Insights Into the Mechanism of Tyrosine Nitration in Preventing β-Amyloid Aggregation in Alzheimer's Disease.

Jie Zhao,Zhonghong Gao,Qiulian Liu,Zhijun He,Ye Zheng,Qiong Liu,Qihui Shi

doi:10.3389/fnmol.2021.619836

Abstract

Nitration of tyrosine at the tenth residue (Tyr10) in amyloid-β (Aβ) has been reported to reduce its aggregation and neurotoxicity in our previous studies. However, the exact mechanism remains unclear. Here, we used Aβ1–42 peptide with differently modified forms at Tyr10 to investigate the molecular mechanism to fill this gap. By using immunofluorescent assay, we confirmed that nitrated Aβ was found in the cortex of 10-month-old female triple transgenic mice of Alzheimer’s disease (AD). And then, we used the surface-enhanced Raman scattering (SERS) method and circular dichroism (CD) to demonstrate that the modification and mutation of Tyr10 in Aβ have little impact on conformational changes. Then, with the aids of fluorescence assays of thioflavin T and 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid, transmission electron microscopy (TEM), atomic force microscopy (AFM), and dynamic light scattering (DLS), we found that adding a large group to the phenolic ring of Tyr10 of Aβ could not inhibit Aβ fibrilization and aggregation. Nitration of Aβ reduces its aggregation mainly because it could induce the deprotonation of the phenolic hydroxyl group of Tyr10 of Aβ at physiological pH. We proposed that the negatively charged Tyr10 caused by nitration at physiological pH could interact with the salt bridge between Glu11 and His6 or His13 and block the kink around Tyr10, thereby preventing Aβ fibrilization and aggregation. These findings provide us new insights into the relationship between Tyr10 nitration and Aβ aggregation, which would help to further understand that keeping the balance of nitric oxide in vivo is important for preventing AD.

Highlights

Alzheimer’s disease (AD) is a neurodegenerative disorder leading to severe memory deficits, progressive cognitive decline, and neuronal death (Miranda et al, 2000; Selkoe and Podlisny, 2002; Goedert and Spillantini, 2006)
Tyrosine nitration alters the bulkiness of the residue, which becomes 30 Å larger than the 205 Å of nonmodified tyrosine, and induces a significant decrease of the ionization constant (pKa) in the phenolic hydroxyl group from 10.1 to the value around 7 in aqueous solution, resulting in the fact that the hydroxyl group of nitrotyrosine is about 50% charged at physiological pH (Zamyatnin, 1972; De Filippis et al, 2006)
To figure out the underlying mechanism, the following Tyr10 modified and mutated Aβ peptides were designed and synthesized: Aβ1–42DM for testing the effect of steric hindrance, Aβ1–42Cl and Aβ1–42I for investigating the effect of pKa change, Aβ1–42NF as a control for testing the effect of the phenolic hydroxyl group in Tyr10 of Aβ1–42NT and its hydrogen bond formed with the nitro group

Summary

Introduction

Alzheimer’s disease (AD) is a neurodegenerative disorder leading to severe memory deficits, progressive cognitive decline, and neuronal death (Miranda et al, 2000; Selkoe and Podlisny, 2002; Goedert and Spillantini, 2006). Its pathophysiology involves extracellular amyloid plaque, in which the primary protein constituents aggregated Aβ (Querfurth and Laferla, 2010). Aβ is derived from amyloid precursor protein (APP) through sequential proteolytic cleavages by β- and γ-secretases, with Aβ1–40 and Aβ1–42 as the predominant species (Roher et al, 1993; Lambert et al, 1998; LaFerla et al, 2007). Under pathological conditions, accumulated Aβ peptides can aggregate into oligomers, protofibrils, and mature fibrils after conformational changes from α-helix to β-sheet (Karran et al, 2011; Knowles et al, 2014). The soluble species of oligomers and protofibrils have been proposed as the primary driving force for AD formation in the amyloid hypothesis (Klein, 2002; Selkoe, 2008; Henry et al, 2015). A detailed understanding of the pathological process of Aβ self-assembly on a molecular level is of fundamental importance to elucidate the risk factors associated with the progression of AD and to develop new and effective intervention strategies

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Conclusion