Dual lipid-modified Thioredoxin-h9 functions as a membrane-associated redox and DNA-interacting protein in...

Thioredoxins (Trxs) are ubiquitous redox proteins that regulate intracellular thiol-disulfide balance and participate in diverse stress response in plants. Although several Arabidopsis h-type Trxs have been functionally characterized, the molecular functional roles of Trx-h9 remain poorly understood. In this study, we investigated the molecular characteristics of Trx-h9 using comparative biochemical analyses with Trx-h2 and Trx-h3. In Arabidopsis thaliana, h-type Trxs are categorized into three subgroups based on their N-terminal extensions and lipid modification. Sub-III Trx-h9 comprises 140 amino acids, including the canonical redox-active motif WCGPC. In addition, Trx-h9 has a N-terminal region containing lipid modification sites for myristoylation and palmitoylation. To elucidate its molecular functions, recombinant Trx-h9 was assayed for disulfide reductase activity (via insulin reduction assay), molecular chaperone capacity (assessed as holdase activity), and nucleic acid-binding specificity. Trx-h9 exhibited concentration-dependent insulin disulfide reductase activity, although its catalytic efficiency was lower than that of Trx-h2 and Trx-h3. Moreover, Trx-h9 displayed molecular chaperone activity similar to that observed in Trx-h3. DNA binding assays further revealed that Trx-h9 preferentially binds double-stranded DNA compared with single-stranded DNA. These multifunctional characteristics imply that Trx-h9 operates as a versatile protein, integrating disulfide reductase activity with holdase chaperone capacity and DNA-binding properties. The coupling of these activities suggests roles in both protein redox regulation and maintenance of macromolecular stability during cellular stress responses.