线粒体F1Fo-ATP酶（ATP合酶）的内源性抑制剂IF1已被证明具有促癌作用，包括细胞能量代谢重编程（伍伯格效应）。IF1的后一作用据报道受其PKA依赖的磷酸化限制，但能量代谢重编程和PKA依赖性磷酸化两者均存在争议。为了阐明这些关键问题，我们制备了稳定的IF1沉默克隆，并将其生物能学与三个亲本IF1表达癌细胞系进行了比较。呼吸速率、ATP合成速率（氧化磷酸化）和线粒体膜电位的所有功能参数在IF1沉默和对照组细胞中都是相似的，明确表明当酶在生理情况下工作时，IF1不能抑制癌细胞中的ATP合酶。此外，不论有无IF1的存在，所有细胞类型在经入PKA调节剂和以NAD依赖底物或琥珀酸酯为能源的条件下都显示类似的氧化磷酸化速率。因此，我们的结果排除了IF1的作用受PKA依赖性磷酸化/去磷酸化状态调节的可能性。值得注意的是，暴露于负PKA调节剂并用NAD依赖底物为能源的细胞显示出明显的氧化磷酸化速率下降，这与已报道的复合物I失活相吻合。总的来说，本研究明确证明了IF1在正氧窒息癌细胞中既不抑制线粒体ATP酶也不抑制氧化磷酸化，也不参与伍伯格效应。因此，目前IF1保护癌细胞免受严重缺氧/缺氧和凋亡的作用仍然是唯一无争议的促癌因素，可以利用这一因素开发治疗方法。版权所有©2023 Elsevier B.V.发布。

The endogenous inhibitor of mitochondrial F1Fo-ATPase (ATP synthase), IF1, has been shown to exert pro-oncogenic actions, including reprogramming of cellular energy metabolism (Warburg effect). The latter action of IF1 has been reported to be hampered by its PKA-dependent phosphorylation, but both reprogramming of metabolism and PKA-dependent phosphorylation are intensely debated. To clarify these critical issues, we prepared stably IF1-silenced clones and compared their bioenergetics with that of the three parental IF1-expressing cancer cell lines. All functional parameters: respiration rate, ATP synthesis rate (OXPHOS), and mitochondrial membrane potential were similar in IF1-silenced and control cells, clearly indicating that IF1 cannot inhibit the ATP synthase in cancer cells when the enzyme works physiologically. Furthermore, all cell types exposed to PKA modulators and energized with NAD+-dependent substrates or succinate showed similar OXPHOS rate regardless of the presence or absence of IF1. Therefore, our results rule out that IF1 action is modulated by its PKA-dependent phosphorylated/dephosphorylated state. Notably, cells exposed to a negative PKA modulator and energized with NAD+-dependent substrates showed a significant decrease of the OXPHOS rate matching previously reported inactivation of complex I. Overall, this study definitively demonstrates that IF1 inhibits neither mitochondrial ATP synthase nor OXPHOS in normoxic cancer cells and does not contribute to the Warburg effect. Thus, currently the protection of cancer cells from severe hypoxia/anoxia and apoptosis remain the only unquestionable actions of IF1 as pro-oncogenic factors that may be exploited to develop therapeutic approaches.Copyright © 2023. Published by Elsevier B.V.