Joshua H. Wong

A strategy for the identification of proteins targeted by thioredoxin

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) nonreducing/reducing or (ii) isoelectric focusing/reducing 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.

A membrane-associated thioredoxin required for plant growth moves from cell to cell, suggestive of a role in intercellular communication

Thioredoxins (Trxs) are small ubiquitous regulatory disulfide proteins. Plants have an unusually complex complement of Trxs composed of six well-defined types (Trxs f, m, x, y, h, and o) that reside in different cell compartments and function in an array of processes. The extraplastidic h type consists of multiple members that in general have resisted isolation of a specific phenotype. In analyzing mutant lines in Arabidopsis thaliana, we identified a phenotype of dwarf plants with short roots and small yellowish leaves for AtTrx h9 (henceforth, Trx h9), a member of the Arabidopsis Trx h family. Trx h9 was found to be associated with the plasma membrane and to move from cell to cell. Controls conducted in conjunction with the localization of Trx h9 uncovered another h-type Trx in mitochondria (Trx h2) and a Trx in plastids earlier described as a cytosolic form in tomato. Analysis of Trx h9 revealed a 17-amino acid N-terminal extension in which the second Gly (Gly2) and fourth cysteine (Cys4) were highly conserved. Mutagenesis experiments demonstrated that Gly2 was required for membrane binding, possibly via myristoylation. Both Gly2 and Cys4 were needed for movement, the latter seemingly for protein structure and palmitoylation. A three-dimensional model was consistent with these predictions as well as with earlier evidence showing that a poplar ortholog is reduced by a glutaredoxin rather than NADP-thioredoxin reductase. In demonstrating the membrane location and intercellular mobility of Trx h9, the present results extend the known boundaries of Trx and suggest a role in cell-to-cell communication.

Unraveling thioredoxin-linked metabolic processes of cereal starchy endosperm using proteomics

Application of a thiol‐specific probe, monobromobimane, with proteomics and enzyme assays led to the identification of 23 thioredoxin targets in the starchy endosperm of mature wheat seeds (Triticum aestivum cv. Butte), almost all containing at least two conserved cysteines. The identified targets, 12 not known to be thioredoxin‐linked, function in a spectrum of processes: metabolism (12 targets), protein storage (three), oxidative stress (three), protein degradation (two), protein assembly/folding (one) and unknown reactions (two). In addition to formulating metabolic pathways functional in the endosperm, the results suggest that thioredoxin acts in redox regulation throughout the life cycle of the seed.

Transgenic barley grain overexpressing thioredoxin shows evidence that the starchy endosperm communicates with the embryo and the aleurone

Homozygous lines of barley overexpressing a wheat thioredoxin h transgene (up to 30-fold) were generated earlier by using a B1-hordein promoter with a signal peptide sequence for targeting to the protein body and found to be enriched in starch debranching enzyme (pullulanase). Here, we describe the effect of biochemically active, overexpressed thioredoxin h on germination and the onset of α-amylase activity. Relative to null segregant controls lacking the transgene, homozygotes overexpressing thioredoxin h effected (i) an acceleration in the rate of germination and appearance of α-amylase activity with a 1.6- to 2.8-fold increase in gibberellin A1 (GA1) content; (ii) a similar acceleration in the appearance of the α-amylase activity in deembryonated transgenic grain incubated with gibberellic acid; (iii) a 35% increase in the ratio of relative reduction (abundance of SH) of the propanol soluble proteins (hordein I fraction); and (iv) an increase in extractable and soluble protein of 5–12% and 11–35%, respectively. Thioredoxin h, which was highly reduced in the dry grain, was degraded in both the null segregant and homozygote after imbibition. The increase in α-amylase activity and protein reduction status was accompanied by a shift in the distribution of protein from the insoluble to the soluble fraction. The results provide evidence that thioredoxin h of the starchy endosperm communicates with adjoining tissues, thereby regulating their activities, notably by accelerating germination of the embryo and the appearance of α-amylase released by the aleurone.

