Nyenhuis David A, Watanabe Susan M, Tjandra Nico, Carter Carol A
Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20982.
Department of Microbiology and Immunology, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY 11794-5222.
Proc Natl Acad Sci U S A. 2025 Jan 7;122(1):e2419542121. doi: 10.1073/pnas.2419542121. Epub 2024 Dec 31.
Tsg101 is a highly conserved protein best known as an early-functioning component of cellular ESCRT machinery participating in recognition, sorting, and trafficking of cellular cargo to various intracellular destinations. It shares sequence and structural homology to canonical ubiquitin-conjugating (E2) enzymes and is linked to diverse events regulated by Ub signaling. How it might fulfill these roles is unclear. Here, we show that Tsg101 E2 mimicry permits interactions with diverse ubiquitin ligating (E3) enzymes and underlies its multifunctional capabilities. Coexpression of Tsg101 with the E3 ligase NNedd4-2s protected the enzyme from degradation and, remarkably, other widely divergent ligases as well. Structural alignment with UbcH5, a canonical E2 enzyme, revealed that recognition at the E2-E3 interface, a region broadly conserved despite sequence and structural differences in both E2 and E3 enzymes, was critical for protection. Nevertheless, UbcH5 failed to protect NNedd4-2s, indicating that the UEV chaperone function is unique to the variant. Studies using Cy5-Ub-VME showed that Tsg101-mediated protection reduced accessibility to Cys residues in the ligase. Access to Tsg101 Ub-binding sites was critical: Rabeprazole, which interferes with Tsg101 Ub-binding, diminished E3 ligase protection. Thus, E2 mimicry permitting control of E3 ligase ubiquitin signaling underlies Tsg101's broad ability to participate in multiple cellular functions. The study provides mechanistic insight into how Tsg101, by partnering with diverse E3 ligases, can contribute to a broad range of cellular activities.
Tsg101是一种高度保守的蛋白质，作为细胞内体分选转运复合体（ESCRT）机制的早期起作用的组分而广为人知，参与细胞货物向细胞内各个目的地的识别、分选和运输。它与典型的泛素结合（E2）酶具有序列和结构同源性，并与由泛素信号调节的多种事件相关联。它如何履行这些功能尚不清楚。在这里，我们表明Tsg101对E2的模拟允许其与多种泛素连接（E3）酶相互作用，并构成其多功能能力的基础。Tsg101与E3连接酶NNedd4-2s共表达可保护该酶不被降解，而且值得注意的是，对其他广泛不同的连接酶也有保护作用。与典型E2酶UbcH5的结构比对显示，在E2-E3界面处的识别对于保护至关重要，尽管E2和E3酶在序列和结构上存在差异，但该区域广泛保守。然而，UbcH5未能保护NNedd4-2s，表明UEV伴侣功能是该变体所特有的。使用Cy5-Ub-VME的研究表明，Tsg101介导的保护降低了连接酶中半胱氨酸残基的可及性。进入Tsg101泛素结合位点至关重要：雷贝拉唑会干扰Tsg101与泛素的结合，从而减弱E3连接酶的保护作用。因此，对E3连接酶泛素信号传导的E2模拟控制是Tsg101广泛参与多种细胞功能的能力基础。这项研究为Tsg101如何通过与多种E3连接酶合作来促进广泛的细胞活动提供了机制上的见解。
