scholarly journals A membrane protein display platform for receptor interactome discovery

2021 ◽  
Vol 118 (39) ◽  
pp. e2025451118
Author(s):  
Shengya Cao ◽  
Sean M. Peterson ◽  
Sören Müller ◽  
Mike Reichelt ◽  
Christian McRoberts Amador ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Cell Surface ◽  
Weak Interactions ◽  
Tumor Stroma ◽  
Extracellular Vesicle ◽  
Receptor Interaction ◽  
Surface Receptors ◽  
Wide Range ◽  
Screening Platform

Cell surface receptors are critical for cell signaling and constitute a quarter of all human genes. Despite their importance and abundance, receptor interaction networks remain understudied because of difficulties associated with maintaining membrane proteins in their native conformation and their typically weak interactions. To overcome these challenges, we developed an extracellular vesicle-based method for membrane protein display that enables purification-free and high-throughput detection of receptor–ligand interactions in membranes. We demonstrate that this platform is broadly applicable to a variety of membrane proteins, enabling enhanced detection of extracellular interactions over a wide range of binding affinities. We were able to recapitulate and expand the interactome for prominent members of the B7 family of immunoregulatory proteins such as PD-L1/CD274 and B7-H3/CD276. Moreover, when applied to the orphan cancer-associated fibroblast protein, LRRC15, we identified a membrane-dependent interaction with the tumor stroma marker TEM1/CD248. Furthermore, this platform enabled profiling of cellular receptors for target-expressing as well as endogenous extracellular vesicles. Overall, this study presents a sensitive and easy to use screening platform that bypasses membrane protein purification and enables characterization of interactomes for any cell surface–expressed target of interest in its native state.

scholarly journals Advancing Cell-Instructive Biomaterials Through Increased Understanding of Cell Receptor Spacing and Material Surface Functionalization

2020 ◽  
Author(s):  
Stephanie A. Maynard ◽  
Charles W. Winter ◽  
Eoghan M. Cunnane ◽  
Molly M. Stevens
Keyword(s):  
Regenerative Medicine ◽  
Cell Surface ◽  
Cell Surface Receptors ◽  
Material Surface ◽  
Surface Receptors ◽  
Native Tissue ◽  
Cell Material ◽  
Wide Range ◽  
Cell Material Interactions ◽  
Regenerative Therapies

Abstract Regenerative medicine is aimed at restoring normal tissue function and can benefit from the application of tissue engineering and nano-therapeutics. In order for regenerative therapies to be effective, the spatiotemporal integration of tissue-engineered scaffolds by the native tissue, and the binding/release of therapeutic payloads by nano-materials, must be tightly controlled at the nanoscale in order to direct cell fate. However, due to a lack of insight regarding cell–material interactions at the nanoscale and subsequent downstream signaling, the clinical translation of regenerative therapies is limited due to poor material integration, rapid clearance, and complications such as graft-versus-host disease. This review paper is intended to outline our current understanding of cell–material interactions with the aim of highlighting potential areas for knowledge advancement or application in the field of regenerative medicine. This is achieved by reviewing the nanoscale organization of key cell surface receptors, the current techniques used to control the presentation of cell-interactive molecules on material surfaces, and the most advanced techniques for characterizing the interactions that occur between cell surface receptors and materials intended for use in regenerative medicine. Lay Summary The combination of biology, chemistry, materials science, and imaging technology affords exciting opportunities to better diagnose and treat a wide range of diseases. Recent advances in imaging technologies have enabled better understanding of the specific interactions that occur between human cells and their immediate surroundings in both health and disease. This biological understanding can be used to design smart therapies and tissue replacements that better mimic native tissue. Here, we discuss the advances in molecular biology and technologies that can be employed to functionalize materials and characterize their interaction with biological entities to facilitate the design of more sophisticated medical therapies.

scholarly journals Understanding the yeast host cell response to recombinant membrane protein production

2011 ◽  
Vol 39 (3) ◽  
pp. 719-723 ◽  
Author(s):  
Zharain Bawa ◽  
Charlotte E. Bland ◽  
Nicklas Bonander ◽  
Nagamani Bora ◽  
Stephanie P. Cartwright ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Drug Targets ◽  
Large Scale ◽  
Protein Production ◽  
Molecular Mechanisms ◽  
New Drugs ◽  
Host Cells ◽  
Host Cell Response ◽  
Wide Range

