title: Domain Mapping of Heat Shock Protein 70 Reveals That Glutamic Acid 446 and Arginine 447 Are Critical for Regulating Superoxide Dismutase 2 Function.
abstract: Stress-inducible heat shock protein 70 (hsp70) interacts with superoxide dismutase 2 (SOD2) in the cytosol after synthesis to transfer the enzyme to the mitochondria for subsequent activation. However, the structural basis for this interaction remains to be defined. To map the SOD2-binding site in hsp70, mutants of hsp70 were made and tested for their ability to bind SOD2. These studies showed that SOD2 binds in the amino acid 393-537 region of the chaperone. To map the hsp70-binding site in SOD2, we used a series of pulldown assays and showed that hsp70 binds to the amino-terminal domain of SOD2. To better define the binding site, we used a series of decoy peptides derived from the primary amino acid sequence in the SOD2-binding site in hsp70. This study shows that SOD2 specifically binds to hsp70 at445GERAMT450Small peptides containing GERAMT inhibited the transfer of SOD2 to the mitochondria and decreased SOD2 activityin vitroandin vivoTo determine the amino acid residues in hsp70 that are critical for SOD2 interactions, we substituted each amino acid residue for alanine or more conservative residues, glutamine or asparagine, in the GERAMT-binding site. Substitutions of E446A/Q and R447A/Q inhibited the ability of the GERAMT peptide to bind SOD2 and preserved SOD2 function more than other substitutions. Together, these findings indicate that the GERAMT sequence is critical for hsp70-mediated regulation of SOD2 and that Glu446and Arg447cooperate with other amino acid residues in the GERAMT-binding site for proper chaperone-dependent regulation of SOD2 antioxidant function.
authors.affiliation: From the Department of Pediatrics, Cardiovascular Research Center, aafolaya@mcw.edu.|Children's Research Institute.
authors.forename: Adeleye J
authors.initials: AJ
authors.lastname: Afolayan
authors.affiliation: From the Department of Pediatrics, Cardiovascular Research Center.|Children's Research Institute.
authors.forename: Maxwell
authors.initials: M
authors.lastname: Alexander
authors.affiliation: Children's Research Institute.|Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Holme
authors.affiliation: From the Department of Pediatrics, Cardiovascular Research Center.|Children's Research Institute.
authors.forename: Teresa
authors.initials: T
authors.lastname: Michalkiewicz
authors.affiliation: From the Department of Pediatrics, Cardiovascular Research Center.|Children's Research Institute.
authors.forename: Ujala
authors.initials: U
authors.lastname: Rana
authors.affiliation: From the Department of Pediatrics, Cardiovascular Research Center.|Children's Research Institute.
authors.forename: Ru-Jeng
authors.initials: RJ
authors.lastname: Teng
authors.affiliation: From the Department of Pediatrics, Cardiovascular Research Center.|Children's Research Institute.
authors.forename: Sara
authors.initials: S
authors.lastname: Zemanovic
authors.affiliation: Children's Research Institute.|Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.
authors.forename: Daisy
authors.initials: D
authors.lastname: Sahoo
authors.affiliation: Children's Research Institute.|Department of Surgery, Division of Pediatric Surgery, and.
authors.forename: Kirkwood A
authors.initials: KA
authors.lastname: Pritchard
authors.affiliation: From the Department of Pediatrics, Cardiovascular Research Center.|Children's Research Institute.
authors.forename: Girija G
authors.initials: GG
authors.lastname: Konduri
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites; D002478:Cells, Cultured; D018698:Glutamic Acid / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D051381:Rats; D012756:Sheep; D013482:Superoxide Dismutase / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism; D000099156:Superoxide Dismutase 2
chemical_list: D001120:Arginine; D018698:Glutamic Acid; D018840:HSP70 Heat-Shock Proteins; D013482:Superoxide Dismutase; D013481:Superoxides; D000099156:Superoxide Dismutase 2
keywords: endothelial cell; mitochondria; oxidative stress; protein import; superoxide dismutase (SOD)