Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064,USA.
Department of Chemistry and Physics, Fayetteville State University, Fayetteville, NC 28301, USA.
Biochim Biophys Acta Gen Subj. 2017 Jul;1861(7):1759-1769. doi: 10.1016/j.bbagen.2017.03.013. Epub 2017 Mar 19.
DJ-1, a small ubiquitously expressed protein implicated in several pathways associated with Parkinson's disease pathogenesis, has been found to interact with α-synuclein and modulate its aggregation, yet the exact mechanisms remain unclear.
The stability and aggregation properties of wild-type DJ-1 under denaturing conditions, such as low pH, high temperature, presence of denaturants were investigated. The interaction between DJ-1 and α-synuclein was tested by SDS-PAGE gel and native gel electrophoresis and by size-exclusion HPLC. Fibrillization was monitored by thioflavin T fluorescence assays and amorphous aggregation was followed by light scattering measurements. The morphology of aggregated species was observed by transmission electron microscopy and atomic force microscopy. Protein secondary structures were characterized by far-UV circular dichroism.
DJ-1 fibrillization was first observed at low pH or by adding denaturants. Amorphous aggregates formed at neutral pH, and the aggregation was dramatically accelerated by elevated temperature and the presence of α-synuclein. Aggregation of DJ-1 were enhanced by heating and perturbed by the co-occurrence of α-synuclein but strong interactions between the two proteins were not found.
Varying environmental factors led to different aggregation pathways of DJ-1 although a simulated physiological condition would not lead to fibrillization. DJ-1 co-aggregating with α-synuclein may result from weak hydrophobic interaction and DJ-1 exhibited chaperon-like activity in the initial time of α-synuclein aggregation at high temperature.
This research on DJ-1 presented its aggregation behavior under denaturing conditions and interaction mechanism with α-synuclein that may help to decipher its potential neuroprotective or neurotoxic role in Parkinson's disease.
DJ-1 是一种小型普遍表达的蛋白质，与帕金森病发病机制相关的几种途径有关，已发现其与α-突触核蛋白相互作用并调节其聚集，但确切的机制仍不清楚。
研究了在变性条件下，如低 pH 值、高温、存在变性剂时，野生型 DJ-1 的稳定性和聚集特性。通过 SDS-PAGE 凝胶和天然凝胶电泳以及排阻 HPLC 测试 DJ-1 与α-突触核蛋白的相互作用。通过硫黄素 T 荧光测定法监测纤维形成，通过光散射测量法跟踪无定形聚集。通过透射电子显微镜和原子力显微镜观察聚集物的形态。通过远紫外圆二色性表征蛋白质二级结构。
首先在低 pH 值或添加变性剂时观察到 DJ-1 纤维形成。在中性 pH 值下形成无定形聚集体，并且升高温度和存在α-突触核蛋白显著加速了聚集。加热增强了 DJ-1 的聚集，并且α-突触核蛋白的共存扰乱了聚集，但未发现两种蛋白质之间存在强烈相互作用。
尽管模拟生理条件不会导致纤维形成，但不同的环境因素导致了 DJ-1 的不同聚集途径。与α-突触核蛋白共聚集的 DJ-1 可能来自弱疏水性相互作用，并且 DJ-1 在高温下α-突触核蛋白聚集的初始时间表现出分子伴侣样活性。
这项关于 DJ-1 的研究展示了其在变性条件下的聚集行为及其与α-突触核蛋白的相互作用机制，这可能有助于破译其在帕金森病中的潜在神经保护或神经毒性作用。
