Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Yonsei-ro 50, Seodaemun-gu, Seoul, 120-749, Korea.
J Neuroinflammation. 2012 Dec 17;9:271. doi: 10.1186/1742-2094-9-271.
Parkinson disease (PD) is characterized by a slow, progressive degeneration of dopaminergic neurons in the substantianigra. The cause of neuronal loss in PD is not well understood, but several genetic loci, including PTEN-induced putative kinase 1 (PINK1), have been linked to early-onset autosomal recessive forms of familial PD. Neuroinflammation greatly contributes to PD neuronal degeneration and pathogenesis. IL-1 is one of the principal cytokines that regulates various immune and inflammatory responses via the activation of the transcription factors NF-κB and activating protein-1. Despite the close relationship between PD and neuroinflammation, the functional roles of PD-linked genes during inflammatory processes remain poorly understood.
To explore the functional roles of PINK1 in response to IL-1β stimulation, HEK293 cells, mouse embryonic fibroblasts derived from PINK1-null (PINK1-/-) and control (PINK1+/+) mice, and 293 IL-1RI cells stably expressing type 1 IL-1 receptor were used. Immunoprecipitation and western blot analysis were performed to detect protein-protein interaction and protein ubiquitination. To confirm the effect of PINK1 on NF-κB activation, NF-κB-dependent firefly luciferase reporter assay was conducted.
PINK1 specifically binds two components of the IL-1-mediated signaling cascade, Toll-interacting protein (Tollip) and IL-1 receptor-associated kinase 1 (IRAK1). The association of PINK1 with Tollip, a negative regulator of IL-1β signaling, increases upon IL-1β stimulation, which then facilitates the dissociation of Tollip from IRAK1 as well as the assembly of the IRAK1-TNF receptor-associated factor 6 (TRAF6) complex. PINK1 also enhances Lys63-linked polyubiquitination of IRAK1, an essential modification of recruitment of NF-κB essential modulator and subsequent IκB kinase activation, and increases formation of the intermediate signalosome including IRAK1, TRAF6, and transforming growth factor-β activated kinase 1. Furthermore, PINK1 stimulates IL-1β-induced NF-κB activity via suppression of Tollip inhibitory action.
These results suggest that PINK1 upregulates IL-1β-mediated signaling through the functional modulation of Tollip and IRAK1. These results further suggest that PINK1 stimulates the ubiquitination of proximal molecules and increases signalosome formation in the IL-1β-mediated signaling pathway. The present study therefore supports the idea of the close relationship between neuroinflammation and PD.
帕金森病（PD）的特征是黑质中多巴胺能神经元的缓慢进行性退化。PD 神经元丢失的原因尚不清楚，但几个遗传位点，包括 PTEN 诱导的假定激酶 1（PINK1），与早发性常染色体隐性家族性 PD 有关。神经炎症对 PD 神经元变性和发病机制有很大贡献。IL-1 是通过激活转录因子 NF-κB 和激活蛋白-1 来调节各种免疫和炎症反应的主要细胞因子之一。尽管 PD 与神经炎症密切相关，但 PD 相关基因在炎症过程中的功能作用仍知之甚少。
为了探讨 PINK1 在 IL-1β刺激下的功能作用，使用了 HEK293 细胞、源自 PINK1 缺失（PINK1-/-）和对照（PINK1+/+）小鼠的小鼠胚胎成纤维细胞以及稳定表达 1 型 IL-1 受体的 293IL-1RI 细胞。进行免疫沉淀和 Western blot 分析以检测蛋白-蛋白相互作用和蛋白泛素化。为了确认 PINK1 对 NF-κB 激活的影响，进行了 NF-κB 依赖性萤火虫荧光素酶报告基因测定。
PINK1 特异性结合 IL-1 介导的信号级联反应的两个组成部分，即 Toll 相互作用蛋白（Tollip）和 IL-1 受体相关激酶 1（IRAK1）。PINK1 与 Tollip 的结合增加，Tollip 是 IL-1β 信号的负调节剂，这种结合增加了 Tollip 与 IRAK1 的解离，以及 IRAK1-TNF 受体相关因子 6（TRAF6）复合物的组装。PINK1 还增强了 IRAK1 的 Lys63 连接多泛素化，这是招募 NF-κB 必需调节剂和随后 IκB 激酶激活的必需修饰，并增加了包括 IRAK1、TRAF6 和转化生长因子-β激活激酶 1 在内的中间信号体的形成。此外，PINK1 通过抑制 Tollip 的抑制作用来刺激 IL-1β 诱导的 NF-κB 活性。
这些结果表明 PINK1 通过 Tollip 和 IRAK1 的功能调节来上调 IL-1β 介导的信号。这些结果进一步表明，PINK1 刺激 IL-1β 介导的信号通路中近端分子的泛素化并增加信号体的形成。因此，本研究支持神经炎症与 PD 密切相关的观点。
