title: Mechanism-based proteomic screening identifies targets of thioredoxin-like proteins.
abstract: Thioredoxin (Trx)-fold proteins are protagonists of numerous cellular pathways that are subject to thiol-based redox control. The best characterized regulator of thiols in proteins is Trx1 itself, which together with thioredoxin reductase 1 (TR1) and peroxiredoxins (Prxs) comprises a key redox regulatory system in mammalian cells. However, there are numerous other Trx-like proteins, whose functions and redox interactors are unknown. It is also unclear if the principles of Trx1-based redox control apply to these proteins. Here, we employed a proteomic strategy to four Trx-like proteins containing CXXC motifs, namely Trx1, Rdx12, Trx-like protein 1 (Txnl1) and nucleoredoxin 1 (Nrx1), whose cellular targets were trapped in vivo using mutant Trx-like proteins, under conditions of low endogenous expression of these proteins. Prxs were detected as key redox targets of Trx1, but this approach also supported the detection of TR1, which is the Trx1 reductant, as well as mitochondrial intermembrane proteins AIF and Mia40. In addition, glutathione peroxidase 4 was found to be a Rdx12 redox target. In contrast, no redox targets of Txnl1 and Nrx1 could be detected, suggesting that their CXXC motifs do not engage in mixed disulfides with cellular proteins. For some Trx-like proteins, the method allowed distinguishing redox and non-redox interactions. Parallel, comparative analyses of multiple thiol oxidoreductases revealed differences in the functions of their CXXC motifs, providing important insights into thiol-based redox control of cellular processes.
authors.affiliation: From the Division of Genetics, Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, the Universidade Federal do Paraná, Departamento de Patologia Básica, 81531-980, Curitiba, PR, Brazil, and.
authors.forename: Lia S
authors.initials: LS
authors.lastname: Nakao
authors.affiliation: the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Everley
authors.affiliation: From the Division of Genetics, Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Stefano M
authors.initials: SM
authors.lastname: Marino
authors.affiliation: the Universidade Federal do Paraná, Departamento de Patologia Básica, 81531-980, Curitiba, PR, Brazil, and.
authors.forename: Sze M
authors.initials: SM
authors.lastname: Lo
authors.affiliation: the Universidade Federal do Paraná, Departamento de Patologia Básica, 81531-980, Curitiba, PR, Brazil, and.
authors.forename: Luiz E
authors.initials: LE
authors.lastname: de Souza
authors.affiliation: the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Steven P
authors.initials: SP
authors.lastname: Gygi
authors.affiliation: From the Division of Genetics, Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, vgladyshev@rics.bwh.harvard.edu.
authors.forename: Vadim N
authors.initials: VN
authors.lastname: Gladyshev
mesh_terms: D051033:Apoptosis Inducing Factor / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002853:Chromatography, Liquid; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D033681:Mitochondrial Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D034622:RNA Interference; D011994:Recombinant Proteins / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*
chemical_list: C494829:AIFM1 protein, human; D051033:Apoptosis Inducing Factor; C507282:CHCHD4 protein, human; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D009687:Nuclear Proteins; D020543:Proteome; D011994:Recombinant Proteins; C491203:TXN protein, human; C504766:TXNL1 protein, human; D013879:Thioredoxins; D010088:Oxidoreductases; C105231:nucleoredoxin
keywords: Mammal; Oxidation-reduction (Redox); Proteomics; Redox Regulation; Selenocysteine; Thiol; Thioredoxin; Thioredoxin Reductase