Modulation of the redox state of tubulin by the glutathione/glutaredoxin reductase system.
Alterations in the redox status of proteins have been implicated in the pathology of several neurodegenerative diseases. We report that peroxynitrite-induced disulfides in porcine brain tubulin are repaired by the glutaredoxin reductase system composed of glutathione reductase, human or Escherichia coli glutaredoxin, reduced glutathione, and NADPH. Reduction of disulfide bonds between the alpha- and beta-tubulin subunits by the glutathione reductase system was assessed by Western blot. Tubulin cysteine oxidation and reduction was quantitated by monitoring the incorporation of 5-iodoacetamido-fluorescein, a thiol-specific labeling reagent. Tubulin disulfide bond reduction by the glutaredoxin reductase system restored tubulin polymerization activity that was lost following peroxynitrite addition. In support of redox modulations of tubulin by glutathione, thiol-disulfide exchange between tubulin and oxidized glutathione was detected and quantitated by HPLC. In addition, glutathionylation of tubulin was detected by dot blot using an anti-GSH antibody.
Landino
Lisa M
LM
Department of Chemistry, The College of William and Mary, P.O. Box 8795, Williamsburg, VA 23187-8795, USA. lmland@wm.edu
Moynihan
Katherine L
KL
Todd
Jonathan V
JV
Kennett
Kelly L
KL
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004396:Coloring Agents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D003553:Cystine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D005452:Fluoresceins / Q000494:pharmacology; D054477:Glutaredoxins; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D005980:Glutathione Reductase / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D010100:Oxygen / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000494:pharmacology; D011506:Proteins / Q000737:chemistry*; D013552:Swine; D013997:Time Factors; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism
D004396:Coloring Agents; D004220:Disulfides; D005452:Fluoresceins; D054477:Glutaredoxins; D011506:Proteins; D014404:Tubulin; D030421:Peroxynitrous Acid; D003553:Cystine; C015296:5-iodoacetamidofluorescein; D010088:Oxidoreductases; D005980:Glutathione Reductase; D005978:Glutathione; D003545:Cysteine; D010100:Oxygen