Thermostability of a Nuclear‐Targeted Luciferase Expressed in Mammalian Cells

Abstract

Protein denaturation and aggregation are most likely the cause for the noxious effects of heat shock. There are some indications that the nucleus is onc of the most sensitive cellular compartments. To test the possibility that the intranuclear microenvirc~nment might be detrimental to the heat stability of proteins, we compared the iu sitct thermal stability of a rcporter protein localized in the nucleus 01-in the cytoplasm. A recombinant firefly (PhoD/nus pyrulis) luciferase carrying a point mutation in the C-terminal domitin remains in the cytoplasm (cyt-luciferase). A nuclear localization sequence wns fused to the N-terminal domain of cyt-luciferase; the resulting nuc-luciferase was efficiently targcted to the cell nucleus.In both cases, decreased luciferase activity and solubility were found in lysates from heat-shocked cells. These characteristics were taken as an indication of thermal denaturation in ,vim. The heat-inactivated luciferases were partially reactivated during recovery after stress, indicating the capacity of both the cytoplastnic and nuclear compartments to reassetnble proteins from an aggregated state.Although both the nuc-itnd the cyt-luciferases were heat inactivated at similar rates in v i m , nucluciferase was more susceptible to thermal denaturation in siiu cotnpared to cyt-luciferase. This ohscrvation suggests that the microenvironment of an intracellular compartment may modulate the thermal stability of proteins. The local concentration might be one element of this microcnvironment affecting the heat-stability of proteins.In cells made thertnotolerant by a priming shock, the thermid inactivation of the recombinant luciferases occurred at a slower rate during a second challenging stress. However, this decreased thermal scnsitivity was less pronounced for the nuc-luciferase (threefold) than for the cyt-luciferase (sevcnfold). The nuclear luciferase might become a useful tool to investigate the action of molecular chapcrones in the nucleus.Keywords: protein denaturation; heat shock; luciferase; nuclear localization ; thermotolcrance.In cells exposed to heat stress, some proteins denature and aggregate [ 1-51, Although proteins distributed in the various intracellular conipartnients have been found to denature, there are some indications that the nucleus is onc of the most sensitive compartments. Most protcin aggregates after heating cells are found within the nucleus 161. A decreasc in solubility of many nuclear proteins has been found lo occur during heat shock. They include the proto-oncogene protcins c-Myc, c-Myb and p53, DNA polymerases, RNA polymerase 11, and topoisoinerase I [7-121. This may reflect an overall reduction in nuclear protein solubility that contributes to the increment in protein content of nuclei isolated frotn heat-treated cells [ 13 1. Such protein aggregation may eventually lead to the killing effects of hyperthermia [ 2, 4, 51. A good correlation was found between h e extent and duration of nuclear protein aggregation and heat killing [I 41. At the beginning of...

Specific protein homeostatic functions of small heat‐shock proteins increase lifespan

“…The luciferase refolding assay (Michels et al ., 1995) was adapted for Schneider's S2 cells as follows. S2 cells were transfected with the pAc5.1‐Luc plasmid, coding for firefly luciferase together with different heat‐shock‐protein‐coding plasmids in a 1:9 ratio.…”

“…After a heat treatment of 30 min at 38 °C, the tubes were cooled down rapidly by placing them in a 25 °C water bath followed by incubation in a 25 °C incubator. Cell lyses and luciferase measurements were performed as described (Michels et al ., 1995). The experiments were performed and measured in triplicate.…”

Firefly luciferase mutants as sensors of proteome stress

“…This could be due to two reasons; either the nuclear environment is highly dense and crowded with macromolecules which enhances the sensitivity of the Fluc variants to stress and/or, this can be due to the differences in the proteostasis capacity to deal with unfolded proteins. Indeed, it has been shown in past by many studies that the nuclear proteome is highly sensitive to stress and that nuclear damage following protein misfolding stress plays a very critical role in stress induced cell death (Hageman et al, 2007;Michels et al, 1997;Michels et al, 1995).…”

Radiation and Transforming Growth Factor-β Cooperate in Transcriptional Activation of the Profibrotic Plasminogen Activator Inhibitor-1 Gene

“…In all cases, after administration of different stimuli, the cells were incubated for another 24 hours and lysed. Cell lysis and luciferase activity measurements were done as previously described (28). Luciferase activity was corrected for cell number and plotted as relative increases compared with the (untreated) control.…”