Urea ameliorates trimethylamine N-oxide-Induced aggregation of intrinsically disordered α-casein protein: the other side of the urea-methylamine counteraction

Trimethylamine N-oxide (TMAO) is generally accumulated by organisms and cells to cope with denaturing effects of urea/hydrodynamic pressure on proteins and can even reverse misfolded or aggregated proteins so as to sustain proteostasis. However, most of the work regarding this urea-TMAO counteraction has been performed on folded proteins. Compelling evidence of aggregation of intrinsically disordered proteins (IDPs) like tau, alpha-synuclein, amyloid beta etc., by TMAO and its potential to impact various protein processes in absence of stressing agents (such as urea) suggests that the contrary feature of interaction profiles of urea and TMAO maximizes their chances of offsetting the perturbing effects of each other. Recently, our lab observed that TMAO induces aggregation of alpha-casein, a model IDP. In this context, the present study, for the first time, evaluated urea for its potential to counteract the TMAO-induced aggregation of alpha-casein. It was observed that, at the biologically relevant ratios of 2:1 or 3:1 (urea:TMAO), urea was able to inhibit TMAO-induced aggregation of alpha-casein. However, urea did not reverse the effects of TMAO on alpha-casein. In addition to this, alpha-casein in presence of 1:1 and 2:1 urea:TMAO working ratios show aggregation-induced cytotoxic effect on HEK-293, Neuro2A and HCT-116 cell lines but not in presence of 3:1 working ratio, as there was no aggregation at all. The study infers that the accumulation of TMAO alone in the cells, in absence of stress (such as urea), might result in loss of conformational flexibility and aggregation of IDPs in TMAO accumulating organisms. Communicated by Ramaswamy H. Sarma