Proteomic analysis of thioredoxin-targeted proteins in Escherichia coli

Abstract

Thioredoxin, a ubiquitous and evolutionarily conserved protein, modulates the structure and activity of proteins involved in a spectrum of processes, such as gene expression, apoptosis, and the oxidative stress response. Here, we present a comprehensive analysis of the thioredoxin-linked Escherichia coli proteome by using tandem affinity purification and nanospray microcapillary tandem mass spectrometry. We have identified a total of 80 proteins associated with thioredoxin, implicating the involvement of thioredoxin in at least 26 distinct cellular processes that include transcription regulation, cell division, energy transduction, and several biosynthetic pathways. We also found a number of proteins associated with thioredoxin that either participate directly (SodA, HPI, and AhpC) or have key regulatory functions (Fur and AcnB) in the detoxification of the cell. Transcription factors NusG, OmpR, and RcsB, not considered to be under redox control, are also associated with thioredoxin.T he role of redox regulatory pathways in signal transduction is well established (1). Thiol-disulfide exchange reactions control the structure and activity of proteins that contain regulatory cysteines (2). This reversible disulfide bond formation is mediated by thiol-dependent proteins, such as thioredoxin and glutaredoxin, that exchange reducing equivalents between their active site cysteines and the cysteines of target proteins (3). The precise molecular mechanisms underlying redox regulation continue to be elucidated. To this end, identification of the cellular targets of thiol-disulfide exchange proteins is an important goal.Thioredoxins are members of a class of small (Ϸ12-kDa) redox active proteins that maintain the reductive intracellular redox potential (4). The thioredoxin fold comprised of five ␤-strands surrounded by four short ␣-helices and the active site cysteines (CXXC) are evolutionarily conserved in all organisms ( Fig. 1) (5). Thioredoxin participates in redox reactions by oxidation of its active-site thiols and is then reduced by NADPH in a reaction catalyzed by thioredoxin reductase (4).Originally isolated from Escherichia coli in 1964 as an electron donor for ribonucleotide reductase (6), thioredoxin is now known to play a role in a multitude of processes (7). Apart from its oxidoreductase activity, thioredoxin exerts control over the activity of its target proteins via reversible thiol-disulfide exchange reactions (Fig. 2a). In plant chloroplasts, thioredoxin regulates the light-activated Calvin cycle by reducing specific regulatory disulfides (8). In eukaryotes, thioredoxin regulates the activity of transcription factors such as NF-B and AP-1 (9, 10). Thioredoxin also plays a critical role in the oxidative stress response; peroxiredoxins that catalyze the reduction of H 2 O 2 are activated in turn by reduction by thioredoxin (11). In E. coli, transient disulfide bonds formed in the presence of reactive oxygen species in proteins such as Hsp33 (12) and the transcriptional regulator OxyR (13) are reduced by th...

Regulation of Translation by the Redox State of Elongation Factor G in the Cyanobacterium Synechocystis sp. PCC 6803

“…EF-G in spinach chloroplasts (28) and E. coli (29) and EF-2 in Arabidopsis thaliana (30) and Chlamydomonas reinhardtii (31) were identified as candidates for targets of thioredoxin, as was EF-G (Slr1463) in Synechocystis (32). In the present study, we demonstrated for the first time, to our knowledge, that EF-G is an actual and functional target of thioredoxin.…”

“…Thioredoxin affinity chromatography captured a number of putative targets of thioredoxin (25)(26)(27)(28)(29)(30)(31)(32). EF-G in spinach chloroplasts (28) and E. coli (29) and EF-2 in Arabidopsis thaliana (30) and Chlamydomonas reinhardtii (31) were identified as candidates for targets of thioredoxin, as was EF-G (Slr1463) in Synechocystis (32).…”

Elucidation of Thioredoxin Target Protein Networks in Mouse

“…Comparison of ICAT and iTRAQ Analysis Results-The importance of Trx1 has spurred enormous interest in identifying its targets, an endeavor that has been aided by the developments of innovative techniques such as fluorescent diagonal electrophoresis (5) and affinity chromatography (28,29). However, these approaches have not been able to provide quantitative information regarding the degree or the specific sites of reduction; both are critical for relating protein redox status and the associated biological functions.…”

A universal entropy-driven mechanism for thioredoxin–target recognition

“…Numerous proteomic surveys have documented the specificity of Trx in target recognition (13)(14)(15). However, the molecular mechanism underlying this process, particularly in the absence of a signature Trx-binding sequence or structural motif, has remained largely unknown.…”