved in a process related to early meningioma recurrence, regardless of their histological classification. Of note, the known tumor invasiveness marker cathepsin B, measured in whole-tumor homogenates, was not prognostic, in contrast to its endogenous inhibitor stefin B, which was highly significant and the only independent prognostic factor to predict meningioma relapse in multivariate analysis and reported herein for the first time. Stefin B inhibition of local invasion was confirmed by in vitro invasion assay, although its other functions cannot be excluded.

scholarly journals Mialostatin, a Novel Midgut Cystatin from Ixodes ricinus Ticks: Crystal Structure and Regulation of Host Blood Digestion

2021 ◽  
Vol 22 (10) ◽  
pp. 5371
Author(s):  
Jan Kotál ◽  
Michal Buša ◽  
Veronika Urbanová ◽  
Pavlína Řezáčová ◽  
Jindřich Chmelař ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Ixodes Ricinus ◽  
Cathepsin L ◽  
Blood Protein ◽  
Hard Tick ◽  
Blood Proteins ◽  
Digestive Cells ◽  
Cysteine Cathepsins ◽  
Host Blood

The hard tick Ixodes ricinus is a vector of Lyme disease and tick-borne encephalitis. Host blood protein digestion, essential for tick development and reproduction, occurs in tick midgut digestive cells driven by cathepsin proteases. Little is known about the regulation of the digestive proteolytic machinery of I. ricinus. Here we characterize a novel cystatin-type protease inhibitor, mialostatin, from the I. ricinus midgut. Blood feeding rapidly induced mialostatin expression in the gut, which continued after tick detachment. Recombinant mialostatin inhibited a number of I. ricinus digestive cysteine cathepsins, with the greatest potency observed against cathepsin L isoforms, with which it co-localized in midgut digestive cells. The crystal structure of mialostatin was determined at 1.55 Å to explain its unique inhibitory specificity. Finally, mialostatin effectively blocked in vitro proteolysis of blood proteins by midgut cysteine cathepsins. Mialostatin is likely to be involved in the regulation of gut-associated proteolytic pathways, making midgut cystatins promising targets for tick control strategies.

scholarly journals A Review on Molecular Functional and Structural Characterization of Human Cysteine Cathepsins in Bacterial Expression Systems

2019 ◽  
Author(s):  
Ruwini Cooray ◽  
Hasanka Madubashetha ◽  
Lakshan Warnakula ◽  
P.D.S.U. Wickramasinghe ◽  
Nimali De Silva
Keyword(s):  
Structural Characterization ◽  
Cancer Metastasis ◽  
Expression System ◽  
Bacterial Expression ◽  
Immune Functions ◽  
Expression Systems ◽  
Biochemical Pathways ◽  
Cysteine Cathepsins ◽  
Bacterial Expression Systems

Cysteine cathepsins, a class of proteinaceous enzymes, regulate a wide variety of metabolic processes in human including protein breakdown and turnover and immune functions. Eleven cysteine cathepsins have been identified so far and a wide array of studies related to identifying their specific functions, regulation and distribution patterns in tissues have been conducted. However, in recent past, the association of cysteine cathepsins in occurrence and progression of cancers have been identified and this has caused unrest in scientists triggering them to investigate the physiology, biochemical pathways and interactions of these cathepsins in cancer metastasis and therefore has become a noteworthy topic of intensive research. This review focusses and collects together the published work on molecular functional and structural characterization studies that have been done so far on in vitro expression of genes encoding for cysteine cathepsins in the Escherichia coli bacterial expression system. Accordingly, it was found out that all cathepsins except for cathepsins K, C, H, X and W have been expressed this way and the majority of them were found to be expressed in E. coli BL21(DE3) pLysS expression host via pET3 expression vector. In addition, it was also noted that in most of the expression studies, the substrate that was used to validate the enzymatic activity of the recombinant enzyme that was produced was a cysteine residue along with a benzyloxy-carbonyl salt. Through this review, the authors suggest that there is a very high need that all cysteine cathepsins need to be characterized both structurally and functionally on a molecular platform to better understand their interactions including the biochemical pathways. It is also momentous that the mass production of the recombinant forms of these enzymes are facilitated via expression in such bacterial expression systems and in turn, would also provide a strong platform for the development and progression of studies related to human physiology including oncological studies such as cancer metastasis. Moreover, as per biochemical features of the enzymes that could be identified, the production of efficient inhibitors or inducers as per the necessity to improve health and promote wellbeing among the mankind could be facilitated.

