Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY 10065, USA.
Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY 10065, USA.
Cell Chem Biol. 2018 Jan 18;25(1):110-120.e3. doi: 10.1016/j.chembiol.2017.10.001. Epub 2017 Nov 5.
Protein-protein interactions mediate essential cellular processes, however the detection of native interactions is challenging since they are often low affinity and context dependent. Here, we develop a chemical proteomics approach in vivo CLASPI [iCLASPI] (in vivo crosslinking-assisted and stable isotope labeling by amino acids in cell culture [SILAC]-based protein identification) relying upon photo-crosslinking, amber suppression, and SILAC-based quantitative proteomics to profile context-dependent protein-protein interactions in living cells. First, we use iCLASPI to profile in vivo binding partners of the N-terminal tails of soluble histone H3 or H4. We identify known histone chaperones and modifying proteins, thereby validating our approach, and find an interaction between soluble histone H3 and UBR7, an E3 ubiquitin ligase, mediated by UBR7's PHD domain. Furthermore, we apply iCLASPI to profile the context-dependent protein-protein interactions of chromatin-associated histone H3 at different cell-cycle stages, and identify ANP32A as a mitosis-specific interactor. Our results demonstrate that the iCLASPI approach can provide a general strategy for identifying native, context-dependent direct protein-protein interactions using photo-crosslinking and quantitative proteomics.
蛋白质-蛋白质相互作用介导着重要的细胞过程，然而，由于它们通常亲和力低且依赖于上下文，因此检测天然相互作用具有挑战性。在这里，我们开发了一种体内化学蛋白质组学方法 iCLASPI（基于细胞培养的交联辅助和稳定同位素标记的氨基酸 [SILAC] 的蛋白质鉴定中的体内交联），该方法依赖于光交联、琥珀酸抑制和 SILAC 定量蛋白质组学来分析活细胞中依赖于上下文的蛋白质-蛋白质相互作用。首先，我们使用 iCLASPI 来分析可溶性组蛋白 H3 或 H4 的 N 端尾部的体内结合伴侣。我们鉴定了已知的组蛋白伴侣和修饰蛋白，从而验证了我们的方法，并发现可溶性组蛋白 H3 与 E3 泛素连接酶 UBR7 之间存在相互作用，这种相互作用是由 UBR7 的 PHD 结构域介导的。此外，我们还应用 iCLASPI 来分析不同细胞周期阶段与染色质相关的组蛋白 H3 的依赖于上下文的蛋白质-蛋白质相互作用，并鉴定出 ANP32A 是有丝分裂特异性相互作用蛋白。我们的结果表明，iCLASPI 方法可以为使用光交联和定量蛋白质组学鉴定天然、依赖于上下文的直接蛋白质-蛋白质相互作用提供一种通用策略。
