Activity profiling of plant serine hydrolases involved in defense

蛋白质组的活性可以通过“基于活性的分析”来显示，这是基于使用标记的、基于机制的抑制物(探针)，这些抑制物以依赖于活性的方式与整个类别的酶反应。这项技术已经成功地应用于医学研究，揭示了人类疾病的许多新方面。我已经介绍了植物中木瓜蛋白酶样半胱氨酸蛋白酶的活性图谱，并展示了它们在植物与病原体相互作用期间的有趣的活性动态。现在，我请求一位经验丰富的化学生物学家为植物丝氨酸蛋白酶建立基于活性的分析。这是植物中最大的一类蛋白水解酶，有超过250种酶，但人们对它们的活性和功能知之甚少。目的是测试五种不同类别的丝氨酸蛋白酶探针。初步实验表明，这些探针针对拟南芥叶片蛋白质组中的多种蛋白质。将在体外(提取物)和体内(在活组织中)对图谱进行优化和表征。探针的靶标将通过质谱学鉴定，并将通过过度表达、反向遗传学和/或抗体的使用来确认。

Activities of proteomes can be displayed through ‘activity-based profiling’, which is based on the use of tagged, mechanism-based inhibitors (probes) that react with whole classes of enzymes in an activity-dependent manner. This technology has been successfully applied in medical research, revealing numerous novel aspects of human diseases. I have introduced activity profiling of papain-like cysteine proteases in plants and displayed intriguing dynamics of their activities during plant-pathogen interactions. Now I am requesting a grant for an experienced chemical biologist to establish activity-based profiling for plant serine proteases. With over 250 enzymes, this is the largest class of proteolytic enzymes in plants, but little is known about their activities and functions. The aim is to test five different classes of serine protease probes. Preliminary experiments demonstrate that these probes target multiple proteins in Arabidopsis leaf proteomes. Profiling will be optimized and characterized in vitro (on extracts) and in vivo (in living tissue). Targets of the probes will be identified by mass spectrometry and will be confirmed through overexpression, reverse genetics and/or use of antibodies.