scholarly journals Procathepsin V Is Secreted in a TSH Regulated Manner from Human Thyroid Epithelial Cells and Is Accessible to an Activity-Based Probe

2020 ◽  
Vol 21 (23) ◽  
pp. 9140
Author(s):  
Alaa Al-Hashimi ◽  
Vaishnavi Venugopalan ◽  
Maren Rehders ◽  
Naphannop Sereesongsaeng ◽  
Zeynep Hein ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Thyroid Tissue ◽  
Chimeric Protein ◽  
Human Thyroid ◽  
Apical Plasma Membrane ◽  
Epithelial Cell Line ◽  
Cysteine Cathepsins ◽  
Cathepsin V

The significance of cysteine cathepsins for the liberation of thyroid hormones from the precursor thyroglobulin was previously shown by in vivo and in vitro studies. Cathepsin L is most important for thyroglobulin processing in mice. The present study aims at specifying the possible contribution of its closest relative, cysteine cathepsin L2/V, to thyroid function. Immunofluorescence analysis on normal human thyroid tissue revealed its predominant localization at the apical plasma membrane of thyrocytes and within the follicle lumen, indicating the secretion of cathepsin V and extracellular tasks rather than its acting within endo-lysosomes. To explore the trafficking pathways of cathepsin V in more detail, a chimeric protein consisting of human cathepsin V tagged with green fluorescent protein (GFP) was stably expressed in the Nthy-ori 3-1 thyroid epithelial cell line. Colocalization studies with compartment-specific markers and analyses of post-translational modifications revealed that the chimeric protein was sorted into the lumen of the endoplasmic reticulum and subsequently transported to the Golgi apparatus, while being N-glycosylated. Immunoblotting showed that the chimeric protein reached endo-lysosomes and it became secreted from the transduced cells. Astonishingly, thyroid stimulating hormone (TSH)-induced secretion of GFP-tagged cathepsin V occurred as the proform, suggesting that TSH upregulates its transport to the plasma membrane before it reaches endo-lysosomes for maturation. The proform of cathepsin V was found to be reactive with the activity-based probe DCG-04, suggesting that it possesses catalytic activity. We propose that TSH-stimulated secretion of procathepsin V is the default pathway in the thyroid to enable its contribution to thyroglobulin processing by extracellular means.

scholarly journals Evaluation of novel cathepsin-X inhibitors in vitro and in vivo and their ability to improve cathepsin-B-directed antitumor therapy

2022 ◽  
Vol 79 (1) ◽  
Author(s):  
Ana Mitrović ◽  
Janja Završnik ◽  
Georgy Mikhaylov ◽  
Damijan Knez ◽  
Urša Pečar Fonović ◽  
...  
Keyword(s):  
Tumor Progression ◽  
Mouse Models ◽  
Cancer Progression ◽  
Cathepsin B ◽  
Cancer Resistance ◽  
Antitumor Therapy ◽  
Cysteine Cathepsins ◽  
Cathepsin X

AbstractNew therapeutic targets that could improve current antitumor therapy and overcome cancer resistance are urgently needed. Promising candidates are lysosomal cysteine cathepsins, proteolytical enzymes involved in various critical steps during cancer progression. Among them, cathepsin X, which acts solely as a carboxypeptidase, has received much attention. Our results indicate that the triazole-based selective reversible inhibitor of cathepsin X named Z9 (1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-((4-isopropyl-4H-1,2,4-triazol-3-yl)thio)ethan-1-one) significantly reduces tumor progression, both in vitro in cell-based functional assays and in vivo in two independent tumor mouse models: the FVB/PyMT transgenic and MMTV-PyMT orthotopic breast cancer mouse models. One of the mechanisms by which cathepsin X contributes to cancer progression is the compensation of cathepsin-B activity loss. Our results confirm that cathepsin-B inhibition is compensated by an increase in cathepsin X activity and protein levels. Furthermore, the simultaneous inhibition of both cathepsins B and X with potent, selective, reversible inhibitors exerted a synergistic effect in impairing processes of tumor progression in in vitro cell-based assays of tumor cell migration and spheroid growth. Taken together, our data demonstrate that Z9 impairs tumor progression both in vitro and in vivo and can be used in combination with other peptidase inhibitors as an innovative approach to overcome resistance to antipeptidase therapy.

