Enzymatic Analysis of WWP2 E3 Ubiquitin Ligase Using Protein Microarrays Identifies Autophagy-Related Substrates

WWP2 is a HECT E3 ligase that targets protein Lys residues for ubiquitination. As a member of the NEDD4 family of E3 ligases, WWP2 is comprised of an N-terminal C2 domain, four central WW domains, and a C-terminal catalytic HECT domain. The peptide segment between the middle WW domains, the 2,3-linker, can autoinhibit the catalytic domain and this can be relieved by phosphorylation at Tyr369. Several protein substrates of WWP2 have been identified, including the tumor suppressor lipid phosphatase PTEN, but the full substrate landscape and biological functions of WWP2 remain to be elucidated. Here we use protein microarray technology and the activated enzyme phosphomimetic mutant WWP2Y369E to identify potential WWP2 substrates. We identified 31 substrate hits for WWP2Y369E using protein microarrays of which three are the autophagy receptors, NDP52, OPTN, and SQSTM1. These three hits were validated with in vitro and cell-based transfection assays and the Lys ubiquitination sites on these proteins were mapped by mass spectrometry. Among the mapped ubiquitin sites on these autophagy receptors, many were previously identified in studies on the endogenous proteins. WWP2 knockout SH-SH5Y neuroblastoma cells using CRISPR-Cas9 showed a defect in mitophagy in a fashion that could be rescued by WWP2Y369E transfection. These studies suggest that WWP2-mediated ubiquitination of the autophagy receptors NDP52, OPTN, and SQSTM1 may positively contribute to the regulation of autophagy.WWP2是一种HECT型E3泛素连接酶（HECT E3 ligase），可靶向蛋白质的赖氨酸残基实施泛素化修饰。作为E3泛素连接酶NEDD4家族的成员，WWP2由N端C2结构域（C2 domain）、四个中间WW结构域（WW domains）以及C端催化型HECT结构域（HECT domain）构成。位于中间WW结构域之间的肽段——2,3连接子（2,3-linker）——可对催化结构域产生自抑制效应，而该自抑制效应可通过酪氨酸369（Tyr369）位点的磷酸化作用得以解除。目前已鉴定出WWP2的多种蛋白质底物，包括肿瘤抑制性脂质磷酸酶PTEN（PTEN），但WWP2完整的底物图谱及其生物学功能仍有待阐明。本研究利用蛋白质微阵列技术（protein microarray technology）以及活化型酶的磷酸化模拟突变体WWP2Y369E，对WWP2的潜在底物进行了筛选。通过蛋白质微阵列实验，我们共鉴定得到31个WWP2Y369E的候选底物，其中3个为自噬受体（autophagy receptors）NDP52（NDP52）、OPTN（OPTN）及SQSTM1（SQSTM1）。我们通过体外（in vitro）实验及细胞水平转染实验（cell-based transfection assays）对这三个候选底物进行了验证，并利用质谱技术（mass spectrometry）定位了这些蛋白质上的赖氨酸泛素化修饰位点。在这些自噬受体上定位到的泛素化位点中，有多个已在针对内源蛋白的相关研究中被鉴定到。我们利用CRISPR-Cas9（CRISPR-Cas9）技术构建了WWP2敲除的SH-SH5Y神经母细胞瘤细胞，该细胞系存在线粒体自噬（mitophagy）功能缺陷，且这一缺陷可通过转染WWP2Y369E得以挽救。上述研究表明，WWP2介导的自噬受体NDP52、OPTN及SQSTM1的泛素化修饰，可能对自噬的调控发挥正向促进作用。