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BACKGROUND

Tumor necrosis factor (TNF) signaling protects against ischemia/reperfusion-induced cardiomyocyte death, in vitro, ex vivo, and in vivo. TNF-receptor-associated factor 2 (TRAF2), an E3 ubiquitin ligase, coordinates cytoprotective signaling downstream of both TNF receptors, via unclear mechanisms. Noting that TRAF2 is recruited to mitochondria, and that autophagic removal of ubiquitin-tagged damaged mitochondria is cytoprotective, we tested the hypothesis that TRAF2 mediates mitochondrial autophagy.

METHODS AND RESULTS

TRAF2 localizes to the mitochondria in neonatal rat cardiac myocytes, and TNF treatment transcriptionally upregulates TRAF2 abundance in the mitochondrial subfraction. TRAF2 colocalizes with ubiquitin, p62 adaptor protein, and mitochondria within LC3-bound autophagosomes; and exogenous TRAF2 enhances autophagic removal of mitochondria. TRAF2 knockdown with adenoviral shRNA transduction induces accumulation of depolarized mitochondria in resting neonatal rat cardiac myocytes, as well as in those treated with TNF or uncoupling agent carbonyl cyanide m-chlorophenyl hydrazone, suggesting an essential role for TRAF2 in homeostatic and stress-induced mitochondrial autophagy. TRAF2 also colocalizes and interacts with PARKIN, a previously described E3 ubiquitin ligase and mitophagy effector, on depolarized mitochondria in neonatal rat cardiac myocytes. Exogenous expression of TRAF2, but not its E3 ligase-deficient mutants, is sufficient to partially restore mitophagy in the setting of PARKIN knockdown, suggesting redundancy in their ubiquitin ligase roles. TRAF2 abundance increases in the mitochondrial subfraction of ischemia/reperfusion-modeled hearts; and exogenous TRAF2, but not its E3 ligase-deficient mutants, reduces depolarized mitochondria and rescues cell death in neonatal rat cardiac myocytes subjected to hypoxia/reoxygenation.

CONCLUSIONS

Taken together, these data indicate an essential role for TRAF2 in concert with PARKIN as a mitophagy effector, which contributes to TRAF2-induced cytoprotective signaling.

BACKGROUND

METHODS AND RESULTS

CONCLUSIONS

Taken together, these data indicate an essential role for TRAF2 in concert with PARKIN as a mitophagy effector, which contributes to TRAF2-induced cytoprotective signaling.

背景

肿瘤坏死因子（TNF）信号转导可防止缺血/再灌注诱导的心肌细胞死亡，在体外、离体和体内均如此。TNF 受体相关因子 2（TRAF2）是一种 E3 泛素连接酶，通过尚不清楚的机制协调两种 TNF 受体下游的细胞保护信号。注意到 TRAF2 被募集到线粒体，并且泛素标记的受损线粒体的自噬去除具有细胞保护作用，我们检验了 TRAF2 介导线粒体自噬的假说。

方法和结果

TRAF2 在新生大鼠心肌细胞中线粒体定位，TNF 处理可使线粒体亚部分中的 TRAF2 丰度转录上调。TRAF2 与泛素、p62 衔接蛋白和 LC3 结合的自噬体中的线粒体共定位；外源性 TRAF2 增强线粒体的自噬清除。用腺病毒 shRNA 转导进行 TRAF2 敲低会导致静息的新生大鼠心肌细胞以及用 TNF 或解偶联剂羰基氰化物 m-氯代苯腙处理的细胞中线粒体去极化的积累，表明 TRAF2 在稳态和应激诱导的线粒体自噬中起关键作用。TRAF2 还与 PARKIN 共定位并相互作用，PARKIN 是先前描述的 E3 泛素连接酶和线粒体自噬效应物，在新生大鼠心肌细胞中线粒体去极化时也是如此。外源性表达 TRAF2（但不是其 E3 连接酶缺陷突变体）足以部分恢复 PARKIN 敲低时的线粒体自噬，表明它们在泛素连接酶作用中存在冗余。在缺血/再灌注模型心脏的线粒体亚部分中，TRAF2 丰度增加；并且外源性 TRAF2（但不是其 E3 连接酶缺陷突变体）可减少去极化的线粒体并挽救缺氧/复氧后新生大鼠心肌细胞的死亡。