Photoaffinity-engineered protein scaffold for systematically exploring native phosphotyrosine signaling complexes in tumor samples
Photoaffinity-engineered protein scaffold for systematically exploring native phosphotyrosine signaling complexes in tumor samples
复制标题
光亲和工程蛋白质支架，用于系统地探索肿瘤样本中的天然磷酸酪氨酸信号复合物
DOI：
10.1073/pnas.1805633115
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发表时间：
2018-09-18
影响因子：
11.1
通讯作者：
Tian, Ruijun
中科院分区：
文献类型：
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作者：
Chu, Bizhu;He, An;Tian, Ruijun
磷酸酪氨酸（pTyr）依赖蛋白复合物是调节癌症信号的关键机制。我们开发了photoptyr -scaffold方法，用于无偏倚地捕获和探索弱和动态pTyr蛋白复合物。通过利用具有纳米摩尔结合亲和力的Src激酶同源性2超结合物，photoptyr -scaffold在分析癌细胞和乳腺肿瘤样本中的天然pTyr蛋白复合物方面表现出优异的敏感性。重要的是，我们发现PDGFRB是介导细胞间癌症信号传导的关键信号节点，它是高表达的，但独立于ERBB2， ERBB2是公认的乳腺癌治疗靶点。我们的研究结果可能会导致新的乳腺癌靶向治疗和探索与其他类型蛋白质翻译后修饰相关的动态蛋白质复合物的通用方法，并从复杂的临床样品中发现生物标志物。磷酸酪氨酸（pTyr）调节的蛋白复合物在癌症信号传导中发挥关键作用。肿瘤样品中这些蛋白复合物的系统表征仍然是一个挑战，因为它们的获取有限和ptyr介导的相互作用的短暂性。我们开发了一种混合化学蛋白质组学方法，称为photoptyr -scaffold，通过工程Src同源性2 （SH2）结构域，特异性结合pTyr蛋白，使用三功能化学探针和基因突变来克服这些挑战。动态SH2结构域支架蛋白复合物在轻度紫外光下有效交联，生物素标签捕获，质谱鉴定。该方法成功地用于在蛋白质组尺度上高选择性地分析乳腺癌组织样本中的天然pTyr蛋白复合物，检测pTyr信号蛋白的灵敏度比传统免疫组织化学方法高约100倍。在已鉴定的1000多个pTyr蛋白中，在癌症相关成纤维细胞上表达的受体酪氨酸激酶PDGFRB被证实是一种重要的细胞间信号调节因子，与ERBB2表达相关性较差，阻断PDGFRB信号传导可有效抑制肿瘤生长。photoptyr -scaffold方法可能成为易于分析临床相关样品中动态pTyr信号复合物的通用工具。
Significance Phosphotyrosine (pTyr)-dependent protein complexes are key machinery for regulating cancer signaling. We developed the Photo-pTyr-scaffold approach for unbiasedly capturing and exploring weak and dynamic pTyr protein complexes. By utilizing the Src kinase Src homology 2 superbinder with nanomolar binding affinity, Photo-pTyr-scaffold showed superior sensitivity for profiling native pTyr protein complexes in cancer cells and breast tumor samples. Importantly, we discovered PDGFRB to be a critical signaling node for mediating intercellular cancer signaling, which is highly expressed but independent of ERBB2, the well-established breast cancer therapeutic target. Our results could lead to new targeted therapies for breast cancer and generic approaches for exploring dynamic protein complexes related to other types of protein posttranslational modifications and discovering biomarkers readily from complex clinical samples. Phosphotyrosine (pTyr)-regulated protein complexes play critical roles in cancer signaling. The systematic characterization of these protein complexes in tumor samples remains a challenge due to their limited access and the transient nature of pTyr-mediated interactions. We developed a hybrid chemical proteomics approach, termed Photo-pTyr-scaffold, by engineering Src homology 2 (SH2) domains, which specifically bind pTyr proteins, with both trifunctional chemical probes and genetic mutations to overcome these challenges. Dynamic SH2 domain-scaffolding protein complexes were efficiently cross-linked under mild UV light, captured by biotin tag, and identified by mass spectrometry. This approach was successfully used to profile native pTyr protein complexes from breast cancer tissue samples on a proteome scale with high selectivity, achieving about 100 times higher sensitivity for detecting pTyr signaling proteins than that afforded by traditional immunohistochemical methods. Among more than 1,000 identified pTyr proteins, receptor tyrosine kinase PDGFRB expressed on cancer-associated fibroblasts was validated as an important intercellular signaling regulator with poor expression correlation to ERBB2, and blockade of PDGFRB signaling could efficiently suppress tumor growth. The Photo-pTyr-scaffold approach may become a generic tool for readily profiling dynamic pTyr signaling complexes in clinically relevant samples.