Posttranslational Modification of Deubiquitinating Enzymes in Neurodegeneration

神经变性与蛋白质质量控制中的扰动密切相关。泛素途径是 通过它们在蛋白质降解中的作用来控制蛋白质质量。在这里，我将研究去泛素化 泛素化酶（DUBs），泛素依赖性途径的组成部分，参与神经退行性变 过程以及DUB本身如何被依次监管。这些研究集中在一个独特的DUB和 脊髓小脑性共济失调3型中的多聚谷氨酰胺疾病蛋白，共济失调蛋白-3（ATS），USP 25和USP 28， 与ATS共享域的其他脑表达DUB。 ATS是一种DUB，在蛋白质质量控制中发挥作用。我以前确定内源性ATS是 在蛋白毒性应激中其泛素化增加。重要的是，泛素化直接 增强ATS的催化活性。在K99阶段，I确定了ATS的泛素化位置， 洞察其泛素化依赖激活背后的机制，并收集证据表明， 泛素化对其某些细胞功能很重要。由于ATS在果蝇模型中具有神经保护作用， 神经变性，在ROO阶段I将使用果蝇神经变性模型 探讨ATS泛素化在其细胞保护功能中的作用。我将生成飞行线表示野生 型或非泛素化的ATS在整个神经系统或眼睛。通过结构、行为 和电生理研究，我将研究ATS泛素化是否对神经保护作用很重要。 在完整动物中的作用。在哺乳动物细胞培养的补充研究将调查的重要性， ATS泛素化在蛋白毒性应激期间保护细胞的能力。 USP 25和USP 28是两个DUB，与ATS一样，具有泛素相互作用基序（UIM）。UIM便于主机 蛋白质泛素化;事实上，USP 25在细胞中被泛素化。我的初步数据表明，USP 25和 USP 28调节阿尔茨海默病引起的突变淀粉样前体蛋白（APP）的细胞水平。 在这里，我将研究USP 25和USP 28调节APP的机制，通过使用基于细胞的和在细胞内的方法。 体外测定。我还将研究USP 25泛素化如何参与这种细胞功能。最后要 开始建立果蝇试验，以检查其他DUB在未来神经变性中的作用。

Neurodegeneration is closely linked to perturbations in protein quality control. Ubiquitin pathways are central to protein quality control through their role in protein degradation. Here, I will investigate how deubiquitinating enzymes (DUBs), integral components of ubiquitin-dependent pathways, are involved in neurodegenerative processes and how DUBs themselves are regulated in turn. These studies focus on a unique DUB and the polyglutamine disease protein in Spinocerebellar Ataxia Type 3, ataxin-3 (ATS), and USP25 and USP28, two other brain-expressed DUBs that share domains with ATS. ATS is a DUB that functions in protein quality control. I previously established that endogenous ATS is ubiquitinated and its ubiquitination increases in proteotoxic stress. Importantly, ubiquitination directly enhances ATS's catalytic activity. During the K99 phase I determined where ATS is ubiquitinated, gathered nsight into the mechanism behind its ubiquitination-dependent activation, and collected evidence that ATS ubiquitination is important for some of its cellular functions. Since ATS is neuroprotective in fly models of neurodegeneration, during the ROO phase I will use Drosophila melanogaster models of neurodegeneration to explore the role of ATS ubiquitination in its cytoprotective functions. I will generate fly lines expressing wild type or non-ubiquitinatable ATS throughout the nervous system or in the eye. Through structural, behavioral and electrophysiological studies I will examine whether ATS ubiquitination is important for its neuroprotective role in intact animals. Complementary studies in mammalian cell culture will investigate the importance of ATS ubiquitination in its ability to protect cells during proteotoxic stress. USP25 and USP28 are two DUBs that, like ATS, have ubiquitin-interacting motifs (UIMs). UIMs facilitate host protein ubiquitination; in fact, USP25 is ubiquitinated in cells. My preliminary data indicate that USP25 and USP28 regulate cellular levels of Alzheimer's Disease-causing, mutant Amyloid Precursor Protein (APP). Here, I will investigate the mechanisms by which USP25 and USP28 regulate APP by using cell-based and in vitro assays. I will also examine how USP25 ubiquitination is involved in this cellular function. Finally, I will begin to establish Drosophila assays to examine the role of other DUBs in neurodegeneration in the future.