Total Chemical Synthesis of ISGylated-Ubiquitin Hybrid Chain Assisted by Acetamidomethyl Derivatives with Dual Functions
Total Chemical Synthesis of ISGylated-Ubiquitin Hybrid Chain Assisted by Acetamidomethyl Derivatives with Dual Functions
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DOI：
10.1021/acs.bioconjchem.0c00026
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发表时间：
2020-03-01
影响因子：
4.7
通讯作者：
Brik, Ashraf
中科院分区：
文献类型：
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作者：
Eid, Emad;Boross, Gabor N.;Brik, Ashraf
干扰素刺激基因15(ISG15)是泛素样修饰物(ULM)家族的成员，它采用与泛素(Ub)相似的β-GRAP折叠结构域(S)，序列同源性很小。ISG15由两个类似Ub的结构域组成，帮助免疫系统中和多种病原体的感染，在防御包括甲型流感在内的许多病毒的细胞中发挥重要作用。最近，人们发现Ub是ISG15的底物，它可以在Lys29和Lys48上ISGyl化，而前者更具优势。这种杂交ISG15-Ub链的发现提出了关于这种结合的性质和效果的各种基本问题。为了进一步研究ISG15-Ub杂交链的作用，在可行的数量上需要这些链的纯均质材料。通过应用先进的蛋白质合成化学策略，我们报道了231个残基的ISG15-Lys29-Ub杂交链的全化学合成。在合成过程中，我们遇到了不可溶的多肽片段，因此我们开发了一种新的基于ACM的可逆增溶标签来有效地解决这一障碍。将这种新的ACM标签与已知的基于Arg的ACM增溶标签进行比较，发现在掺入和效率方面更可靠，如合成天然ISG15-Ub杂交链所示。
Interferon-stimulated gene 15 (ISG15) is a member of the ubiquitin-like modifiers (ULM) family, which adopts a beta-grasp fold domain(s) similar to ubiquitin (Ub) with only minor sequence homology. ISG15 consists of two Ub-like domains and aids the immune system in neutralizing infections by numerous pathogens and plays an important role in defending cells against many viruses including influenza A. Recently, Ub was found to be a substrate for ISG15, which can be ISGylated on Lys29 and Lys48, while the former is more dominant. The discovery of such hybrid ISG15-Ub chains brought forward various fundamental questions regarding the nature and effect of this conjugation. To further investigate the role of hybrid ISG15-Ub chains, the pure homogeneous material of these chains is needed in workable quantities. By applying advanced chemical strategies for protein synthesis, we report the total chemical synthesis of a 231-residue ISG15-Lys29-Ub hybrid chain. During the synthesis we encountered insoluble peptide fragments, and therefore we developed a new reversible Acm based solubilizing tag to efficiently tackle this hurdle. This new Acm tag was compared with the known Arg based Acm solubilizing tag and was found to be more reliable in terms of incorporation and efficiency as demonstrated in the synthesis of the native ISG15-Ub hybrid chain.