scholarly journals Simultaneous targeting of CD44 and MMP9 catalytic and hemopexin domains as a therapeutic strategy

2021 ◽  
Author(s):  
Gal Yosef ◽  
Hezi Hayun ◽  
Niv Papo
Keyword(s):  
Cell Surface ◽  
Cancer Cell ◽  
Cancer Progression ◽  
Catalytic Domain ◽  
Surface Display ◽  
Specific Inhibitor ◽  
Yeast Surface Display ◽  
Great Promise ◽  
Mmp Inhibitor ◽  
Neoplastic Process

Crosstalk of the oncogenic matrix metalloproteinase-9 (MMP9) and one of its ligands, CD44, involves cleavage of CD44 by the MMP9 catalytic domain, with the CD44–MMP9 interaction on the cell surface taking place through the MMP9 hemopexin domain (PEX). This interaction promotes cancer cell migration and invasiveness. In concert, MMP9-processed CD44 induces the expression of MMP9, which degrades ECM components and facilitates growth factor release and activation, cancer cell invasiveness, and metastasis. Since both MMP9 and CD44 contribute to cancer progression, we have developed a new strategy to fully block this neoplastic process by engineering a multi-specific inhibitor that simultaneously targets CD44 and both the catalytic and PEX domains of MMP9. Using a yeast surface display technology, we first obtained a high-affinity inhibitor for the MMP9 catalytic domain, which we termed C9, by modifying a natural non-specific MMP inhibitor, N-TIMP2. We then conjugated C9 via a flexible linker to PEX, thereby creating a multi-specific inhibitor (C9-PEX) that simultaneously targets the MMP9 catalytic and PEX domains and CD44. It is likely that, via its co-localization with CD44, C9-PEX may compete with MMP9 localization on the cell surface, thereby inhibiting MMP9 catalytic activity, reducing MMP9 cellular levels, interfering with MMP9 homodimerization, and reducing the activation of downstream MAPK/ERK pathway signaling. The developed platform could be extended to other oncogenic MMPs as well as to other important target proteins, thereby offering great promise for creating novel multi-specific therapeutics for cancer and other diseases.

scholarly journals Converting a broad matrix metalloproteinase family inhibitor into a specific inhibitor of MMP-9 and MMP-14

2017 ◽  
Author(s):  
Jason Shirian ◽  
Valeria Arkadash ◽  
Itay Cohen ◽  
Tamila Sapir ◽  
Evette S. Radisky ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Surface Display ◽  
Specific Inhibitor ◽  
Computational Design ◽  
Yeast Surface Display ◽  
Physiological Conditions ◽  
Mmp Inhibitor ◽  
Single Target ◽  
Display Technology ◽  
Yeast Surface

AbstractMMP-14 and MMP-9 are two well established cancer targets for which no specific clinically relevant inhibitor is available. Using a powerful combination of computational design and yeast surface display technology, we engineered such an inhibitor starting from a non-specific MMP inhibitor, N-TIMP2. The engineered purified N-TIMP2 variants showed enhanced specificity towards MMP-14 and MMP-9 relative to a panel of off-target MMPs. MMP-specific N-TIMP2 sequence signatures were obtained that could be understood from the structural perspective of MMP/N-TIMP2 interactions. Our MMP-9 inhibitor exhibited 1000-fold preference for MMP-9 vs. MMP-14, which is likely to translate into significant differences under physiological conditions. Our results provide new insights regarding evolution of promiscuous proteins and optimization strategies for design of inhibitors with single-target specificities.

scholarly journals Development of High Affinity and High Specificity Inhibitors of Matrix Metalloproteinase 14 through Computational Design and Directed Evolution

2017 ◽  
Vol 292 (8) ◽  
pp. 3481-3495 ◽  
Author(s):  
Valeria Arkadash ◽  
Gal Yosef ◽  
Jason Shirian ◽  
Itay Cohen ◽  
Yuval Horev ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Surface Display ◽  
High Specificity ◽  
Computational Design ◽  
Computational Method ◽  
Yeast Surface Display ◽  
Mmp Inhibitor ◽  
Starting Point ◽  
Yeast Surface

