Translational thermotolerance provided by small heat shock proteins is limited to cap-dependent initiation and inhibited by 2-aminopurine.
Heat shock results in inhibition of general protein synthesis. In thermotolerant cells, protein synthesis is still rapidly inhibited by heat stress, but protein synthesis recovers faster than in naive heat-shocked cells, a phenomenon known as translational thermotolerance. Here we investigate the effect of overexpressing a single heat shock protein on cap-dependent and cap-independent initiation of translation during recovery from a heat shock. When overexpressing alphaB-crystallin or Hsp27, cap-dependent initiation of translation was protected but no effect was seen on cap-independent initiation of translation. When Hsp70 was overexpressed however, both cap-dependent and -independent translation were protected. This finding indicates a difference in the mechanism of protection mediated by small or large heat shock proteins. Phosphorylation of alphaB-crystallin and Hsp27 is known to significantly decrease their chaperone activity; therefore, we tested phosphorylation mutants of these proteins in this system. AlphaB-crystallin needs to be in its non-phosphorylated state to give protection, whereas phosphorylated Hsp27 is more potent in protection than the unphosphorylatable form. This indicates that chaperone activity is not a prerequisite for protection of translation by small heat shock proteins after heat shock. Furthermore, we show that in the presence of 2-aminopurine, an inhibitor of kinases, among which is double-stranded RNA-activated kinase, the protective effect of overexpressing alphaB-crystallin is abolished. The synthesis of the endogenous Hsps induced by the heat shock to test for thermotolerance is also blocked by 2-aminopurine. Most likely the protective effect of alphaB-crystallin requires synthesis of the endogenous heat shock proteins. Translational thermotolerance would then be a co-operative effect of different heat shock proteins.
Doerwald
Linda
L
Department of Biochemistry, Faculty of Science, University of Nijmegen, 6500HB Nijmegen, The Netherlands.
Onnekink
Carla
C
van Genesen
Siebe T
ST
de Jong
Wilfried W
WW
Lubsen
Nicolette H
NH
D015075:2-Aminopurine / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D017930:Genes, Reporter; D055551:HSP27 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry; D006367:HeLa Cells; D006360:Heat-Shock Proteins; D006358:Hot Temperature; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008957:Models, Genetic; D018832:Molecular Chaperones; D009154:Mutation; D009363:Neoplasm Proteins / Q000502:physiology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013696:Temperature; D013997:Time Factors; D014162:Transfection; D038203:alpha-Crystallin B Chain / Q000502:physiology
D055551:HSP27 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; C486113:Hsbp1 protein, mouse; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D038203:alpha-Crystallin B Chain; D015075:2-Aminopurine; D008156:Luciferases