BioID screening of biotinylation sites using the avidin-like protein Tamavidin 2-REV identifies global interactors of stimulator of interferon genes (STING)
BioID screening of biotinylation sites using the avidin-like protein Tamavidin 2-REV identifies global interactors of stimulator of interferon genes (STING)
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DOI：
10.1074/jbc.ra120.014323
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发表时间：
2020-08-07
影响因子：
4.8
通讯作者：
Kosako, Hidetaka
中科院分区：
文献类型：
--
作者：
Motani, Kou;Kosako, Hidetaka
干扰素基因刺激因子（STING）通过膜运输介导胞质DNA诱导的先天免疫信号传导。与STING时空相互作用的蛋白质的全球鉴定将提供对其运输机制和STING信号通路的更好理解。邻近依赖性生物素识别（BioID）是一种强大的技术，用于识别活细胞中生理相关的蛋白质-蛋白质相互作用。然而，在传统的BioID方法中很少检测到生物素化的肽，该方法使用链霉亲和素珠来拉下生物素化的蛋白质，因为生物素-链霉亲和素相互作用太强。结果，仅鉴定了非生物素化肽，其不能与非特异性下拉蛋白质的肽区分开。在这里，我们开发了一种简单的方法，有效地和特异性地富集生物素化的肽使用Tamavidin 2-REV，一种工程化的抗生物素蛋白样蛋白，具有可逆的生物素结合能力。使用稳定表达TurboID融合STING的RAW 264.7巨噬细胞，我们鉴定并定量了STING近端蛋白的> 4，000种生物素化肽。各种内质网相关蛋白在未受刺激的细胞中被生物素化，STING激活引起位于高尔基体和内体的许多蛋白的生物素化。这些蛋白质包括已知与活化的STING相互作用的蛋白质，例如TANK结合激酶1（TBK1）、几种棕榈酰转移酶和p62/螯合体1（SQSTM 1）。此外，干扰素诱导的跨膜蛋白3（IFITM3），一个内溶酶体定位的抗病毒蛋白，结合STING在后期激活阶段。这些动态相互作用概况将提供详细的见解STING信号;我们建议，我们的方法使用Tamavidin 2-REV将是有用的BioID为基础的和其他基于生物素化的肽鉴定方法。
Stimulator of interferon genes (STING) mediates cytosolic DNA-induced innate immune signaling via membrane trafficking. The global identification of proteins that spatiotemporally interact with STING will provide a better understanding of its trafficking mechanisms and of STING signaling pathways. Proximity-dependent biotin identification (BioID) is a powerful technology to identify physiologically relevant protein-protein interactions in living cells. However, biotinylated peptides are rarely detected in the conventional BioID method, which uses streptavidin beads to pull down biotinylated proteins, because the biotin-streptavidin interaction is too strong. As a result, only nonbiotinylated peptides are identified, which cannot be distinguished from peptides of nonspecifically pull-downed proteins. Here, we developed a simple method to efficiently and specifically enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. Using RAW264.7 macrophages stably expressing TurboID-fused STING, we identified and quantified >4,000 biotinylated peptides of STING-proximal proteins. Various endoplasmic reticulum-associated proteins were biotinylated in unstimulated cells, and STING activation caused biotinylation of many proteins located in the Golgi and endosomes. These proteins included those known to interact with activated STING, such as TANK-binding kinase 1 (TBK1), several palmitoyl transferases, and p62/sequestosome 1 (SQSTM1). Furthermore, interferon-induced transmembrane protein 3 (IFITM3), an endolysosome-localized antiviral protein, bound to STING at the late activation stage. These dynamic interaction profiles will provide detailed insights into STING signaling; we propose that our approach using Tamavidin 2-REV would be useful for BioID-based and other biotinylation-based peptide identification methods.