Singh Rajesh K, Sundar Adithya, Fushman David
Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742-3360 (USA).
Angew Chem Int Ed Engl. 2014 Jun 10;53(24):6120-5. doi: 10.1002/anie.201402642. Epub 2014 Apr 24.
Uncovering the mechanisms that allow conjugates of ubiquitin (Ub) and/or Ub-like (UBL) proteins such as Rub1 to serve as distinct molecular signals requires the ability to make them with native connectivity and defined length and linkage composition. A novel, effective, and affordable strategy for controlled chemical assembly of fully natural UBL-Ub, Ub-UBL, and UBL-UBL conjugates from recombinant monomers is presented. Rubylation of Ub and Rub1 and ubiquitination of Rub1 was achieved without E2/E3 enzymes. New residue-specific information was obtained on the interdomain contacts in naturally-occurring K48-linked Rub1-Ub and Ub-Rub1, and K29-linked Rub1-Ub heterodimers, and their recognition by a K48-linkage-specific Ub receptor. The disassembly of these heterodimers by major deubiquitinating enzymes was examined and it was discovered that some deubiquitinases also possess derubylase activity. This unexpected result suggests possible crosstalk between Ub and Rub1/Nedd8 signaling pathways.
揭示泛素（Ub）和/或类泛素（UBL）蛋白（如Rub1）的缀合物作为独特分子信号的机制，需要具备以天然连接性、确定长度和连接组成来合成它们的能力。本文提出了一种新颖、有效且经济的策略，用于从重组单体可控化学组装完全天然的UBL-Ub、Ub-UBL和UBL-UBL缀合物。在没有E2/E3酶的情况下实现了Ub和Rub1的Rubylation以及Rub1的泛素化。获得了关于天然存在的K48连接的Rub1-Ub和Ub-Rub1以及K29连接的Rub1-Ub异二聚体中结构域间接触的新的残基特异性信息，以及它们被K48连接特异性Ub受体识别的信息。研究了主要去泛素化酶对这些异二聚体的拆解，发现一些去泛素酶也具有去Rubylase活性。这一意外结果表明Ub和Rub1/Nedd8信号通路之间可能存在串扰。
