Posttranslational Modification of Deubiquitinating Enzymes in Neurodegeneration

神经退行性变与蛋白质质量控制中的扰动密切相关。泛素途径是中心 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，我将研究去泛素化是如何 酶(DUBS)是泛素依赖通路的组成部分，参与神经退行性变 过程以及配音本身是如何依次受到监管的。这些研究集中在一种独特的配音和 脊髓小脑性共济失调3型ATS和USP25和USP28中的多谷氨酰胺病蛋白 与ATS共享结构域的其他大脑表达的配音。 ATS是一种在蛋白质质量控制中起作用的配音。我之前已经确定内源性ATS是 泛素化及其泛素化在蛋白毒性应激中增加。重要的是，泛素化直接 增强ATS的催化活性。在K99阶段，我确定了ATS泛素化、聚集的位置 研究其泛素化依赖的激活机制，并收集证据表明ATS 泛素化对于它的一些细胞功能很重要。由于ATS在果蝇模型中具有神经保护作用 神经退行性变，在Roo阶段I将使用果蝇黑腹果蝇神经退行性变模型 探讨ATS泛素化在其细胞保护功能中的作用。我将生成表达狂野的苍蝇线条 神经系统或眼睛中的类型性或非普遍存在的ATS。通过结构、行为 和电生理学研究我将检查ATS泛素化是否对其神经保护重要 在完好无损的动物身上扮演的角色。在哺乳动物细胞培养中的补充研究将调查 ATS泛素化在蛋白毒性应激中保护细胞的能力。 USP25和USP28是两个DUB，与ATS一样，具有泛素相互作用基序(UIM)。UIM促进主机 蛋白质泛素化；事实上，USP25在细胞中泛素化。我的初步数据显示，USP25和 USP28调节导致阿尔茨海默病的突变淀粉样前体蛋白(APP)的细胞水平。 在这里，我将研究USP25和USP28通过使用基于细胞的和在 体外试验。我还将研究USP25泛素化是如何参与这种细胞功能的。最后，我会 开始建立果蝇试验，以检查其他DUBS在未来神经退化中的作用。

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.