Screening for Interacting Proteins with Peptide Biomarker of Blood-Brain Barrier Alteration under Inflammatory Conditions.

Karina Vargas-Sanchez,Vaneza Lorett-Velásquez,Susana Novoa-Herrán,Maria Mogilevskaya,Monica Losada-Barragán,David Ramírez,Yehidi Medina,Cristian Buendía-Atencio,Klaus G Petry,Rodrigo E Gonzalez-Reyes,Jessica Martínez-Bernal

doi:10.3390/ijms22094725

Abstract

Neurodegenerative diseases are characterized by increased permeability of the blood–brain barrier (BBB) due to alterations in cellular and structural components of the neurovascular unit, particularly in association with neuroinflammation. A previous screening study of peptide ligands to identify molecular alterations of the BBB in neuroinflammation by phage-display, revealed that phage clone 88 presented specific binding affinity to endothelial cells under inflammatory conditions in vivo and in vitro. Here, we aimed to identify the possible target receptor of the peptide ligand 88 expressed under inflammatory conditions. A cross-link test between phage-peptide-88 with IL-1β-stimulated human hCMEC cells, followed by mass spectrometry analysis, was used to identify the target of peptide-88. We modeled the epitope–receptor molecular interaction between peptide-88 and its target by using docking simulations. Three proteins were selected as potential target candidates and tested in enzyme-linked immunosorbent assays with peptide-88: fibronectin, laminin subunit α5 and laminin subunit β-1. Among them, only laminin subunit β-1 presented measurable interaction with peptide-88. Peptide-88 showed specific interaction with laminin subunit β-1, highlighting its importance as a potential biomarker of the laminin changes that may occur at the BBB endothelial cells under pathological inflammation conditions.

Highlights

The breakdown of the blood–brain barrier (BBB) and its associated increased permeability is an important feature of neurodegenerative diseases [1]
Phage 88 displayed a high affinity for target proteins in hCMEC/D3 cells stimulated with IL-1β (Figure 1D), whereas no specific binding was observed with non-stimulated cells (Figure 1C)
We applied a crosslinking strategy and MS analysis to isolate and identify the target proteins interacting with phage 88 on the HCMEC/D3 cells under inflammatory conditions

Summary

Introduction

The breakdown of the blood–brain barrier (BBB) and its associated increased permeability is an important feature of neurodegenerative diseases [1]. The BBB does not act as an isolated system, as it performs its functions within a neurovascular unit (NVU), which includes cellular components such as endothelial cells, pericytes, neurons, astrocytes and microglia [2], as well as structures of the extracellular matrix (ECM) [3]. Astrocytes, in particular, due to their special cellular organization, are crucial to preserve functional aspects of both the BBB and the NVU. Astrocytes are the main regulators of brain energetic metabolism, providing neurons with necessary lactate and other sources of energy [4]. Alterations in any of these structural components could cause BBB disruption and facilitate the infiltration of peripheral immune cells into the brain parenchyma, leading to the development of abnormal neuroinflammatory reactions [6]

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Conclusion