MUTATIONAL ANALYSIS OF THE PARKIN GENE AND FUNCTIONAL ANALYSIS OF THE PARKIN PROTEIN

我们调查了500多个帕金森病家族中parkin基因的突变，该基因是我们小组克隆的。我们在大约50%的常染色体隐性遗传家系中发现了帕金突变。此外，在parkin阴性家族中，我们发现8个家族具有PINK 1突变（PARK 6），10个家族具有LRRK 2突变（PARK 8），和2个家族具有α-突触核蛋白基因的重复（PARK 1）。我们没有发现DJ-1突变（PARK 7）。关于Parkin蛋白的功能，我们发现14-3-3蛋白是Parkin的调节蛋白，因为14-3-3附着在Parkin的Linger区域上，并且作为泛素系统的E3负调节Parkin的酶活性。在氧化应激时，α-突触核蛋白上调并附着于14-3-3;然后这两种蛋白质的缀合物从帕金分离，释放帕金的活性作为E3。我们进一步揭示了parkin基因敲除的SH-SY 5 Y细胞在多巴胺合成过程中表现出多巴胺醌的增加。 关于我们 导致氧化应激。这两个观察结果彼此一致，表明PARK 2中黑质神经元选择性损伤的可能机制，即未暴露于氧化应激的神经元不需要太多的Parkin活性。为什么PARK 2患者的黑质神经元中没有积累Parkin相互作用蛋白，这一直是一个问题。作为这个问题的答案，我们发现了一个证据，表明赖氨酸-63介导的多聚泛素化帕金。我们发现LMO 4是一种转录调控蛋白，它经历了Lys-63介导的多聚泛素化，但没有传递被26 S蛋白酶体降解的信号。Lys-63介导的多泛素化蛋白具有不同的生物学作用，例如DNA修复和内吞作用等。这一发现为研究PARK 2的发病机制开辟了新的途径。此外，我们还成功地用AAV载体转染α-synuclein基因，建立了偏侧帕金森病大鼠模型。parkin的共转染部分防止了该模型中黑质神经元的损失。这种方法可以扩展到帕金森病患者的基因治疗。少

We investigated mutations in the parkin gene, which had been cloned by our group, in more than 500 families with Parkinsons's disease. We found parkin mutations in approximately 50% of autosomal recessive families. In addition, among parkin-negative families, we found 8 families with PINK] mutations (PARK6), 10 families with LRRK2 mutations (PARK8), and 2 families with duplication of the alpha-synuclein gene (PARK 1). We did not find DJ-1 mutations (PARK7). Regarding the function of Parkin protein, we found that 14-3-3 protein was a regulatory protein for Parkin, in that 14-3-3 was attaching the linger region of Parkin and negatively regulating the enzymatic activity of Parkin as E3 of the ubiquitin system. Upon oxidative stress, alpha-synuclein is upregulated and attaches to 14-3-3; then the conjugate of these two proteins detaches from Parkin releasing the activity of Parkin as E3. We further revealed that parkin knockout SH-SY5Y cells showed increase in dopamine quinone formation in … More ducing oxidative stress. These two observations are consistent with each other indicating a possible mechanism for selective damage of nigral neurons in PARK2, in that neurons not exposed to oxidative stress do not require much of Parkin activity. It has been a question why Parkin-interacting proteins were not accumulated in nigral neurons of PARK2 patients. As an answer for this question, we found an evidence to indicate lysine-63 mediated polyubiquitilation by Parkin. We found that LMO4, a transcription regulatory protein, underwent Lys-63 mediated polyubiqutilationm which did not confer a signal for degradation by 26S proteasome. Lys-63 mediated polyubiquitylated proteins have different biological roles such as DNA repair and endocytosis among others. This observation opens new approaches for the investigation of the pathogenesis of PARK2. Furthermore, we succeeded in the production of hemi-parkinsonism rat model by transfecting alpha-synuclein gene using AAV vector. Co-transfection of parkin partially prevented the loss of nigral neurons in this model. This approach can be extended to gene therapy of human patients with Parkinson's disease. Less