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Domain Mapping of Heat Shock Protein 70 Reveals That Glutamic Acid 446 and Arginine 447 Are Critical for Regulating Superoxide Dismutase 2 Function. J Biol Chem 2017 02 10;292(6):2369-2378

Date

12/29/2016

Pubmed ID

28028182

Pubmed Central ID

PMC5313107

DOI

10.1074/jbc.M116.756122

Scopus ID

2-s2.0-85012108909   4 Citations

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 at 445GERAMT450 Small peptides containing GERAMT inhibited the transfer of SOD2 to the mitochondria and decreased SOD2 activity in vitro and in vivo To 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 Glu446 and Arg447 cooperate with other amino acid residues in the GERAMT-binding site for proper chaperone-dependent regulation of SOD2 antioxidant function.

Author List

Afolayan AJ, Alexander M, Holme RL, Michalkiewicz T, Rana U, Teng RJ, Zemanovic S, Sahoo D, Pritchard KA Jr, Konduri GG

Authors

Adeleye James Afolayan MD Associate Professor in the Pediatrics department at Medical College of Wisconsin
Girija Ganesh Konduri MD Chief, Professor in the Pediatrics department at Medical College of Wisconsin
Kirkwood A. Pritchard PhD Professor in the Surgery department at Medical College of Wisconsin
Daisy Sahoo PhD Vice Chair, Professor in the Medicine department at Medical College of Wisconsin
Ru-Jeng Teng MD Associate Professor in the Pediatrics department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Amino Acid Substitution
Animals
Arginine
Binding Sites
Cells, Cultured
Glutamic Acid
HSP70 Heat-Shock Proteins
Mitochondria
Rats
Sheep
Superoxide Dismutase
Superoxides
jenkins-FCD Prod-482 91ad8a360b6da540234915ea01ff80e38bfdb40a