A strategy for the identification of proteins targeted by thioredoxin

Abstract

Thioredoxins are 12-kDa proteins functional in the regulation of cellular processes throughout the animal, plant, and microbial kingdoms. Growing evidence with seeds suggests that an h-type of thioredoxin, reduced by NADPH via NADP-thioredoxin reductase, reduces disulfide bonds of target proteins and thereby acts as a wakeup call in germination. A better understanding of the role of thioredoxin in seeds as well as other systems could be achieved if more were known about the target proteins. To this end, we have devised a strategy for the comprehensive identification of proteins targeted by thioredoxin. Tissue extracts incubated with reduced thioredoxin are treated with a fluorescent probe (monobromobimane) to label sulfhydryl groups. The newly labeled proteins are isolated by conventional two-dimensional electrophoresis: (i) nonreducing͞reducing or (ii) isoelectric focusing͞reducing SDS/PAGE. The isolated proteins are identified by amino acid sequencing. Each electrophoresis system offers an advantage: the first method reveals the specificity of thioredoxin in the reduction of intramolecular vs. intermolecular disulfide bonds, whereas the second method improves the separation of the labeled proteins. By application of both methods to peanut seed extracts, we isolated at least 20 thioredoxin targets and identified 5-three allergens (Ara h2, Ara h3, and Ara h6) and two proteins not known to occur in peanut (desiccation-related and seed maturation protein). These findings open the door to the identification of proteins targeted by thioredoxin in a wide range of systems, thereby enhancing our understanding of its function and extending its technological and medical applications.peanut allergens ͉ desiccation-related protein ͉ seed maturation protein ͉ Ara h allergens ͉ proglycinin T here is a growing body of evidence that, as in photosynthesis, the regulatory protein thioredoxin (1-4) plays a role in heterotropic processes in plants. In this capacity, the disulfide group of a thioredoxin of the h-type is reduced by NADPH via the flavin enzyme, NADP-thioredoxin reductase (NTR) (1, 5, 6) (Eq. 1).Biochemical studies initiated a decade ago with wheat have provided evidence for a function of thioredoxin h in germination and seedling development. The results suggest that thioredoxin h, reduced via NTR with metabolically generated NADPH, acts early in the imbibed seed to initiate the mobilization of nitrogen and carbon in the endosperm, the major repository of storage protein and carbohydrate in cereals (7,8). The NADPH needed for this reduction can be generated enzymatically from carbohydrate stored in the endosperm via glucose 6-phophate and 6-phosphogluconate dehydrogenases (8).Through the reduction of intramolecular disulfide bonds (Eq. 2), thioredoxin h was shown to promote the degradation of major storage proteins, the inactivation of small proteins that inhibit amylolytic enzymes, and the activation of a novel calciumdependent substrate-specific protease (1,7,9,10 It has become clear that more complete information o...

A complete ferredoxin/thioredoxin system regulates fundamental processes in amyloplasts

“…The finding of a complete ferredoxin͞Trx system prompted us to look for Trx-regulated proteins in amyloplasts. To this end, we applied recently devised proteomic approaches based on fluorescent labeling (2,8) and affinity chromatography (1,5). As observed previously (8), the two techniques were complementary.…”

“…Recent approaches make it possible to identify proteins linked to Trx by combining proteomics with affinity chromatography (1) or thiol probes (2)(3)(4). These capabilities have defined an extended role of Trx in chloroplasts (1,5) and helped elucidate its function in other plant systems: mitochondria (6), seeds (2,3,7,8), and seedlings (4,9). Currently almost 200 proteins appear to be linked to Trx in plants (10).…”

A yeast two-hybrid knockout strain to explore thioredoxin-interacting proteinsin vivo

“…To answer these questions, much recent work has been devoted to large-scale searches for their putative targets. Various improved in vitro proteomic approaches [such as affinity chromatography (16)(17)(18)(19)(20)(21), labeled gel electrophoresis in which target proteins are revealed after reduction of protein extract by a TRX͞NADPH thioredoxin reductase (NTR) system (22)(23)(24), or coimmunoprecipitation (25)] suggested Ͼ100 putative TRX targets, but for which further investigations will be required for target validation regarding TRX specificity. Other methods devoted to the large-scale isolation of TRX͞target complexes established in vivo have also been developed, including affinity chromatography of TRX͞target complexes established in vivo during cell culture or the yeast͞mammalian two-hybrid methods (26)(27)(28)(29)(30)(31).…”

Update and challenges on proteomics in rice

“…Recently, Yano et al [56] developed a subtraction display strategy in which free sulfhydryl groups of proteins are labeled with fluorescence before or after in vivo/in vitro redox reactions. In this procedure, control and reduced protein samples are incubated with an sulfhydryl-specific probe, usually monobromobimane, separated by 2-DE and their fluorescent patterns compared.…”

“…Targets of thioredoxin are detected as increased fluorescent spots under UV illumination. Application of the protocol to dry seed allowed identification of putative targets of thioredoxin to give insights into its role in redox regulation [56]. Monitoring the dynamics of cellular redox change is a major field of interest.…”