Degradation of the extracellular matrices in the human body is controlled by matrix metalloproteinases (MMPs), a family of more than 20 homologous enzymes. Imbalance in MMP activity can result in many diseases, such as arthritis, cardiovascular diseases, neurological disorders, fibrosis, and cancers. Thus, MMPs present attractive targets for drug design and have been a focus for inhibitor design for as long as 3 decades. Yet, to date, all MMP inhibitors have failed in clinical trials because of their broad activity against numerous MMP family members and the serious side effects of the proposed treatment. In this study, we integrated a computational method and a yeast surface display technique to obtain highly specific inhibitors of MMP-14 by modifying the natural non-specific broad MMP inhibitor protein N-TIMP2 to interact optimally with MMP-14. We identified an N-TIMP2 mutant, with five mutations in its interface, that has an MMP-14 inhibition constant (Ki) of 0.9 pm, the strongest MMP-14 inhibitor reported so far. Compared with wild-type N-TIMP2, this variant displays ∼900-fold improved affinity toward MMP-14 and up to 16,000-fold greater specificity toward MMP-14 relative to other MMPs. In an in vitro and cell-based model of MMP-dependent breast cancer cellular invasiveness, this N-TIMP2 mutant acted as a functional inhibitor. Thus, our study demonstrates the enormous potential of a combined computational/directed evolution approach to protein engineering. Furthermore, it offers fundamental clues into the molecular basis of MMP regulation by N-TIMP2 and identifies a promising MMP-14 inhibitor as a starting point for the development of protein-based anticancer therapeutics.

scholarly journals Yeast Surface Display of Trifunctional Minicellulosomes for Simultaneous Saccharification and Fermentation of Cellulose to Ethanol

2009 ◽  
Vol 76 (4) ◽  
pp. 1251-1260 ◽  
Author(s):  
Fei Wen ◽  
Jie Sun ◽  
Huimin Zhao
Keyword(s):  
Cell Surface ◽  
Consolidated Bioprocessing ◽  
Surface Display ◽  
Cellulose Hydrolysis ◽  
Cellulase Production ◽  
Single Step ◽  
Yeast Surface Display ◽  
Biofuel Production ◽  
Yeast Cells ◽  
Recombinant Yeast Strain

ABSTRACT By combining cellulase production, cellulose hydrolysis, and sugar fermentation into a single step, consolidated bioprocessing (CBP) represents a promising technology for biofuel production. Here we report engineering of Saccharomyces cerevisiae strains displaying a series of uni-, bi-, and trifunctional minicellulosomes. These minicellulosomes consist of (i) a miniscaffoldin containing a cellulose-binding domain and three cohesin modules, which was tethered to the cell surface through the yeast a-agglutinin adhesion receptor, and (ii) up to three types of cellulases, an endoglucanase, a cellobiohydrolase, and a β-glucosidase, each bearing a C-terminal dockerin. Cell surface assembly of the minicellulosomes was dependent on expression of the miniscaffoldin, indicating that formation of the complex was dictated by the high-affinity interactions between cohesins and dockerins. Compared to the unifunctional and bifunctional minicellulosomes, the quaternary trifunctional complexes showed enhanced enzyme-enzyme synergy and enzyme proximity synergy. More importantly, surface display of the trifunctional minicellulosomes gave yeast cells the ability to simultaneously break down and ferment phosphoric acid-swollen cellulose to ethanol with a titer of ∼1.8 g/liter. To our knowledge, this is the first report of a recombinant yeast strain capable of producing cell-associated trifunctional minicellulosomes. The strain reported here represents a useful engineering platform for developing CBP-enabling microorganisms and elucidating principles of cellulosome construction and mode of action.

