Thermostability of a nuclear-targeted luciferase expressed in mammalian cells. Destabilizing influence of the intranuclear microenvironment.

Details

Serval ID
serval:BIB_C2D110579E03
Type
Article: article from journal or magazin.
Collection
Publications
Title
Thermostability of a nuclear-targeted luciferase expressed in mammalian cells. Destabilizing influence of the intranuclear microenvironment.
Journal
European Journal of Biochemistry
Author(s)
Michels A.A., Nguyen V.T., Konings A.W., Kampinga H.H., Bensaude O.
ISSN
0014-2956
Publication state
Published
Issued date
1995
Peer-reviewed
Oui
Volume
234
Number
2
Pages
382-389
Language
english
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 one of the most sensitive cellular compartments. To test the possibility that the intranuclear microenvironment might be detrimental to the heat stability of proteins, we compared the in situ thermal stability of a reporter protein localized in the nucleus or in the cytoplasm. A recombinant firefly (Photynus pyralis) luciferase carrying a point mutation in the C-terminal domain remains in the cytoplasm (cyt-luciferase). A nuclear localization sequence was fused to the N-terminal domain of cyt-luciferase; the resulting nuc-luciferase was efficiently targeted 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 situ. The heat-inactivated luciferases were partially reactivated during recovery after stress, indicating the capacity of both the cytoplasmic and nuclear compartments to reassemble proteins from an aggregated state. Although both the nuc- and the cyt-luciferases were heat inactivated at similar rates in vitro, nuc-luciferase was more susceptible to thermal denaturation in situ compared to cyt-luciferase. This observation suggests that the microenvironment of an intracellular compartment may modulate the thermal stability of proteins. The local concentration might be one element of this microenvironment affecting the heat-stability of proteins. In cells made thermotolerant by a priming shock, the thermal inactivation of the recombinant luciferases occurred at a slower rate during a second challenging stress. However, this decreased thermal sensitivity was less pronounced for the nuc-luciferase (threefold) than for the cyt-luciferase (sevenfold). The nuclear luciferase might become a useful tool to investigate the action of molecular chaperones in the nucleus.
Keywords
Base Sequence, Cell Nucleus/enzymology, Enzyme Stability, Heat-Shock Proteins/biosynthesis, Hot Temperature, Luciferases/biosynthesis, Luciferases/chemistry, Molecular Sequence Data, Recombinant Proteins/biosynthesis
Pubmed
Web of science
Open Access
Yes
Create date
28/07/2008 16:11
Last modification date
20/08/2019 15:37
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