Research Article
Exploration of the cysteine reactivity of human inducible Hsp70 and cognate Hsc70

https://doi.org/10.1016/j.jbc.2022.102723Get rights and content
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Hsp70s are multifunctional proteins and serve as the central hub of the protein quality control network. Hsp70s are also related to a number of diseases and have been established as drug targets. Human HspA1A (hHsp70) and HspA8 (hHsc70) are the major cytosolic Hsp70s, and they have both overlapping and distinct functions. hHsp70 contains five cysteine residues, and hHsc70 contains four cysteine residues. Previous studies have shown these cysteine residues can undergo different cysteine modifications such as oxidation or reaction with electrophiles to regulate their function, and hHsp70 and hHsc70 have different cysteine reactivity. To address the mechanism of the differences in cysteine reactivity between hHsp70 and hHsc70, we studied the factors that determine this reactivity by Ellman assay for the quantification of accessible free thiols and NMR analysis for the assessment of structural dynamics. We found the lower cysteine reactivity of hHsc70 is probably due to its lower structural dynamics and the stronger inhibition effect of interaction between the α-helical lid subdomain of the substrate-binding domain (SBDα) and the β-sheet substrate-binding subdomain (SBDβ) on cysteine reactivity of hHsc70. We determined that Gly557 in hHsp70 contributes significantly to the higher structural dynamics and cysteine reactivity of hHsp70 SBDα. Exploring the cysteine reactivity of hHsp70 and hHsc70 facilitates an understanding of the effects of redox reactions and electrophiles on their chaperone activity and regulation mechanisms, and how these differences allow them to undertake distinct cellular roles.

Keywords

cysteine reactivity
Hsp70
glutathionylation
disulfide bond formation
oxidative stress

Abbreviations

CD
circular dichroism
DTNB
5,5′-dithiobis-(2-nitrobenzoic acid)
FP
fluorescence polarization
MB
methylene blue
NBD
nucleotide-binding domain
PES
2-phenylethynesulfonamide
PTM
posttranslational modification
RT
room temperature
SBD
substrate-binding domain
SEC
size-exclusion chromatography

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Present addresses for: Zhouping Hong, Department of Cell Biology, Yale School of Medicine, New Haven, CT 06520, USA; Jie Yang, Leewe Biopharmaceutical Co., Ltd, Xianlin University Town, Jiangsu Life Park seventh floor F2 building, nine Weidi Road, Qixia District, Nanjing, 210,033, China; Sainan Li, Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Laoshan District, Qingdao, Shandong 266,101, China; Hong Zhang, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing 100005, China.

These authors contributed equally to this work.