Liu Chia-Chi, Kim Yeon Jae, Teh Rachel, Garcia Alvaro, Hamilton Elisha J, Cornelius Flemming, Baxter Robert C, Rasmussen Helge H
North Shore Heart Research Group, Kolling Medical Research Institute, University of Sydney, St Leonards, NSW, Australia.
School of Chemistry, University of Sydney, Camperdown, NSW, Australia.
Front Oncol. 2022 Mar 17;12:859216. doi: 10.3389/fonc.2022.859216. eCollection 2022.
The seven mammalian FXYD proteins associate closely with α/β heterodimers of Na/K-ATPase. Most of them protect the β1 subunit against glutathionylation, an oxidative modification that destabilizes the heterodimer and inhibits Na/K-ATPase activity. A specific cysteine (Cys) residue of FXYD proteins is critical for such protection. One of the FXYD proteins, FXYD3, confers treatment resistance when overexpressed in cancer cells. We developed two FXYD3 peptide derivatives. FXYD3-pep CKCK retained the Cys residue that can undergo glutathionylation and that is critical for protecting the β1 subunit against glutathionylation. FXYD3-pep SKSK had all Cys residues mutated to Serine (Ser). The chemotherapeutic doxorubicin induces oxidative stress, and suppression of FXYD3 with siRNA in pancreatic- and breast cancer cells that strongly express FXYD3 increased doxorubicin-induced cytotoxicity. Exposing cells to FXYD3-pep SKSK decreased co-immunoprecipitation of FXYD3 with the α1 Na/K-ATPase subunit. FXYD3-pep SKSK reproduced the increase in doxorubicin-induced cytotoxicity seen after FXYD3 siRNA transfection in pancreatic- and breast cancer cells that overexpressed FXYD3, while FXYD3-pep CKCK boosted the native protein's protection against doxorubicin. Neither peptide affected doxorubicin's cytotoxicity on cells with no or low FXYD3 expression. Fluorescently labeled FXYD3-pep SKSK was detected in a perinuclear distribution in the cells overexpressing FXYD3, and plasmalemmal Na/K-ATPase turnover could not be implicated in the increased sensitivity to doxorubicin that FXYD3-pep SKSK caused. FXYD peptide derivatives allow rapid elimination or amplification of native FXYD protein function. Here, their effects implicate the Cys residue that is critical for countering β1 subunit glutathionylation in the augmentation of cytotoxicity with siRNA-induced downregulation of FXYD3.
七种哺乳动物FXYD蛋白与钠钾ATP酶的α/β异二聚体紧密结合。它们中的大多数能保护β1亚基免受谷胱甘肽化修饰，这种氧化修饰会使异二聚体不稳定并抑制钠钾ATP酶的活性。FXYD蛋白的一个特定半胱氨酸（Cys）残基对于这种保护作用至关重要。FXYD蛋白之一FXYD3在癌细胞中过表达时会赋予治疗抗性。我们开发了两种FXYD3肽衍生物。FXYD3 - pep CKCK保留了可发生谷胱甘肽化修饰且对保护β1亚基免受谷胱甘肽化修饰至关重要的半胱氨酸残基。FXYD3 - pep SKSK的所有半胱氨酸残基都突变为丝氨酸（Ser）。化疗药物阿霉素会诱导氧化应激，在高表达FXYD3的胰腺癌细胞和乳腺癌细胞中，用小干扰RNA（siRNA）抑制FXYD3可增加阿霉素诱导的细胞毒性。将细胞暴露于FXYD3 - pep SKSK会降低FXYD3与α1钠钾ATP酶亚基的共免疫沉淀。FXYD3 - pep SKSK重现了在过表达FXYD3的胰腺癌细胞和乳腺癌细胞中转染FXYD3 siRNA后阿霉素诱导的细胞毒性增加的现象，而FXYD3 - pep CKCK增强了天然蛋白对阿霉素的保护作用。这两种肽对FXYD3无表达或低表达的细胞的阿霉素细胞毒性均无影响。在过表达FXYD3的细胞中，荧光标记的FXYD3 - pep SKSK呈核周分布，且FXYD3 - pep SKSK导致的对阿霉素敏感性增加与质膜钠钾ATP酶的周转无关。FXYD肽衍生物能够快速消除或增强天然FXYD蛋白的功能。在此，它们的作用表明，在siRNA诱导FXYD3下调从而增强细胞毒性的过程中，对抵抗β1亚基谷胱甘肽化修饰至关重要的半胱氨酸残基发挥了作用。
