Defining a Molecular Link between Parkinson and Gaucher Diseases

定义帕金森病和戈谢病之间的分子联系

• 批准号: 8746694
• 负责人: Jennifer Lee
• 金额: $ 39.77万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8746694
• 关键词: Adopted; Affect; Affinity; Amyloid; Binding; Binding Proteins; Biochemical; C-terminal; Cell model; Charge; Clinical Research; Complex; Data; Development; Disease; Dissociation; Electrostatics; Energy Transfer; Enzymes; Fluorescence; Gaucher Disease; Genes; Homeostasis; Inhibitory Concentration 50; Lewy Bodies; Link; Lipids; Maps; Membrane; Minority; Molecular; Molecular Conformation; Mutation; N-terminal; Parkinson Disease; Pathogenesis; Patients; Proteins; Research; Risk; Role; Site; Solutions; Surface; Tail; Time; Variant; Vesicle; Work; alpha synuclein; disorder risk; enzyme activity; enzyme deficiency; genetic risk factor; glucosylceramidase; inhibitor/antagonist; insight; interest; novel therapeutics; protein degradation; synucleinopathy; unilamellar vesicle

Mutations in the GBA1 gene, encoding the enzyme glucocerebrosidase (GCase), cause the lysosomal storage disorder, Gaucher disease (GD), and are associated with the development of Parkinsons disease (PD) and other Lewy body disorders. Interestingly, GBA1 variants are the most common genetic risk factor associated with PD. While clinical studies argue a strong case towards a link between GBA1 mutations and the development of PD, mechanistic insights have been lacking. Recent research suggests a relationship between GCase and the PD-related amyloid-forming protein, alpha-synuclein; however, the specific molecular mechanisms responsible for association remain elusive. While the effects of GCase on alpha-synuclein homeostasis are the subject of considerable work, a role for alpha-synuclein in enzyme function has not been established. Such information could have further implications and indicate other mechanisms responsible for the increased PD risk. In prior work, we showed that alpha-synuclein and GCase interact selectively under lysosomal conditions, and proposed that this newly identified interaction might influence cellular levels of alpha-synuclein by either promoting protein degradation and/or inhibiting aggregation. We now extend our study to consider how membranes would modulate this complex formation. Lipids function not only as the substrate for GCase in intralysosomal vesicles, but also a surface for alpha-synuclein conformation alteration. For the first time, we demonstrate that the two proteins associate both on and off the membrane with comparable affinity, with apparent dissociation constants in the micromolar range. Using site-specific fluorescence and Forster energy transfer probes, we mapped the protein-enzyme interacting regions on unilamellar vesicles. Our data suggest that on the membrane surface, the GCase-alpha-synuclein interaction involves a larger alpha-synuclein region compared to that found in solution. Binding of alpha-synuclein to the membrane is critical for complex formation. In the absence of the N-terminal membrane binding domain, alpha-synuclein and GCase do not associate on the membrane, whereas in solution, this complex stays intact. Due to the likelihood of electrostatic repulsion between the negatively charged membrane surface and the acidic C-terminal tail, we suggest that at least some of the N-terminal membrane-binding residues, 1-95, are required to anchor alpha-synuclein to the membrane, thereby promoting and/or stabilizing its binding to GCase at the C-terminal. Despite the current interest in defining the role of GCase in PD, our study is the first to investigate whether alpha-synuclein has a direct effect on GCase function. We show that alpha-synuclein is a potent GCase inhibitor (an apparent IC50 in the submicromolar range) only when it adopts an alpha-helical conformation, supporting the concept that not only is membrane binding critical in modulating activity, but it also affects the specific conformational change of alpha-synuclein upon lipid and enzyme association. The observed mixed mode of inhibition suggests that at the membrane interface, alpha-synuclein binding influences both substrate accessibility and turnover. This study supports the notion that GCase deficiency can contribute to the pathogenesis of the synucleinopathies. The observed interplay between alpha-synuclein-GCase association and activity can be a potential self-perpetuating mechanism connecting enzyme deficiency and the accumulation of alpha-synuclein and substrate. This link between membrane-bound alpha-synuclein and GCase could be one of many possible mechanisms that connect GD and PD. However, since only a minority of GD patients or carriers develops PD, other pathological factors are likely involved. Nevertheless, it is now possible to search for proteins and other molecules that could modulate this alpha-synuclein-GCase interaction and to evaluate their effects on enzyme activity.

