Ortho-methylated 3-hydroxypyridines hinder hen egg-white lysozyme fibrillogenesis

Abstract

Protein aggregation with the concomitant formation of amyloid fibrils is related to several neurodegenerative diseases, but also to non-neuropathic amyloidogenic diseases and non-neurophatic systemic amyloidosis. Lysozyme is the protein involved in the latter, and it is widely used as a model system to study the mechanisms underlying fibril formation and its inhibition. Several phenolic compounds have been reported as inhibitors of fibril formation. However, the anti-aggregating capacity of other heteroaromatic compounds has not been studied in any depth. We have screened the capacity of eleven different hydroxypyridines to affect the acid-induced fibrillization of hen lysozyme. Although most of the tested hydroxypyridines alter the fibrillation kinetics of HEWL, only 3-hydroxy-2-methylpyridine, 3-hydroxy-6-methylpyridine and 3-hydroxy-2,6-dimethylpyridine completely abolish fibril formation. Different biophysical techniques and several theoretical approaches are combined to elucidate their mechanism of action. O-methylated 3-hydroxypyridines bind non-cooperatively to two distinct but amyloidogenic regions of monomeric lysozyme. This stabilises the protein structure, as evidenced by enhanced thermal stability, and results in the inhibition of the conformational transition that precedes fibril assembly. Our results point to o-methylated 3-hydroxypyridines as a promising molecular scaffold for the future development of novel fibrillization inhibitors.

Vitamin k3 inhibits protein aggregation: Implication in the treatment of amyloid diseases

“…As shown in Fig. 1b , the aggregation kinetics of HEWL exhibit a typical sigmoidal appearance containing a lag phase associated with nucleation, a fast growth phase linked to the elongation and propagation of fibrils, and a final stationary phase 28 . Further, these results are in agreement with the nucleation dependent polymerisation for amyloidogenic proteins.…”

Inhibition of HEWL fibril formation by taxifolin: Mechanism of action

“…Aliquots of the samples without or with taxifolin were withdrawn at 0 and 7 days of incubation and subjected to SDS-PAGE. As shown in Fig 4 , the protein incubated alone displayed both high molecular weight assemblies (corresponding to amyloid fibrils) trapped in the well of the stacking gel and a range of low-molecular weight bands corresponding to acid-induced hydrolysis of the monomeric structure[ 46 , 47 ]. A similar pattern was observed for samples containing 25 and 50 μM taxifolin, indicating that the polyphenol did not protect the protein against acidic proteolysis at these concentrations.…”

“…There are many reports demonstrating that polyphenols are effective inhibitors of protein fibrillation [ 12 , 13 , 17 – 20 ], through interaction with one or more of the amyloidogenic species, produced during the course of the aggregation process. For instance, some polyphenols prevent amyloid formation by interacting with and stabilizing native structure of proteins [ 47 , 58 , 61 ]; while others bind to prefibrillar structures and redirect amyloidogenic polypeptides into unstructured, off-pathways oligomers [ 62 ], or toward an alternative non-toxic disordered (amorphous) aggregation pathway [ 20 ]. Recently, Hirohata et al .…”

“…We suggest this binding of taxifolin to hydrophobic surfaces, exposed in the course of the fibrillation process, shifts the equilibrium in the aggregation pathway by promoting the formation of very large globular aggregates, with low content of surface-exposed hydrophobic regions (Figs 3D, 3E and 7 ). It remains unclear how taxifolin promotes conversion of HEWL prefibrillar species into very large globular, chain-like aggregates, instead of formation of off-pathway conformers, or large amorphous aggregates, a mechanism displayed by some other polyphenols [ 20 , 47 , 58 , 60 – 62 ]. As the protein bears a net positive charge under acidic condition used for amyloid fibrillation, a probable reason for generation of such chain-like assemblies is the existence of repulsive forces between the aggregate species that cause the aggregates to find the configuration in which repulsive forces are minimized, i.e., the arrangement of aggregates in a linear chain.…”

Elevated temperatures accelerate the formation of toxic amyloid fibrils of hen egg‐white lysozyme

“…Hence, the objective of this study was to investigate the effect of high temperatures on the formation of amyloid fibrils using the protein model of HEWL. Previous studies have demonstrated that HEWL protein could form into amyloid fibrils at 54°C in 50 mM glycine of pH 2.2 (Seraj et al., 2019), 60°C in NaCl and KCl solution of pH 2.0 (Marino et al., 2015), and 65°C in 500 mM glycine of pH 2.0 (Sivalingam et al., 2016). All these previously reported higher temperatures used to generate HEWL amyloid fibrils are specific.…”