CAREER: Degron-based substrates: A novel toolkit for biosensing and targeted inhibition

控制蛋白质降解是健康细胞功能所必需的。失去控制会导致癌症和神经退行性疾病。蛋白质降解的一个中央控制系统被称为UPS（泛素-蛋白酶体系统）。UPS的活性在群体中的单个细胞之间是高度可变的。因此，能够在单个细胞的基础上测量活动是很重要的。该项目将开发荧光传感器来测量单个细胞的UPS活性。这些传感器可以提高研发新药的力度，并在逐个患者的基础上优化治疗策略。实践学习模块也将被开发并呈现给巴吞鲁日地区的中学生。本科生和高中生也将被招募参加扩展的研究经验，以努力发展和充实一个强大的生物制造劳动力。泛素-蛋白酶体系统（UPS）控制DNA修复和蛋白质降解。它的特异性是由E3泛素连接酶控制的，它识别目标蛋白上的降解序列或降解序列。不幸的是，我们对E3连接酶和蛋白酶体活性的了解是不完整的。这就导致无法获得整个细胞群中蛋白酶体活动的完整图像。UPS可以利用靶向嵌合体（proteolysis targeting chimeras, PROTACs）将蛋白质引导到蛋白酶体进行降解，从而抑制蛋白质。目前的PROTACs虽然有效，但由于细胞渗透性低和蛋白酶敏感性高，其效力降低。因此，需要将长寿命的、细胞渗透性的肽结合到下一代PROTACs中。我们的总体假设是，基于降解的底物可以用作(1)直接量化蛋白酶体活性的下一代方法；(2)具有更高寿命和灵敏度的新型PROTAC。拟议的工作将评估含有&amp；#946；-发夹基序作为一种新的定量完整细胞中蛋白酶体活性和降解难以抑制靶标的方法。我们还将研究使用高度稳定的非天然氨基酸序列作为一类新的降解基底物。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Control of protein degradation is required for healthy cellular function. Loss of control can lead to cancer and neurodegenerative diseases. One central control system for protein degradation is referred to as UPS (ubiquitin-proteasome system). The activity of the UPS is highly variable across individual cells in a population. It is important therefore to be able to measure activity on a single cell basis. This project will develop fluorescent sensors to measure the UPS activity of individual cells. These sensors could enhance efforts to develop new drugs and optimal treatment strategies on a patient by patient basis. Hands-on learning modules will also be developed and presented to middle school students in the Baton Rouge area. Undergraduates and high school students will also be recruited to participate in extended research experiences in an effort to develop and populate a robust biomanufacturing workforce.The ubiquitin-proteasome system (UPS) governs DNA repair and protein degradation. Its specificity is governed by E3 ubiquitin ligases, which recognize select degradation sequences, or degrons, on target proteins. Unfortunately, our understanding of E3 ligase and proteasome activity is incomplete. This translates into an inability to obtain a complete picture of proteasome activity across a population of cells. The UPS could be harnessed to inhibit proteins by directing them to the proteasome for degradation using proteolysis targeting chimeras (PROTACs). While effective, current PROTACs suffer from reduced potency due to low cell permeability and high protease susceptibility. Thus, there is a need for long-lived, cell permeable peptides to be incorporated into the next generation of PROTACs. Our overarching hypothesis is that degron-based substrates can be utilized as (1) a next generation method to directly quantify proteasome activity and (2) a novel PROTAC with increased lifetime and sensitivity. The proposed work will evaluate peptides containing a β-hairpin motif as a new approach for quantifying proteasome activity in intact cells and degrading difficult to inhibit targets. We will also investigate the use of highly stable, non-natural amino acid sequences as a new class of degron-based substrates.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.