Bondalapati Somasekhar, Eid Emad, Mali Sachitanand M, Wolberger Cynthia, Brik Ashraf
Schulich Faculty of Chemistry , Technion-Israel Institute of Technology , Haifa 3200008 , Israel . Email:
Department of Biophysics and Biophysical Chemistry , Johns Hopkins University School of Medicine , Baltimore , MD 21205 , USA.
Chem Sci. 2017 May 1;8(5):4027-4034. doi: 10.1039/c7sc00488e. Epub 2017 Apr 5.
Small ubiquitin like modifier (SUMO) proteins are known to regulate many important cellular processes such as transcription and apoptosis. Recently, hybrid SUMO-ubiquitin chains containing SUMO-2 linked to Lys63-di-ubiquitin were found to play a major role in DNA repair. Despite some progress in understanding the role of these hybrid chains in DNA repair, there are various fundamental questions remaining to be answered. To further investigate the importance of hybrid SUMO-ubiquitin chains in DNA repair, the homogenous material of these chains, and their unique analogues, are needed in workable quantities. By applying advanced chemical strategies for protein synthesis, we report the first total chemical synthesis of four different SUMO-2-Lys63-linked di-ubiquitin hybrid chains, in which the di-ubiquitin is linked to different lysines in SUMO. In these syntheses, the usefulness of removable solubilizing tags is demonstrated, and two different approaches were examined in terms of reliability and efficiency. In the first approach, a poly-Arg tag was attached to the C-terminus of SUMO a 3,4-diaminobenzoic acid cleavable linker, whereas in the second we attached the tag a phenylacetamidomethyl linker, which can be cleaved by PdCl. The comparison between these different strategies offers guidelines for future scale-up preparation of these analogues and other proteins, which currently use synthetic peptide intermediates that are difficult to handle and purify. The availability of the SUMO-ubiquitin hybrid chains opens up new opportunities for studying the role of these chains in DNA repair and other cellular processes.
已知小泛素样修饰物（SUMO）蛋白可调节许多重要的细胞过程，如转录和细胞凋亡。最近发现，含有与赖氨酸63-二聚泛素相连的SUMO-2的杂合SUMO-泛素链在DNA修复中起主要作用。尽管在理解这些杂合链在DNA修复中的作用方面取得了一些进展，但仍有各种基本问题有待解答。为了进一步研究杂合SUMO-泛素链在DNA修复中的重要性，需要有可操作数量的这些链的同质材料及其独特类似物。通过应用先进的蛋白质合成化学策略，我们报道了四种不同的SUMO-2-赖氨酸63相连的二聚泛素杂合链的首次全化学合成，其中二聚泛素与SUMO中的不同赖氨酸相连。在这些合成中，证明了可去除的增溶标签的有用性，并从可靠性和效率方面研究了两种不同的方法。在第一种方法中，将聚精氨酸标签连接到SUMO的C末端——一个3,4-二氨基苯甲酸可裂解连接子，而在第二种方法中，我们连接了标签——一个苯乙酰氨基甲基连接子，其可被氯化钯裂解。这些不同策略之间的比较为这些类似物和其他蛋白质的未来放大制备提供了指导方针，目前这些制备使用难以处理和纯化的合成肽中间体。SUMO-泛素杂合链的可得性为研究这些链在DNA修复和其他细胞过程中的作用开辟了新机会。
