Feng Yue, Goncalves Monica M, Jitkova Yulia, Keszei Alexander F A, Yan Yongran, Sarathy Chaitra, St-Germain Jonathan, Kenney Tristan M G, Tcheng Matthew, Trudel Vincent, Mancini Ross S, Upadhyay Rahul, Hurren Rose, Gronda Marcela, Schultz Matthew, Soriano Kaylen, Lees Kaitlin, Pomroy Neil C, Currie S Quinn W, Privé Gilbert G, Reed Mark A, Yudin Andrei K, Penn Linda Z, Arrowsmith Cheryl H, Raught Brian, Mazhab-Jafari Mohammad T, Vahidi Siavash, Schimmer Aaron D
Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 1L7, Canada.
Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada.
Proc Natl Acad Sci U S A. 2025 Feb 4;122(5):e2422447122. doi: 10.1073/pnas.2422447122. Epub 2025 Jan 29.
ClpXP is a two-component mitochondrial matrix protease. The caseinolytic mitochondrial matrix peptidase chaperone subunit X (ClpX) recognizes and translocates protein substrates into the degradation chamber of the caseinolytic protease P (ClpP) for proteolysis. ClpXP degrades damaged respiratory chain proteins and is necessary for cancer cell survival. Despite the critical role of ClpXP in mitochondrial protein quality control, the specific degrons, or modifications that tag substrate proteins for degradation by human ClpXP, are still unknown. We demonstrated that phosphorylated serine (pSer) targets substrates to ClpX and facilitates their degradation by ClpXP in biochemical assays. In contrast, ClpP hyperactivated by the small-molecule drug ONC201 lost the preference for phosphorylated substrates. Hydrogen deuterium exchange mass spectrometry combined with biochemical assays showed that pSer binds the RKL loop of ClpX. ClpX variants with substitutions in the RKL loop failed to recognize phosphorylated substrates. In intact cells, ClpXP also preferentially degraded substrates with pSer. Moreover, ClpX substrates with the pSer were selectively found in aggregated mitochondrial proteins. Our work uncovers a mechanism for substrate recognition by ClpXP, with implications for targeting acute myeloid leukemia and other disorders involving ClpXP dysfunction.
ClpXP是一种双组分线粒体基质蛋白酶。酪蛋白水解线粒体基质肽酶伴侣亚基X（ClpX）识别蛋白质底物并将其转运到酪蛋白水解蛋白酶P（ClpP）的降解腔中进行蛋白水解。ClpXP可降解受损的呼吸链蛋白，是癌细胞存活所必需的。尽管ClpXP在线粒体蛋白质质量控制中起关键作用，但人类ClpXP用于标记底物蛋白进行降解的特定降解信号或修饰仍不清楚。我们在生化分析中证明，磷酸化丝氨酸（pSer）将底物靶向ClpX，并促进它们被ClpXP降解。相比之下，小分子药物ONC201过度激活的ClpP失去了对磷酸化底物的偏好。氢氘交换质谱结合生化分析表明，pSer结合ClpX的RKL环。RKL环发生取代的ClpX变体无法识别磷酸化底物。在完整细胞中，ClpXP也优先降解含有pSer的底物。此外，含有pSer的ClpX底物选择性地存在于聚集的线粒体蛋白中。我们的工作揭示了ClpXP识别底物的机制，并对靶向急性髓系白血病和其他涉及ClpXP功能障碍的疾病具有启示意义。
