Department of Physiology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, TX 75390-9040, USA.
J Bioenerg Biomembr. 2010 Feb;42(1):85-95. doi: 10.1007/s10863-009-9258-y. Epub 2010 Feb 11.
A hallmark of Parkinson disease (PD) is the formation of intracellular protein inclusions called Lewy bodies that also contain mitochondria. alpha-Synuclein (alpha Syn) is a major protein component of Lewy bodies, where it is in an amyloid conformation and a significant fraction is truncated by poorly understood proteolytic events. Previously, we demonstrated that the 20S proteasome cleaves alpha Syn in vitro to produce fragments like those observed in Lewy bodies and that the fragments accelerate the formation of amyloid fibrils from full-length alpha Syn. Three point mutations in alpha Syn are associated with early-onset familial PD: A30P, E46K, and A53T. However, these mutations have very different effects on the amyloidogenicity and vesicle-binding activity of alpha Syn, suggesting neither of these processes directly correlate with neurodegeneration. Here, we evaluate the effect of the disease-associated mutations on the fragmentation, conformation, and association reactions of alpha Syn in the presence of the 20S proteasome and liposomes. The 20S proteasome produced the C-terminal fragments from both the mutant and wildtype alpha Syn. These truncations accelerated fibrillization of all alpha-synucleins, but again there was no clear correlation between the PD-associated mutations and amyloid formation in the presence of liposomes. Recent data suggests that cellular toxicity is caused by a soluble oligomeric species, which is a precursor to the amyloid form and is immunologically distinguishable from both soluble monomeric and amyloid forms of alpha Syn. Notably, the rate of formation of the soluble, presumptively cytotoxic oligomers correlated with the disease-associated mutations when both 20S proteasome and liposomes were present. Under these conditions, the wildtype protein was also cleaved and formed the oligomeric structures, albeit at a slower rate, suggesting that 20S-mediated truncation of alpha Syn may play a role in sporadic PD as well. Evaluation of the biochemical reactions of the PD-associated alpha-synuclein mutants in our in vitro system provides insight into the possible pathogenetic mechanism of both familial and sporadic PD.
帕金森病 (PD) 的一个特征是形成称为路易体的细胞内蛋白包涵体，其中也含有线粒体。α-突触核蛋白 (α Syn) 是路易体的主要蛋白成分，在其中处于淀粉样构象，并且相当一部分被未知的蛋白水解事件截断。以前，我们证明 20S 蛋白酶体在体外切割 α Syn 以产生类似于在路易体中观察到的片段，并且这些片段加速全长 α Syn 从纤维状淀粉样形成纤维。α Syn 中的三个点突变与早发性家族性 PD 相关：A30P、E46K 和 A53T。然而，这些突变对 α Syn 的淀粉样形成和囊泡结合活性有非常不同的影响，这表明这两个过程都与神经退行性变没有直接关系。在这里，我们评估疾病相关突变对 20S 蛋白酶体和脂质体存在下 α Syn 的碎片化、构象和结合反应的影响。20S 蛋白酶体从突变和野生型 α Syn 中均产生 C 端片段。这些截断加速了所有 α-突触核蛋白的纤维形成，但在存在脂质体的情况下，PD 相关突变与淀粉样形成之间仍然没有明显的相关性。最近的数据表明，细胞毒性是由可溶性寡聚物引起的，该寡聚物是淀粉样形式的前体，并且与可溶性单体和 α Syn 的淀粉样形式在免疫学上可区分。值得注意的是，当 20S 蛋白酶体和脂质体都存在时，可溶性、假定的毒性寡聚物的形成速度与疾病相关突变相关。在这些条件下，野生型蛋白也被切割并形成寡聚物结构，尽管速度较慢，这表明 20S 介导的 α Syn 截断可能在散发性 PD 中也起作用。在我们的体外系统中评估与 PD 相关的 α-突触核蛋白突变体的生化反应为家族性和散发性 PD 的可能发病机制提供了深入了解。
