scholarly journals Novel Cysteine Protease Inhibitor Derived from the Haementeria vizottoi Leech: Recombinant Expression, Purification, and Characterization

Toxins ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 857
Author(s):  
Débora do Carmo Linhares ◽  
Fernanda Faria ◽  
Roberto Tadashi Kodama ◽  
Adriane Michele Xavier Prado Amorim ◽  
Fernanda Calheta Vieira Portaro ◽  
...  
Keyword(s):  
Cancer Progression ◽  
Cysteine Protease ◽  
Biochemical Characterization ◽  
Recombinant Expression ◽  
Cathepsin L ◽  
Cysteine Protease Inhibitor ◽  
In Vivo Models ◽  
Lysosomal Cysteine Protease ◽  
First Time

Cathepsin L (CatL) is a lysosomal cysteine protease primarily involved in the terminal degradation of intracellular and endocytosed proteins. More specifically, in humans, CatL has been implicated in cancer progression and metastasis, as well as coronary artery diseases and others. Given this, the search for potent CatL inhibitors is of great importance. In the search for new molecules to perform proteolytic activity regulation, salivary secretions from hematophagous animals have been an important source, as they present protease inhibitors that evolved to disable host proteases. Based on the transcriptome of the Haementeria vizzotoi leech, the cDNA of Cystatin-Hv was selected for this study. Cystatin-Hv was expressed in Pichia pastoris and purified by two chromatographic steps. The kinetic results using human CatL indicated that Cystatin-Hv, in its recombinant form, is a potent inhibitor of this protease, with a Ki value of 7.9 nM. Consequently, the present study describes, for the first time, the attainment and the biochemical characterization of a recombinant cystatin from leeches as a potent CatL inhibitor. While searching out for new molecules of therapeutic interest, this leech cystatin opens up possibilities for the future use of this molecule in studies involving cellular and in vivo models.

scholarly journals Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Miao-Miao Zhao ◽  
Wei-Li Yang ◽  
Fang-Yuan Yang ◽  
Li Zhang ◽  
Wei-Jin Huang ◽  
...  
Keyword(s):  
Drug Development ◽  
Cathepsin L ◽  
Human Cells ◽  
New Drugs ◽  
Disease Course ◽  
Promising Target ◽  
First Time

AbstractTo discover new drugs to combat COVID-19, an understanding of the molecular basis of SARS-CoV-2 infection is urgently needed. Here, for the first time, we report the crucial role of cathepsin L (CTSL) in patients with COVID-19. The circulating level of CTSL was elevated after SARS-CoV-2 infection and was positively correlated with disease course and severity. Correspondingly, SARS-CoV-2 pseudovirus infection increased CTSL expression in human cells in vitro and human ACE2 transgenic mice in vivo, while CTSL overexpression, in turn, enhanced pseudovirus infection in human cells. CTSL functionally cleaved the SARS-CoV-2 spike protein and enhanced virus entry, as evidenced by CTSL overexpression and knockdown in vitro and application of CTSL inhibitor drugs in vivo. Furthermore, amantadine, a licensed anti-influenza drug, significantly inhibited CTSL activity after SARS-CoV-2 pseudovirus infection and prevented infection both in vitro and in vivo. Therefore, CTSL is a promising target for new anti-COVID-19 drug development.

scholarly journals Studies of Inhibitory Mechanisms of Propeptide-Like Cysteine Protease Inhibitors

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Bui T. T. Nga ◽  
Yuki Takeshita ◽  
Misa Yamamoto ◽  
Yoshimi Yamamoto
Keyword(s):  
Protease Inhibitors ◽  
Cysteine Protease ◽  
Cathepsin L ◽  
Cysteine Residue ◽  
Cysteine Protease Inhibitor ◽  
Inhibition Mechanism ◽  
Cysteine Protease Inhibitors ◽  
Inhibitory Potency ◽  
Inhibitory Mechanisms