A redox-active FKBP-type immunophilin functions in accumulation of the photosystem II supercomplex in Arabidopsis thaliana

Photosystem II (PSII) catalyzes the first of two photosynthetic reactions that convert sunlight into chemical energy. Native PSII is a supercomplex consisting of core and light-harvesting chlorophyll proteins. Although the structure of PSII has been resolved by x-ray crystallography, the mechanism underlying its assembly is poorly understood. Here, we report that an immunophilin of the chloroplast thylakoid lumen is required for accumulation of the PSII supercomplex in Arabidopsis thaliana. The immunophilin, FKBP20-2, belongs to the FK-506 binding protein (FKBP) subfamily that functions as peptidyl-prolyl isomerases (PPIases) in protein folding. FKBP20-2 has a unique pair of cysteines at the C terminus and was found to be reduced by thioredoxin (Trx) (itself reduced by NADPH by means of NADP-Trx reductase). The FKBP20-2 protein, which contains only two of the five amino acids required for catalysis, showed a low level of PPIase activity that was unaffected on reduction by Trx. Genetic disruption of the FKBP20-2 gene resulted in reduced plant growth, consistent with the observed lower rate of PSII activity determined by fluorescence (using leaves) and oxygen evolution (using isolated chloroplasts). Analysis of isolated thylakoid membranes with blue native gels and immunoblots showed that accumulation of the PSII supercomplex was compromised in mutant plants, whereas the levels of monomer and dimer building blocks were elevated compared with WT. The results provide evidence that FKBP20-2 participates specifically in the accumulation of the PSII supercomplex in the chloroplast thylakoid lumen by means of a mechanism that has yet to be determined.

New evidence for a role for thioredoxin h in germination and seedling development

Thioredoxin of the h-type — earlier linked to the reduction of wheat (Triticum durum Desf. cv. Monroe) endosperm proteins — was converted from an oxidized to a partially reduced state during germination and seedling development. While the abundance of thioredoxin progressively decreased during this period, the availability of reducing equivalents, defined as the product of the relative abundance of thioredoxin and the percent reduction, increased. The amount of the enzyme catalyzing the reduction of thioredoxin h (NADP-thioredoxin reductase) remained constant. The activities of enzymes generating the NADPH needed for the reduction of thioredoxin (glucose 6-phosphate and 6-phosphogluconate dehydrogenases) increased. The level of thioredoxin h in the endosperm appeared to be controlled by the embryo via hormones. Gibberellic acid enhanced the disappearance of thioredoxin, whereas abscisic acid showed the opposite effect. Moreover, uniconazole, an inhibitor of gibberellic acid synthesis, slowed seedling growth and inhibited the disappearance of thioredoxin in a manner reversible by gibberellic acid. The results are consistent with a role for thioredoxin h in initiating the mobilization of nitrogen and carbon needed for germination and seedling development.

The Level of Expression of Thioredoxin is Linked to Fundamental Properties and Applications of Wheat Seeds

Work with cereals (barley and wheat) and a legume (Medicago truncatula) has established thioredoxin h (Trx h) as a central regulatory protein of seeds. Trx h acts by reducing disulfide (S-S) groups of diverse seed proteins (storage proteins, enzymes, and enzyme inhibitors), thereby facilitating germination. Early in vitro protein studies were complemented with experiments in which barley seeds with Trx h overexpressed in the endosperm showed accelerated germination and early or enhanced expression of associated enzymes (alpha-amylase and pullulanase). The current study extends the transgenic work to wheat. Two approaches were followed to alter the expression of Trx h genes in the endosperm: (1) a hordein promoter and its protein body targeting sequence led to overexpression of Trx h5, and (2) an antisense construct of Trx h9 resulted in cytosolic underexpression of that gene (Arabidopsis designation). Underexpression of Trx h9 led to effects opposite to those observed for overexpression Trx h5 in barley-retardation of germination and delayed or reduced expression of associated enzymes. Similar enzyme changes were observed in developing seeds. The wheat lines with underexpressed Trx showed delayed preharvest sprouting when grown in the greenhouse or field without a decrease in final yield. Wheat with overexpressed Trx h5 showed changes commensurate with earlier in vitro work: increased solubility of disulfide proteins and lower allergenicity of the gliadin fraction. The results are further evidence that the level of Trx h in cereal endosperm determines fundamental properties as well as potential applications of the seed.

