S-thiolation of HSP27 regulates its multimeric aggregate size independently of phosphorylation.
HSP27 exists as large aggregates that breakdown after phosphorylation. We show rat cardiac HSP27 is S-thiolated during oxidant stress, and this modification, without phosphorylation, disaggregates multimeric HSP27. Biotinylated cysteine acts as a probe for thiolated proteins, which are detected using non-reducing Western blots probed with streptavidin-horseradish peroxidase. Controls show a low level of S-thiolation, which is increased 3.6-fold during post-ischemic reperfusion. S-thiolated proteins were purified using streptavidin-agarose, and Western immunoblotting showed HSP27 was present. We increased protein S-thiolation 10-fold with 10 microm H2O2 with or without a kinase inhibitor mixture (staurosporine, genistein, bisindolylmaleimide, SB203580, and PD98059). H2O2 alone induced the phosphorylation of HSP27 Ser-86 and Ser-45/Ser-59 of its homologue alphaB crystallin. However, kinase inhibition reduced phosphorylation of these sites below basal. Despite effective kinase inhibition, H2O2 still disaggregated HSP27, but not alphaB crystallin. This is consistent with the lack of an S-thiolation site on alphaB crystallin. Thus, we have demonstrated a novel mechanism of HSP27 multimeric size regulation. S-thiolation must occur at Cys-141, the only cysteine in rat HSP27.
Eaton
Philip
P
Centre for Cardiovascular Biology and Medicine, The Rayne Institute, St Thomas' Hospital, London SE1 7EH, United Kingdom. philip.eaton@kcl.ac.uk
Fuller
William
W
Shattock
Michael J
MJ
D000818:Animals; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D019921:Biotinylation; D015153:Blotting, Western; D003545:Cysteine / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D055551:HSP27 Heat-Shock Proteins; D006321:Heart / Q000502:physiology; D006360:Heat-Shock Proteins; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008297:Male; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism*; D018384:Oxidative Stress; D010477:Perfusion; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D012694:Serine / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors
D004791:Enzyme Inhibitors; D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C486114:Hspb1 protein, rat; D009363:Neoplasm Proteins; D013438:Sulfhydryl Compounds; D012694:Serine; D001710:Biotin; D006861:Hydrogen Peroxide; D003545:Cysteine