scholarly journals Crystal structure and functional characterization of an immunomodulatory salivary cystatin from the soft tick Ornithodoros moubata

2010 ◽  
Vol 429 (1) ◽  
pp. 103-112 ◽  
Author(s):  
Jiří Salát ◽  
Guido C. Paesen ◽  
Pavlína Řezáčová ◽  
Michalis Kotsyfakis ◽  
Zuzana Kovářová ◽  
...  
Keyword(s):  
Crystal Structure ◽  
African Swine Fever Virus ◽  
Functional Characterization ◽  
Interleukin 12 ◽  
Soft Tick ◽  
Ornithodoros Moubata ◽  
Cysteine Cathepsins

The saliva of blood-feeding parasites is a rich source of peptidase inhibitors that help to overcome the host's defence during host–parasite interactions. Using proteomic analysis, the cystatin OmC2 was demonstrated in the saliva of the soft tick Ornithodoros moubata, an important disease vector transmitting African swine fever virus and the spirochaete Borrelia duttoni. A structural, biochemical and biological characterization of this peptidase inhibitor was undertaken in the present study. Recombinant OmC2 was screened against a panel of physiologically relevant peptidases and was found to be an effective broad-specificity inhibitor of cysteine cathepsins, including endopeptidases (cathepsins L and S) and exopeptidases (cathepsins B, C and H). The crystal structure of OmC2 was determined at a resolution of 2.45 Å (1 Å=0.1 nm) and was used to describe the structure–inhibitory activity relationship. The biological impact of OmC2 was demonstrated both in vitro and in vivo. OmC2 affected the function of antigen-presenting mouse dendritic cells by reducing the production of the pro-inflammatory cytokines tumour necrosis factor α and interleukin-12, and proliferation of antigen-specific CD4+ T-cells. This suggests that OmC2 may suppress the host's adaptive immune response. Immunization of mice with OmC2 significantly suppressed the survival of O. moubata in infestation experiments. We conclude that OmC2 is a promising target for the development of a novel anti-tick vaccine to control O. moubata populations and combat the spread of associated diseases.

scholarly journals Processing and Maturation of Cathepsin C Zymogen: A Biochemical and Molecular Modeling Analysis

2019 ◽  
Vol 20 (19) ◽  
pp. 4747 ◽  
Author(s):  
Anne-Sophie Lamort ◽  
Yveline Hamon ◽  
Cezary Czaplewski ◽  
Artur Gieldon ◽  
Seda Seren ◽  
...  
Keyword(s):  
Molecular Modeling ◽  
Immune Cell ◽  
Serine Proteinase ◽  
Cathepsin G ◽  
Cathepsin C ◽  
Cysteine Cathepsins ◽  
Modeling Analysis ◽  
Cysteine Cathepsin ◽  
Proteolytic Cleavages

Cysteine cathepsin C (CatC) is a ubiquitously expressed, lysosomal aminopeptidase involved in the activation of zymogens of immune-cell-associated serine proteinases (elastase, cathepsin G, proteinase 3, neutrophil serine proteinase 4, lymphocyte granzymes, and mast cell chymases). CatC is first synthetized as an inactive zymogen containing an intramolecular chain propeptide, the dimeric form of which is processed into the mature tetrameric form by proteolytic cleavages. A molecular modeling analysis of proCatC indicated that its propeptide displayed a similar fold to those of other lysosomal cysteine cathepsins, and could be involved in dimer formation. Our in vitro experiments revealed that human proCatC was processed and activated by CatF, CatK, and CatV in two consecutive steps of maturation, as reported for CatL and CatS previously. The unique positioning of the propeptide domains in the proCatC dimer complex allows this order of cleavages to be understood. The missense mutation Leu172Pro within the propeptide region associated with the Papillon–Lefèvre and Haim–Munk syndrome altered the proform stability as well as the maturation of the recombinant Leu172Pro proform.

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