Intracellular refolding assay.

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds with inhibitory/stimulating activity. Molecular chaperones are proteins involved in regulation of protein folding and have a crucial role in promoting cell survival upon stress insults like heat shock, nutrient starvation and exposure to chemicals/poisons. For this reason chaperones are found to be involved in events like tumor development, chemioresistance of cancer cells as well as neurodegeneration. Design of small molecules able to inhibit or stimulate the activity of these enzymes is therefore one of the most studied strategies for cancer therapy and neurodegenerative disorders. The assay here described offers the possibility to measure the refolding activity of a particular molecular chaperone and to study the effect of compounds on its activity. In this method the gene of the molecular chaperone investigated is transfected together with an expression vector encoding for the firefly luciferase gene. It has been already described that denaturated firefly luciferase can be refolded by molecular chaperones. As normalizing transfection control, a vector encoding for the renilla luciferase gene is transfected. All transfections described in this protocol are performed with X-treme Gene (Roche) in HEK-293 cells. In the first step, protein synthesis is inhibited by treating the cells with cycloheximide. Thereafter protein unfolding is induced by heat shock at 45°C for 30 minutes. Upon recovery at 37°C, proteins are re-folded into their active conformation and the activity of the firefly luciferase is used as read-out: the more light will be produced, the more protein will have re-gained the original conformation. Non-heat shocked cells are set as reference (100% of refolded luciferase).