Department of Chemistry , University of California Riverside , Riverside , California 92521-0403 , United States.
Anal Chem. 2018 Oct 16;90(20):11751-11755. doi: 10.1021/acs.analchem.8b03142. Epub 2018 Sep 26.
The 90-kDa heat shock protein (HSP90) is a molecular chaperone that maintains the proper folding of its client proteins including protein kinases and steroid hormone receptors. Helicases are a group of nucleic acid-binding ATPases that can unwind DNA and/or RNA and function in almost every aspect of nucleic acid metabolism. Not much, however, is known about the interactions between HSP90 and helicase proteins. Herein, we developed a parallel-reaction monitoring (PRM)-based targeted proteomic method that allows for quantifying >80% of the human helicase proteome. By employing this method, we demonstrated that a large number of helicase proteins exhibited diminished expression in cultured human cells upon treatment with two small-molecule inhibitors of HSP90. We further introduced a tandem affinity tag to the C-terminus of endogenous HSP90β protein by using the CRISPR-Cas9 genome editing method. Affinity purification followed by LC-PRM analysis revealed an enrichment of 40 out of the 66 quantified helicases from the lysate of cells expressing tagged HSP90β. Together, we developed a high-throughput targeted proteomic method for assessing quantitatively the human helicase proteome, and our results support that helicases may constitute an important group of client proteins for HSP90.
90kDa 热休克蛋白 (HSP90) 是一种分子伴侣，它可以维持其客户蛋白（包括蛋白激酶和甾体激素受体）的正确折叠。解旋酶是一组核酸结合 ATP 酶，能够解开 DNA 和/或 RNA，并在核酸代谢的几乎所有方面发挥作用。然而，关于 HSP90 和解旋酶蛋白之间的相互作用，人们知之甚少。在此，我们开发了一种基于平行反应监测（PRM）的靶向蛋白质组学方法，该方法能够定量检测 >80%的人类解旋酶蛋白质组。通过使用该方法，我们证明在培养的人类细胞中，用两种 HSP90 的小分子抑制剂处理后，大量的解旋酶蛋白表达减少。我们进一步通过 CRISPR-Cas9 基因组编辑方法在 HSP90β 内源性蛋白的 C 末端引入串联亲和标签。亲和纯化后进行 LC-PRM 分析显示，在表达标记 HSP90β 的细胞裂解物中，有 40 种定量检测到的解旋酶得到了富集。总之，我们开发了一种高通量靶向蛋白质组学方法来定量评估人类解旋酶蛋白质组，我们的结果支持解旋酶可能是 HSP90 的一个重要客户蛋白组。