scholarly journals ERK1/2-dependent activation of FCHSD2 drives cancer cell-selective regulation of clathrin-mediated endocytosis

2018 ◽  
Author(s):  
Guan-Yu Xiao ◽  
Aparna Mohanakrishnan ◽  
Sandra L. Schmid
Keyword(s):  
Cell Surface ◽  
Cancer Patient ◽  
Cancer Cells ◽  
Cancer Cell ◽  
Cancer Progression ◽  
Patient Survival ◽  
Expression Level ◽  
Downstream Signaling ◽  
And Migration ◽  
Proliferation And Migration

AbstractClathrin-mediated endocytosis (CME) regulates the uptake of cell surface receptors, as well as their downstream signaling activities. We recently reported that signaling reciprocally regulates CME in cancer cells and that the crosstalk can contribute to cancer progression. To further explore the nature and extent of the crosstalk between signaling and CME in cancer cell biology, we analyzed a panel of oncogenic signaling kinase inhibitors for their effects on CME. Inhibition of several kinases selectively affected CME function in cancer cells. Among these, ERK1/2 inhibition selectively inhibited CME in cancer cells by decreasing the rate of CCP initiation. We identified an ERK1/2 substrate, the FCH/F-BAR and SH3 domain-containing protein, FCHSD2, as being essential for the ERK1/2-dependent effects on CME and CCP initiation. ERK1/2 phosphorylation activates FCHSD2 and regulates EGFR endocytic trafficking as well as downstream signaling activities. Loss of FCHSD2 activity in non-small-cell lung cancer cells leads to increased cell surface expression and altered signaling downstream of EGFR, resulting in enhanced cell proliferation and migration. The expression level of FCHSD2 is positively correlated with higher cancer patient survival rate, suggesting that FCHSD2 negatively affects cancer progression. These findings provide new insight into the mechanisms and consequences of the reciprocal regulation of signaling and CME in cancer cells.SignificanceClathrin-mediated endocytosis (CME) determines the internalization of receptors and their downstream signaling. We discovered that CME is differentially regulated by specific signaling kinases in cancer cells. In particular, ERK1/2-mediated phosphorylation of the FCH/F-BAR and double SH3 domains-containing protein 2 (FCHSD2) regulates CME, and the trafficking and signaling activities of EGF receptors. This reciprocal interaction negatively regulates cancer proliferation and migration. The expression level of FCHSD2 is positively correlated with higher cancer patient survival rates. This study identifies signaling pathways that impinge on the endocytic machinery and reveals a molecular nexus for crosstalk between intracellular signaling and CME. Cancer cells specifically adapt this crosstalk as a determinant for tumor progression, which has implications for novel therapeutics against cancers.

scholarly journals Role for ERK1/2-dependent activation of FCHSD2 in cancer cell-selective regulation of clathrin-mediated endocytosis

2018 ◽  
Vol 115 (41) ◽  
pp. E9570-E9579 ◽  
Author(s):  
Guan-Yu Xiao ◽  
Aparna Mohanakrishnan ◽  
Sandra L. Schmid
Keyword(s):  
Cell Surface ◽  
Cancer Cells ◽  
Cancer Cell ◽  
Cancer Progression ◽  
Cell Biology ◽  
Egf Receptor ◽  
Nsclc Patient ◽  
Nonsmall Cell Lung Cancer ◽  
Cell Surface Expression ◽  
Downstream Signaling