Membrane proteins are drug targets for a wide range of diseases. Having access to appropriate samples for further research underpins the pharmaceutical industry's strategy for developing new drugs. This is typically achieved by synthesizing a protein of interest in host cells that can be cultured on a large scale, allowing the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to that of the native human source cells of many proteins of interest, while also being quick, easy and cheap to grow and process. Even in these cells, the production of human membrane proteins can be plagued by low functional yields; we wish to understand why. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast host strains. By relieving the bottlenecks to recombinant membrane protein production in yeast, we aim to contribute to the drug discovery pipeline, while providing insight into translational processes.

scholarly journals Cell-surface discoidin in aggregating cells of Dictyostelium discoideum

1982 ◽  
Vol 204 (3) ◽  
pp. 787-794 ◽  
Author(s):  
I C Madley ◽  
M J Cook ◽  
B D Hames
Keyword(s):  
Membrane Protein ◽  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Electrophoretic Analysis ◽  
Integral Membrane Protein ◽  
Polyacrylamide Gel ◽  
Two Dimensional ◽  
Surface Receptors

Both discoidin I and discoidin II have been detected on the surface of aggregating (10 h developmental stage) cells of Dictyostelium discoideum NC4 by radioiodination of the cell-surface followed by immunoprecipitation and sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic analysis. Approx. 92% of cell-surface discoidin I and 72% of cell-surface discoidin II can be eluted with 0.5 M-galactose, showing that most of each endogenous lectin is not present as integral membrane protein but rather is bound to cell-surface discoidin receptors. Two-dimensional polyacrylamide-gel-electrophoretic analysis of discoidin I suggests that the native tetramer may be a hetero-multimer composed of both Ia and Ib subunits. Cell-surface discoidin I also contains both types of subunit, but it is not clear whether both subunits have corresponding cell-surface receptors.

scholarly journals Lipoprotein receptors – an evolutionarily ancient multifunctional receptor family

2010 ◽  
Vol 391 (11) ◽  
Author(s):  
Marco Dieckmann ◽  
Martin Frederik Dietrich ◽  
Joachim Herz
Keyword(s):  
Cell Surface ◽  
Receptor Gene ◽  
Ldl Receptor ◽  
Amyloid Peptide ◽  
Cell Surface Receptors ◽  
Lipoprotein Receptors ◽  
Surface Receptors ◽  
Wide Range ◽  
Extracellular Environment ◽  
Receptor Family

Abstract The evolutionarily ancient low-density lipoprotein (LDL) receptor gene family represents a class of widely expressed cell surface receptors. Since the dawn of the first primitive multicellular organisms, several structurally and functionally distinct families of lipoprotein receptors have evolved. In accordance with the now obsolete ‘one-gene-one-function’ hypothesis, these cell surface receptors were orginally perceived as mere transporters of lipoproteins, lipids, and nutrients or as scavenger receptors, which remove other kinds of macromolecules, such as proteases and protease inhibitors from the extracellular environment and the cell surface. This picture has since undergone a fundamental change. Experimental evidence has replaced the perception that these receptors serve merely as cargo transporters. Instead it is now clear that the transport of macromolecules is inseparably intertwined with the molecular machinery by which cells communicate with each other. Lipoprotein receptors are essentially sensors of the extracellular environment that participate in a wide range of physiological processes by physically interacting and coevolving with primary signal transducers as co-regulators. Furthermore, lipoprotein receptors modulate cellular trafficking and localization of the amyloid precursor protein (APP) and the β-amyloid peptide (Aβ), suggesting a role in the pathogenesis of Alzheimer's disease. Moreover, compelling evidence shows that LDL receptor family members are involved in tumor development and progression.

scholarly journals Membrane-active Polymers: NCMNP13-x, NCMNP21-x and NCMNP21b-x for Membrane Protein Structural Biology

2022 ◽  
Author(s):  
Thi Kim Hoang Trinh ◽  
Claudio Catalano ◽  
Youzhong Guo
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Structural Biology ◽  
Maleic Acid ◽  
Drug Targets ◽  
Design Synthesis ◽  
Native Cell ◽  
Wide Range ◽  
Lipid Particles ◽  
High Resolution Structure

