scholarly journals Designing a Cleavable Cell Surface Protein for Cytotherapy and Drug Delivery Applications

2021 ◽  
Vol 11 (6) ◽  
pp. 2792
Author(s):  
Matthew T. Basel ◽  
Tej B. Shrestha ◽  
Marla Pyle ◽  
Tuyen Duong Thanh Nguyen ◽  
Santosh Aryal ◽  
...  
Keyword(s):  
Cell Surface ◽  
Plasminogen Activator ◽  
Neural Progenitor Cells ◽  
Surface Protein ◽  
Surface Expression ◽  
Urokinase Plasminogen Activator ◽  
Plga Nanoparticles ◽  
Cell Surface Expression ◽  
Disease Site ◽  
Plga Nanoparticle

Many cytotherapy applications focus on delivering a therapeutic molecule or nanoparticle to a disease site. One challenging step in this delivery is releasing the therapeutic molecule from the delivery cell upon arrival at the delivery sight. Here a protein is designed and expressed that can bind a biotin-labeled cargo and release that cargo in response to the presence of urokinase plasminogen activator. A gene was designed that coded for a protein that contained a streptavidin domain for binding biotin-labeled cargo, a urokinase cleavage domain for release by urokinase plasminogen activator, and a PLAP domain for cell-surface expression. The utility of the resultant protein was tested with biotin (5-fluorescein) and a biotinylated PLGA nanoparticle to test the performance of the delivery systems with models for small molecule drugs and nanoformulations. When expressed in neural progenitor cells (C17.2), the designed protein was able to bind both the biotin (5-fluorescein) and the biotinylated PLGA nanoparticles and was able to release the biotin (5-fluorescein) in response to urokinase plasminogen activator. This designed, multi-domain protein may prove useful as a method for specifically releasing a cargo from delivery cells at a target site.

scholarly journals Involvement of LFA-1 in lymphoma invasion and metastasis demonstrated with LFA-1-deficient mutants.

1989 ◽  
Vol 108 (5) ◽  
pp. 1979-1985 ◽  
Author(s):  
F F Roossien ◽  
D de Rijk ◽  
A Bikker ◽  
E Roos
Keyword(s):  
Cell Surface ◽  
Surface Protein ◽  
Metastatic Potential ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Lymphocyte Function ◽  
Lymphoma Cells ◽  
Mutant Cells

Lymphocyte function-associated antigen-1 (LFA-1) is a leukocyte and lymphoma cell surface protein that promotes intercellular adhesion. We have previously shown that the invasion of hepatocyte cultures by lymphoma cells is inhibited by anti-LFA-1 antibodies (Roos, E., and F. F. Roossien. 1987. J. Cell Biol. 105:553-559). In addition, we now report that LFA-1 is also involved in invasion of lymphoma cells into fibroblast monolayers. To investigate the role of LFA-1 in metastasis of these lymphoma cells, we have generated mutants that are deficient in LFA-1 cell surface expression because of impaired synthesis of either the alpha or beta subunit precursor of LFA-1. We identified at least three distinct mutant clones. The invasive potential of the mutant cells in vitro, in both hepatocyte and fibroblast cultures, was considerably lower than that of parental cells. The metastatic potential of the mutants was much reduced, indicating that LFA-1 expression is required for efficient metastasis formation by certain lymphoma cells.

scholarly journals The Tetraspan Molecule Cd151, a Novel Constituent of Hemidesmosomes, Associates with the Integrin α6β4 and May Regulate the Spatial Organization of Hemidesmosomes

2000 ◽  
Vol 149 (4) ◽  
pp. 969-982 ◽  
Author(s):  
Lotus M.Th. Sterk ◽  
Cecile A.W. Geuijen ◽  
Lauran C.J.M. Oomen ◽  
Jero Calafat ◽  
Hans Janssen ◽  
...  
Keyword(s):  
Cell Surface ◽  
Spatial Organization ◽  
Transmembrane Domain ◽  
Focal Adhesions ◽  
Surface Protein ◽  
K562 Cells ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Junctional Epidermolysis Bullosa ◽  
Integrin Α6β4

