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 inducible human hsp70 and its parental cell line Rat-1 were used for in vivo studies to analyse the role of hsp70 during thermal protein denaturation and the subsequent renaturation. In order to monitor denaturation and renaturation of a cellular protein in vivo, both cell lines were transiently transfected with firefly luciferase (Luc). The continuous monitoring of Luc activity during and after heat stress allowed a detailed analysis of the inactivation and reactivation kinetics in cells grown in monolayers. The aim of these studies was to distinguish a protective effect of increased hsp70 levels during heat shock-induced protein inactivation from a stimulation of reactivation. In this paper we show that in cells that are stably transfected with hsp70, thermal Luc inactivation decreased, and subsequent reactivation yielded higher activity levels, compared with the parental cells. The difference in early inactivation kinetics observed in the two cell lines suggests an immediate effect of the presence of an extra amount of hsp70 on enzyme inactivation. Using different mathematical models, the heat-induced inactivation and reactivation kinetics was compared with simulations of denaturation and renaturation. It is concluded that the model in which it is assumed that hsp70 is able to interact with partially denatured proteins, which did not yet lose their enzymatic activity, most optimally explains the experimental observations.
Souren
J E
JE
Department of Molecular Cell Biology, Utrecht University, The Netherlands. J.E.M.Souren@bio.uu.nl
Wiegant
F A
FA
Van Wijk
R
R
D000818:Animals; D002460:Cell Line; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008433:Mathematics; D008954:Models, Biological; D011489:Protein Denaturation; D051381:Rats; D014162:Transfection
D018840:HSP70 Heat-Shock Proteins; D008156:Luciferases