The role of hsp70 in protection and repair of luciferase activity in vivo;experimental data and mathematical modelling

The stably transfected rat cell line HR24 expressing high levels of the ind ucible human hsp70 and its parental cell line Rat-1 were used for in vivo s tudies to analyse the role of hsp70 during thermal protein denaturation and the subsequent renaturation. In order to monitor denaturation and renatura tion of a cellular protein in vivo, both cell lines were transiently transf ected with firefly luciferase (Luc). The continuous monitoring of Luc activ ity during and after heat stress allowed a detailed analysis of the inactiv ation and reactivation kinetics in cells grown in monolayers. The aim of th ese studies was to distinguish a protective effect of increased hsp70 level s during heat shock-induced protein inactivation from a stimulation of reac tivation. In this paper we show that in cells that are stably transfected w ith hsp70, thermal Luc inactivation decreased, and subsequent reactivation yielded higher activity levels, compared with the parental cells. The diffe rence in early inactivation kinetics observed in the two cell lines suggest s an immediate effect of the presence of an extra amount of hsp70 on enzyme inactivation. Using different mathematical models, the heat-induced inacti vation and reactivation kinetics was compared with simulations of denaturat ion and renaturation. It is concluded that the model in which it is assumed that hsp70 is able to interact with partially denatured proteins, which di d not yet lose their enzymatic activity, most optimally explains the experi mental observations.