BACKGROUND Mitochondrial dysfunction is a key factor in the development of atherogenesis. METTL4 (methyltransferase-like protein 4) mediates N6- methyldeoxyadenosine (6mA) of mammalian mitochondrial DNA (mtDNA). However, the role of METTL4-mediated mitoepigenetic regulation in atherosclerosis is still unknown. This study aims to investigate the potential involvement of METTL4 in atherosclerosis, explore the underlying mechanism, and develop targeted strategies for treating atherosclerosis. METHODS Expression levels of mtDNA 6mA and METTL4 were determined in atherosclerotic lesions. We explored the mechanism of METTL4 involvement in atherosclerosis using Mettl4Mac-KO-Apoe-/- and Mettl4MUT-Apoe-/- mice and cell models, as well as bone marrow transplantation. Natural compound libraries were screened to identify potent METTL4 antagonists. In addition, bioinspired proteolysis targeting chimera technology targeting macrophages within plaques was used to increase the efficacy of the METTL4 antagonist. RESULTS The expression levels of mtDNA 6mA and METTL4 were significantly increased in plaque macrophages. Mettl4Mac-KO-Apoe-/- mice displayed suppressed mtDNA 6mA levels and atherosclerotic progression, which were reversed by METTL4 restoration through bone marrow transplantation (n=6). Mechanistically, elevated METTL4 expression reduces MT-ATP6 expression by suppressing its transcription, thereby impairing the activity of mitochondrial respiration chain complex V. This disruption leads to the accumulation of excess protons in the mitochondrial intermembrane space, causing mitochondrial dysfunction. Consequently, mtDNA is released into the cytoplasm, ultimately triggering inflammasome activation. All results were reversed by the mutation in the METTL4 methyltransferase active site. Mettl4MUT-Apoe-/- mice showed suppressed mtDNA 6mA levels and atherosclerotic progression and repaired mitochondrial function of macrophage, which were reversed by METTL4 restoration through bone marrow transplantation (n=6). Pemetrexed was identified as the first METTL4 antagonist to effectively alleviate atherosclerotic progression. Furthermore, we generated a proteolysis targeting chimera drug based on pemetrexed that specifically targeted METTL4 in macrophages within plaques, showing a promising therapeutic effect on atherosclerosis. CONCLUSIONS This study revealed a novel mechanism by which mtDNA 6mA orchestrated mitochondrial function-related gene expression in macrophages, thereby promoting atherosclerosis. Through various experimental techniques, such as gene manipulation, pharmacological inhibition, and proteolysis targeting chimera, this study demonstrated that mtDNA 6mA and its specific enzyme METTL4 hold potential as therapeutic targets for atherosclerosis.

中文翻译：

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METTL4 介导的线粒体 DNA N6-甲基脱氧腺苷促进巨噬细胞炎症和动脉粥样硬化。


背景 线粒体功能障碍是动脉粥样硬化发生的关键因素。METTL4 （甲基转移酶样蛋白 4） 介导哺乳动物线粒体 DNA （mtDNA） 的 N6-甲基脱氧腺苷 （6mA）。然而，METTL4 介导的有丝分裂表观遗传调控在动脉粥样硬化中的作用仍然未知。本研究旨在探讨 METTL4 在动脉粥样硬化中的潜在参与，探讨其潜在机制，并制定治疗动脉粥样硬化的靶向策略。方法 测定动脉粥样硬化病灶中 mtDNA 6mA 和 METTL4 的表达水平。我们使用 Mettl4Mac-KO-Apoe-/- 和 Mettl4MUT-Apoe-/- 小鼠和细胞模型以及骨髓移植探索了 METTL4 参与动脉粥样硬化的机制。筛选天然化合物库以鉴定有效的 METTL4 拮抗剂。此外，针对斑块内巨噬细胞的生物启发蛋白水解靶向嵌合体技术用于提高 METTL4 拮抗剂的疗效。结果 噬菌斑巨噬细胞中 mtDNA 6mA 和 METTL4 的表达水平显著升高。Mettl4Mac-KO-Apoe-/- 小鼠表现出 mtDNA 6mA 水平抑制和动脉粥样硬化进展，这些进展被骨髓移植的 METTL4 修复逆转 （n=6）。从机制上讲，METTL4 表达升高通过抑制 MT-ATP6 的转录来降低 MT-ATP6 的表达，从而损害线粒体呼吸链复合物 V 的活性。这种破坏导致过量质子在线粒体膜间空间中积累，导致线粒体功能障碍。因此，mtDNA 被释放到细胞质中，最终触发炎性小体激活。 所有结果均被 METTL4 甲基转移酶活性位点的突变逆转。Mettl4MUT-Apoe-/- 小鼠表现出 mtDNA 6mA 水平抑制和动脉粥样硬化进展，巨噬细胞线粒体功能修复，这些功能被骨髓移植的 METTL4 恢复逆转 （n=6）。培美曲塞被确定为第一个有效缓解动脉粥样硬化进展的 METTL4 拮抗剂。此外，我们生成了一种基于培美曲塞的蛋白水解靶向嵌合体药物，该药物特异性靶向斑块内巨噬细胞中的 METTL4，显示出对动脉粥样硬化的有希望的治疗效果。结论 本研究揭示了 mtDNA 6mA 协调巨噬细胞线粒体功能相关基因表达，从而促进动脉粥样硬化的新机制。通过基因操作、药理抑制和蛋白水解靶向嵌合体等各种实验技术，本研究证明 mtDNA 6mA 及其特异性酶 METTL4 具有作为动脉粥样硬化治疗靶点的潜力。