Institute of Systems and Physical Biology, Shenzhen Bay Laboratory, Shenzhen, 518132, China.
Department of Chemistry, UF Scripps Biomedical Research, 130 Scripps Way, Jupiter, FL, 33458, USA.
Nat Commun. 2022 Aug 20;13(1):4906. doi: 10.1038/s41467-022-32689-z.
Enzymatic-based proximity labeling approaches based on activated esters or phenoxy radicals have been widely used for mapping subcellular proteome and protein interactors in living cells. However, activated esters are poorly reactive which leads to a wide labeling radius and phenoxy radicals generated by peroxide treatment may disturb redox-sensitive pathways. Herein, we report a photoactivation-dependent proximity labeling (PDPL) method designed by genetically attaching photosensitizer protein miniSOG to a protein of interest. Triggered by blue light and tunned by irradiation time, singlet oxygen is generated, thereafter enabling spatiotemporally-resolved aniline probe labeling of histidine residues. We demonstrate its high-fidelity through mapping of organelle-specific proteomes. Side-by-side comparison of PDPL with TurboID reveals more specific and deeper proteomic coverage by PDPL. We further apply PDPL to the disease-related transcriptional coactivator BRD4 and E3 ligase Parkin, and discover previously unknown interactors. Through over-expression screening, two unreported substrates Ssu72 and SNW1 are identified for Parkin, whose degradation processes are mediated by the ubiquitination-proteosome pathway.
基于活化酯或苯氧自由基的酶促邻近标记方法已被广泛用于在活细胞中绘制亚细胞蛋白质组和蛋白质相互作用体图谱。然而，活化酯的反应性较差，导致标记半径较宽，而过氧化氢处理产生的苯氧自由基可能会干扰氧化还原敏感途径。在此，我们报告了一种光激活依赖性邻近标记 (PDPL) 方法，该方法通过将光敏蛋白 miniSOG 基因附着到感兴趣的蛋白质上来设计。在蓝光的触发下，并通过辐照时间进行调节，生成单线态氧，随后能够对组氨酸残基进行时空分辨的苯胺探针标记。我们通过对细胞器特异性蛋白质组的作图证明了其高保真度。PDPL 与 TurboID 的并排比较表明，PDPL 具有更特异和更深层次的蛋白质组覆盖度。我们进一步将 PDPL 应用于与疾病相关的转录共激活因子 BRD4 和 E3 连接酶 Parkin，并发现了以前未知的相互作用体。通过过表达筛选，鉴定出 Parkin 的两个未报道的底物 Ssu72 和 SNW1，其降解过程由泛素-蛋白酶体途径介导。