CD151 is a cell surface protein that belongs to the tetraspan superfamily. It associates with other tetraspan molecules and certain integrins to form large complexes at the cell surface. CD151 is expressed by a variety of epithelia and mesenchymal cells. We demonstrate here that in human skin CD151 is codistributed with α3β1 and α6β4 at the basolateral surface of basal keratinocytes. Immunoelectron microscopy showed that CD151 is concentrated in hemidesmosomes. By immunoprecipitation from transfected K562 cells, we established that CD151 associates with α3β1 and α6β4. In β4-deficient pyloric atresia associated with junctional epidermolysis bullosa (PA-JEB) keratinocytes, CD151 and α3β1 are clustered together at the basal cell surface in association with patches of laminin-5. Focal adhesions are present at the periphery of these clusters, connected with actin filaments, and they contain both CD151 and α3β1. Transient transfection studies of PA-JEB cells with β4 revealed that the integrin α6β4 becomes incorporated into the α3β1-CD151 clusters where it induces the formation of hemidesmosomes. As a result, the amount of α3β1 in the clusters diminishes and the protein becomes restricted to the peripheral focal adhesions. Furthermore, CD151 becomes predominantly associated with α6β4 in hemidesmosomes, whereas its codistribution with α3β1 in focal adhesions becomes partial. The localization of α6β4 in the pre-hemidesmosomal clusters is accompanied by a strong upregulation of CD151, which is at least partly due to increased cell surface expression. Using β4 chimeras containing the extracellular and transmembrane domain of the IL-2 receptor and the cytoplasmic domain of β4, we found that for recruitment of CD151 into hemidesmosomes, the β4 subunit must be associated with α6, confirming that integrins associate with tetraspans via their α subunits. CD151 is the only tetraspan identified in hemidesmosomal structures. Others, such as CD9 and CD81, remain diffusely distributed at the cell surface. In conclusion, we show that CD151 is a major component of (pre)-hemidesmosomal structures and that its recruitment into hemidesmosomes is regulated by the integrin α6β4. We suggest that CD151 plays a role in the formation and stability of hemidesmosomes by providing a framework for the spatial organization of the different hemidesmosomal components.

scholarly journals Trafficking of Kv1.4 potassium channels: interdependence of a pore region determinant and a cytoplasmic C-terminal VXXSL determinant in regulating cell-surface trafficking

2003 ◽  
Vol 375 (3) ◽  
pp. 761-768 ◽  
Author(s):  
Jing ZHU ◽  
Itaru WATANABE ◽  
Barbara GOMEZ ◽  
William B. THORNHILL
Keyword(s):  
Cell Surface ◽  
Cell Function ◽  
Cell Signalling ◽  
Surface Protein ◽  
Surface Expression ◽  
Inhibitory Effect ◽  
Cell Surface Expression ◽  
Pore Region ◽  
Expression Levels ◽  
Protein Levels

Kv1.4 and Kv1.1 potassium channel homomers have been shown to exhibit different intracellular trafficking programmes and cell-surface expression levels in cell lines: a determinant in the pore region of Kv1.4 and Kv1.1 [Zhu, Watanabe, Gomez and Thornhill (2001) J. Biol. Chem. 276, 39419–39427] and a cytoplasmic C-terminal VXXSL determinant on Kv1.4 [Li, Takimoto and Levitan (2000) J. Biol. Chem. 275, 11597–11602] have been described, which affected trafficking and cell-surface expression levels. In the present study, we examined whether trafficking pore determinants influenced any cytoplasmic C-terminal trafficking determinant. We found that removal of VXXSL from a Kv1.4 chimaera that contained the pore of Kv1.1 did not affect cell-surface trafficking. Therefore removal of the C-terminal VXXSL of Kv1.4 inhibited protein surface levels only in the presence of the Kv1.4 pore. In contrast, truncating the cytoplasmic C-terminus of Kv1.1 or truncating a Kv1.1 chimaera with the pore of Kv1.4, had little effect on surface protein levels. Furthermore, the subregion of the Kv1.4 pore trafficking determinant that was required for the inhibitory effect of VXXSL removal was mapped to a threonine residue in the deep pore region. Therefore the Kv1.4 pore determinant affected the trafficking and cell-surface levels directed by the C-terminal VXXSL determinant. Different Kv1 trafficking programmes would affect cell-surface expression levels either positively or negatively and also cell signalling. Cells may use differential trafficking programmes of membrane proteins as a post-translational mechanism to regulate surface protein levels and cell function.

scholarly journals Structural Determinants in the Calcitonin Receptor-Like Receptor (Crlr) Important for Cgrp and Adrenomedullin (Am) Receptor Function of Crlr/Receptor-Activity-Modifying Protein (Ramp) 1 and Crlr/Ramp2 Heterodimers

2001 ◽  
Vol 1 ◽  
pp. 8-8
Author(s):  
W. Born ◽  
K. Leuthauser ◽  
R. Gujer ◽  
R. Muff ◽  
J.A. Fischer
Keyword(s):  
Cell Surface ◽  
Surface Protein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Cross Linking ◽  
Cell Surface Protein ◽  
Calcitonin Receptor ◽  
Camp Production ◽  
Structural Determinants ◽  
Structural Alterations

Cell surface protein cross-linking, coimmmunoprecipitation, and confocal microscopy identified CRLR/RAMP1-, CRLR/RAMP2-, and calcitonin receptor isotype 2 (CTR2)/RAMP1 heterodimers as CGRP-, AM-, and CGRP/amylin receptors, linked to cAMP production. Along these lines, effects of structural alterations in the N-terminal extracellular domain of the human CRLR on cell surface expression as well as the association with RAMP and CGRP or AM have been investigated.

uPAR regulates pericellular proteolysis through a mechanism involving integrins and fMLF-receptors