Membrane proteins are a ubiquitous group of bio-macromolecules responsible for many crucial biological processes and serve as drug targets for a wide range of modern drugs. Detergent-free technologies such as styrene-maleic acid lipid particles (SMALP), diisobutylene-maleic acid lipid particles (DIBMALP), and native cell membrane nanoparticles (NCMN) systems have recently emerged as revolutionary alternatives to the traditional detergent-based approaches for membrane protein research. NCMN systems aim to create a membrane-active polymer library suitable for high-resolution structure determination. Herein, we report our design, synthesis, characterization and comparative application analyses of three novel classes of NCMN polymers, NCMNP13-x, NCMNP21-x and NCMNP21b-x. Although each NCMN polymer can solubilize various model membrane proteins and conserve native lipids into NCMN particles, only the NCMNP21b-x series reveals lipid-protein particles with good buffer compatibility and high homogeneity suitable for single-particle cryo-EM analysis. Consequently, the NCMNP21b-x polymers that bring out high-quality NCMN particles are particularly attractive for membrane protein structural biology.

scholarly journals Folding of Insulin Receptor Monomers Is Facilitated by the Molecular Chaperones Calnexin and Calreticulin and Impaired by Rapid Dimerization

1998 ◽  
Vol 141 (3) ◽  
pp. 637-646 ◽  
Author(s):  
Joseph Bass ◽  
Gavin Chiu ◽  
Yair Argon ◽  
Donald F. Steiner
Keyword(s):  
Membrane Proteins ◽  
Cell Surface ◽  
Insulin Receptor ◽  
Tyrosine Kinases ◽  
Normal Growth ◽  
Surface Expression ◽  
Growth Conditions ◽  
Cell Surface Expression ◽  
Surface Receptors

Many complex membrane proteins undergo subunit folding and assembly in the ER before transport to the cell surface. Receptors for insulin and insulin-like growth factor I, both integral membrane proteins and members of the family of receptor tyrosine kinases (RTKs), are unusual in that they require homodimerization before export from the ER. To better understand chaperone mechanisms in endogenous membrane protein assembly in living cells, we have examined the folding, assembly, and transport of the human insulin receptor (HIR), a dimeric RTK. Using pulse-chase labeling and nonreducing SDS-PAGE analysis, we have explored the molecular basis of several sequential maturation steps during receptor biosynthesis. Under normal growth conditions, newly synthesized receptor monomers undergo disulfide bond formation while associated with the homologous chaperones calnexin (Cnx) and calreticulin (Crt). An inhibitor of glucose trimming, castanospermine (CST), abolished binding to Cnx/Crt but also unexpectedly accelerated receptor homodimerization resulting in misfolded oligomeric proreceptors whose processing was delayed and cell surface expression was also decreased by ∼30%. Prematurely-dimerized receptors were retained in the ER and more avidly associated with the heat shock protein of 70 kD homologue binding protein. In CST-treated cells, receptor misfolding followed disordered oligomerization. Together, these studies demonstrate a chaperone function for Cnx/Crt in HIR folding in vivo and also provide evidence that folding efficiency and homodimerization are counterbalanced.

scholarly journals Cytosolic aggregates perturb the degradation of nontranslocated secretory and membrane proteins

2011 ◽  
Vol 22 (10) ◽  
pp. 1625-1637 ◽  
Author(s):  
Oishee Chakrabarti ◽  
Neena S. Rane ◽  
Ramanujan S. Hegde
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Signal Sequence ◽  
Proteasome Inhibition ◽  
Cellular Protein ◽  
Protein Homeostasis ◽  
Rapid Degradation ◽  
Wide Range ◽  
A Minor ◽  
Sequence Efficiency

