Scalia Pierluigi, Williams Stephen J
ISOPROG-Somatolink EPFP Research Network, Philadelphia, PA, USA and Caltanissetta, Italy.
Sbarro Institute for Cancer Research and Molecular Medicine, Division of Biology, CST, Temple University, Philadelphia, USA.
Bio Protoc. 2023 Sep 20;13(18):e4816. doi: 10.21769/BioProtoc.4816.
The identification and characterization of the ubiquitin E-ligase complexes involved in specific proteins' degradation via the ubiquitin-proteasome system (UPS) can be challenging and require biochemical purification processes and in vitro reconstitution assays. Likewise, evaluating the effect of parallel phosphorylation and ubiquitination events occurring in vivo at dual phospho/ubiquitin-regulated motifs (called Phospho-Degrons or pDegrons) driving UPS degradation of the targeted protein has remained elusive. Indeed, the functional study of such E1-E2-E3 complexes acting on a protein-specific level requires previously or otherwise acquired knowledge of the nature of such degradation complex components. Furthermore, the molecular basis of the interaction between an E3 ligase and its pDegron binding motif on a target protein would require individually optimized in vitro kinase and ubiquitination assays. Here, we describe a novel enzymatically enhanced pull-down method to functionally streamline the discovery and functional validation of the ubiquitin E-ligase components interacting with a phospho-degron containing protein domain and/or sub-domain. The protocol combines key features of a protein kinase and ubiquitination in vitro assay by including them in a pull-down step exerted by a known or putative pDegron-tagged peptide using the cell extracts as a source of enzymatically active post-translational modification (PTM) modifying/binding native proteins. The same method allows studying specific stimuli or treatments towards the recruitment of the molecular degradation complex at the target protein's phospho-degron site, reflecting in vivo-initiated events further enhanced through the assay design. In order to take full advantage of the method over traditional protein-protein interaction methods, we propose to use this PTM-enhanced (PTMe) pull down both towards the degradation complex discovery/ID phase as well as for the functional pDegron recruitment validation phase, which is the one described in the present protocol both graphically and in a stepwise fashion for reproduceable results. Key features • Suitable to study UPS-regulated (a) cytosolic and/or nuclear proteins, (b) intracellular region of transmembrane proteins, and (c) protein sub-domains bearing a known/putative pDegron motif. • Requires a biotin-tagged recombinant version of the target protein and/or sub-domain. • Allows the qualitative and quantitative analysis of endogenous ubiquitin (Ub) E-ligases recruitment to a known or putative pDegron bearing protein/sub-domain. • Allows simultaneous testing of various treatments and/or conditions affecting the phosphorylative and/or ubiquitylation status of the studied pDegron bearing protein/sub-domain and the recruited factors. Graphical overview.
通过泛素 - 蛋白酶体系统（UPS）参与特定蛋白质降解的泛素E连接酶复合物的鉴定和表征具有挑战性，需要生化纯化过程和体外重组分析。同样，评估在体内发生的平行磷酸化和泛素化事件对驱动靶蛋白UPS降解的双磷酸/泛素调节基序（称为磷酸化降解基序或pDegrons）的影响仍然难以捉摸。事实上，对作用于蛋白质特异性水平的此类E1 - E2 - E3复合物进行功能研究需要事先了解或通过其他方式获取此类降解复合物成分的性质。此外，E3连接酶与其在靶蛋白上的pDegron结合基序之间相互作用的分子基础需要分别优化体外激酶和泛素化分析。在这里，我们描述了一种新型的酶促增强下拉方法，以功能上简化与含磷酸化降解基序的蛋白质结构域和/或亚结构域相互作用的泛素E连接酶成分的发现和功能验证。该方案结合了蛋白激酶和泛素化体外分析的关键特征，将它们包含在由已知或推定的pDegron标记肽进行的下拉步骤中，使用细胞提取物作为酶促活性翻译后修饰（PTM）修饰/结合天然蛋白质的来源。相同的方法允许研究针对靶蛋白磷酸化降解基序位点处分子降解复合物募集的特定刺激或处理，反映通过分析设计进一步增强的体内起始事件。为了充分利用该方法相对于传统蛋白质 - 蛋白质相互作用方法的优势，我们建议将这种PTM增强（PTMe）下拉用于降解复合物发现/鉴定阶段以及功能性pDegron募集验证阶段，本方案以图形方式并逐步描述了该阶段以获得可重复的结果。关键特征 • 适用于研究UPS调节的（a）胞质和/或核蛋白，（b）跨膜蛋白的细胞内区域，以及（c）带有已知/推定pDegron基序的蛋白质亚结构域。 • 需要靶蛋白和/或亚结构域的生物素标记重组版本。 • 允许对内源性泛素（Ub）E连接酶募集到已知或推定的含pDegron的蛋白质/亚结构域进行定性和定量分析。 • 允许同时测试影响所研究含pDegron的蛋白质/亚结构域和募集因子的磷酸化和/或泛素化状态的各种处理和 / 或条件。图形概述。
