[P007] Poster

12:10 PM - 1:00 PM JST(3:10 AM - 4:00 AM UTC)

[P04-597] Contrasting lipid-binding properties of the closely related isozymes Prx1 and Prx2

○Hinaho Hatanaka¹, Chisato Endo^1,2, Tran Ngoc Trang^2,3, Hiroki Konno^1,4 (1. Graduate School of Natural Science & Technology, Kanazawa University (Japan), 2. WISE Program for Nano-Precision Medicine, Science, and Technology (HaKaSe+ for WISE), Kanazawa University (Japan), 3. Graduate School of Frontier Science Initiative, Kanazawa University (Japan), 4. WPI Nano Life Science Institute (WPI-NanoLSI), Kanazawa University (Japan))

Keywords:

Isozyme-specific oligomerization,Lipid-protein interaction,Phosphatidylserine,Peroxiredoxin 2,Site-directed mutagenesis,Surface electrostatic properties

Peroxiredoxin 2 (Prx2) is a typical 2-Cys peroxiredoxin that plays an essential role in cellular redox homeostasis through reduction of hydrogen peroxide. In addition to its peroxidase activity, Prx2 undergoes stress-dependent oligomerization accompanied by functional switching toward a molecular chaperone-like state. Previous studies have shown that negatively charged phospholipids such as phosphatidylserine (PS) are essential for lipid-dependent oligomer formation of Prx2, suggesting that lipid interaction contributes to its structural and functional regulation. However, the molecular determinants underlying the lipid-binding specificity of Prx2 remain unclear. In this study, we investigated the lipid-binding properties of Prx2 in comparison with its closely related isozyme Prx1 to clarify the basis of isozyme-specific lipid recognition.
Recombinant human Prx1, Prx2, and Prx2 mutants were expressed in E. coli and purified for biochemical characterization. Lipid-binding properties were examined by incubating proteins with negatively charged phospholipids (DOPS), followed by size-exclusion chromatography to evaluate lipid-dependent oligomer formation. Site-directed mutagenesis was used to examine the contribution of positively charged residues unique to Prx2. In addition, electrostatic surface properties of Prx1 and Prx2 were compared to investigate structural determinants underlying lipid-binding specificity.
Sequence analysis revealed that positively charged amino acid residues previously identified as critical for interaction with negatively charged phospholipids in Prx2 are fully conserved in Prx1. However, biochemical analyses demonstrated that Prx1 did not interact with negatively charged phospholipids and failed to form lipid-dependent oligomers under conditions in which Prx2 readily assembled into lipid-containing complexes. Furthermore, mutational analyses showed that additional positively charged residues unique to Prx2 are not required for lipid-dependent oligomerization. Comparison of electrostatic surface properties also indicated that static charge distribution alone cannot explain the observed lipid-binding specificity.
These results suggest that lipid recognition by Prx2 cannot be explained solely by primary sequence conservation or surface electrostatic properties and instead likely depends on more subtle structural or dynamic features unique to Prx2. Because lipid-dependent oligomerization of Prx2 is closely associated with stress-responsive functional switching, isozyme-specific lipid interaction may represent an important mechanism for regulating membrane-associated redox protein behavior.
Our findings reveal previously unrecognized functional divergence between closely related peroxiredoxin isozymes and provide new insight into the molecular basis of lipid-dependent regulation of Prx2. These results contribute to a better understanding of membrane-associated regulation of antioxidant proteins and may support future biotechnology applications involving engineering of stimulus-responsive redox protein assemblies and lipid-interacting regulators associated with intracellular membrane dynamics.