Mouse cytotoxic T-lymphocyte antigen-2α (CTLA-2α), Drosophila CTLA-2-like protein (crammer), and Bombyx cysteine protease inhibitor (BCPI) belong to a novel family of cysteine protease inhibitors (I29). Their inhibitory mechanisms were studied comparatively. CTLA-2α contains a cysteine residue (C75), which is essential for its inhibitory potency. The CTLA-2α monomer was converted to a disulfide-bonded dimer in vitro and in vivo. The dimer was fully inhibitory, but the monomer, which possessed a free thiol residue, was not. A disulfide-bonded CTLA-2α/cathepsin L complex was isolated, and a cathepsin L subunit with a molecular weight of 24,000 was identified as the interactive enzyme protein. Crammer also contains a cysteine residue (C72). Both dimeric and monomeric forms of crammer were inhibitory. A crammer mutant with Cys72 to alanine (C72A) was fully inhibitory, while the replacement of Gly73 with alanine (G73A) caused a significant loss in inhibitory potency, which suggests a different inhibition mechanism from CTLA-2α. BCPI does not contain cysteine residue. C-terminal region (L77-R80) of BCPI was essential for its inhibitory potency. CTLA-2α was inhibitory in the acidic pH condition but stabilized cathepsin L under neutral pH conditions. The different inhibition mechanisms and functional considerations of these inhibitors are discussed.

scholarly journals Effect of phenyl vinyl sulphone cysteine protease inhibitor on Schistosoma mansoni: in vitro and in vivo experimental studies

2017 ◽  
Vol 41 (4) ◽  
pp. 1049-1058
Author(s):  
Manal Salah El-Din Mahmoud ◽  
Ayman Nabil Ibrahim ◽  
Abeer Fathy Badawy ◽  
Nourhan Mohamed Abdelmoniem
Keyword(s):  
Protease Inhibitor ◽  
Schistosoma Mansoni ◽  
Cysteine Protease ◽  
Experimental Studies ◽  
Cysteine Protease Inhibitor ◽  
Phenyl Vinyl

scholarly journals Biochemical Characterization of Individual Components of the Allochromatium vinosum DsrMKJOP Transmembrane Complex Aids Understanding of Complex Function In Vivo

2010 ◽  
Vol 192 (24) ◽  
pp. 6369-6377 ◽  
Author(s):  
Fabian Grein ◽  
Inês A. C. Pereira ◽  
Christiane Dahl
Keyword(s):  
Biochemical Characterization ◽  
Sulfur Metabolism ◽  
Complex Function ◽  
Redox Potentials ◽  
Allochromatium Vinosum ◽  
Purple Sulfur Bacterium ◽  
Membrane Anchoring ◽  
Integral Proteins ◽  
First Time

ABSTRACT The DsrMKJOP transmembrane complex has a most important function in dissimilatory sulfur metabolism and consists of cytoplasmic, periplasmic, and membrane integral proteins carrying FeS centers and b- and c-type cytochromes as cofactors. In this study, the complex was isolated from the purple sulfur bacterium Allochromatium vinosum and individual components were characterized as recombinant proteins. The two integral membrane proteins DsrM and DsrP were successfully produced in Escherichia coli C43(DE3) and C41(DE3), respectively. DsrM was identified as a diheme cytochrome b, and the two hemes were found to be in low-spin state. Their midpoint redox potentials were determined to be +60 and +110 mV. Although no hemes were predicted for DsrP, it was also clearly identified as a b-type cytochrome. To the best of our knowledge, this is the first time that heme binding has been experimentally proven for a member of the NrfD protein family. Both cytochromes were partly reduced after addition of a menaquinol analogue, suggesting interaction with quinones in vivo. DsrO and DsrK were both experimentally proven to be FeS-containing proteins. In addition, DsrK was shown to be membrane associated, and we propose a monotopic membrane anchoring for this protein. Coelution assays provide support for the proposed interaction of DsrK with the soluble cytoplasmic protein DsrC, which might be its substrate. A model for the function of DsrMKJOP in the purple sulfur bacterium A. vinosum is presented.

scholarly journals PL3.6 Targeting GSK-3 activity promotes mitotic catastrophe via centrosome destabilisation and enhances the effect of radiotherapy in glioma models

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii4-iii4
Author(s):  
A Bruning-Richardson ◽  
H Sanganee ◽  
S Barry ◽  
D Tams ◽  
T Brend ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Progression ◽  
Combination Treatment ◽  
Single Agent ◽  
Drug Penetration ◽  
In Vivo Models ◽  
Human Astrocytes ◽  
Normal Human

