Rebecca A. Sager , Sarah J. Backe , Jennifer Heritz , Mark R. Woodford , Dimitra Bourboulia , Mehdi Mollapour
{"title":"流式细胞仪 FRET 揭示翻译后修饰驱动蛋白磷酸酶-5 在哺乳动物细胞中的构象变化","authors":"Rebecca A. Sager , Sarah J. Backe , Jennifer Heritz , Mark R. Woodford , Dimitra Bourboulia , Mehdi Mollapour","doi":"10.1016/j.cstres.2024.10.002","DOIUrl":null,"url":null,"abstract":"<div><div>The serine/threonine Protein Phosphatase-5 (PP5) plays an essential role in regulating hormone and stress-induced signaling networks as well as extrinsic apoptotic pathways in cells. Unlike other Protein Phosphatases, PP5 possesses both regulatory and catalytic domains, and its function is further modulated through post-translational modifications (PTMs). PP5 contains a tetratricopeptide repeat (TPR) domain, which usually inhibits its phosphatase activity by blocking the active site (closed conformation). Certain activators bind to the PP5–TPR domain, alleviating this inhibition and allowing the catalytic domain to adopt an active (open) conformation. While this mechanism has been proposed based on structural and biophysical studies, PP5 conformational changes and activity have yet to be observed in cells. Here, we designed and developed a flow cytometry-based fluorescence resonance energy transfer (FC-FRET) method, enabling real-time observation of PP5 autoinhibition and activation within live mammalian cells. By quantifying FRET efficiency using sensitized emission, we established a standardized and adaptable data acquisition workflow. Our findings revealed that, in a cellular context, PP5 exists in multiple conformational states, none of which alone fully predicts its activity. Additionally, we have demonstrated that PTMs such as phosphorylation and SUMOylation impact PP5 conformational changes, representing a significant advancement in our understanding of its regulatory mechanisms.</div></div>","PeriodicalId":9684,"journal":{"name":"Cell Stress & Chaperones","volume":"29 6","pages":"Pages 709-717"},"PeriodicalIF":3.3000,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Flow cytometry FRET reveals post-translational modifications drive Protein Phosphatase-5 conformational changes in mammalian cells\",\"authors\":\"Rebecca A. Sager , Sarah J. Backe , Jennifer Heritz , Mark R. Woodford , Dimitra Bourboulia , Mehdi Mollapour\",\"doi\":\"10.1016/j.cstres.2024.10.002\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><div>The serine/threonine Protein Phosphatase-5 (PP5) plays an essential role in regulating hormone and stress-induced signaling networks as well as extrinsic apoptotic pathways in cells. Unlike other Protein Phosphatases, PP5 possesses both regulatory and catalytic domains, and its function is further modulated through post-translational modifications (PTMs). PP5 contains a tetratricopeptide repeat (TPR) domain, which usually inhibits its phosphatase activity by blocking the active site (closed conformation). Certain activators bind to the PP5–TPR domain, alleviating this inhibition and allowing the catalytic domain to adopt an active (open) conformation. While this mechanism has been proposed based on structural and biophysical studies, PP5 conformational changes and activity have yet to be observed in cells. Here, we designed and developed a flow cytometry-based fluorescence resonance energy transfer (FC-FRET) method, enabling real-time observation of PP5 autoinhibition and activation within live mammalian cells. By quantifying FRET efficiency using sensitized emission, we established a standardized and adaptable data acquisition workflow. Our findings revealed that, in a cellular context, PP5 exists in multiple conformational states, none of which alone fully predicts its activity. Additionally, we have demonstrated that PTMs such as phosphorylation and SUMOylation impact PP5 conformational changes, representing a significant advancement in our understanding of its regulatory mechanisms.</div></div>\",\"PeriodicalId\":9684,\"journal\":{\"name\":\"Cell Stress & Chaperones\",\"volume\":\"29 6\",\"pages\":\"Pages 709-717\"},\"PeriodicalIF\":3.3000,\"publicationDate\":\"2024-10-10\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Cell Stress & Chaperones\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S1355814524001196\",\"RegionNum\":3,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"CELL BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cell Stress & Chaperones","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1355814524001196","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"CELL BIOLOGY","Score":null,"Total":0}
Flow cytometry FRET reveals post-translational modifications drive Protein Phosphatase-5 conformational changes in mammalian cells
The serine/threonine Protein Phosphatase-5 (PP5) plays an essential role in regulating hormone and stress-induced signaling networks as well as extrinsic apoptotic pathways in cells. Unlike other Protein Phosphatases, PP5 possesses both regulatory and catalytic domains, and its function is further modulated through post-translational modifications (PTMs). PP5 contains a tetratricopeptide repeat (TPR) domain, which usually inhibits its phosphatase activity by blocking the active site (closed conformation). Certain activators bind to the PP5–TPR domain, alleviating this inhibition and allowing the catalytic domain to adopt an active (open) conformation. While this mechanism has been proposed based on structural and biophysical studies, PP5 conformational changes and activity have yet to be observed in cells. Here, we designed and developed a flow cytometry-based fluorescence resonance energy transfer (FC-FRET) method, enabling real-time observation of PP5 autoinhibition and activation within live mammalian cells. By quantifying FRET efficiency using sensitized emission, we established a standardized and adaptable data acquisition workflow. Our findings revealed that, in a cellular context, PP5 exists in multiple conformational states, none of which alone fully predicts its activity. Additionally, we have demonstrated that PTMs such as phosphorylation and SUMOylation impact PP5 conformational changes, representing a significant advancement in our understanding of its regulatory mechanisms.
期刊介绍:
Cell Stress and Chaperones is an integrative journal that bridges the gap between laboratory model systems and natural populations. The journal captures the eclectic spirit of the cellular stress response field in a single, concentrated source of current information. Major emphasis is placed on the effects of climate change on individual species in the natural environment and their capacity to adapt. This emphasis expands our focus on stress biology and medicine by linking climate change effects to research on cellular stress responses of animals, micro-organisms and plants.