Modelling HSP60 deficiency using an inducible transgenic HSP60 ATPase deficient mutant
To investigate the immediate effect of HSP60 dysfunction on mitochondrial functions, we induced the expression of a dominant-negative ATPase-deficient HSP60 mutant (HSP60-D423A) in HEK293 cells using tetracycline . Incorporation of HSP60-D423A subunits into HSP60 heptamer rings leads to dysfunction of the chaperonin complex. We titrated tetracycline levels to have a biologically relevant cellular model that can be monitored over time. Expression of the HSP60-D432A protein resulted in significantly reduced cell counts at 72 hours induction compared to uninduced and HSP60-WT co-expressing cells. To study the effects of onset of HSP60 deficiency, we monitored transcriptional changes using RNASeq. Overall design: HEK293-HSP60_D423A Flp-in cells were induced with 50µg/ml tetracyclin for 48h or 72h. As controls/reference, uninduced cells were used. 3 biological replicates for each condition were prepared.