Cameron Crissey, Clark R Mason, Metts Adam M, Jiang Runze M, Scaggs Toya D, Kim Kwangho, Sulikowski Gary A, Plate Lars
Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37235, United States.
Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235, United States.
ACS Chem Biol. 2025 Sep 19;20(9):2229-2242. doi: 10.1021/acschembio.5c00377. Epub 2025 Sep 1.
Dynamic protein-protein interactions are key drivers of many cellular processes. Determining the relative sequence and precise timing of these interactions is crucial for elucidating the functional dynamics of biological processes. Here, we developed a time-resolved analysis of protein-protein ensembles using a destabilizing domain (TRAPPED) to study protein-protein interactions in a temporal manner. We have taken advantage of a dihydrofolate reductase-destabilizing domain (DHFR(DD)) that can be fused to a protein of interest and is constitutively degraded by the proteosome. Addition of the ligand trimethoprim (TMP) can stabilize DHFR(DD), preventing proteasomal degradation of the fusion protein and thereby inducing accumulation in cells. We synthesized and optimized TRimethoprim Analog Probes that maintain stabilization activity and contain a terminal alkyne for Click functionalization and a thiol reactive group to covalently tag DHFR(DD). Click reaction with a biotin tag and subsequent streptavidin enrichment enable time-resolved mass spectrometric identification of interacting partners. We evaluated the timing of protein interactions of SARS-CoV-2 and SARS-CoV nonstructural protein 15 (nsp15) over a 2 h period. We found interactors GEMIN5 and YBX3, known regulators of SARS-CoV-2 infection that bind viral RNA, as well as CACYBP and FHL1 that implicate nsp15 in the disruption of host ERK1/2 signaling. We further revealed that these interactions remain relatively steady from 0 to 2 h post translation of nsp15. TRAPPED methodology can be applied to determine the sequence and timing of protein-protein interactions of temporally regulated biological processes such as viral infection or signal transduction.
动态蛋白质-蛋白质相互作用是许多细胞过程的关键驱动因素。确定这些相互作用的相对序列和精确时间对于阐明生物过程的功能动态至关重要。在这里，我们开发了一种使用去稳定结构域的蛋白质-蛋白质聚集体的时间分辨分析方法（TRAPPED），以时间分辨的方式研究蛋白质-蛋白质相互作用。我们利用了一种二氢叶酸还原酶-去稳定结构域（DHFR(DD)），它可以与感兴趣的蛋白质融合，并被蛋白酶体持续降解。添加配体甲氧苄啶（TMP）可以稳定DHFR(DD)，防止融合蛋白的蛋白酶体降解，从而诱导其在细胞中积累。我们合成并优化了甲氧苄啶类似物探针，这些探针保持稳定活性，并含有用于点击功能化的末端炔烃和用于共价标记DHFR(DD)的硫醇反应性基团。与生物素标签的点击反应和随后的链霉亲和素富集能够对相互作用的伙伴进行时间分辨质谱鉴定。我们在2小时内评估了严重急性呼吸综合征冠状病毒2（SARS-CoV-2）和严重急性呼吸综合征冠状病毒（SARS-CoV）非结构蛋白15（nsp15）的蛋白质相互作用时间。我们发现了相互作用蛋白GEMIN5和YBX3，它们是已知的与病毒RNA结合的SARS-CoV-2感染调节因子，以及CACYBP和FHL1，它们表明nsp15参与宿主细胞外信号调节激酶1/2（ERK1/2）信号通路的破坏。我们进一步揭示，这些相互作用在nsp15翻译后0至2小时内保持相对稳定。TRAPPED方法可用于确定诸如病毒感染或信号转导等时间调节生物过程中蛋白质-蛋白质相互作用的序列和时间。