Metabolite-mediated catalyst conversion of PFK and PFP: a mechanism of enyme regulation in green plants

Metabolites known to occur in the cytosol of photosynthetic leaf cells were found to mediate the reversible conversion of pyrophosphate—D‐fructose‐6‐phosphate 1‐phosphotransferase (PFP) to phosphofructokinase (PFK) in partially purified preparations from spinach leaves. Preincubation of PFP with fructose 2,6‐bisphosphate, ATP or fructose 6‐phosphate converted PFP to PFK. The reverse reaction (PFK → PFP) was promoted by UDP‐glucose plus pyrophosphate. These conversions in catalytic capability were accompanied by changes in molecular mass and charge. The results are in accord with the view that the alterations in PFP and PFK activity, provisionally called ‘metabolite‐mediated catalyst conversion’, represent a regulatory mechanism to direct left cytosolic carbon flux in either the biosynthetic or degradatory direction.

Thioredoxin targets fundamental processes in a methane-producing archaeon, Methanocaldococcus jannaschii

Significance This study extends thioredoxin (Trx)-based oxidative redox regulation to the archaea, the third domain of life. Our study suggests that Trx is nearly ubiquitous in anaerobic methanogens, enabling them to recover from oxidative stress and synchronize cellular processes, including methane biogenesis, with the availability of reductants. As methane is a valuable fuel, an end product of anaerobic biodegradation and a potent greenhouse gas, Trx may now be considered a critical participant in the global carbon cycle, climate change, and bioenergy production. Because methanogenesis developed before the oxygenation of the earth, our work raises the possibility that Trx functioned in a complex redox regulatory network in anaerobic prokaryotes at least 2.5 billion years ago. Thioredoxin (Trx), a small redox protein, controls multiple processes in eukaryotes and bacteria by changing the thiol redox status of selected proteins. The function of Trx in archaea is, however, unexplored. To help fill this gap, we have investigated this aspect in methanarchaea—strict anaerobes that produce methane, a fuel and greenhouse gas. Bioinformatic analyses suggested that Trx is nearly universal in methanogens. Ancient methanogens that produce methane almost exclusively from H2 plus CO2 carried approximately two Trx homologs, whereas nutritionally versatile members possessed four to eight. Due to its simplicity, we studied the Trx system of Methanocaldococcus jannaschii—a deeply rooted hyperthermophilic methanogen growing only on H2 plus CO2. The organism carried two Trx homologs, canonical Trx1 that reduced insulin and accepted electrons from Escherichia coli thioredoxin reductase and atypical Trx2. Proteomic analyses with air-oxidized extracts treated with reduced Trx1 revealed 152 potential targets representing a range of processes—including methanogenesis, biosynthesis, transcription, translation, and oxidative response. In enzyme assays, Trx1 activated two selected targets following partial deactivation by O2, validating proteomics observations: methylenetetrahydromethanopterin dehydrogenase, a methanogenesis enzyme, and sulfite reductase, a detoxification enzyme. The results suggest that Trx assists methanogens in combating oxidative stress and synchronizing metabolic activities with availability of reductant, making it a critical factor in the global carbon cycle and methane emission. Because methanogenesis developed before the oxygenation of Earth, it seems possible that Trx functioned originally in metabolic regulation independently of O2, thus raising the question whether a complex biological system of this type evolved at least 2.5 billion years ago.

Thioredoxin and seed proteins.

Publisher Summary This chapter focuses on the thioredoxin and seed proteins. Storage proteins, which are formed and stored within seeds, serve as the main source of nitrogen and as a primary source of carbon for the germination and growth of seedlings. Storage proteins are synthesized after pollination and accumulate during seed maturation. The proteins are mobilized, proteolytically degraded, and utilized when conditions are favorable for germination. In the case of cereals, the bulk of the storage proteins are water insoluble after the grains mature. In most other types of seeds, the proteins remain soluble and enclosed within a membrane, thereby creating a structure known as a protein body. In some cereals, the protein body membrane is disrupted during maturation and drying of the grain, thereby exposing the storage proteins to endogenous proteases of the endosperm. Disulfide bonds are one of the main stabilizing forces in storage proteins. The formation of disulfide bonds appears to function in cereals to provide increased structural stability on the one hand and decreased solubility on the other. Both features provide protection against proteolysis.