title: Thioredoxin-1 regulates cellular heme insertion by controlling S-nitrosation of glyceraldehyde-3-phosphate dehydrogenase.
abstract: NO generated by inducible NOS (iNOS) causes buildup of S-nitrosated GAPDH (SNO-GAPDH) in cells, which then inhibits further iNOS maturation by limiting the heme insertion step (Chakravarti, R., Aulak, K. S., Fox, P. L., and Stuehr, D. J. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 18004-18009). We investigated what regulates this process utilizing a slow-release NO donor (NOC-18) and studying changes in cellular SNO-GAPDH levels during and after NO exposure. Culturing macrophage-like cells with NOC-18 during cytokine activation caused buildup of heme-free (apo) iNOS and SNO-GAPDH. Upon NOC-18 removal, the cells quickly recovered their heme insertion capacity in association with rapid SNO-GAPDH denitrosation, implying that these processes are linked. We then altered cell expression of thioredoxin-1 (Trx1) or S-nitrosoglutathione reductase, both of which can function as a protein denitrosylase. Trx1 knockdown increased SNO-GAPDH levels in cells, made heme insertion hypersensitive to NO, and increased the recovery time, whereas Trx1 overexpression greatly diminished SNO-GAPDH buildup and protected heme insertion from NO inhibition. In contrast, knockdown of S-nitrosoglutathione reductase expression had little effect on these parameters. Experiments utilizing C152S GAPDH confirmed that the NO effects are all linked to S-nitrosation of GAPDH at Cys-152. We conclude (i) that NO inhibition of heme insertion and its recovery can be rapid and dynamic processes and are inversely linked to the S-nitrosation of GAPDH and (ii) that the NO sensitivity of heme insertion can vary depending on the Trx1 expression level due to Trx1 acting as an SNO-GAPDH denitrosylase. Together, our results identify a new way that cells regulate heme protein maturation during inflammation.
authors.affiliation: Department of Pathobiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
authors.forename: Ritu
authors.initials: R
authors.lastname: Chakravarti
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Stuehr
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D055785:Gene Knockdown Techniques; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D020125:Mutation, Missense; D009569:Nitric Oxide / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*
chemical_list: C515559:Txn1 protein, mouse; D009569:Nitric Oxide; D006418:Heme; D013879:Thioredoxins; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D000445:Aldehyde Oxidoreductases; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; C087006:formaldehyde dehydrogenase, glutathione-independent