"Affimer" synthetic protein scaffolds block oxidized LDL binding to the LOX-1 scavenger receptor and inhibit ERK1/2 activation.

Autor:	Roper BWR; School of Molecular & Cellular Biology, University of Leeds, Leeds, UK., Tiede C; School of Molecular & Cellular Biology, University of Leeds, Leeds, UK., Abdul-Zani I; School of Molecular & Cellular Biology, University of Leeds, Leeds, UK., Cuthbert GA; School of Molecular & Cellular Biology, University of Leeds, Leeds, UK; Leeds Vascular Institute, Leeds General Infirmary, Leeds, UK., Jade D; School of Biomedical Sciences, University of Leeds, Leeds, UK., Al-Aufi A; School of Molecular & Cellular Biology, University of Leeds, Leeds, UK; Leeds Vascular Institute, Leeds General Infirmary, Leeds, UK., Critchley WR; School of Molecular & Cellular Biology, University of Leeds, Leeds, UK., Saikia Q; School of Molecular & Cellular Biology, University of Leeds, Leeds, UK., Homer-Vanniasinkam S; Leeds Vascular Institute, Leeds General Infirmary, Leeds, UK., Sawamura T; Department of Physiology, Shinshu University, Nagano, Japan., McPherson MJ; School of Molecular & Cellular Biology, University of Leeds, Leeds, UK., Harrison MA; School of Biomedical Sciences, University of Leeds, Leeds, UK., Tomlinson DC; School of Molecular & Cellular Biology, University of Leeds, Leeds, UK., Ponnambalam S; School of Molecular & Cellular Biology, University of Leeds, Leeds, UK. Electronic address: s.ponnambalam@leeds.ac.uk.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2023 Nov; Vol. 299 (11), pp. 105325. Date of Electronic Publication: 2023 Oct 05.
DOI:	10.1016/j.jbc.2023.105325
Abstrakt:	In multicellular organisms, a variety of lipid-protein particles control the systemic flow of triacylglycerides, cholesterol, and fatty acids between cells in different tissues. The chemical modification by oxidation of these particles can trigger pathological responses, mediated by a group of membrane proteins termed scavenger receptors. The lectin-like oxidized low-density lipoprotein (LOX-1) scavenger receptor binds to oxidized low-density lipoprotein (oxLDL) and mediates both signaling and trafficking outcomes. Here, we identified five synthetic proteins termed Affimers from a phage display library, each capable of binding recombinant LOX-1 extracellular (oxLDL-binding) domain with high specificity. These Affimers, based on a phytocystatin scaffold with loop regions of variable sequence, were able to bind to the plasma membrane of HEK293T cells exclusively when human LOX-1 was expressed. Binding and uptake of fluorescently labeled oxLDL by the LOX-1-expressing cell model was inhibited with subnanomolar potency by all 5 Affimers. ERK1/2 activation, stimulated by oxLDL binding to LOX-1, was also significantly inhibited (p < 0.01) by preincubation with LOX-1-specific Affimers, but these Affimers had no direct agonistic effect. Molecular modeling indicated that the LOX-1-specific Affimers bound predominantly via their variable loop regions to the surface of the LOX-1 lectin-like domain that contains a distinctive arrangement of arginine residues previously implicated in oxLDL binding, involving interactions with both subunits of the native, stable scavenger receptor homodimer. These data provide a new class of synthetic tools to probe and potentially modulate the oxLDL/LOX-1 interaction that plays an important role in vascular disease. Competing Interests: Conflicts of interest M. J. M. and D. C. T. are named inventors of the Affimer technology and this is filed under U.S. patent number #10,844,370 assigned to the University of Leeds on “Scaffold proteins derived from plant cystatins”. (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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