Reversible cysteine-targeted oxidation of proteins during renal oxidative stress.
Biotin-cysteine was used to study protein S-thiolation in isolated rat kidneys subjected to ischemia and reperfusion. After 40 min of ischemia, total protein S-thiolation increased significantly (P < 0.05), by 311%, and remained significantly elevated (P < 0.05), 221% above control, after 5 min of postischemic reperfusion. Treatment of protein samples with 2-mercaptoethanol abolished the S-thiolation signals detected, consistent with the dependence of the signal on the presence of a disulfide bond. With the use of gel filtration chromatography followed by affinity purification with streptavidin-agarose, S-thiolated proteins were purified from CHAPS-soluble kidney homogenate. The proteins were then separated by SDS-PAGE and stained with Coomassie blue. With a combination of matrix-assisted laser desorption ionization time of flight mass spectrometry and LC/MS/MS analysis of protein bands digested with trypsin, a number of S-thiolation substrates were identified. These included the LDL receptor-related protein 2, ATP synthase alpha chain, heat shock protein 90 beta, hydroxyacid oxidase 3, serum albumin precursor, triose phosphate isomerase, and lamin. These represent proteins that may be functionally regulated by S-thiolation and thus could undergo a change in activity or function after renal ischemia and reperfusion.
Eaton
Philip
P
The Centre for Cardiovascular Biology and Medicine, The Rayne Institute, St Thomas' Hospital, King's College London, London, United Kingdom. philip.eaton@kcl.ac.uk
Jones
Miriam E
ME
McGregor
Emma
E
Dunn
Michael J
MJ
Leeds
Nicola
N
Byers
Helen L
HL
Leung
Kit-Yi
KY
Ward
Malcolm A
MA
Pratt
Julian R
JR
Shattock
Michael J
MJ
D000818:Animals; D003545:Cysteine / Q000378:metabolism*; D066298:In Vitro Techniques; D007668:Kidney / Q000378:metabolism*; D008297:Male; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012079:Renal Circulation; D015427:Reperfusion Injury / Q000378:metabolism*; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism
D011506:Proteins; D013438:Sulfhydryl Compounds; D003545:Cysteine