Clathrin-mediated endocytosis (CME) regulates the uptake of cell-surface receptors as well as their downstream signaling activities. We recently reported that signaling can reciprocally regulate CME in cancer cells and that this crosstalk can contribute to cancer progression. To further explore the nature and extent of the crosstalk between signaling and CME in cancer cell biology, we analyzed a panel of oncogenic signaling kinase inhibitors for their effects on CME across a panel of normal and cancerous cells. Inhibition of several kinases selectively affected CME in cancer cells, including inhibition of ERK1/2, which selectively inhibited CME by decreasing the rate of clathrin-coated pit (CCP) initiation. We identified an ERK1/2 substrate, the FCH/F-BAR and SH3 domain-containing protein FCHSD2, as being essential for the ERK1/2-dependent effects on CME and CCP initiation. Our data suggest that ERK1/2 phosphorylation activates FCHSD2 and regulates EGF receptor (EGFR) endocytic trafficking as well as downstream signaling activities. Loss of FCHSD2 activity in nonsmall cell lung cancer (NSCLC) cells leads to increased cell-surface expression and altered signaling downstream of EGFR, resulting in enhanced cell proliferation and migration. The expression level of FCHSD2 is positively correlated with higher NSCLC patient survival rates, suggesting that FCHSD2 can negatively affect cancer progression. These findings provide insight into the mechanisms and consequences of the reciprocal regulation of signaling and CME in cancer cells.

scholarly journals Preferential Identification of Agonistic OX40 Antibodies by Using Cell Lysate to Pan Natively Paired, Humanized Mouse-Derived Yeast Surface Display Libraries

Antibodies ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 17 ◽  
Author(s):  
Angélica Medina-Cucurella ◽  
Rena Mizrahi ◽  
Michael Asensio ◽  
Robert Edgar ◽  
Jackson Leong ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Display ◽  
Yeast Surface Display ◽  
Soluble Antigen ◽  
Single Chain ◽  
Humanized Mouse ◽  
Modern Approach ◽  
Cell Lysate ◽  
Scfv Library ◽  
Mouse Immunization

To discover therapeutically relevant antibody candidates, many groups use mouse immunization followed by hybridoma generation or B cell screening. One modern approach is to screen B cells by generating natively paired single chain variable fragment (scFv) display libraries in yeast. Such methods typically rely on soluble antigens for scFv library screening. However, many therapeutically relevant cell-surface targets are difficult to express in a soluble protein format, complicating discovery. In this study, we developed methods to screen humanized mouse-derived yeast scFv libraries using recombinant OX40 protein in cell lysate. We used deep sequencing to compare screening with cell lysate to screening with soluble OX40 protein, in the context of mouse immunizations using either soluble OX40 or OX40-expressing cells and OX40-encoding DNA vector. We found that all tested methods produce a unique diversity of scFv binders. However, when we reformatted forty-one of these scFv as full-length monoclonal antibodies (mAbs), we observed that mAbs identified using soluble antigen immunization with cell lysate sorting always bound cell surface OX40, whereas other methods had significant false positive rates. Antibodies identified using soluble antigen immunization and cell lysate sorting were also significantly more likely to activate OX40 in a cellular assay. Our data suggest that sorting with OX40 protein in cell lysate is more likely than other methods to retain the epitopes required for antibody-mediated OX40 agonism.

Constructon of Yeast Surface Display of ProROL by Using Flo1p Anchor System

2012 ◽  
Vol 512-515 ◽  
pp. 356-360 ◽  
Author(s):  
Ye Wang ◽  
Wen Qian Li ◽  
Xun Li ◽  
Hao Shi ◽  
Fei Wang
Keyword(s):  
Cell Surface ◽  
Fluorescent Protein ◽  
Surface Display ◽  
Yeast Surface Display ◽  
Functional Domain ◽  
Display System ◽  
Fermentation Time ◽  
Anchor System ◽  
Pastoris Cell ◽  
Yeast Surface

A Pichia pastoris cell-surface display system was constructed using a Flo1p anchor system containing N-terminal flocculation functional domain (874 residues, FS), derived from Saccharomyces cerevisiae. The lipase from Rhizopus oryzae with a pro sequence (ProROL) and green fluorescent protein (GFP) gene were successfully cloned and genetically fused to the anchor system with their C-terminus free. Fluorescence microscope was used to detect the GFP displayed on the recombinant P. pastoris cell surface. The results showed that the yeast surface display system using Flo1p as the anchor protein was successfully constructed, and the activity of ProROL displayed on KM71H reached to 217.15 U/g, much higher than 61.30 U/g reported by Matsumoto. Besides, the yeast surface display system could effectively shorten the fermentation time compared with traditional fermentation.