Abstract BACKGROUND Targeting kinases as regulators of cellular processes that drive cancer progression is a promising approach to improve patient outcome in GBM management. The glycogen synthase kinase 3 (GSK-3) plays a role in cancer progression and is known for its pro-proliferative activity in gliomas. The anti-proliferative and cytotoxic effects of the GSK-3 inhibitor AZD2858 were assessed in relevant in vitro and in vivo glioma models to confirm GSK-3 as a suitable target for improved single agent or combination treatments. MATERIAL AND METHODS The immortalised cell line U251 and the patient derived cell lines GBM1 and GBM4 were used in in vitro studies including MTT, clonogenic survival, live cell imaging, immunofluorescence microscopy and flow cytometry to assess the cytotoxic and anti-proliferative effects of AZD2858. Observed anti-proliferative effects were investigated by microarray technology for the identification of target genes with known roles in cell proliferation. Clinical relevance of targeting GSK-3 with the inhibitor either for single agent or combination treatment strategies was determined by subcutaneous and orthotopic in vivo modelling. Whole mount mass spectroscopy was used to confirm drug penetration in orthotopic tumour models. RESULTS AZD2858 was cytotoxic at low micromolar concentrations and at sub-micromolar concentrations (0.01 - 1.0 μM) induced mitotic defects in all cell lines examined. Prolonged mitosis, centrosome disruption/duplication and cytokinetic failure leading to cell death featured prominently among the cell lines concomitant with an observed S-phase arrest. No cytotoxic or anti-proliferative effect was observed in normal human astrocytes. Analysis of the RNA microarray screen of AZD2858 treated glioma cells revealed the dysregulation of mitosis-associated genes including ASPM and PRC1, encoding proteins with known roles in cytokinesis. The anti-proliferative and cytotoxic effect of AZD2858 was also confirmed in both subcutaneous and orthotopic in vivo models. In addition, combination treatment with AZD2858 enhanced clinically relevant radiation doses leading to reduced tumour volume and improved survival in orthotopic in vivo models. CONCLUSION GSK-3 inhibition with the small molecule inhibitor AZD2858 led to cell death in glioma stem cells preventing normal centrosome function and promoting mitotic failure. Normal human astrocytes were not affected by treatment with the inhibitor at submicromolar concentrations. Drug penetration was observed alongside an enhanced effect of clinical radiotherapy doses in vivo. The reported aberrant centrosomal duplication may be a direct consequence of failed cytokinesis suggesting a role of GSK-3 in regulation of mitosis in glioma. GSK-3 is a promising target for combination treatment with radiation in GBM management and plays a role in mitosis-associated events in glioma biology.

scholarly journals Presence of Anti-cystatin C–positive Dendritic Cells or Macrophages and Localization of Cysteine Proteases in the Apical Bud of the Enamel Organ in the Rat Incisor

2005 ◽  
Vol 53 (5) ◽  
pp. 643-651 ◽  
Author(s):  
Sumio Nishikawa
Keyword(s):  
Dendritic Cells ◽  
Cystatin C ◽  
Cysteine Protease ◽  
Cathepsin L ◽  
Cysteine Proteases ◽  
Cysteine Protease Inhibitor ◽  
Enamel Organ ◽  
Apical Bud ◽  
Apical Buds ◽  
Proliferation And Differentiation

Cystatin C, a cysteine protease inhibitor, was examined in the apical buds of rat incisors by immunohistochemistry, because in transition and maturation zones most of the dendritic cells in the papillary layer are anti-cystatin C–positive. Anti-cystatin C–labeled cells were sparse and localized to the proliferation and differentiation zones, constituting the apical bud of 5-week-old rat incisors. These cells were considered macrophages or dendritic cells, based on their reactivity with OX6 and ED1, as well as their ultrastructure. Basement membrane at the periphery of apical bud was also labeled by anti-cystatin C antibody. The apical buds included a few apoptotic fragments and weak reactivity with antibody to cathepsin L, a cysteine protease. Reactivity to anti-cystatin C and anti-cathepsin ∗∗∗L antibodies was also detected in the apical bud of newborn rat incisors. These results suggest that the cystatin C–positive macrophages or dendritic cells are involved in normal incisor formation. They may be related to the clearance of apoptotic cells or protection from putative cysteine protease activity.

scholarly journals Atovaquone Suppresses Triple-Negative Breast Tumor Growth by Reducing Immune-Suppressive Cells

2021 ◽  
Vol 22 (10) ◽  
pp. 5150
Author(s):  
Nehal Gupta ◽  
Shreyas Gaikwad ◽  
Itishree Kaushik ◽  
Stephen E. Wright ◽  
Maciej M. Markiewski ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Cancer Progression ◽  
Breast Tumor ◽  
Triple Negative ◽  
Immune Surveillance ◽  
Suppressor Cells ◽  
Myeloid Derived Suppressor Cells ◽  
In Vivo Models ◽  
Ribosomal Protein S19

