Modification of tubulin cysteines by nitric oxide and nitroxyl donors alters tubulin polymerization activity.
The modification of reduced cysteines of proteins by nitric oxide alters protein function, structure, and potentially, interactions with downstream signaling targets. We assessed the effect of the S-nitroso compounds S-nitrosoglutathione and S-nitroso-N-acetyl-penicillamine, the NO donor 2-(N,N-diethylamino)-diazenolate 2-oxide, and the nitroxyl donor Angeli's salt on the cysteines of the abundant cytoskeletal protein, tubulin. Total cysteine modification by each compound was quantitated and compared to peroxynitrite anion, an oxidant that we have studied previously. Angeli's salt was most effective at modifying the cysteines of tubulin and at inducing the formation of tubulin interchain disulfide bonds followed by peroxynitrite anion, S-nitrosoglutathione, S-nitroso-N-acetyl-penicillamine, and 2-(N,N-diethylamino)-diazenolate 2-oxide. S-nitrosation of tubulin by S-nitrosoglutathione and S-nitroso-N-acetyl-penicillamine was detected by the Saville assay. Our data show that tubulin interchain disulfide bond formation by these molecules correlated with inhibition of tubulin polymerization. Closer examination of the reaction of tubulin with S-nitrosoglutathione showed a concentration-dependent shift in the type of cysteine modification detected. More tubulin disulfides were detected at lower concentrations of S-nitrosoglutathione than at higher concentrations, suggesting that reduced glutathione, generated by the reaction of S-nitrosoglutathione with tubulin cysteines, reduced disulfides initially formed by S-nitrosoglutathione.
Landino
Lisa M
LM
Department of Chemistry, The College of William and Mary, P.O. Box 8795, Williamsburg, Virginia 23187-8795, USA. lmland@wm.edu
Koumas
Maria T
MT
Mason
Courtney E
CE
Alston
Jane A
JA
D003545:Cysteine / Q000737:chemistry*; D005978:Glutathione / Q000378:metabolism; D009569:Nitric Oxide / Q000494:pharmacology*; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D011108:Polymers / Q000737:chemistry*; D026422:S-Nitrosoglutathione / Q000494:pharmacology; D014404:Tubulin / Q000737:chemistry*
D020030:Nitric Oxide Donors; D011108:Polymers; D014404:Tubulin; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D005978:Glutathione; D003545:Cysteine