The generation and testing of a cell-permeable constitutively active recombinant parkin protein for its neuroprotective properties

帕金森病（PD）是加拿大第二常见的神经退行性疾病，约50%的早发病例与编码帕金蛋白的PARK 2基因突变有关。Parkin作为E3泛素（Ub）连接酶发挥功能，在一系列PD细胞和动物模型中发挥神经保护作用。有趣的是，我的实验室最近的一项研究提供了第一个证据，证明帕金存在于自动抑制状态。因此，帕金的基础E3活性低，需要激活。值得注意的是，将parkin中的W403改变为丙氨酸消除了这种抑制，导致parkin的过度激活。考虑到帕金蛋白的神经保护特性，开发活性帕金蛋白提供了一种新的和令人兴奋的治疗途径，以探索保护PD患者的神经元免受应激。在这里，我们建议与iProgen合作，利用他们专有的iProgen蛋白转导结构域（iPTD）来产生新的细胞渗透性帕金蛋白，否则我们将无法产生。使用该iPTD标签，iProgen将产生W403A组成型活性parkin蛋白，我们将在PD细胞模型中将其保护特性与基础失活的野生型parkin进行比较。在项目的第一部分，iProgen将工程化，表达和纯化重组帕金蛋白。在第二部分中，我们将测试parkin蛋白的体外泛素连接酶活性，并研究其在PD细胞模型中的生物学活性。 如果目前的研究表明，去阻遏帕金蛋白可以增强细胞保护相对于野生型蛋白，这些结果提供了动力，进一步研究探索这种组成型活性帕金蛋白的保护特性。对于iProgen，这些结果使他们能够继续进行下一阶段的生产更大数量的帕金蛋白用于未来的试验。

Parkinson's disease (PD) is the second most common neurodegenerative disease in Canada, with ~50% of cases with an early onset linked to mutations in the PARK2 gene, which encodes the parkin protein. Parkin functions as an E3 ubiquitin (Ub) ligase that plays a neuroprotective role in a range of cellular and animal models of PD. Intriguingly, a recent study from my lab has provided the first evidence that parkin exists in an autoinhibited state. Thus, basal E3 activity of parkin is low, requiring activation. Remarkably, changing the W403 in parkin to an alanine abolished this inhibition, causing hyperactivation of parkin. Considering the neuroprotective properties of parkin, developing an active parkin protein presents a novel and exciting therapeutic avenue to explore in protecting neurons against stress in PD patients. Here we propose to partner with iProgen to take advantage of their proprietary iProgen Protein Transduction Domain (iPTD) to generate novel cell-permeable parkin proteins, that we would otherwise be unable to generate. Using this iPTD tag, iProgen will generate a W403A constitutively active parkin protein whose protective properties we will compare against a basally inactive wild-type parkin in cellular models of PD. In the first part of the project, iProgen will engineer, express and purify recombinant parkin proteins. In the second part of the project, we will test the in vitro ubiquitin ligase activity of the parkin proteins, and investigate their biological activity in cellular models of PD. If the present study demonstrates that the derepressed parkin protein can enhance cellular protection relative to the wild-type protein, these results provide impetus for further studies exploring the protective properties of this constitutively active parkin. For iProgen these results enable them to proceed towards the next stage of producing greater quanitites of the parkin protein for use in future trials.