Bregnard Thomas A, Fairchild Daniel, Chen Xiang, Erlandsen Heidi, Tarasov Sergey G, Walters Kylie J, Korzhnev Dmitry M, Bezsonova Irina
Department of Molecular Biology and Biophysics, UCONN Health, Farmington, Connecticut, USA.
Protein Processing Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland, USA.
J Biol Chem. 2025 Feb;301(2):108149. doi: 10.1016/j.jbc.2024.108149. Epub 2024 Dec 30.
Abnormalities in the expression of the ubiquitin ligase UBE3A (ubiquitin-protein ligase E3A)/E6AP (human papillomavirus E6-associated protein) are implicated in neurological disorders including Angelman syndrome and autism. Human UBE3A is expressed as three protein isoforms that differ in their abundance and subcellular localization. While previous studies indicate isoform-specific functions, the distinct roles of each isoform in human development remain unknown. The isoforms differ only by an extension at the N-terminal end of the AZUL (N-terminal zinc [Zn]-binding domain Amino-terminal Zn finger of the UBE3A Ligase) domain, which tethers UBE3A to the proteasome by interaction with proteasomal subunit Rpn10. Differences in the structure and biophysical properties of UBE3A isoforms likely contribute to their individual functions. Here, we use a combination of NMR spectroscopy and other biophysical and biochemical techniques to identify differences in structure, dynamics, and the Rpn10 binding of the AZUL isoforms. We show that the AZUL domain structure is retained in all three isoforms with an extended N-terminal helix in longer isoforms 2 and 3. Accordingly, all isoforms could effectively associate with the Rpn10. Significant differences between the isoforms were found in their propensities to multimerize where only the longer isoforms 2 and 3 of the AZUL domain could form dimers, which may play a role in the previously observed oligomerization-dependent activation of the UBE3A. Moreover, our NMR relaxation dispersion experiments revealed a dynamic Zn-coordination site in isoforms 1 and 3, but not in isoform 2 of UBE3A, suggesting its possible isoform-specific sensitivity to oxidative stress. This structural and biophysical characterization of the isoforms will advance our understanding of isoform-specific functions of UBE3A and may contribute to future treatment strategies for Angelman syndrome and other UBE3A-related diseases.
泛素连接酶UBE3A（泛素 - 蛋白连接酶E3A）/E6AP（人乳头瘤病毒E6相关蛋白）表达异常与包括天使综合征和自闭症在内的神经疾病有关。人UBE3A以三种蛋白异构体形式表达，它们在丰度和亚细胞定位上存在差异。虽然先前的研究表明异构体具有特定功能，但每种异构体在人类发育中的独特作用仍不清楚。这些异构体仅在AZUL（UBE3A连接酶的N端锌[Zn]结合结构域氨基末端锌指）结构域的N末端延伸部分有所不同，该延伸部分通过与蛋白酶体亚基Rpn10相互作用将UBE3A连接到蛋白酶体上。UBE3A异构体在结构和生物物理性质上的差异可能有助于其各自的功能。在这里，我们结合使用核磁共振光谱以及其他生物物理和生化技术，来识别AZUL异构体在结构、动力学和Rpn10结合方面的差异。我们表明，在所有三种异构体中都保留了AZUL结构域结构，较长的异构体2和3中具有延伸的N末端螺旋。因此，所有异构体都能有效地与Rpn10结合。在异构体的多聚倾向方面发现了显著差异，其中只有AZUL结构域较长的异构体2和3能够形成二聚体，这可能在先前观察到的UBE3A寡聚化依赖性激活中发挥作用。此外，我们的核磁共振弛豫色散实验揭示了异构体1和3中存在动态锌配位位点，而在UBE3A的异构体2中不存在，这表明其可能对氧化应激具有异构体特异性敏感性。对这些异构体的结构和生物物理特性进行表征将增进我们对UBE3A异构体特异性功能的理解，并可能有助于未来针对天使综合征和其他UBE3A相关疾病的治疗策略。