A major contributing factor in triple-negative breast cancer progression is its ability to evade immune surveillance. One mechanism for this immunosuppression is through ribosomal protein S19 (RPS19), which facilitates myeloid-derived suppressor cells (MDSCs) recruitment in tumors, which generate cytokines TGF-β and IL-10 and induce regulatory T cells (Tregs), all of which are immunosuppressive and enhance tumor progression. Hence, enhancing the immune system in breast tumors could be a strategy for anticancer therapeutics. The present study evaluated the immune response of atovaquone, an antiprotozoal drug, in three independent breast-tumor models. Our results demonstrated that oral administration of atovaquone reduced HCC1806, CI66 and 4T1 paclitaxel-resistant (4T1-PR) breast-tumor growth by 45%, 70% and 42%, respectively. MDSCs, TGF-β, IL-10 and Tregs of blood and tumors were analyzed from all of these in vivo models. Our results demonstrated that atovaquone treatment in mice bearing HCC1806 tumors reduced MDSCs from tumor and blood by 70% and 30%, respectively. We also observed a 25% reduction in tumor MDSCs in atovaquone-treated mice bearing CI66 and 4T1-PR tumors. In addition, a decrease in TGF-β and IL-10 in tumor lysates was observed in atovaquone-treated mice with a reduction in tumor Tregs. Moreover, a significant reduction in the expression of RPS19 was found in tumors treated with atovaquone.

scholarly journals LpCat1 Promotes Malignant Transformation of Hepatocellular Carcinoma Cells by Directly Suppressing STAT1

2021 ◽  
Vol 11 ◽  
Author(s):  
Weidan Ji ◽  
Zhangxiao Peng ◽  
Bin Sun ◽  
Lei Chen ◽  
Qin Zhang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cancer Progression ◽  
Cell Cycle Progression ◽  
Migration And Invasion ◽  
Cycle Progression ◽  
Clinical Gene Therapy ◽  
First Time ◽  
Hcc Cells

Hepatocellular carcinoma (HCC) is a malignant cancer with rapid proliferation and high metastasis ability. To explore the crucial genes that maintain the aggressive behaviors of cancer cells is very important for clinical gene therapy of HCC. LpCat1 was reported to be highly expressed and exert pro-tumorigenic effect in a variety of cancers, including HCC. However, its detailed molecular mechanism remained unclear. In this study, we confirmed that LpCat1 was up-regulated in HCC tissues and cancer cell lines. The overexpressed LpCat1 promoted the proliferation, migration and invasion of HCC cells, and accelerated cell cycle progression, while knocking down LpCat1 significantly inhibited cell proliferation, migration and invasion in vitro and in vivo, and arrested HCC cells at G0/G1 phase. Moreover, we proved for the first time that LpCat1 directly interacted with STAT1 which was generally recognized as a tumor suppressor in HCC. High levels of LpCat1 in HCC could inhibit STAT1 expression, up-regulate CyclinD1, CyclinE, CDK4 and MMP-9, and decrease p27kip1 to promote cancer progression. Conversely, down-regulation of LpCat1 would cause the opposite changes to repress the viability and motility of HCC cells. Consequently, we concluded that LpCat1 was a contributor to progression and metastasis of HCC by interacting with STAT1.

scholarly journals Structure, interdomain dynamics and pH-dependent autoactivation of pro-rhodesain, the main lysosomal cysteine protease from African trypanosomes

2020 ◽  
Author(s):  
Patrick Johé ◽  
Elmar Jaenicke ◽  
Hannes Neuweiler ◽  
Tanja Schirmeister ◽  
Christian Kersten ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cysteine Protease ◽  
Cathepsin L ◽  
Salt Bridge ◽  
Correlation Spectroscopy ◽  
African Trypanosomes ◽  
Lysosomal Cysteine Protease ◽  
Control Switch ◽  
Α Helix ◽  
Dynamics Simulations

AbstractRhodesain is the lysosomal cathepsin L-like cysteine protease of T. brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood-brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating pro-domain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression of T. brucei rhodesiense pro-rhodesain in E. coli and determined its crystal structure. The trypanosomal pro-domain differs from non-parasitic pro-cathepsins by a unique, extended α-helix that blocks the active site and whose interactions resemble that of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence correlation spectroscopy increase at low pH, where pro-rhodesain also undergoes autocleavage. Using the crystal structure, molecular dynamics simulations and mutagenesis, we identify a conserved interdomain salt bridge that prevents premature intramolecular cleavage at higher pH values and may thus present a control switch for the observed pH-sensitivity of pro-enzyme cleavage in (trypanosomal) CathL-like proteases.

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