Bekturova Aizat, Makaros Yaara, Ben-David Shahar, Koren Itay
The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 5290002, Israel.
Proc Natl Acad Sci U S A. 2025 Aug 19;122(33):e2501681122. doi: 10.1073/pnas.2501681122. Epub 2025 Aug 12.
Hypoxia, a condition characterized by insufficient oxygen supply, challenges cellular homeostasis and energy production, triggering adaptive responses to promote survival under these stressful conditions. One key strategy involves enzymatic oxidation of N-terminal cysteine residues coupled with proteolysis through the Cys-Arg/N-degron pathway. Despite hundreds of human proteins possessing N-terminal cysteine, very few have been identified as substrates of this pathway, and its substrate selectivity remains unclear. Moreover, the biological role of this pathway in the cellular response to hypoxia is not well defined. Here, by systematically screening protein stability using an N-terminome library, we reveal a broad set of cysteine-initiating proteins regulated by this pathway. Mutagenesis experiments further revealed the specificity of Cys-Arg/N-degron pathway, showing a preference for hydrophobic and positively charged residues following cysteine. Additionally, we uncovered full-length substrates that are regulated by this pathway during hypoxia, including IP6K1. Loss of IP6K1 impaired glucose uptake, glycolytic ATP production, and overall mitochondrial function. Consequently, IP6K1-deficient cells exhibited disrupted metabolic adaptation under hypoxic conditions and reduced survival under stress. These findings underscore the importance of the Cys-Arg/N-degron pathway in regulating metabolic responses and highlight its potential importance in hypoxia-related disorders.
缺氧是一种以氧气供应不足为特征的状态，它挑战细胞内稳态和能量产生，引发适应性反应以促进在这些应激条件下的存活。一种关键策略涉及通过Cys-Arg/N-降解子途径对N端半胱氨酸残基进行酶促氧化并伴随蛋白水解。尽管数百种人类蛋白质含有N端半胱氨酸，但很少有被鉴定为该途径的底物，其底物选择性仍不清楚。此外，该途径在细胞对缺氧反应中的生物学作用尚未明确界定。在这里，通过使用N端蛋白质组文库系统地筛选蛋白质稳定性，我们揭示了受该途径调控的广泛的半胱氨酸起始蛋白。诱变实验进一步揭示了Cys-Arg/N-降解子途径的特异性，表明对半胱氨酸之后的疏水和带正电荷残基有偏好。此外，我们发现了在缺氧期间受该途径调控的全长底物，包括IP6K1。IP6K1的缺失损害了葡萄糖摄取、糖酵解ATP产生和整体线粒体功能。因此，IP6K1缺陷细胞在缺氧条件下表现出代谢适应紊乱，在应激下存活率降低。这些发现强调了Cys-Arg/N-降解子途径在调节代谢反应中的重要性，并突出了其在缺氧相关疾病中的潜在重要性。
