Substrate binding specificity of enzymatic components of the N-end rule pathway

细胞内多种功能的直接执行者是蛋白质而不是基因。因此，作为所有活细胞的主要成分之一的蛋白质分析是生物学、生物技术和医学中最重要的任务之一。蛋白质起着核心作用，例如作为支架或储存成分（细胞骨架，能量储存），作为生物化学催化剂，大力促进生物化学反应，作为许多其他蛋白质及其修饰以及信号分子的“生物合成工厂”。保持细胞内所有蛋白质（蛋白质组）的生理功能平衡是至关重要的。这意味着1)必须调节蛋白质的浓度和丰度，2)必要时它们的降解。在我们的实验室里，我们专注于一种非常特殊的降解途径实际上是一种处理蛋白质的方法。蛋白质识别和降解的整个过程包括细胞生物学和生化途径以及各种额外的酶活性蛋白质，并消耗大量可用的细胞内能量。例如，所谓的n端规则识别需要被降解的蛋白质，因为它们的作用时间已经过去，或者它们变成了细胞毒性。我们关注的是这种信号和降解途径，它是泛素蛋白酶体系统的一部分，因此是蛋白质质量控制的一部分。在我们的工作中，我们使用了目前最了解的模式植物，小型十字花科植物拟南芥。目前，我们对植物通过n端规则途径参与蛋白质降解的酶组分知之甚少，只有少数蛋白质靶点被降解，即所谓的底物。目前的研究出版物提示，这些调节蛋白质稳定性的过程在发育和细胞生物学中起着核心作用。这些过程既包括植物的内源分子通讯，也包括环境分子通讯。例如，植物被病原体感染、洪水期间、细胞分裂、种子发芽和储存蛋白质和脂质的分解，即与基础和应用植物科学以及基于植物的生物技术和农业高度相关的所有领域。这里提出的项目涉及所谓的E3泛素蛋白连接酶，PROTEOLYSIS 1的分析，该酶识别底物并将其降解。我们的研究旨在通过n端规则途径的酶来表征分子识别机制，并首次对植物中该蛋白质修饰途径的酶成分进行功能分析。

Proteins rather than genes are the direct executers of manifold functions within a cell. The analysis of proteins as one of the main constituents of all living cells is therefore one of the most important tasks in biology, biotechnology and medicine. Proteins play central roles e.g. as scaffold or storage components (cytoskeleton, energy storage), as biochemical catalysts to energetically facilitate biochemical reactions and as "biosynthetic factories" for numerous additional proteins and their modifications as well as signaling molecules. It is crucial to keep the entity of all proteins within a cell (the proteome) in a physiologically functional balance. This implies that 1) the protein concentration and abundance must be regulated and 2) their degradation if necessary. In our lab, we focus on a very specific degradation pathway virtually a way to dispose of proteins. The overall process of protein recognition and degradation comprises cell biological and biochemical pathways and diverse additional enzymatically active proteins and consume a lion's share of available intracellular energy. For example, the so-called N-end rule recognizes specifically proteins that need to be degraded because their time-of-action is past or they turned to be cytotoxic. We focus on this signaling and degradation pathway which is part of the Ubiquitin proteasome system and thus of the protein quality control. For our work, we use the currently best-understood model plant, the small crucifer Arabidopsis thaliana. Currently, only very few in known about enzymatic components involved in protein degradation via the N-end rule pathway in plants and only a small number of protein targets to be degraded, the so-called substrates. Current research publications give hints that these processes regulating protein stability play central roles in developmental and cellular biology. These processes comprise both the endogenous as well as environmental molecular communication of the plant. Examples are amongst plant infection by pathogens, during flooding situations, in cell division, seed germination and breakdown of storage proteins and lipids, i.e. altogether areas with high relevance for basic and applied plant science but also for plant-based biotechnology and agriculture. The here proposed projects deal with the analysis of a so-called E3 Ubiquitin protein ligase, PROTEOLYSIS 1, which recognizes substrates and routing them for degradation. Our studies aim at characterizing the molecular recognition mechanism by enzymes of the N-end rule pathway and by this deliver for the first time a functional analysis of enzymatic components of this protein modification pathway in plants.