Defining a Molecular Link between Parkinson and Gaucher Diseases

GBA1基因突变是帕金森病(PD)最常见的已知危险因素。虽然临床研究证明了GBA1突变与帕金森病的发生之间存在联系的有力证据，但一直缺乏机械性的见解。GBA1编码葡萄糖脑苷酶(GCase)，是一种将葡萄糖神经酰胺(GluCer)分解为葡萄糖和神经酰胺的溶酶体酶，在高谢病(GD)中缺乏。最近的研究表明，GCase与帕金森病相关的淀粉样蛋白-α-突触核蛋白(α-SYN)之间存在关系；然而，导致这种联系的具体分子机制仍然不清楚。 在我们的工作中，我们重点研究了溶酶体中α-syn和GCase相互作用的结构-功能关系。我们评估了酶的活性，用中子反射法表征了膜结合蛋白复合体，并评价了GCase激活剂皂苷C对复合体形成和GCase活性的影响。在定义分子相互作用，驱动在体内的GCase和α-SYN水平之间的相互关系，我们已经转向研究如何在溶酶体中降解α-SYN。随着溶酶体移除易聚集的物种或过量水平的α-syn，发生在溶酶体内的分子相互作用，如与GCase的相互作用将是相关的。这种相互作用可以通过改变α-syn裂解位点的可用性来调节蛋白分解效率，并决定了蛋白酶的特异性和有效性。 我们正在测试关于溶酶体如何在健康和疾病相关的条件下促进α-syn蛋白稳定的假设。具体地说，我们正在研究α-syn和溶酶体酶、半胱氨酸组织蛋白和GCase之间的关系。我们的工作指出了半胱氨酸组织蛋白在溶酶体清除α-syn中的直接作用。我们感兴趣的是如何将这些酶作为帕金森病进展的干预策略。对于半胱氨酸组织蛋白酶，我们的数据尤其令人信服，组织蛋白酶L(CTSL)具有降解α-突触蛋白纤维的潜力。对于GCase来说，在基于细胞和动物的PD模型中，其水平或活性的增强似乎可以改善α-syn毒性；然而，这种情况发生的分子基础还不是很清楚。我们的研究正在努力解决这两个方面：评估CTSL是否可以作为一种可行的治疗药物来清除α-SYN的细胞，并阐明观察到的PD风险与GCase浓度之间的相关性的机制(S)。 为了了解CTSL是如何降解α-SYN纤维的，利用肽图和电子显微镜(EM)对在纯化的CTSL存在下α-SYN纤维的降解过程进行了详细的动力学研究。使用质谱仪，指示α-突触纤维的早期时间点首先在N-末端和C-末端被截断，产生如1-114、6-114、10-114、1-126、1-134等肽，这与先前的文献显示的C-末端和N-末端在纤维状态下是柔性和无序的一致。延长孵育时间会发现α-syn的淀粉样蛋白生成区域(残基30-100之间)有裂解位点。有趣的是，这些位置中的大多数都含有甘氨酸，已知甘氨酸参与原纤维中的β-转折，很可能更容易被蛋白酶访问。通过EM可视化，纤维沿纤维轴显示不完善，这与显示CTSL在淀粉样变核心内切割的肽图数据相吻合。其中一些图像揭示了原纤维内密度的缺失，表明蛋白酶正在侵蚀原纤维。这项研究的结果将是发展这种蛋白水解酶作为帕金森病治疗剂的关键。

Mutations in the GBA1 gene are the most common of the known risk factors for Parkinson disease (PD). While clinical studies argue a strong case towards a link between GBA1 mutations and the development of PD, mechanistic insights have been lacking. GBA1 encodes glucocerebrosidase (GCase), a lysosomal enzyme which hydrolyzes glucosylceramide (GluCer) into glucose and ceramide and is deficient in Gaucher disease (GD). Recent research suggests a relationship between GCase and the PD-related amyloid-forming protein, alpha-synuclein (alpha-syn); however, the specific molecular mechanisms responsible for association remain elusive. In our work, we focused on the structure-function relationship of alpha-syn and GCase interaction in the lysosome. We have evaluated enzymatic activity, characterized the membrane-bound protein complex by neutron reflectometry, and assessed the effect of Saposin C, an activator for GCase, on complex formation and GCase activity. In defining the molecular interactions that drive the reciprocal relationship between GCase and alpha-syn levels in vivo, we have turned to investigate how alpha-syn is degraded in the lysosome. As the lysosome removes aggregation-prone species or excess levels of alpha-syn, molecular interactions that occur within the lysosome such as with GCase would be pertinent. Such interactions could modulate proteolysis efficiency by altering availability of alpha-syn cleavage sites and dictate protease specificity and efficacy. We are testing hypotheses on how lysosomes contribute to alpha-syn proteostasis under healthy and disease-related conditions. Specifically, we are investigating the relationship between alpha-syn and lysosomal enzymes, cysteine cathepsins and GCase. Our work points to a direct role of cysteine cathepsins in the lysosomal clearance of alpha-syn. We are interested in how these enzymes could be targeted as an intervention strategy in PD progression. With cysteine cathepsins, our data are especially compelling for the potential for cathepsin L (CtsL) to degrade alpha-syn fibrils. For GCase, the enhancement of its levels or activity appears to ameliorate alpha-syn toxicity in cell-based and animal PD models; however, the molecular basis for why this occurs is not well understood. Our research efforts are addressing both these fronts: to evaluate whether CtsL could be a viable therapeutic agent towards cellular clearance of alpha-syn and to elucidate the mechanism(s) responsible for the observed correlation between risk for PD and GCase concentration. To understand how CtsL degrades alpha-syn fibrils, a detailed kinetics study has been carried out by tracking the degradation process of alpha-syn fibrils in the presence of purified CtsL using peptide mapping and electron microscopy (EM). Using mass spectrometry, early time points indicated alpha-syn fibrils are first truncated at the N- and C-termini, generating peptides such as 1-114, 6-114, 10-114, 1-126, 1-134, consistent with prior literature showing that the C- and N-termini are flexible and disordered in the fibrillar state. Prolonging the incubation reveals cleavage sites within the amyloidogenic region (between residues 30-100) of alpha-syn. Interestingly, the majority of these positions contains a glycine, which is known to be involved in beta-turns in a fibril and likely to be more accessible to the protease. By EM visualization, fibrils show imperfections along the fibril axis, which coincides with peptide mapping data showing CtsL cutting within the amyloidogenic core. Some of the images reveal missing density within the fibril, suggesting the protease is eating away at the fibril. Results from this study will be pivotal in developing this protease as a therapeutic agent for PD.