Ubiquitination and ubiquitin-like protein post-translational modifications play an enormous number of roles in cellular processes. These modifications are constituted of multistep reaction cascades. Readily implementable and robust methods to evaluate each step of the overall process, while presently limited, are critical to the understanding and modulation of the reaction sequence at any desired level, both in terms of basic research and potential therapeutic drug discovery and development. We developed multiple robust and reliable high-throughput assays to interrogate each of the sequential discrete steps in the reaction cascade leading to protein ubiquitination. As models for the E1 ubiquitin-activating enzyme, the E2 ubiquitin-conjugating enzyme, the E3 ubiquitin ligase, and their ultimate substrate of ubiquitination in a cascade, we examined Uba1, Rad6, Rad18, and proliferating cell nuclear antigen (PCNA), respectively, in reconstituted systems. Identification of inhibitors of this pathway holds promise in cancer therapy since PCNA ubiquitination plays a central role in DNA damage tolerance and resulting mutagenesis. The luminescence-based assays we developed allow for the quantitative determination of the degree of formation of ubiquitin thioester conjugate intermediates with both E1 and E2 proteins, autoubiquitination of the E3 protein involved, and ubiquitination of the final substrate. Thus, all covalent adducts along the cascade can be individually probed. We tested previously identified inhibitors of this ubiquitination cascade, finding generally good correspondence between compound potency trends determined by more traditional low-throughput methods and the present high-throughput ones. These approaches are readily adaptable to other E1, E2, and E3 systems, and their substrates in both ubiquitination and ubiquitin-like post-translational modification cascades.

中文翻译：

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强大的高通量分析方法可评估泛素化和相关级联反应中的离散步骤

泛素化和类泛素蛋白的翻译后修饰在细胞过程中起着巨大的作用。这些修饰由多步反应级联组成。在基础研究和潜在的治疗药物发现与开发方面，目前受限制的易于实施且健壮的方法可用于评估整个过程的每个步骤，但对于了解和调节反应序列处于任何所需水平至关重要。我们开发了多种强大且可靠的高通量检测方法，以探查导致蛋白质泛素化的反应级联反应中的每个连续离散步骤。作为E1泛素激活酶的模型，E2泛素结合酶，E3泛素连接酶及其最终级联泛素化的底物，我们在重组系统中分别检查了Uba1，Rad6，Rad18和增殖细胞核抗原（PCNA）。由于PCNA泛素化在DNA损伤耐受性和导致的诱变中起着核心作用，因此鉴定这种途径的抑制剂在癌症治疗中具有广阔的前景。我们开发的基于发光的分析方法可以定量确定E1和E2蛋白与泛素硫酯共轭中间体的形成程度，所涉及的E3蛋白的自泛素化以及最终底物的泛素化。因此，沿着级联反应的所有共价加合物都可以被单独探测。我们测试了先前确定的泛素化级联抑制剂，发现由更传统的低通量方法确定的复合效价趋势与当前的高通量方法之间的总体良好趋势对应。这些方法很容易适用于其他E1，E2和E3系统，以及它们在泛素化和泛素样翻译后修饰级联反应中的底物。