Hippuric acid, a uremic toxin, binds to aggregation prone region of human lysozyme and potentiates the fibrillation: A biophysical insight

Abstract

End-stage renal disease (ESRD) and human lysozyme (HL) amyloidosis are often interconnected, typically marked by elevated levels of uremic toxins in patients' blood. In this context, hippuric acid (HA), a uremic toxin derived from the metabolism of aromatic compounds, was investigated in vitro for its effect on HL fibrillation using spectroscopic, calorimetric, and computational methods. The results indicate that HA enhances HL fibrillation in a concentration-dependent manner, evidenced by increased ThT fluorescence and the detection of amyloid fibrils with a hydrodynamic radius of approximately 840.8 nm through dynamic light scattering and right-angle light scattering. Furthermore, HA promotes the conversion of HL's α+β structure into a predominantly β-sheet configuration, as confirmed by far-UV CD spectroscopy. This interaction occurs through the formation of a complex between HA and HL, stabilized by hydrogen bonds and hydrophobic interactions, as demonstrated by isothermal titration calorimetry (ITC) and computational studies. Specifically, HA binds to Q58 and N60 in the aggregation-prone region 2 (APR2) and Trp64 in non-aggregation-prone region, inducing conformational changes that favours fibrillation. The relative lytic activity of HL increase in presence of HA which further confirm the non-involvement of key residues, D35 and E53 in binding of HA to HL. Also, HL fibrils formed in presence of HA increases the hemolysis of RBCs and the appearance of more mis-shaped RBCs. Consequently, HA significantly enhances amyloid fibrillation in HL which provides valuable insights for future research focusing on in vivo studies, pre-clinical trials, and clinical applications.