title: Peroxiredoxin 6 homodimerization and heterodimerization with glutathione S-transferase pi are required for its peroxidase but not phospholipase A2 activity.
abstract: Peroxiredoxin 6 (Prdx6) is a unique 1-Cys member of the peroxiredoxin family with both GSH peroxidase and phospholipase A2 (PLA2) activities. It is highly expressed in the lung where it plays an important role in antioxidant defense and lung surfactant metabolism. Glutathionylation of Prdx6 mediated by its heterodimerization with GSH S-transferase π (πGST) is required for its peroxidatic catalytic cycle. Recombinant human Prdx6 crystallizes as a homodimer and sedimentation equilibrium analysis confirmed that this protein exists as a high affinity dimer in solution. Based on measurement of molecular mass, dimeric Prdx6 that was oxidized to the sulfenic acid formed a sulfenylamide during storage. After examination of the dimer interface in the crystal structure, we postulated that the hydrophobic amino acids L145 and L148 play an important role in homodimerization of Prdx6 as well as in its heterodimerization with πGST. Oxidation of Prdx6 also was required for its heterodimerization. Sedimentation equilibrium analysis and the Duolink proximity ligation assay following mutation of the L145 and L148 residues of Prdx6 to Glu indicated greatly decreased dimerization propensity reflecting the loss of hydrophobic interactions between the protein monomers. Peroxidase activity was markedly reduced by mutation at either of the Leu sites and was essentially abolished by the double mutation, while PLA2 activity was unaffected. Decreased peroxidase activity following mutation of the interfacial leucines presumably is mediated via impaired heterodimerization of Prdx6 with πGST that is required for reduction and re-activation of the oxidized enzyme.
authors.affiliation: Institute for Environmental Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: Suiping
authors.initials: S
authors.lastname: Zhou
authors.affiliation: Institute for Environmental Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: Elena M
authors.initials: EM
authors.lastname: Sorokina
authors.affiliation: Center for Systems and Computational Biology, The Wistar Institute, Philadelphia, PA 19104, USA.
authors.forename: Sandra
authors.initials: S
authors.lastname: Harper
authors.affiliation: Institute for Environmental Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: Haitao
authors.initials: H
authors.lastname: Li
authors.affiliation: Institute for Environmental Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: Luis
authors.initials: L
authors.lastname: Ralat
authors.affiliation: Institute for Environmental Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: Chandra
authors.initials: C
authors.lastname: Dodia
authors.affiliation: Center for Systems and Computational Biology, The Wistar Institute, Philadelphia, PA 19104, USA.
authors.forename: David W
authors.initials: DW
authors.lastname: Speicher
authors.affiliation: Institute for Environmental Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: Sheldon I
authors.initials: SI
authors.lastname: Feinstein
authors.affiliation: Institute for Environmental Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. Electronic address: abf@mail.med.upenn.edu.
authors.forename: Aron B
authors.initials: AB
authors.lastname: Fisher
mesh_terms: D000975:Antioxidants / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D018360:Crystallography, X-Ray; D019281:Dimerization; D051549:Glutathione S-Transferase pi / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000235:genetics*; D009195:Peroxidase / Q000235:genetics / Q000378:metabolism; D054465:Peroxiredoxin VI / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2 / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011663:Pulmonary Surfactants / Q000737:chemistry / Q000378:metabolism
chemical_list: D000975:Antioxidants; D011663:Pulmonary Surfactants; C515977:PRDX6 protein, human; D054465:Peroxiredoxin VI; D009195:Peroxidase; D051549:Glutathione S-Transferase pi; D054467:Phospholipases A2
keywords: Duolink assay; Hydrophobic interactions; Leucine mutation; Mass spectroscopy; Phospholipid hydroperoxide glutathione peroxidase activity; Sedimentation equilibrium analysis; Sulfenylamide