Active nNOS Is Required for Grp94-Induced Antioxidant Cytoprotection: A Lesson from Myogenic to Cancer Cells
The endoplasmic reticulum (ER) chaperone Grp94/gp96 appears to be involved in cytoprotection without being required for cell survival. This study compared the effects of Grp94 protein levels on Ca<sup>2+</sup> homeostasis, antioxidant cytoprotection and protein–protein interactions betwe...
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MDPI AG
2022-03-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/6/2915 |
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| author | Filippo Fornasiero Cristina Scapin Maurizio Vitadello Paola Pizzo Luisa Gorza |
| author_facet | Filippo Fornasiero Cristina Scapin Maurizio Vitadello Paola Pizzo Luisa Gorza |
| author_sort | Filippo Fornasiero |
| collection | DOAJ |
| description | The endoplasmic reticulum (ER) chaperone Grp94/gp96 appears to be involved in cytoprotection without being required for cell survival. This study compared the effects of Grp94 protein levels on Ca<sup>2+</sup> homeostasis, antioxidant cytoprotection and protein–protein interactions between two widely studied cell lines, the myogenic C2C12 and the epithelial HeLa, and two breast cancer cell lines, MDA-MB-231 and HS578T. In myogenic cells, but not in HeLa, Grp94 overexpression exerted cytoprotection by reducing ER Ca<sup>2+</sup> storage, due to an inhibitory effect on SERCA2. In C2C12 cells, but not in HeLa, Grp94 co-immunoprecipitated with non-client proteins, such as nNOS, SERCA2 and PMCA, which co-fractionated by sucrose gradient centrifugation in a distinct, medium density, ER vesicular compartment. Active nNOS was also required for Grp94-induced cytoprotection, since its inhibition by L-NNA disrupted the co-immunoprecipitation and co-fractionation of Grp94 with nNOS and SERCA2, and increased apoptosis. Comparably, only the breast cancer cell line MDA-MB-231, which showed Grp94 co-immunoprecipitation with nNOS, SERCA2 and PMCA, increased oxidant-induced apoptosis after nNOS inhibition or Grp94 silencing. These results identify the Grp94-driven multiprotein complex, including active nNOS as mechanistically involved in antioxidant cytoprotection by means of nNOS activity and improved Ca<sup>2+</sup> homeostasis. |
| first_indexed | 2024-03-09T19:44:34Z |
| format | Article |
| id | doaj.art-946a4be4591b40a89e9892ac90acef37 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T19:44:34Z |
| publishDate | 2022-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-946a4be4591b40a89e9892ac90acef372023-11-24T01:29:12ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-03-01236291510.3390/ijms23062915Active nNOS Is Required for Grp94-Induced Antioxidant Cytoprotection: A Lesson from Myogenic to Cancer CellsFilippo Fornasiero0Cristina Scapin1Maurizio Vitadello2Paola Pizzo3Luisa Gorza4Department of Biomedical Sciences, University of Padova, 35131 Padova, ItalyDepartment of Biomedical Sciences, University of Padova, 35131 Padova, ItalyCNR-Neuroscience Institute, National Research Council, 35131 Padova, ItalyDepartment of Biomedical Sciences, University of Padova, 35131 Padova, ItalyDepartment of Biomedical Sciences, University of Padova, 35131 Padova, ItalyThe endoplasmic reticulum (ER) chaperone Grp94/gp96 appears to be involved in cytoprotection without being required for cell survival. This study compared the effects of Grp94 protein levels on Ca<sup>2+</sup> homeostasis, antioxidant cytoprotection and protein–protein interactions between two widely studied cell lines, the myogenic C2C12 and the epithelial HeLa, and two breast cancer cell lines, MDA-MB-231 and HS578T. In myogenic cells, but not in HeLa, Grp94 overexpression exerted cytoprotection by reducing ER Ca<sup>2+</sup> storage, due to an inhibitory effect on SERCA2. In C2C12 cells, but not in HeLa, Grp94 co-immunoprecipitated with non-client proteins, such as nNOS, SERCA2 and PMCA, which co-fractionated by sucrose gradient centrifugation in a distinct, medium density, ER vesicular compartment. Active nNOS was also required for Grp94-induced cytoprotection, since its inhibition by L-NNA disrupted the co-immunoprecipitation and co-fractionation of Grp94 with nNOS and SERCA2, and increased apoptosis. Comparably, only the breast cancer cell line MDA-MB-231, which showed Grp94 co-immunoprecipitation with nNOS, SERCA2 and PMCA, increased oxidant-induced apoptosis after nNOS inhibition or Grp94 silencing. These results identify the Grp94-driven multiprotein complex, including active nNOS as mechanistically involved in antioxidant cytoprotection by means of nNOS activity and improved Ca<sup>2+</sup> homeostasis.https://www.mdpi.com/1422-0067/23/6/2915oxidative stressGrp94gp96nNOSPMCASERCA2 |
| spellingShingle | Filippo Fornasiero Cristina Scapin Maurizio Vitadello Paola Pizzo Luisa Gorza Active nNOS Is Required for Grp94-Induced Antioxidant Cytoprotection: A Lesson from Myogenic to Cancer Cells International Journal of Molecular Sciences oxidative stress Grp94 gp96 nNOS PMCA SERCA2 |
| title | Active nNOS Is Required for Grp94-Induced Antioxidant Cytoprotection: A Lesson from Myogenic to Cancer Cells |
| title_full | Active nNOS Is Required for Grp94-Induced Antioxidant Cytoprotection: A Lesson from Myogenic to Cancer Cells |
| title_fullStr | Active nNOS Is Required for Grp94-Induced Antioxidant Cytoprotection: A Lesson from Myogenic to Cancer Cells |
| title_full_unstemmed | Active nNOS Is Required for Grp94-Induced Antioxidant Cytoprotection: A Lesson from Myogenic to Cancer Cells |
| title_short | Active nNOS Is Required for Grp94-Induced Antioxidant Cytoprotection: A Lesson from Myogenic to Cancer Cells |
| title_sort | active nnos is required for grp94 induced antioxidant cytoprotection a lesson from myogenic to cancer cells |
| topic | oxidative stress Grp94 gp96 nNOS PMCA SERCA2 |
| url | https://www.mdpi.com/1422-0067/23/6/2915 |
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