The effect of macromolecular crowding on protein misfolding as well as oxidative aggregation and its corresponding mechanisms under carbamylation conditions.

Abstract

Protein carbamylation is a non-enzymatic post-translational modification, potentially increasing the risk of misfolding diseases. However, studies on carbamylation in recent years have focused only on amyloid aggregation, ignoring its effect on more toxic oligomers. Additionally, the behavior of proteins in dilute solutions does not fully reflect how these molecules function in their physiological environment. To address these concerns, this study developed a protein carbamylation system using potassium cyanate and used dextran 70 (300 g/L) as a crowding agent to clarify the effects of macromolecular crowding on carbamylated protein homeostasis, especially its effect on oligomers with membrane-damaging capacity, thereby elucidating the role of carbamylation in the onset and progression of protein misfolding diseases and achieving extracellular biomimetic mimicry of intracellular protein behavior. The results indicate that potassium cyanate can form hydrogen bonds and van der Waals interactions with proteins, undergoing nucleophilic addition reactions, which increases the free radical content in the system and induces protein misfolding, oxidative damage, amyloid aggregation and oligomerization. Moreover, under the macromolecular crowding environment, protein carbamylation and its induced misfolding and oxidative aggregation are aggravated accompanied by a reduction in amyloid aggregation and an increase in oligomer formation. These findings provide insights into the molecular and biochemical basis of protein misfolding diseases triggered by carbamylation and may offer potential biomarkers for the early diagnosis and treatment of such diseases, further achieving high-throughput screening of drugs for the prevention and treatment of age-related diseases.