2013 ◽  
Vol 109 (02) ◽  
pp. 309-318 ◽  
Author(s):  
Loredana Rinaldi ◽  
Vincenza Elena Anna Rea ◽  
Nunzia Montuori ◽  
Vincenzo Cosimato ◽  
Daniela Alfano ◽  
...  
Keyword(s):  
Cell Surface ◽  
Phosphatidyl Inositol ◽  
Surface Protein ◽  
Surface Expression ◽  
Surface Proteins ◽  
Cell Surface Expression ◽  
Type Plasminogen Activator ◽  
Mitogen Activated Protein ◽  
Gi Protein ◽  
Tumour Promoters

SummaryThe expression of the urokinase-type plasminogen activator (uPA) and its receptor (uPAR) can be regulated by several hormones, cytokines, and tumour promoters. uPAR is a glycosyl-phosphatidyl inositol (GPI)- linked cell-surface protein; however, it is capable to transduce signals inside the cell by interacting with other cell-surface proteins, such as integrins and G-protein coupled (GPC) receptors. We previously reported that uPAR cell-surface expression can be positively regulated by its ligand, uPA, independently of its proteolytic activity. We now demonstrate that uPAR overexpression induces or increases uPA secretion both in uPAR-negative and in uPAR-expressing cells. Accordingly, uPAR depletion impairs uPA expression in cells which constitutively express both uPA and its receptor. uPAR exerts its regulatory effect through the activation of the ERK mitogen-activated protein kinases (MAPKs), whereas the p-38 MAPK is not involved. Overexpression of truncated forms of uPAR, lacking the N-terminal domain (DI) and not able to interact with membrane co-receptors, failed to increase uPA expression. Inhibition of uPAR-integrin interaction by the specific P-25 peptide, as well as Gi-protein inhibition by cholera pertussin toxin or depletion of the GPC receptors for fMLF (fMLF-Rs) also impaired uPAR capability to regulate uPA expression. These findings demonstrate that uPAR, whose expression is regulated by uPA, can, in turn, regulate uPA expression through a mechanism involving its functional interaction with integrins and fMLF-Rs.

Analysis of the Cell Surface Expression of Cytokine Receptors Using the Surface Protein Biotinylation Method

2014 ◽  
pp. 185-192 ◽  
Author(s):  
Mahmud Arif Pavel ◽  
Clarissa Lam ◽  
Parul Kashyap ◽  
Zahra Salehi-Najafabadi ◽  
Gurpreet Singh ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Protein ◽  
Surface Expression ◽  
Cytokine Receptors ◽  
Cell Surface Expression

Persistent HIV Transcription Induces Sialyl-Lewis-X Glyco-Antigen Cell-Surface Expression

2020 ◽  
Author(s):  
Florent Colomb ◽  
Leila B. Giron ◽  
Leticia Kuri Cervantes ◽  
Tongcui Ma ◽  
Samson Adeniji ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Sialyl Lewis X ◽  
Cell Surface Expression ◽  
Lewis X ◽  
Hiv Transcription ◽  
Sialyl Lewis

Influence of β-D-mannuronic acid, as a new member of non-steroidal anti-inflammatory drugs family, on expression pattern of chemokines and their receptors in rheumatoid arthritis

2019 ◽  
Vol 16 ◽  
Author(s):  
Mona Aslani ◽  
Arman Ahmadzadeh ◽  
Zahra Aghazadeh ◽  
Majid Zaki-Dizaji ◽  
Laleh Sharifi ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mrna Expression ◽  
Surface Expression ◽  
Phase Iii ◽  
Cell Surface Expression ◽  
Optimum Dose ◽  
High Dose ◽  
Mannuronic Acid ◽  
Anti Inflammatory ◽  
High Doses

Background: : Based on the encouraging results of phase III clinical trial of β-D-mannuronic acid (M2000) (as a new anti-inflammatory drug) in patients with RA, in this study, we aimed to evaluate the effects of this drug on the expression of chemokines and their receptors in PBMCs of RA patients. Methods:: PBMCs of RA patients and healthy controls were separated and the patients' cells were treated with low, moderate and high doses (5, 25 and 50 μg/mL) of M2000 and optimum dose (1 μg/mL) of diclofenac, as a control in RPMI-1640 medium. Real-time PCR was used for evaluating the mRNA expression of CXCR3, CXCR4, CCR2, CCR5 and CCL2/MCP-1. Cell surface expression of CCR2 was investigated using flow cytometry. Results:: CCR5 mRNA expression reduced significantly, after treatment of the patients' cells with all three doses of M2000 and optimum dose of diclofenac. CXCR3 mRNA expression down-regulated significantly followed by treatment of these cells with moderate and high doses of M2000 and optimum dose of diclofenac. CXCR4 mRNA expression declined significantly after treatment of these cells with moderate and high doses of M2000. CCL2 mRNA expression significantly reduced only followed by treatment of these cells with high dose of M2000, whereas, mRNA and cell surface expressions of CCR2 diminished significantly followed by treatment of these cells with high dose of M2000 and optimum dose of diclofenac. Conclusion:: According to our results, M2000 through the down-regulation of chemokines and their receptors may restrict the infiltration of immune cells into the synovium.

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