Tolmachova Kateryna A, Farnung Jakob, Liang Jin Rui, Corn Jacob E, Bode Jeffrey W
Laboratory for Organic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zürich, 8093 Zürich, Switzerland.
Institute of Molecular Health Sciences, Department of Biology, ETH Zürich, 8093 Zürich, Switzerland.
ACS Cent Sci. 2022 Jun 22;8(6):756-762. doi: 10.1021/acscentsci.2c00203. Epub 2022 May 17.
Aberrations in protein modification with ubiquitin-fold modifier (UFM1) are associated with a range of diseases, but the biological function and regulation of this post-translational modification, known as UFMylation, remain enigmatic. To provide activity-based probes for UFMylation, we have developed a new method for the installation of electrophilic warheads at the C-terminus of recombinant UFM1. A C-terminal UFM1 acyl hydrazide was readily produced by selective intein cleavage and chemoselectively acylated by a variety of carboxylic acid anhydrides at pH 3, without detriment to the folded protein or reactions at unprotected amino acid side chains. The resulting UFM1 activity-based probes show a range of tunable reactivity and high selectivity for proteins involved in UFMylation processes; structurally related E1s, E2s, and proteases associated with Ub or other Ubls were unreactive. The UFM1 probes were active both in cell lysates and in living cells. A previously inaccessible α-chloroacetyl probe was remarkably selective for covalent modification of the active-site cysteine of de-UFMylase UFSP2 .
泛素样修饰因子1（UFM1）介导的蛋白质修饰异常与一系列疾病相关，但这种称为UFMylation的翻译后修饰的生物学功能和调控机制仍不清楚。为了开发基于活性的UFMylation探针，我们开发了一种在重组UFM1的C末端安装亲电弹头的新方法。通过选择性内含肽切割可轻松制备C末端UFM1酰肼，并在pH 3条件下通过多种羧酸酐进行化学选择性酰化，而不会损害折叠蛋白或未保护氨基酸侧链的反应。所得基于UFM1活性的探针显示出一系列可调节的反应性，并且对参与UFMylation过程的蛋白质具有高选择性；与泛素（Ub）或其他泛素样蛋白（Ubl）相关的结构相关的E1、E2和蛋白酶无反应性。UFM1探针在细胞裂解物和活细胞中均具有活性。一种以前无法获得的α-氯乙酰探针对去UFMylase UFSP2活性位点半胱氨酸的共价修饰具有显著的选择性。
