Stress protection by a fluorescent Hsp27 chimera that is independent of nuclear translocation or multimeric dissociation.

Borrelli MJ, Bernock LJ, Landry J, Spitz DR, Weber LA, Hickey E, Freeman ML, Corry PM
Cell Stress Chaperones. 2002 7 (3): 281-96

PMID: 12482204 · PMCID: PMC514828 · DOI:10.1379/1466-1268(2002)007<0281:spbafh>;2

A chimeric protein consisting of enhanced green fluorescent protein (EGFP) fused to the N-terminus of human Hsp27 conferred stress protection in human A549 lung carcinoma and murine L929 cells that were stably transfected to express the chimera constitutively. The resultant protection was comparable with that in the same cell lines when they were transfected to express corresponding levels of Hsp27. Unlike L929 cells, A549 cells exhibit endogenous Hsp27 expression, whose expression was inhibited in proportion to the amount of fluorescent chimera expressed, suggesting that the A549 cells recognized the latter as Hsp27. Upregulation of Hsp27 or chimeric Hsp27 in all transfected cell lines (stable or transient transfection) caused no measurable change in cellular glutathione levels, indicating that glutathione played no role in the stress protection associated with either protein. Chimeric Hsp27 had a monomeric molecular weight of 55 kDa (that of Hsp27 plus EGFP) in both cell types and formed a 16-mer complex twice as massive as that formed by Hsp27. Heat shock or sodium arsenite induced phosphorylation of both chimeric Hsp27 and Hsp27, which resulted in the disaggregation of Hsp27 multimers in both cell types and disaggregation of 20% of the chimeric multimers in L929 cells. But chimeric Hsp27 multimers did not disaggregate after stress in A549 cells. Epifluorescence and confocal microscopy demonstrated that chimeric Hsp27 was restricted to the cytoplasm under normal growth conditions and after heat shock in all cells. This study supports the conclusions that Hsp27 stress protection requires neither its translocation into the nucleus nor the dissociation of its multimeric complex. Furthermore, it demonstrates that fluorescent chimeras of heat shock proteins can be functional and used to observe the protein's distribution within living cells.

MeSH Terms (18)

Animals Cell Nucleus Fibroblasts Green Fluorescent Proteins Heat-Shock Proteins Heat-Shock Response Hot Temperature HSP27 Heat-Shock Proteins Humans Indicators and Reagents Luminescent Proteins Lung Neoplasms Mammals Neoplasm Proteins Oxidative Stress Phosphorylation Recombinant Fusion Proteins Tumor Cells, Cultured

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