Presentation Information
[P04-483]Hydroxyurea Modulates Thiol–Disulfide Homeostasis and Selectively Impairs ER-Associated Degradation in Yeast
○Kunio Nakatsukasa1 (1. Nagoya City University (Japan))
Keywords:
Hydroxyurea,Saccharomyces cerevisiae,endoplasmic reticulum,protein degradation,thiol-disulfide homeostasis
[Purpose]
Hydroxyurea (HU) is widely used to induce replication stress through inhibition of ribonucleotide reductase; however, its impact on intracellular protein quality control remains unclear. The endoplasmic reticulum (ER) maintains proteostasis via ER-associated degradation (ERAD), a process closely linked to thiol–disulfide homeostasis. Here, we aimed to determine whether HU affects ERAD pathways and ER redox balance in budding yeast.
[Method]
We analyzed degradation kinetics of representative misfolded proteins categorized as ERAD-L (luminal substrates), ERAD-M (membrane substrates), and cytosolic substrates. Protein turnover was assessed using cycloheximide chase assays followed by immunoblotting. To distinguish HU-specific effects from cell-cycle arrest, we compared degradation under synchronized cell-cycle conditions and alternative replication stress. ER redox homeostasis was further examined using the ero1-1 mutant, defective in oxidative protein folding.
[Results]
HU selectively impaired degradation of ERAD-L substrates, while ERAD-M and cytosolic substrate degradation were largely unaffected. This effect was independent of S-phase arrest, indicating a pathway-specific action beyond replication stress. Mechanistically, HU likely promotes disulfide bond formation in cysteine-containing luminal substrates, thereby inhibiting the reductive processing required for retrotranslocation. Consistently, HU partially rescued growth defects of ero1-1 cells under reductive stress conditions.
[Consideration]
These findings suggest that HU directly modulates ER thiol–disulfide homeostasis and reveal an unexpected connection between replication stress-inducing agents and ER proteostasis. The selective impairment of the ERAD-L pathway underscores the importance of redox balance in regulating substrate-specific ER quality control.
[Conclusion]
HU exerts a previously unrecognized function in controlling ER proteostasis by modulating thiol–disulfide homeostasis, thereby selectively regulating the ERAD-L pathway. This study provides new insights into ER quality control mechanisms and highlights potential strategies for their targeted modulation.
Hydroxyurea (HU) is widely used to induce replication stress through inhibition of ribonucleotide reductase; however, its impact on intracellular protein quality control remains unclear. The endoplasmic reticulum (ER) maintains proteostasis via ER-associated degradation (ERAD), a process closely linked to thiol–disulfide homeostasis. Here, we aimed to determine whether HU affects ERAD pathways and ER redox balance in budding yeast.
[Method]
We analyzed degradation kinetics of representative misfolded proteins categorized as ERAD-L (luminal substrates), ERAD-M (membrane substrates), and cytosolic substrates. Protein turnover was assessed using cycloheximide chase assays followed by immunoblotting. To distinguish HU-specific effects from cell-cycle arrest, we compared degradation under synchronized cell-cycle conditions and alternative replication stress. ER redox homeostasis was further examined using the ero1-1 mutant, defective in oxidative protein folding.
[Results]
HU selectively impaired degradation of ERAD-L substrates, while ERAD-M and cytosolic substrate degradation were largely unaffected. This effect was independent of S-phase arrest, indicating a pathway-specific action beyond replication stress. Mechanistically, HU likely promotes disulfide bond formation in cysteine-containing luminal substrates, thereby inhibiting the reductive processing required for retrotranslocation. Consistently, HU partially rescued growth defects of ero1-1 cells under reductive stress conditions.
[Consideration]
These findings suggest that HU directly modulates ER thiol–disulfide homeostasis and reveal an unexpected connection between replication stress-inducing agents and ER proteostasis. The selective impairment of the ERAD-L pathway underscores the importance of redox balance in regulating substrate-specific ER quality control.
[Conclusion]
HU exerts a previously unrecognized function in controlling ER proteostasis by modulating thiol–disulfide homeostasis, thereby selectively regulating the ERAD-L pathway. This study provides new insights into ER quality control mechanisms and highlights potential strategies for their targeted modulation.
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