Madden Michael M, Song Wenjiao, Martell Paul G, Ren Yong, Feng Jian, Lin Qing
Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, New York 14260-3000, USA.
Biochemistry. 2008 Mar 25;47(12):3636-44. doi: 10.1021/bi702078m. Epub 2008 Feb 29.
Protein ubiquitination is a widespread protein posttranslational modification in eukaryotes that regulates essentially every aspect of cellular processes. The attachment of ubiquitin to a protein substrate is accomplished through an enzymatic cascade involving the actions of an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3). There are more than 600 E3 ligases estimated to exist in the human genome that regulate the targeting specificity of protein ubiquitination. To understand the dynamic role of protein ubiquitination in biological processes, robust tools need to be developed which can be employed to establish the substrate specificity of each of these E3 ligases. In this report, we show that the ubiquitin carboxyl-terminally derived peptide probes can serve as modest ubiquitin surrogates for the ubiquitination pathway. In the E1-catalyzed probe adenylation assay, peptide probe 3 with a RLRGG recognition sequence exhibited the highest activity, with the k cat/ K 1/2 determined to be 1.1 x 10 (4) M (-1) s (-1), roughly 470-fold lower than that of ubiquitin. The rate of transfer from the E1 peptide probe thioesters to E2 showed clear sequence dependency, with peptide probe 4 with an LRLRGG recognition sequence showed the fastest rate ( t 1/2 = 0.9 min), essentially identical to that of ubiquitin ( t 1/2 = 0.8 min) under our assay conditions. Furthermore, peptide probes 4 and 8 also exhibited the selective, parkin-mediated labeling of tubulins in a semipurified tubulin-parkin complex. Finally, these carboxyl-terminally derived peptide probes were shown to label the ubiquitination substrates in fraction II of the rabbit reticulocyte lysate with an efficiency parallel to their substrate properties. The selective use of these ubiquitin carboxyl-terminally derived peptide probes by the ubiquitination pathway suggests that perhaps more potent peptide ubiquitination probes based on the ubiquitin C-terminal scaffold can be developed through additional structural optimization.
蛋白质泛素化是真核生物中一种广泛存在的蛋白质翻译后修饰，它基本上调控着细胞过程的方方面面。泛素与蛋白质底物的连接是通过一个酶促级联反应完成的，该反应涉及激活酶（E1）、缀合酶（E2）和连接酶（E3）的作用。据估计，人类基因组中存在600多种E3连接酶，它们调控着蛋白质泛素化的靶向特异性。为了理解蛋白质泛素化在生物过程中的动态作用，需要开发强大的工具，用于确定这些E3连接酶各自的底物特异性。在本报告中，我们表明泛素羧基末端衍生的肽探针可作为泛素化途径中适度的泛素替代物。在E1催化的探针腺苷酸化测定中，具有RLRGG识别序列的肽探针3表现出最高活性，其催化常数/米氏常数（kcat/K1/2）测定为1.1×10⁴ M⁻¹ s⁻¹，大约比泛素低470倍。从E1肽探针硫酯转移到E2的速率显示出明显的序列依赖性，具有LRLRGG识别序列的肽探针4显示出最快的速率（半衰期t1/2 = 0.9分钟），在我们的测定条件下与泛素的速率基本相同（t1/2 = 0.8分钟）。此外，肽探针4和8在半纯化的微管蛋白 - 帕金复合物中也表现出对微管蛋白的选择性、帕金介导的标记。最后，这些羧基末端衍生的肽探针被证明能够以与其底物性质平行的效率标记兔网织红细胞裂解物第二组分中的泛素化底物。泛素化途径对这些泛素羧基末端衍生肽探针的选择性使用表明，也许通过进一步的结构优化，可以开发出基于泛素C末端支架的更有效的肽泛素化探针。
