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Published on: November 17, 2018
"Affimer" synthetic protein scaffolds block oxidized LDL binding to the LOX-1 scavenger receptor and inhibit ERK1/2
Barnaby W R Roper1, Christian Tiede1, Izma Abdul-Zani1
1School of Molecular & Cellular Biology, University of Leeds, Leeds, UK.
Abstract:
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.
Insights
Researchers developed synthetic Affimer proteins that specifically bind to LOX-1, a scavenger receptor involved in vascular disease. These Affimers effectively block oxidized low-density lipoprotein (oxLDL) uptake and signaling, offering potential therapeutic tools for cardiovascular conditions.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Lipid-protein particles like oxidized low-density lipoprotein (oxLDL) play roles in cellular transport.
- Scavenger receptors, such as LOX-1, mediate pathological responses to modified lipids.
- LOX-1 is crucial for oxLDL binding, signaling, and cellular trafficking, impacting vascular health.
Purpose of the Study:
- To identify novel synthetic molecules targeting the LOX-1 scavenger receptor.
- To characterize the binding specificity and inhibitory potential of these molecules against oxLDL/LOX-1 interactions.
- To explore the therapeutic implications of targeting the oxLDL/LOX-1 pathway in vascular disease.
Main Methods:
- Phage display library screening to identify Affimer proteins against the LOX-1 extracellular domain.
- Cell-based assays using HEK293T cells expressing human LOX-1 to assess Affimer binding and oxLDL uptake.
- Western blot analysis to evaluate the inhibition of ERK1/2 signaling pathway activation by Affimers.
- Molecular modeling to predict the binding interactions of Affimers with the LOX-1 homodimer.
Main Results:
- Five synthetic Affimer proteins were identified with high specificity for the extracellular domain of LOX-1.
- Affimers demonstrated specific binding to LOX-1 on the plasma membrane of expressing cells and inhibited oxLDL binding and uptake with subnanomolar potency.
- Affimers significantly inhibited oxLDL-induced ERK1/2 activation without direct agonistic effects.
- Molecular modeling suggested Affimers bind to the LOX-1 lectin-like domain, interacting with arginine residues crucial for oxLDL binding.
Conclusions:
- A novel class of synthetic Affimer proteins specifically targets the oxLDL-binding site of LOX-1.
- These Affimers serve as potent inhibitors of oxLDL uptake and downstream signaling, demonstrating therapeutic potential.
- The Affimers represent valuable tools for further investigation and potential modulation of the oxLDL/LOX-1 axis in vascular pathologies.
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