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

职业：基于降解决定子的底物：用于生物传感和靶向抑制的新型工具包

• 批准号: 2416519
• 负责人: Adam Melvin
• 金额: $ 50万
• 依托单位: Clemson University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-10-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2416519&HistoricalAwards=false
• 关键词: CAREER; Degron; based; substrates; novel

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.

控制蛋白质降解是健康细胞功能所必需的。失控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的活性在群体中的各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性是重要的。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，开发新的药物和最佳的治疗策略，对病人的基础上。还将开发实践学习模块，并向巴吞鲁日地区的中学生介绍。大学生和高中生也将被招募参加扩展的研究经验，努力发展和填充一个强大的生物制造劳动力。泛素-蛋白酶体系统（UPS）管理DNA修复和蛋白质降解。其特异性由E3泛素连接酶控制，该酶识别靶蛋白上的选择降解序列或降解决定子。 不幸的是，我们对E3连接酶和蛋白酶体活性的理解是不完整的。这意味着无法获得整个细胞群中蛋白酶体活性的全貌。UPS可以通过使用蛋白水解靶向嵌合体（PROTAC）将蛋白质引导至蛋白酶体进行降解来抑制蛋白质。虽然有效，但目前的PROTAC由于低细胞渗透性和高蛋白酶敏感性而遭受降低的效力。因此，需要将长寿命的细胞可渗透肽掺入下一代PROTAC中。我们的总体假设是，基于脱氮基的底物可以用作（1）直接定量蛋白酶体活性的下一代方法和（2）具有增加的寿命和灵敏度的新型PROTAC。拟议的工作将评估含有发夹基序的肽作为一种新的方法，用于定量蛋白酶体活性在完整的细胞和降解难以抑制的目标。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脱氮基底物。该奖项反映了NSF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。