Wu Qin, Ploegh Hidde L, Truttmann Matthias C
Whitehead Institute for Biomedical Research , 9 Cambridge Center, Cambridge, Massachusetts 02142, United States.
Department of Biology, Massachusetts Institute of Technology (MIT) , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
ACS Chem Biol. 2017 Mar 17;12(3):664-673. doi: 10.1021/acschembio.6b00998. Epub 2017 Jan 18.
In vivo protein ligation is of emerging interest as a means of endowing proteins with new properties in a controlled fashion. Tools to site-specifically and covalently modify proteins with small molecules, peptides, or other proteins in living cells are few and far between. Here, we describe the development of a Staphylococcus aureus sortase (SrtA)-based protein ligation approach for site-specific conjugation of fluorescent dyes and ubiquitin (Ub) to modify proteins in Caenorhabditis elegans. Hepta-mutant SrtA (SrtA) expressed in C. elegans is functional and supports in vitro sortase reactions in a low-Ca environment. Feeding SrtA-expressing C. elegans with small peptide-based probes such as (Gly)- biotin or (Gly)-fluorophores enables in vivo target protein modification. SrtA also catalyzes the circularization of suitably modified linear target proteins in vivo and allows the installation of F-box domains on targets to induce their degradation in a ubiquitin-dependent manner. This is a noninvasive method to achieve in vivo protein labeling, protein circularization, and targeted degradation in C. elegans. This technique should improve our ability to monitor and alter the function of intracellular proteins in vivo.
作为一种以可控方式赋予蛋白质新特性的手段，体内蛋白质连接正日益受到关注。在活细胞中用小分子、肽或其他蛋白质对蛋白质进行位点特异性和共价修饰的工具寥寥无几。在此，我们描述了一种基于金黄色葡萄球菌分选酶（SrtA）的蛋白质连接方法的开发，用于将荧光染料和泛素（Ub）位点特异性偶联，以修饰秀丽隐杆线虫中的蛋白质。在秀丽隐杆线虫中表达的七突变分选酶（SrtA）具有功能，并在低钙环境中支持体外分选酶反应。用基于小肽的探针（如（甘氨酸）-生物素或（甘氨酸）-荧光团）喂养表达SrtA的秀丽隐杆线虫可实现体内靶蛋白修饰。SrtA还能在体内催化适当修饰的线性靶蛋白的环化，并允许在靶标上安装F-box结构域，以泛素依赖的方式诱导其降解。这是一种在秀丽隐杆线虫中实现体内蛋白质标记、蛋白质环化和靶向降解的非侵入性方法。这项技术应能提高我们在体内监测和改变细胞内蛋白质功能的能力。
