Domain Mapping of Heat Shock Protein 70 Reveals That Glutamic Acid 446 and Arginine 447 Are Critical for Regulating Superoxide Dismutase 2 Function.

Publisher: Elsevier Inc. on behalf of American Society for Biochemistry and Molecular Biology Country of Publication: United States NLM ID: 2985121R Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1083-351X (Electronic) Linking ISSN: 00219258 NLM ISO Abbreviation: J Biol Chem Subsets: MEDLINE

Publication: 2021- : [New York, NY] : Elsevier Inc. on behalf of American Society for Biochemistry and Molecular Biology
Original Publication: Baltimore, MD : American Society for Biochemistry and Molecular Biology

Arginine/*metabolism ; Glutamic Acid/*metabolism ; HSP70 Heat-Shock Proteins/*metabolism ; Superoxide Dismutase/*metabolism; Amino Acid Substitution ; Animals ; Binding Sites ; Cells, Cultured ; HSP70 Heat-Shock Proteins/chemistry ; Mitochondria/metabolism ; Rats ; Sheep ; Superoxides/metabolism

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 ⁴⁴⁵ GERAMT ⁴⁵⁰ 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 Glu ⁴⁴⁶ and Arg ⁴⁴⁷ cooperate with other amino acid residues in the GERAMT-binding site for proper chaperone-dependent regulation of SOD2 antioxidant function.
(© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Proc Natl Acad Sci U S A. 2015 Aug 18;112(33):10389-94. (PMID: 26240360)
Molecules. 2013 Jan 24;18(2):1383-93. (PMID: 23348992)
RNA Biol. 2016 Sep;13(9):872-82. (PMID: 27388791)
Amino Acids. 2015 May;47(5):885-98. (PMID: 25646959)
Histochem Cell Biol. 2013 Aug;140(2):105-17. (PMID: 23744526)
Am J Physiol Lung Cell Mol Physiol. 2008 Dec;295(6):L979-87. (PMID: 18790993)
PLoS One. 2014 Jul 24;9(7):e103518. (PMID: 25058147)
J Biol Chem. 2007 Dec 28;282(52):37567-74. (PMID: 17971446)
Oncogene. 2015 Aug 6;34(32):4229-37. (PMID: 25362851)
Mol Cancer Res. 2013 Mar;11(3):219-29. (PMID: 23303345)
Respir Physiol Neurobiol. 2010 Dec 31;174(3):272-81. (PMID: 20804862)
Science. 2016 Jul 1;353(6294):aac4354. (PMID: 27365453)
Proc Natl Acad Sci U S A. 2016 May 17;113(20):E2794-801. (PMID: 27140645)
Arterioscler Thromb Vasc Biol. 2012 Mar;32(3):745-55. (PMID: 22199367)
Am J Physiol Heart Circ Physiol. 2007 Apr;292(4):H1812-20. (PMID: 17142346)
Science. 1993 Jun 25;260(5116):1902-3. (PMID: 8100364)
Vascul Pharmacol. 2006 May;44(5):299-308. (PMID: 16527547)
Circulation. 2010 Jun 22;121(24):2661-71. (PMID: 20529999)
Am J Physiol Lung Cell Mol Physiol. 2010 Jul;299(1):L109-16. (PMID: 20400523)
J Biochem Mol Biol. 2005 Jan 31;38(1):111-4. (PMID: 15715955)
Front Mol Biosci. 2015 Oct 20;2:58. (PMID: 26539440)
Am J Physiol Lung Cell Mol Physiol. 2014 Feb 15;306(4):L351-60. (PMID: 24375796)
PLoS One. 2013 Nov 12;8(11):e78443. (PMID: 24265689)
Subcell Biochem. 2015;78:243-73. (PMID: 25487025)
Nature. 1988 Apr 28;332(6167):800-5. (PMID: 3282178)
Free Radic Biol Med. 2015 Oct;87:36-47. (PMID: 26073127)
Proc Natl Acad Sci U S A. 2015 Aug 18;112(33):10395-400. (PMID: 26240333)
J Appl Physiol (1985). 2007 Jan;102(1):255-60. (PMID: 17023572)
Plant Cell. 2016 May;28(5):1002-8. (PMID: 27099259)
Am J Physiol Lung Cell Mol Physiol. 2012 Nov 15;303(10):L870-9. (PMID: 22962015)
J Biol Chem. 1990 Jul 5;265(19):11069-76. (PMID: 2193031)
Cell Physiol Biochem. 2014;34(5):1614-25. (PMID: 25402826)
Mol Cell. 2009 Oct 9;36(1):15-27. (PMID: 19818706)
Biochemistry. 2016 Aug 2;55(30):4140-53. (PMID: 27433847)
J Am Chem Soc. 2014 Nov 26;136(47):16683-8. (PMID: 25393077)
Mol Cancer Res. 2008 Dec;6(12):1881-93. (PMID: 19074833)
Front Mol Biosci. 2015 Jun 05;2:29. (PMID: 26097841)
Biochem Soc Trans. 2014 Aug;42(4):1012-6. (PMID: 25109995)
PLoS One. 2012;7(9):e44627. (PMID: 22970272)
Cell. 1990 Nov 2;63(3):447-50. (PMID: 2225059)
Cancer Epidemiol Biomarkers Prev. 2008 Dec;17(12):3298-305. (PMID: 19064542)
J Biomol Screen. 2016 Dec;21(10 ):1100-1111. (PMID: 27455993)

K08 HL133379 United States HL NHLBI NIH HHS; R01 HL057268 United States HL NHLBI NIH HHS; R01 HL112270 United States HL NHLBI NIH HHS; R01 HL128371 United States HL NHLBI NIH HHS

Keywords: endothelial cell; mitochondria; oxidative stress; protein import; superoxide dismutase (SOD)

PDB 4PO2; 2ADQ

0 (HSP70 Heat-Shock Proteins)
11062-77-4 (Superoxides)
3KX376GY7L (Glutamic Acid)
94ZLA3W45F (Arginine)
EC 1.15.1.1 (Superoxide Dismutase)
EC 1.15.1.1 (superoxide dismutase 2)

Date Created: 20161229 Date Completed: 20170704 Latest Revision: 20220330

20221213

PMC5313107

10.1074/jbc.M116.756122

28028182