Thompson Joel W, Nagel Jane, Hoving Sjouke, Gerrits Bertran, Bauer Andreas, Thomas Jason R, Kirschner Marc W, Schirle Markus, Luchansky Sarah J
From the Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts 02139.
Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, CH-4056 Basel, Switzerland, and.
J Biol Chem. 2014 Oct 17;289(42):28942-55. doi: 10.1074/jbc.M114.573352. Epub 2014 Aug 21.
Targeted degradation of proteins through the ubiquitin-proteasome system (UPS) via the activities of E3 ubiquitin ligases regulates diverse cellular processes, and misregulation of these enzymes contributes to the pathogenesis of human diseases. One of the challenges facing the UPS field is to delineate the complete cohort of substrates for a particular E3 ligase. Advances in mass spectrometry and the development of antibodies recognizing the Lys-ϵ-Gly-Gly (diGly) remnant from ubiquitinated proteins following trypsinolysis have provided a tool to address this question. We implemented an inducible loss of function approach in combination with quantitative diGly proteomics to find novel substrates of HUWE1 (HECT, UBA, and WWE domain containing 1, E3 ubiquitin protein ligase), an E3 ligase implicated in cancer and intellectual disabilities. diGly proteomics results led to the identification of DNA damage-inducible transcript 4 (DDIT4) as a putative HUWE1 substrate. Cell-based assays demonstrated that HUWE1 interacts with and regulates ubiquitination and stability of DDIT4. Together these data suggest a model in which HUWE1 mediates DDIT4 proteasomal degradation. Our results demonstrate proof of concept that inducible knockdown of an E3 ligase in combination with diGly proteomics provides a potentially advantageous method for identifying novel E3 substrates that may help to identify candidates for therapeutic modulation in the UPS.
通过E3泛素连接酶的活性，经由泛素-蛋白酶体系统（UPS）对蛋白质进行靶向降解，可调节多种细胞过程，而这些酶的调节异常会导致人类疾病的发病机制。UPS领域面临的挑战之一是确定特定E3连接酶的完整底物群体。质谱技术的进步以及识别胰蛋白酶消化后泛素化蛋白质中赖氨酸-ε-甘氨酰-甘氨酸（二甘氨酸）残基的抗体的开发，为解决这个问题提供了一种工具。我们采用了功能诱导缺失方法，并结合定量二甘氨酸蛋白质组学，以寻找HUWE1（含HECT、UBA和WWE结构域的1型，E3泛素蛋白连接酶）的新底物，该E3连接酶与癌症和智力残疾有关。二甘氨酸蛋白质组学结果导致鉴定出DNA损伤诱导转录本4（DDIT4）作为假定的HUWE1底物。基于细胞的实验表明，HUWE1与DDIT4相互作用并调节其泛素化和稳定性。这些数据共同表明了一种模型，即HUWE1介导DDIT4的蛋白酶体降解。我们的结果证明了这样一个概念验证，即E3连接酶的诱导性敲低与二甘氨酸蛋白质组学相结合，为识别新的E3底物提供了一种潜在的有利方法，这可能有助于识别UPS中治疗调节的候选物。
