TFEB的SUMO化修饰障碍导致衰老心肌自噬功能缺陷

The expression of autophagy related gene is decreased in aged cardiomyocyte. Age-associated reduction in autophagy flux involves autophagosome, lysosome and autolysosome fusion impairment. The newly discovered transcription factor EB (TFEB) is implicated in lysosomal biogenesis as well as autophagy induction. However, the influence of post-translational modification of TFEB on its transcriptional activity and myocardial autophagy remain to be elucidated. Through the exploratory experiment, we find that ① cardiac nuclear TFEB is SUMOylated;② reduction of SUMOylation decreases stability and transcriptional activity of TFEB;③the nuclear sumoylated TFEB was significantly reduced in aged hearts. We hypothesize that SUMOylation is important for the autophagic transcriptional regulation of TFEB in the myocardial nucleus. Impaired SUMOylation of TFEB plays a critical (mTOR-independent) role in the aging-related autophagic decline. The present study will focus on the mechanism of TFEB SUMOylation deficiency in aged heart. How dose SUMOylation influences the stability of TFEB in aged heart? Targeting TFEB SUMOylation may provide a novel therapeutic strategy for the treatment of aging-related autophagic decline. The present study will provide new evidence for basic strategy research in age-related cardiovascular disease.

衰老心肌自噬体、溶酶体和自噬溶酶体融合等多环节自噬流相关基因表达减退。而新近发现的转录因子EB（TFEB）对自噬体和溶酶体途径均具有重要的调控作用。但是，TFEB自身所发生的翻译后修饰对其转录活性和心肌自噬的影响尚待阐明。我们通过探索性实验发现：①心肌细胞核内，TFEB可以被SUMO化修饰（SUMOylated TFEB）；②抑制SUMOylation可破坏心肌细胞核内TFEB的稳定性和转录活性。③衰老心肌细胞核内SUMO化的TFEB显著减退。据此提出科学假设：心肌细胞核内SUMO化修饰对于TFEB发挥自噬调控至关重要，TFEB的SUMO化障碍可能是衰老心肌自噬缺陷（mTOR非依赖性的）新机制。本项目拟从衰老心肌TFEB的SUMO化减退机制，SUMO化修饰对衰老心肌TFEB的稳定性的影响机制以及衰老心肌自噬流缺陷的干预新策略三个方面进行深入研究，为老年病防治的基础策略研究提供新证据。

转录因子EB（TFEB）对自噬体和溶酶体途径均具有重要的调控作用，本项目旨在探讨衰老心肌中TFEB翻译后修饰对其转录活性和衰老心肌自噬的影响和作用机制。.SUMOylation（小泛素相关修饰物，Small Ubiquitin-like Modifier, SUMO）作为一种重要的翻译后修饰方式对蛋白质的功能有广泛的调节作用，主要包括调节蛋白质的细胞定位，蛋白质之间的相互作用以及蛋白质的活性等方面。本研究在前期研究基础上，主要围绕TFEB-SUMO化修饰减退是否参与衰老心肌自噬功能障碍并探讨自噬功能减退对衰老心肌缺血再灌注损伤的影响，目前取得的研究结果证实：.1)与成年对照小鼠相比，老年小鼠MI/R损伤加重；与之对应，衰老小鼠MI/R损伤区心肌自噬功能障碍，自噬流受阻，自噬蛋白相关基因转录水平显著减退。.2)成年小鼠心肌遭受MI/R损伤后，TFEB出现核转位，进入心肌细胞核内。衰老MI/R心肌细胞核内TFEB水平较成年组相比显著降低。.3)心肌细胞核内，TFEB可被SUMO化修饰。衰老MI/R心肌核内TFEB-SUMO化较成年MI/R心肌显著减退，与TFEB总量减退趋势一致。检测SUMOylation的转移酶UBC9结果发现，衰老MI/R心肌中UBC9水平减退。.4)在培养的心肌细胞中敲低心肌SUMOylation水平可显著降低心肌细胞核内TFEB水平并降低心肌自噬功能。在体给予AAV9-shRNA-SUMO-1敲低心肌SUMOylation水平。发现抑制SUMOylation可导致心肌核内TFEB水平显著降低。采用MG132实验发现，SUMOylation可保持核内TFEB蛋白水平。.5) TFEB的347赖氨酸位点为 SUMOylation修饰位点。.6) 增强SUMOylation可有效维持衰老MI/R心肌核内TFEB水平，有效保护MI/R心肌功能性自噬流。.7) 发现衰老心肌CARM1-TFEB复合体水平下降导致自噬功能障碍。老年心肌CARM1的水平受AMPK-p-FoxO3a-Skp2信号调控。.8) 新发现衰老心肌自噬功能障碍可触发p62与RIP1及RIP3结合进而促进necroptosis。自噬功能障碍导致心肌程序性坏死增加是衰老心肌缺血易损的新机制。