Department of Biology, Tor Vergata University of Rome, 00133 Rome, Italy.
Computational Biology Laboratory, Center of Excellence in Autophagy, Recycling and Disease (CARD), Danish Cancer Society Research Center, 2100 Copenhagen, Denmark.
Biochem Pharmacol. 2020 Jun;176:113869. doi: 10.1016/j.bcp.2020.113869. Epub 2020 Feb 21.
The mitochondrial chaperone TRAP1 has been involved in several mitochondrial functions, and modulation of its expression/activity has been suggested to play a role in the metabolic reprogramming distinctive of cancer cells. TRAP1 posttranslational modifications, i.e. phosphorylation, can modify its capability to bind to different client proteins and modulate its oncogenic activity. Recently, it has been also demonstrated that TRAP1 is S-nitrosylated at Cys501, a redox modification associated with its degradation via the proteasome. Here we report molecular dynamics simulations of TRAP1, together with analysis of long-range structural communication, providing a model according to which Cys501 S-nitrosylation induces conformational changes to distal sites in the structure of the protein. The modification is also predicted to alter open and closing motions for the chaperone function. By means of colorimetric assays and site directed mutagenesis aimed at generating C501S variant, we also experimentally confirmed that selective S-nitrosylation of Cys501 decreases ATPase activity of recombinant TRAP1. Coherently, C501S mutant was more active and conferred protection to cell death induced by staurosporine. Overall, our results provide the first in silico, in vitro and cellular evidence of the relevance of Cys501 S-nitrosylation in TRAP1 biology.
线粒体伴侣蛋白 TRAP1 参与了多种线粒体功能，其表达/活性的调节被认为在癌细胞特有的代谢重编程中发挥作用。TRAP1 的翻译后修饰，如磷酸化，可以改变其与不同客户蛋白结合的能力，并调节其致癌活性。最近，还证明 TRAP1 在 Cys501 处发生 S-亚硝基化，这是一种与通过蛋白酶体降解相关的氧化还原修饰。在这里，我们报告了 TRAP1 的分子动力学模拟，以及对远程结构通信的分析，提供了一个模型，根据该模型，Cys501 S-亚硝基化诱导蛋白质结构中远端位点的构象变化。该修饰还预计会改变伴侣功能的开放和关闭运动。通过比色测定法和旨在生成 C501S 变体的定点诱变，我们还通过实验证实，Cys501 的选择性 S-亚硝基化降低了重组 TRAP1 的 ATP 酶活性。一致地，C501S 突变体更活跃，并赋予了对 staurosporine 诱导的细胞死亡的保护作用。总的来说，我们的结果提供了 TRAP1 生物学中 Cys501 S-亚硝基化的首个计算、体外和细胞证据。
