Department of Neurosurgery, Huashan Hospital, the Shanghai Key Laboratory of Medical Epigenetics, State Key Laboratory of Medical Neurobiology and Ministry of Education Frontiers Center for Brain Science, National Center for Neurological Disorders, Institutes of Biomedical Sciences, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China.
State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200032, China.
Proc Natl Acad Sci U S A. 2023 Aug 15;120(33):e2302478120. doi: 10.1073/pnas.2302478120. Epub 2023 Aug 7.
Lipid homeostasis is essential for normal cellular functions and dysregulation of lipid metabolism is highly correlated with human diseases including neurodegenerative diseases. In the ubiquitin-dependent autophagic degradation pathway, Troyer syndrome-related protein Spartin activates and recruits HECT-type E3 Itch to lipid droplets (LDs) to regulate their turnover. In this study, we find that Spartin promotes the formation of Itch condensates independent of LDs. Spartin activates Itch through its multiple PPAY-motif platform generated by self-oligomerization, which targets the WW12 domains of Itch and releases the autoinhibition of the ligase. Spartin-induced activation and subsequent autoubiquitination of Itch lead to liquid-liquid phase separation (LLPS) of the poly-, but not oligo-, ubiquitinated Itch together with Spartin and E2 both in vitro and in living cells. LLPS-mediated condensation of the reaction components further accelerates the generation of polyubiquitin chains, thus forming a positive feedback loop. Such Itch-Spartin condensates actively promote the autophagy-dependent turnover of LDs. Moreover, we show that the catalytic HECT domain of Itch is sufficient to interact and phase separate with poly-, but not oligo-ubiquitin chains. HECT domains from other HECT E3 ligases also exhibit LLPS-mediated the promotion of ligase activity. Therefore, LLPS and ubiquitination are mutually interdependent and LLPS promotes the ligase activity of the HECT family E3 ligases.
脂质稳态对于正常细胞功能至关重要，脂质代谢的失调与包括神经退行性疾病在内的人类疾病高度相关。在泛素依赖性自噬降解途径中，Troyer 综合征相关蛋白 Spartin 激活并募集 HECT 型 E3 连接酶 Itch 到脂滴（LDs）上，以调节它们的周转。在本研究中，我们发现 Spartin 独立于 LDs 促进了 Itch 凝聚物的形成。Spartin 通过其自身寡聚化产生的多个 PPAY-motif 平台激活 Itch，该平台靶向 Itch 的 WW12 结构域并释放该连接酶的自身抑制。Spartin 诱导的 Itch 激活及其随后的自泛素化导致多聚而非寡聚泛素化的 Itch 与 Spartin 和 E2 一起在体外和活细胞中发生液-液相分离（LLPS）。反应成分的 LLPS 介导的凝聚进一步加速了多泛素链的产生，从而形成正反馈环。这种 Itch-Spartin 凝聚物积极促进了 LDs 的自噬依赖性周转。此外，我们表明，Itch 的催化 HECT 结构域足以与多聚而非寡聚泛素链相互作用并发生液-液相分离。其他 HECT E3 连接酶的 HECT 结构域也表现出 LLPS 介导的连接酶活性促进作用。因此，LLPS 和泛素化是相互依存的，LLPS 促进了 HECT 家族 E3 连接酶的连接酶活性。
