Pang Eric Z, Zhao Boyu, Flowers Cameron, Oroudjeva Elizabeth, Winter Jasmine B, Pandey Vijaya, Sawaya Michael R, Wohlschlegel James, Loo Joseph A, Rodriguez Jose A, Clarke Steven G
Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA.
Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA.
bioRxiv. 2025 May 21:2025.05.21.654933. doi: 10.1101/2025.05.21.654933.
A major type of spontaneous protein damage that accumulates with age is the formation of kinked polypeptide chains with L-isoaspartyl residues. Mitigating this damage is necessary for maintaining proteome stability and prolonging organismal survival. While repair through methylation by PCMT1 has been previously shown to suppress L-isoaspartyl accumulation, we provide an additional mechanism for L-isoaspartyl maintenance through PCMTD1, a cullin-RING ligase (CRL). We combined cryo-EM, native mass spectrometry, and biochemical assays to provide insight on how the assembly and architecture of PCMTD1 in the context of a CRL complex fulfils this alternative mechanism. We show that the PCMTD1 CRL complex specifically binds L-isoaspartyl residues when bound to AdoMet. This work provides evidence for a growing class of E3 ubiquitin ligases that recognize spontaneous covalent modifications as potential substrates for ubiquitylation and subsequent proteasomal degradation.
随着年龄增长而积累的一种主要的自发性蛋白质损伤是形成带有L-异天冬氨酰残基的扭结多肽链。减轻这种损伤对于维持蛋白质组稳定性和延长机体寿命是必要的。虽然先前已表明通过PCMT1进行甲基化修复可抑制L-异天冬氨酰的积累，但我们提供了一种通过PCMTD1（一种cullin-RING连接酶（CRL））来维持L-异天冬氨酰的额外机制。我们结合了冷冻电镜、天然质谱和生化分析，以深入了解在CRL复合物背景下PCMTD1的组装和结构如何实现这种替代机制。我们表明，当与腺苷甲硫氨酸结合时，PCMTD1 CRL复合物特异性结合L-异天冬氨酰残基。这项工作为越来越多的E3泛素连接酶提供了证据，这些连接酶将自发的共价修饰识别为泛素化和随后蛋白酶体降解的潜在底物。
