The ubiquitin E3 ligase parkin regulates the proapoptotic function of Bax
The ubiquitin E3 ligase parkin regulates the proapoptotic function of Bax
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DOI：
10.1073/pnas.1113248109
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发表时间：
2012-04-17
影响因子：
11.1
通讯作者：
LaVoie, Matthew J.
中科院分区：
文献类型：
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作者：
Johnson, Bethann N.;Berger, Alison K.;LaVoie, Matthew J.
PARK2基因内的常染色体隐性功能缺失突变使E3泛素连接酶帕金（parkin）功能失活，导致儿茶酚胺能神经元的神经变性以及一种家族性帕金森病。目前的证据表明帕金具有线粒体功能以及神经保护作用，而这两者实际上可能相互关联。帕金的抗凋亡作用已被广泛报道，可能涉及细胞凋亡细胞色素c释放阈值的根本变化，但参与帕金依赖性保护的底物尚未确定。在此，我们通过比较野生型（WT）和帕金基因敲除（KO）小鼠的原代培养神经元，并使用多个帕金过表达细胞培养系统，证明了内源性Bax的帕金依赖性泛素化。在与重组帕金一起孵育后，在体外也观察到纯化的Bax的直接泛素化。我们发现帕金阻止了基础状态和凋亡应激诱导的Bax向线粒体的转位。此外，一种经过改造的抗泛素化形式的Bax保留了其凋亡功能，但用赖氨酸突变型Bax补充的Bax基因敲除细胞没有表现出在表达野生型Bax的细胞中观察到的帕金的抗凋亡作用。这些数据表明Bax是负责帕金抗凋亡作用的主要底物，并为帕金的至少一部分线粒体作用提供了机制上的见解。
Autosomal recessive loss-of-function mutations within the PARK2 gene functionally inactivate the E3 ubiquitin ligase parkin, resulting in neurodegeneration of catecholaminergic neurons and a familial form of Parkinson disease. Current evidence suggests both a mitochondrial function for parkin and a neuroprotective role, which may in fact be interrelated. The antiapoptotic effects of parkin have been widely reported, and may involve fundamental changes in the threshold for apoptotic cytochrome c release, but the substrate(s) involved in parkin dependent protection had not been identified. Here, we demonstrate the parkin-dependent ubiquitination of endogenous Bax comparing primary cultured neurons from WT and parkin KO mice and using multiple parkin-overexpressing cell culture systems. The direct ubiquitination of purified Bax was also observed in vitro following incubation with recombinant parkin. We found that parkin prevented basal and apoptotic stress-induced translocation of Bax to the mitochondria. Moreover, an engineered ubiquitination-resistant form of Bax retained its apoptotic function, but Bax KO cells complemented with lysine-mutant Bax did not manifest the antiapoptotic effects of parkin that were observed in cells expressing WT Bax. These data suggest that Bax is the primary substrate responsible for the antiapoptotic effects of parkin, and provide mechanistic insight into at least a subset of the mitochondrial effects of parkin.