1
|
Yang J, Zhang H, Gong W, Liu Z, Wu H, Hu W, Chen X, Wang L, Wu S, Chen C, Perrett S. S-Glutathionylation of human inducible Hsp70 reveals a regulatory mechanism involving the C-terminal α-helical lid. J Biol Chem 2020; 295:8302-8324. [PMID: 32332101 PMCID: PMC7294093 DOI: 10.1074/jbc.ra119.012372] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2019] [Revised: 04/13/2020] [Indexed: 12/23/2022] Open
Abstract
Heat shock protein 70 (Hsp70) proteins are a family of ancient and conserved chaperones. Cysteine modifications have been widely detected among different Hsp70 family members in vivo, but their effects on Hsp70 structure and function are unclear. Here, we treated HeLa cells with diamide, which typically induces disulfide bond formation except in the presence of excess GSH, when glutathionylated cysteines predominate. We show that in these cells, HspA1A (hHsp70) undergoes reversible cysteine modifications, including glutathionylation, potentially at all five cysteine residues. In vitro experiments revealed that modification of cysteines in the nucleotide-binding domain of hHsp70 is prevented by nucleotide binding but that Cys-574 and Cys-603, located in the C-terminal α-helical lid of the substrate-binding domain, can undergo glutathionylation in both the presence and absence of nucleotide. We found that glutathionylation of these cysteine residues results in unfolding of the α-helical lid structure. The unfolded region mimics substrate by binding to and blocking the substrate-binding site, thereby promoting intrinsic ATPase activity and competing with binding of external substrates, including heat shock transcription factor 1 (Hsf1). Thus, post-translational modification can alter the structure and regulate the function of hHsp70.
Collapse
Affiliation(s)
- Jie Yang
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Chaoyang District, Beijing, China.,University of the Chinese Academy of Sciences, Shijingshan District, Beijing, China
| | - Hong Zhang
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Chaoyang District, Beijing, China .,University of the Chinese Academy of Sciences, Shijingshan District, Beijing, China
| | - Weibin Gong
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Chaoyang District, Beijing, China
| | - Zhenyan Liu
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Chaoyang District, Beijing, China
| | - Huiwen Wu
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Chaoyang District, Beijing, China.,University of the Chinese Academy of Sciences, Shijingshan District, Beijing, China
| | - Wanhui Hu
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Chaoyang District, Beijing, China.,University of the Chinese Academy of Sciences, Shijingshan District, Beijing, China
| | - Xinxin Chen
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Chaoyang District, Beijing, China.,University of the Chinese Academy of Sciences, Shijingshan District, Beijing, China
| | - Lei Wang
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Chaoyang District, Beijing, China.,University of the Chinese Academy of Sciences, Shijingshan District, Beijing, China
| | - Si Wu
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Chaoyang District, Beijing, China.,University of the Chinese Academy of Sciences, Shijingshan District, Beijing, China
| | - Chang Chen
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Chaoyang District, Beijing, China .,University of the Chinese Academy of Sciences, Shijingshan District, Beijing, China.,Beijing Institute for Brain Disorders, Youanmen, Beijing, China
| | - Sarah Perrett
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Chaoyang District, Beijing, China .,University of the Chinese Academy of Sciences, Shijingshan District, Beijing, China
| |
Collapse
|
3
|
Ulbrich F, Kaufmann KB, Coburn M, Lagrèze WA, Roesslein M, Biermann J, Buerkle H, Loop T, Goebel U. Neuroprotective effects of Argon are mediated via an ERK-1/2 dependent regulation of heme-oxygenase-1 in retinal ganglion cells. J Neurochem 2015; 134:717-27. [PMID: 25876941 DOI: 10.1111/jnc.13115] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2015] [Revised: 03/31/2015] [Accepted: 04/02/2015] [Indexed: 12/22/2022]
Abstract
Retinal ischemia and reperfusion injuries (R-IRI) damage neuronal tissue permanently. Recently, we demonstrated that Argon exerts anti-apoptotic and protective properties. The molecular mechanism remains unclear. We hypothesized that Argon inhalation exert neuroprotective effects in rats retinal ganglion cells (RGC) via an ERK-1/2 dependent regulation of heat-shock proteins. Inhalation of Argon (75 Vol%) was performed after R-IRI on the rats' left eyes for 1 h immediately or with delay. Retinal tissue was harvested after 24 h to analyze mRNA and protein expression of heat-shock proteins -70, -90 and heme-oxygenase-1, mitogen-activated protein kinases (p38, JNK, ERK-1/2) and histological changes. To analyze ERK dependent effects, the ERK inhibitor PD98059 was applicated prior to Argon inhalation. RGC count was analyzed 7 days after injury. Statistics were performed using anova. Argon significantly reduced the R-IRI-affected heat-shock protein expression (p < 0.05). While Argon significantly induced ERK-1/2 expression (p < 0.001), inhibition of ERK-1/2 before Argon inhalation resulted in significantly lower vital RGCs (p < 0.01) and increase in heme-oxygenase-1 (p < 0.05). R-IRI-induced RGC loss was reduced by Argon inhalation (p < 0.001). Immunohistochemistry suggested ERK-1/2 activation in Müller cells. We conclude, that Argon treatment protects R-IRI-induced apoptotic loss of RGC via an ERK-1/2 dependent regulation of heme-oxygenase-1. We proposed the following possible mechanism for Argon-mediated neuroprotection: Argon exerts its protective effects via an induction of an ERK with subsequent suppression of the heat shock response. In conclusion, ischemia and reperfusion injuries and subsequent neuronal apoptosis are attenuated. These novel findings may open up new opportunities for Argon as a therapeutic option, especially since Argon is not toxic.
Collapse
Affiliation(s)
- Felix Ulbrich
- Department of Anesthesiology and Intensive Care Medicine, University Medical Center, Freiburg, Germany
| | - Kai B Kaufmann
- Department of Anesthesiology and Intensive Care Medicine, University Medical Center, Freiburg, Germany
| | - Mark Coburn
- Department of Anesthesiology, University Hospital RWTH Aachen, Aachen, Germany
| | | | - Martin Roesslein
- Department of Anesthesiology and Intensive Care Medicine, University Medical Center, Freiburg, Germany
| | - Julia Biermann
- Eye Center, University Medical Center, Freiburg, Germany
| | - Hartmut Buerkle
- Department of Anesthesiology and Intensive Care Medicine, University Medical Center, Freiburg, Germany
| | - Torsten Loop
- Department of Anesthesiology and Intensive Care Medicine, University Medical Center, Freiburg, Germany
| | - Ulrich Goebel
- Department of Anesthesiology and Intensive Care Medicine, University Medical Center, Freiburg, Germany
| |
Collapse
|