Substrates of the N-end rule of targeted protein degradation

蛋白质是细胞的主力，执行的任务范围之广令人着迷。为了正确地完成它们的工作，蛋白质需要在正确的时间、正确的位置和正确的数量。有几种方法可以在空间和时间上调节蛋白质的数量。许多注意力都集中在控制蛋白质合成的不同过程上，但蛋白质分解的重要性直到最近才得到认识。以前，蛋白质分解（称为蛋白质水解）被认为是负面的，与细胞损伤和死亡有关。然而，现在人们知道，受控的蛋白质水解是一种极其重要的机制，它使蛋白质丰度能够被优雅和精确地调节。在几乎所有的生物体中，都有一种特殊形式的受控、靶向蛋白水解，称为n端规则途径。这一途径已经通过人工“报告蛋白”被阐明，并且已经证明，一个给定的蛋白质可以被酶切割（被酶破坏）以显示一个新的前端（“n端”）。新n端氨基酸的身份决定了蛋白质在细胞中的命运：它是否成为蛋白质水解的目标，因此被称为n端规则。在动物中，参与神经和心脏发育的蛋白质以这种方式受到控制，n端规则通路在生育和DNA修复中也起着重要作用。最近的证据表明，这一途径调节了植物发育和代谢的关键阶段。我们知道这一点，因为如果缺乏蛋白质水解所需的特定酶，植物就会表现出一系列显著的缺陷：种子不能正常发芽，幼苗不能分解储存的油并长成成年植物，幼苗不能对改变的氧气水平做出适当的反应，叶子不能正常发育。这告诉我们，n端规则通路控制着重要蛋白质的水平，包括调节植物发育的蛋白质。然而，通过该途径被分解的蛋白质（“底物”）的身份是未知的，很难用常规方法发现。本研究试图通过采用三种互补的蛋白质鉴定方法来比较正常植物和缺乏n端规则酶的植物来解决这一问题。在这个项目中开发的方法和获得的信息可以更广泛地用于研究一系列不同的生物体。从长远来看，该项目还将提供有关植物生理学的重要信息，这些信息可用于帮助开发更好的作物。

Proteins are the workhorses of the cell and perform a fascinatingly wide range of tasks. To perform their jobs correctly, proteins need to be in the right place, at the right time and in the right amount. There are several ways in which protein amounts can be regulated in space and in time. Much attention has been focussed on different processes which control protein synthesis, but the importance of protein breakdown has only relatively recently been appreciated. Previously, protein breakdown (known as proteolysis) was seen in a negative light, associated with cell damage and death. However, it is now known that controlled proteolysis is an extremely important mechanism which enables protein abundance to be modulated with elegance and precision.In almost all organisms, there is a specialised form of controlled, targeted proteolysis, called the N-end rule pathway. The pathway has been elucidated using artificial "reporter" proteins and it has been shown that a given protein can be cleaved (broken by an enzyme) to reveal a new front end (the "N-terminus"). The identity of the amino acid at the new N-terminus determines the fate of the protein in the cell: whether it is targeted for proteolysis or not, hence the name, N-end rule. In animals, proteins involved in the development of nerves and the heart are controlled in this manner and the N-end rule pathway also plays important roles in fertility and DNA repair. Recent evidence has shown that this pathway regulates key stages of plant development and metabolism. We know this because plants display a range of dramatic defects if the specific enzymes needed for proteolysis are absent: seeds cannot germinate properly, seedlings cannot break down stored oil and grow into adult plants, seedlings cannot respond appropriately to altered oxygen levels and leaves do not develop normally. This tells us that the N-end rule pathway controls the levels of important proteins, including proteins which regulate plant development. However, the identities of the proteins which are broken down by the pathway (the "substrates") are unknown and difficult to discover by conventional methods. This proposal seeks to solve this problem by adapting three complementary methods for protein identification to compare normal plants with plants which lack N-end rule enzymes.The methods developed in this project and the information obtained can be more widely useful in studying a range of different organisms. In the longer term, this project will also provide important information about plant physiology which can be applied to help develop better crops.