Expression Cassette and Plasmid Construction for Yeast Surface Display in Saccharomyces Cerevisiae

2021 ◽  
Author(s):  
Renan E A Piraine ◽  
Vitória S Gonçalves ◽  
Alceu GS dos Santos Junior ◽  
Rodrigo C Cunha ◽  
Pedro MM Albuquerque ◽  
...  
Keyword(s):  
Cell Surface ◽  
Recombinant Protein Expression ◽  
Polyclonal Antibodies ◽  
Surface Display ◽  
Cell Surface Display ◽  
Yeast Surface Display ◽  
Expression Cassette ◽  
Dot Blot ◽  
Herpesvirus Type ◽  
Yeast Cell Surface

Abstract Objectives. Develop a Cell Surface Display system in S. cerevisiae, based on the construction of an expression cassette for pYES2 plasmid. Results. The construction of an expression cassette containing the α-factor signal peptide and the C-terminal portion of the α-agglutinin protein was made and its sequence inserted into a plasmid named pYES2/gDαAgglutinin, allowing cell surface display of bovine herpesvirus type 5 (BoHV-5) glycoprotein D (gD) on S. cerevisiae BY4741 strain. Recombinant protein expression was confirmed by dot blot, and indirect immunofluorescence using monoclonal anti-histidine antibodies and polyclonal antibodies from mice experimentally vaccinated with a recombinant gD. Conclusions. These results demonstrate that the approach and plasmid used represent not only an effective system for immobilizing proteins on the yeast cell surface, as well as a platform for immunobiologicals development.

Expression of Urokinase and Its Receptor in Invasive and Non-Invasive Prostate Cancer Cell Lines

1992 ◽  
Vol 68 (06) ◽  
pp. 662-666 ◽  
Author(s):  
W Hollas ◽  
N Hoosein ◽  
L W K Chung ◽  
A Mazar ◽  
J Henkin ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Steady State ◽  
Cell Surface ◽  
Cell Lines ◽  
Cancer Cell ◽  
Prostate Cancer Cell ◽  
Cancer Cell Lines ◽  
Lncap Cells ◽  
Prostate Cancer Cell Lines ◽  
Non Invasive

SummaryWe previously reported that extracellular matrix invasion by the prostate cancer cell lines, PC-3 and DU-145 was contingent on endogenous urokinase being bound to a specific cell surface receptor. The present study was undertaken to characterize the expression of both urokinase and its receptor in the non-invasive LNCaP and the invasive PC-3 and DU-145 prostate cells. Northern blotting indicated that the invasive PC-3 cells, which secreted 10 times more urokinase (680 ng/ml per 106 cells per 48 h) than DU-145 cells (63 ng/ml per 106 cells per 48 h), had the most abundant transcript for the plasminogen activator. This, at least, partly reflected a 3 fold amplification of the urokinase gene in the PC-3 cells. In contrast, urokinase-specific transcript could not be detected in the non-invasive LNCaP cells previously characterized as being negative for urokinase protein. Southern blotting indicated that this was not a consequence of deletion of the urokinase gene. Crosslinking of radiolabelled aminoterminal fragment of urokinase to the cell surface indicated the presence of a 51 kDa receptor in extracts of the invasive PC-3 and DU-145 cells but not in extracts of the non-invasive LNCaP cells. The amount of binding protein correlated well with binding capacities calculated by Scatchard analysis. In contrast, the steady state level of urokinase receptor transcript was a poor predictor of receptor display. PC-3 cells, which were equipped with 25,000 receptors per cell had 2.5 fold more steady state transcript than DU-145 cells which displayed 93,000 binding sites per cell.

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