Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana, 47907, USA.
Chembiochem. 2021 Aug 3;22(15):2553-2560. doi: 10.1002/cbic.202100117. Epub 2021 Jun 15.
Aging is characterized by changes in several cellular processes, including dysregulation of proteostasis. Current research has shown long-lived rodents display elevated proteasome activity throughout life and proteasome dysfunction is linked to shorter lifespans in a transgenic mouse model. The ubiquitin proteasome system (UPS) is one of the main pathways leading to cellular protein clearance and quality maintenance. Reduction in proteasome activity is associated with aging and its related pathologies. Small molecule stimulators of the proteasome have been proposed to help alleviate cellular stress related to unwanted protein accumulation. Here we have described the development of techniques to monitor the impact of proteasome stimulation in wild-type yeast and a strain that has impaired proteasome expression. We validated our chronological lifespan assay using both types of yeast with a variety of small molecule stimulators at different concentrations. By modifying the media conditions for the yeast, molecules can be evaluated for their potential to increase chronological lifespan in five days. Additionally, our assay conditions can be used to monitor the activity of proteasome stimulators in modulating the degradation of a YFP-α-synuclein fusion protein produced by yeast. We anticipate these methods to be valuable for those wishing to study the impact of increasing proteasome-mediated degradation of proteins in a eukaryotic model organism.
衰老是由几个细胞过程的变化引起的，包括蛋白质稳态的失调。目前的研究表明，长寿啮齿动物在整个生命过程中表现出较高的蛋白酶体活性，而蛋白酶体功能障碍与转基因小鼠模型中寿命缩短有关。泛素蛋白酶体系统 (UPS) 是导致细胞蛋白清除和质量维持的主要途径之一。蛋白酶体活性的降低与衰老及其相关病理有关。蛋白酶体的小分子刺激物已被提出用于帮助减轻与不需要的蛋白质积累相关的细胞应激。在这里，我们描述了监测野生型酵母和一种蛋白酶体表达受损的酵母中蛋白酶体刺激影响的技术的发展。我们使用两种酵母类型以及不同浓度的各种小分子刺激物验证了我们的时序寿命测定法。通过修改酵母的培养基条件，可以评估分子在五天内增加时序寿命的潜力。此外，我们的测定条件可用于监测蛋白酶体刺激物在调节酵母产生的 YFP-α-突触核蛋白融合蛋白降解中的活性。我们预计这些方法对那些希望在真核模式生物中研究增加蛋白酶体介导的蛋白质降解的影响的人将非常有价值。