编码葡萄糖脑苷脂酶 (GCase) 的 GBA1 基因突变会导致溶酶体贮积症、戈谢病 (GD)，并与帕金森病 (PD) 和其他路易体疾病的发生有关。 有趣的是，GBA1 变异是与 PD 相关的最常见的遗传风险因素。 尽管临床研究有力证明 GBA1 突变与 PD 发展之间存在联系，但仍缺乏机制见解。 最近的研究表明 GCase 与 PD 相关的淀粉样蛋白形成蛋白 α-突触核蛋白之间存在关系；然而，负责关联的具体分子机制仍然难以捉摸。虽然 GCase 对 α-突触核蛋白稳态的影响是大量工作的主题，但 α-突触核蛋白在酶功能中的作用尚未确定。此类信息可能具有进一步的影响，并表明导致局部放电风险增加的其他机制。 在之前的工作中，我们表明α-突触核蛋白和GCase在溶酶体条件下选择性地相互作用，并提出这种新发现的相互作用可能通过促进蛋白质降解和/或抑制聚集来影响α-突触核蛋白的细胞水平。我们现在扩展我们的研究，考虑膜如何调节这种复杂的形成。脂质不仅充当溶酶体内囊泡中 GCase 的底物，而且充当 α-突触核蛋白构象改变的表面。 我们首次证明这两种蛋白质在膜上和膜外均以相当的亲和力结合，表观解离常数在微摩尔范围内。使用位点特异性荧光和福斯特能量转移探针，我们绘制了单层囊泡上的蛋白质-酶相互作用区域。我们的数据表明，与溶液中发现的相比，在膜表面上，GCase-α-突触核蛋白相互作用涉及更大的α-突触核蛋白区域。 α-突触核蛋白与膜的结合对于复合物的形成至关重要。在缺乏 N 端膜结合结构域的情况下，α-突触核蛋白和 GCase 不会在膜上结合，而在溶液中，该复合物保持完整。由于带负电的膜表面和酸性 C 末端尾部之间可能存在静电排斥，我们建议至少需要一些 N 末端膜结合残基 1-95 将 α-突触核蛋白锚定到膜，从而促进和/或稳定其在 C 端与 GCase 的结合。 尽管目前人们对定义 GCase 在 PD 中的作用感兴趣，但我们的研究是第一个调查 α-突触核蛋白是否对 GCase 功能有直接影响的研究。我们证明，只有当 α-突触核蛋白采用 α-螺旋构象时，它才是一种有效的 GCase 抑制剂（表观 IC50 在亚微摩尔范围内），这支持了这样的概念：膜结合不仅在调节活性中至关重要，而且还影响特异性脂质和酶结合后α-突触核蛋白的构象变化。观察到的混合抑制模式表明，在膜界面，α-突触核蛋白结合影响底物可及性和周转。 这项研究支持了 GCase 缺乏可能导致突触核蛋白病发病机制的观点。观察到的 α-突触核蛋白-GCase 关联和活性之间的相互作用可能是一种潜在的自我延续机制，将酶缺乏与 α-突触核蛋白和底物的积累联系起来。膜结合 α-突触核蛋白和 GCase 之间的这种联系可能是连接 GD 和 PD 的多种可能机制之一。然而，由于只有少数 GD 患者或携带者发生 PD，因此可能还涉及其他病理因素。然而，现在可以寻找可以调节这种 α-突触核蛋白-GCase 相互作用的蛋白质和其他分子，并评估它们对酶活性的影响。