Department of Biological Sciences, Hunter College and Graduate Center, City University of New York, NY 10065, USA.
Department of Biological Sciences, Hunter College and Graduate Center, City University of New York, NY 10065, USA.
Biochim Biophys Acta Mol Basis Dis. 2019 Jun 1;1865(6):1436-1450. doi: 10.1016/j.bbadis.2019.02.016. Epub 2019 Feb 21.
Mitochondrial impairment and calcium (Ca) dyshomeostasis are associated with Parkinson's disease (PD). When intracellular ATP levels are lowered, Ca-ATPase pumps are impaired causing cytoplasmic Ca to be elevated and calpain activation. Little is known about the effect of calpain activation on Parkin integrity. To address this gap, we examined the effects of mitochondrial inhibitors [oligomycin (Oligo), antimycin and rotenone] on endogenous Parkin integrity in rat midbrain and cerebral cortical cultures. All drugs induced calpain-cleavage of Parkin to ~36.9/43.6 kDa fragments. In contrast, treatment with the proinflammatory prostaglandin J2 (PGJ2) and the proteasome inhibitor epoxomicin induced caspase-cleavage of Parkin to fragments of a different size, previously shown by others to be triggered by apoptosis. Calpain-cleaved Parkin was enriched in neuronal mitochondrial fractions. Pre-treatment with the phosphatase inhibitor okadaic acid prior to Oligo-treatment, stabilized full-length Parkin phosphorylated at Ser, and reduced calpain-cleavage of Parkin. Treatment with the Ca ionophore A23187, which facilitates Ca transport across the plasma membrane, mimicked the effect of Oligo by inducing calpain-cleavage of Parkin. Removing extracellular Ca from the media prevented oligomycin- and ionophore-induced calpain-cleavage of Parkin. Computational analysis predicted that calpain-cleavage of Parkin liberates its UbL domain. The phosphagen cyclocreatine moderately mitigated Parkin cleavage by calpain. Moreover, the pituitary adenylate cyclase activating peptide (PACAP27), which stimulates cAMP production, prevented caspase but not calpain-cleavage of Parkin. Overall, our data support a link between Parkin phosphorylation and its cleavage by calpain. This mechanism reflects the impact of mitochondrial impairment and Ca-dyshomeostasis on Parkin integrity and could influence PD pathogenesis.
线粒体功能障碍和钙（Ca）稳态失调与帕金森病（PD）有关。当细胞内 ATP 水平降低时，Ca-ATP 酶泵受损，导致细胞质 Ca 升高和钙蛋白酶激活。关于钙蛋白酶激活对 Parkin 完整性的影响知之甚少。为了解决这一差距，我们研究了线粒体抑制剂[寡霉素（Oligo）、抗霉素和鱼藤酮]对大鼠中脑和大脑皮质培养物中内源性 Parkin 完整性的影响。所有药物均诱导钙蛋白酶将 Parkin 切割为~36.9/43.6 kDa 片段。相比之下，用促炎前列腺素 J2（PGJ2）和蛋白酶体抑制剂环氧霉素处理诱导 Parkin 发生 caspase 切割，产生不同大小的片段，之前的研究表明这是由细胞凋亡引发的。钙蛋白酶切割的 Parkin 富集在神经元线粒体部分。寡霉素处理前用磷酸酶抑制剂 okadaic acid 预处理可稳定全长 Parkin 磷酸化 Ser 并减少钙蛋白酶对 Parkin 的切割。用 Ca 离子载体 A23187 处理，促进质膜 Ca 转运，可模拟寡霉素的作用，诱导钙蛋白酶切割 Parkin。从培养基中去除细胞外 Ca 可防止寡霉素和离子载体诱导的钙蛋白酶切割 Parkin。计算分析预测钙蛋白酶切割 Parkin 可释放其 UbL 结构域。磷酸肌酸磷酸化酶可适度减轻钙蛋白酶对 Parkin 的切割。此外，刺激 cAMP 产生的脑垂体腺苷酸环化酶激活肽（PACAP27）可防止 caspase 但不能防止钙蛋白酶切割 Parkin。总的来说，我们的数据支持 Parkin 磷酸化与其被钙蛋白酶切割之间的联系。这种机制反映了线粒体功能障碍和 Ca 稳态失调对 Parkin 完整性的影响，并可能影响 PD 的发病机制。