A wide range of diseases are associated with the accumulation of cytosolic protein aggregates. The effects of these aggregates on various aspects of normal cellular protein homeostasis remain to be determined. Here we find that cytosolic aggregates, without necessarily disrupting proteasome function, can markedly delay the normally rapid degradation of nontranslocated secretory and membrane protein precursors. In the case of mammalian prion protein (PrP), the nontranslocated fraction is recruited into preexisting aggregates before its triage for degradation. This recruitment permits the growth and persistence of cytosolic PrP aggregates, explaining their apparent “self-conversion” seen in earlier studies of transient proteasome inhibition. For other proteins, the aggregate-mediated delay in precursor degradation led to aggregation and/or soluble residence in the cytosol, often causing aberrant cellular morphology. Remarkably, improving signal sequence efficiency mitigated these effects of aggregates. These observations identify a previously unappreciated consequence of cytosolic aggregates for nontranslocated secretory and membrane proteins, a minor but potentially disruptive population the rapid disposal of which is critical to maintaining cellular homeostasis.

scholarly journals Selective and immediate effects of clathrin heavy chain mutations on Golgi membrane protein retention in Saccharomyces cerevisiae.

1992 ◽  
Vol 118 (3) ◽  
pp. 531-540 ◽  
Author(s):  
M Seeger ◽  
G S Payne
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Cell Surface ◽  
Heavy Chain ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Golgi Membrane ◽  
Alpha Factor ◽  
Mutant Cells

The role of clathrin in retention of Golgi membrane proteins has been investigated. Prior work showed that a precursor form of the peptide mating pheromone alpha-factor is secreted by Saccharomyces cerevisiae cells which lack the clathrin heavy chain gene (CHC1). This defect can be accounted for by the observation that the Golgi membrane protein Kex2p, which initiates maturation of alpha-factor precursor, is mislocalized to the cell surface of mutant cells. We have examined the localization of two additional Golgi membrane proteins, dipeptidyl aminopeptidase A (DPAP A) and guanosine diphosphatase (GDPase) in clathrin-deficient yeast strains. Our findings indicate that DPAP A is aberrantly transported to the cell surface but GDPase is not. In mutant cells carrying a temperature-sensitive allele of CHC1 (chc1-ts), alpha-factor precursor appears in the culture medium within 15 min, and Kex2p and DPAP A reach the cell surface within 30 min, after imposing the nonpermissive temperature. In contrast to these immediate effects, a growth defect is apparent only after 2 h at the nonpermissive temperature. Also, sorting of the vacuolar membrane protein, alkaline phosphatase, is not affected in chc1-ts cells until 2 h after the temperature shift. A temperature-sensitive mutation which blocks a late stage of the secretory pathway, sec1, prevents the appearance of mislocalized Kex2p at the cell surface of chc1-ts cells. We propose that clathrin plays a direct role in the retention of specific proteins in the yeast Golgi apparatus, thereby preventing their transport to the cell surface.

scholarly journals Intracellular and Extracellular Leukemia Inhibitory Factor Proteins Have Different Cellular Activities That Are Mediated by Distinct Protein Motifs

2000 ◽  
Vol 11 (4) ◽  
pp. 1369-1383 ◽  
Author(s):  
Bryan P. Haines ◽  
Roger B. Voyle ◽  
Peter D. Rathjen
Keyword(s):  
Cell Surface ◽  
Leukemia Inhibitory Factor ◽  
Cellular Localization ◽  
Mutational Analysis ◽  
Biological Response ◽  
Inhibitory Factor ◽  
Receptor Interaction ◽  
Cell Surface Receptors ◽  
Protein Motifs ◽  
Surface Receptors

Although many growth factors and cytokines have been shown to be localized within the cell and nucleus, the mechanism by which these molecules elicit a biological response is not well understood. The cytokine leukemia inhibitory factor (LIF) provides a tractable experimental system to investigate this problem, because translation of alternatively spliced transcripts results in the production of differentially localized LIF proteins, one secreted from the cell and acting via cell surface receptors and the other localized within the cell. We have used overexpression analysis to demonstrate that extracellular and intracellular LIF proteins can have distinct cellular activities. Intracellular LIF protein is localized to both nucleus and cytoplasm and when overexpressed induces apoptosis that is inhibited by CrmA but not Bcl-2 expression. Mutational analysis revealed that the intracellular activity was independent of receptor interaction and activation and reliant on a conserved leucine-rich motif that was not required for activation of cell surface receptors by extracellular protein. This provides the first report of alternate intracellular and extracellular cytokine activities that result from differential cellular localization of the protein and are mediated by spatially distinct motifs.

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