Centre for Neuromuscular and Neurological Disorders, The University of Western Australia, Australian Neuro-Muscular Research Institute, Nedlands, Western Australia, Australia.
J Neurosci Res. 2014 Apr;92(4):506-16. doi: 10.1002/jnr.23331. Epub 2013 Dec 24.
Mutations in the leucine-rich repeat kinase 2 (lrrk2) gene are the leading genetic cause of Parkinson's disease (PD). In characterizing the novel ROC domain mutant A1442P, we compared its steady-state protein levels, propensity to aggregate, and toxicity with the pathogenic R1441C mutant and wild-type (WT) LRRK2. Mutant (R1441C and A1442P) and WT LRRK2 fused to green fluorescent protein (GFP) and FLAG were transiently expressed in HEK293 cells using plasmid constructs. Western analysis and fluorescence microscopy consistently demonstrated lower mutant LRRK2 protein levels compared with WT. A time-course expression study using flow cytometry showed that WT LRRK2 expression increased initially but then plateaued by 72 hr. Conversely, R1441C and A1442P mutant expression attained 85% and 74% of WT levels at 24 hr but fell to 68% and 55% of WT levels by 72 hr, respectively. We found that proteasome inhibition markedly increased mutant LRRK2 to levels approaching those of WT. Taken together, our findings reveal increased intracellular degradation for both mutants. Furthermore, the impact of mutant and WT LRRK2 expression on HEK293 cell viability was assessed under normative and oxidative (hydrogen peroxide) conditions and found not to differ. Expression of WT and mutant LRRK2 protein gave rise to intracellular aggregates of similar appearance and cellular localization. In summary, we provide evidence that the novel A1442P mutant and the previously investigated R1441C pathogenic mutant exhibit increased intracellular degradation, a property reportedly demonstrated for the pathogenic LRRK2 kinase domain mutant I2020T.
LRRK2 基因中的亮氨酸丰富重复激酶 2 (LRRK2) 突变是帕金森病 (PD) 的主要遗传原因。在表征新型 ROC 结构域突变 A1442P 时，我们比较了其稳定态蛋白水平、聚集倾向和毒性与致病性 R1441C 突变体和野生型 (WT) LRRK2。使用质粒构建体，将突变 (R1441C 和 A1442P) 和 WT LRRK2 与绿色荧光蛋白 (GFP) 和 FLAG 融合，瞬时表达在 HEK293 细胞中。Western 分析和荧光显微镜一致表明，与 WT 相比，突变 LRRK2 蛋白水平较低。使用流式细胞术进行的时间过程表达研究表明，WT LRRK2 表达最初增加，但在 72 小时时达到平台期。相反，R1441C 和 A1442P 突变体的表达在 24 小时时达到 WT 水平的 85%和 74%，但在 72 小时时分别降至 WT 水平的 68%和 55%。我们发现蛋白酶体抑制显著增加了突变 LRRK2 的水平，接近 WT 水平。综上所述，我们的研究结果揭示了两种突变体的细胞内降解增加。此外，还在正常和氧化 (过氧化氢) 条件下评估了突变和 WT LRRK2 表达对 HEK293 细胞活力的影响，发现没有差异。WT 和突变 LRRK2 蛋白的表达导致类似外观和细胞定位的细胞内聚集。总之，我们提供了证据表明新型 A1442P 突变体和先前研究的致病性 R1441C 突变体表现出增加的细胞内降解，据报道，致病性 LRRK2 激酶结构域突变 I2020T 具有这种特性。
