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%的早发性病例与PARK2基因突变有关，PARK2基因编码帕金森蛋白。Parkin作为一种E3泛素(Ub)连接酶，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，证明帕金存在自我抑制状态。因此，PARKIN的基础E3活性较低，需要激活。值得注意的是，将Parkin中的W403改变为丙氨酸，取消了这种抑制，导致了Parkin的过度激活。考虑到parkin的神经保护特性，开发一种具有活性的parkin蛋白为探索帕金森病患者神经细胞应激保护提供了一条新的、令人兴奋的治疗途径。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)来产生我们原本无法产生的新型细胞渗透性Parkin蛋白。使用这个iPTD标签，iProgen将产生一种W403A结构性活性parkin蛋白，我们将在帕金森病的细胞模型中将其保护特性与碱基不活跃的野生型parkin进行比较。在该项目的第一部分，iProgen将设计、表达和纯化重组parkin蛋白。在项目的第二部分，我们将检测Parkin蛋白的体外泛素连接酶活性，并在PD细胞模型中研究它们的生物学活性。如果本研究表明，相对于野生型蛋白，去抑制的parkin蛋白可以增强细胞保护，这些结果为进一步研究这种结构性活性parkin的保护特性提供了动力。对于iProgen来说，这些结果使他们能够继续进行下一阶段生产更多数量的parkin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.