The effect of temperature and protein synthesis on the renaturation of firefly luciferase in intact H9c2 cells

Abstract

A mild increase in temperature that does not exert an effect on tolerance development or synthesis of heat shock proteins (Hsps) in control cells can stimulate these processes when applied to cells that have previously been heat shocked. To study the underlying mechanism of this effect, H9c2 cells were stably transfected with the gene encoding firefly luciferase (Luc). Heat-shock-induced inactivation of Luc and its subsequent reactivation is frequently used as a model for cellular protein denaturation and renaturation. Luc reactivation was determined following a damaging heat shock (43 or 44 degrees C for 30 min) in cells that were subsequently exposed to either control temperatures (37 degrees C) or various mild hyperthermic conditions (from 38.5 to 41.5 degrees C for 1 h). To prevent changes in Luc activity consequent to new synthesis of Luc, Luc reactivation was monitored in the presence of cycloheximide, an inhibitor of protein synthesis. The results showed that reactivation of Luc was inhibited when heat-treated cells were post-treated under mild hyperthermic conditions. The observed increase in Hsp synthesis under mild hyperthermic post-heat shock conditions therefore appears to be the result of an increase in the period during which denatured proteins are present. In addition, we studied Luc reactivation in the absence of protein synthesis inhibitors. This condition led to much higher Luc activity. By estimating half-life times of Luc, the contribution of new Luc synthesis in this recovery could be determined, and only partially explained the observed increase in Luc reactivation after heat shock. Thus the synthesis of other proteins must be important for the renaturation of heat-damaged proteins.

Thermostabilization of Firefly Luciferases Using Genetic Engineering

“…Similar results were obtained for L. mingrelica luciferase (Lundovskikh et al, 1998;Ugarova et al, 2000). It was demonstrated that inactivated luciferase is virtually unable to restore activity after cooling and usually aggregates (Minami & Minami, 1999;Schumacher et al, 1996;Souren et al, 1999aSouren et al, , 1999b. Appreciable spontaneous reactivation was only observed for diluted solutions of luciferase that was fully inactivated by guanidine chloride.…”

“…Substrate and competitive inhibitors change the conformation of luciferase, when introduced to prokaryotic and eukaryotic cells, leading to a several-fold decrease of enzyme degradation (Thompson et al, 1991). P. pyralis luciferase quickly inactivates in eukaryotic cells at 40-45°C with a half-life of 4-20 min (Forreitor et al, 1997;Souren et al, 1999b). Nevertheless, the stability can be relatively high at moderate temperatures.…”

HIV Gag mRNA Transfection of Dendritic Cells (DC) Delivers Encoded Antigen to MHC Class I and II Molecules, Causes DC Maturation, and Induces a Potent Human In Vitro Primary Immune Response

“…1C). As luciferase protein denatures quickly after translation and loses enzymatic activity (50), the measure of enzymatic activity corresponds to active translation of encoded mRNA, suggesting that mRNA transfection of DC results in transient, high level protein production.…”

Development of a High-Throughput Screening Cancer Cell-Based Luciferase Refolding Assay for Identifying Hsp90 Inhibitors

“…This finding is in complete agreement with previous studies that estimated the synthesis rate of luciferase to be 2.6% per hour relative to the basal amount of luciferase already present within a cell. 22,23 After miniaturizing the cancer cell-based assay to a 384-well format, the Z-factor and S/N observed were 0.5 and 7, respectively.…”

“…Others have described the use of luciferase as a reporter of chaperone activity in Arabidopsis and in the rat myoblast cell, H9c2. [21][22][23] Assays based on rabbit reticulocyte lysates have been successfully used to biochemically characterize the refolding kinetics of the Hsp70/Hsp90 system as well as a screening tool to identify compounds that inhibit Hsp90 activity. 24 While the rabbit reticulocyte assay is quite sensitive and robust, questions remain as to the physiological relevance of the active chaperone complexes in this system, as it represents a species more related to normal tissue rather than disease.…”