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

控制蛋白质降解是细胞健康功能所必需的。失控可能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的活动在种群中的各个细胞中是高度不同的。因此，能够在单个细胞的基础上测量活动是重要的。该项目将开发荧光传感器来测量单个细胞的UPS活性。这些传感器可以加强努力，根据患者的情况开发新药和最佳治疗策略。还将开发实践学习模块，并向巴吞鲁日地区的中学生介绍。本科生和高中生也将被招募参加扩展的研究经验，以努力开发和填充一支强大的生物制造工作力量。泛素-蛋白酶体系统(UPS)管理DNA修复和蛋白质降解。它的特异性由E3泛素连接酶控制，E3泛素连接酶识别靶蛋白上的选定降解序列或降解。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。这意味着无法获得一组细胞中蛋白酶体活动的完整图像。UPS可以通过利用蛋白水解靶向嵌合体(PROTAC)将蛋白质引导到蛋白酶体进行降解来抑制蛋白质。目前的PROTAC虽然有效，但由于细胞通透性低和对蛋白酶的敏感性高，其效力降低。因此，需要在下一代PROTAC中引入长寿命、细胞透透性的多肽。我们的主要假设是，基于降解素的底物可以被用作(1)直接量化蛋白酶体活性的下一代方法和(2)具有更长寿命和灵敏度的新型PROTAC。这项拟议的工作将评估含有发夹基序的多肽，作为一种新的方法来量化完整细胞中的蛋白酶体活性，并降解难以抑制的目标。我们还将调查高度稳定的非天然氨基酸序列作为一种新的降解型底物的使用。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.