SUMO proteomics in C. elegans; germ line, meiosis and the DNA damage response

在细胞和生物体中，蛋白质需要被调节以适当地履行其功能。在活细胞内传递各种信号需要一组特殊的蛋白质。这些信号蛋白通过将不同的分子附着在目标蛋白上来传递信号。这些分子的附着，被称为修饰，作为一种“细胞语言”。现代分子生物学的研究集中在这些信号蛋白上，并试图解决它们在改变其靶标活性方面的重要性。磷酸化或泛素化等信号的修饰很早以前就已为人所知。然而，只有在过去的十年中，人们才清楚地认识到sumoylation提供了一种重要的细胞内通讯方式。Hay实验室详细阐述了与SUMO修饰所需的基本生化反应有关的重要分子细节。SUMO本身是一种相当大的蛋白质，它附着在其靶蛋白的某些残基上，并且需要几种蛋白质来实现SUMO化。所谓的“系统方法”旨在提供对“相扑语言”的全面分析和理解。这种分析需要识别所有的SUMO修饰蛋白，并详细分析SUMO修饰变化如何以及在何种程度上响应各种生理和病理生理条件。第一个评估SUMO修饰的全局作用的实验是在细胞培养中完成的。虽然这些系统具有明显的实验优势，但这些细胞并不一定反映完整生物体中细胞的行为。这就是为什么我们计划使用小蛔虫秀丽隐杆线虫作为一个模型系统，以全球范围内评估生物体中酰化的范围和重要性。秀丽隐杆线虫被认为是最简单的动物系统。由于该生物在发育和解剖水平上的简单性，由于其易于维护，以及由于可以操纵其遗传信息程序以研究SUMO修饰的重要性所必需的方法的可用性，促进了研究。尽管它很简单，秀丽隐杆线虫是一种多细胞生物，与人类共享许多基本的遗传程序。因此，在秀丽隐杆线虫系统中获得的结果可能适用于人类，并且与蠕虫一起工作不会引起伦理问题。作为我们研究的一部分，我们想要在蠕虫中表达SUMO的修饰版本，这样我们就可以最好地应用我们开发的先进方法来分离所有的SUMO修饰蛋白。为了鉴定这些蛋白质，也为了评估SUMO修饰的程度，我们将使用我们最近开发的一种方法，允许鉴定和定量数千种蠕虫蛋白质。我们不仅想找到所有的SUMO修饰蛋白，还想评估SUMO修饰的重要性。首先，我们选择了一个在实验室中广泛研究的重要生物过程。减数分裂是一种特殊的细胞分裂，需要生殖细胞的产生。在分裂过程中，正常细胞中复制的染色体组减少为一个拷贝。随后的生殖细胞融合恢复了一组重复的染色体一条来自父亲，另一条来自母亲。不仅如此，在减数分裂过程中，称为重组的过程也混合了单个染色体，使染色体包含来自祖父母的元素。重组确保了遗传多样性。2)重组是指DNA分子的断裂和连接。当DNA损伤剂如电离辐射破坏染色体时，使用重组。我们已经知道SUMO修饰对减数分裂和重组很重要，现在我们想对此进行系统的分析。我们的研究对于理解遗传信息是如何从一代传递到下一代很重要。

Proteins need to be regulated to properly fulfil their function in cells and living organisms. Specialized sets of proteins are required to convey a variety of signals within living cells. Such signalling proteins confer signals by attaching distinct sets of molecules to their target proteins. The attachment of such molecules, referred to as modification serves as a 'cellular language'. Modern molecular biology research focuses on such signalling proteins and tries to address their importance in modifying the activity of their targets. Modifications that confer signals such as phosphorylation or ubiquitylation have been known for a long time. However, only in the past decade, it became clear that sumoylation provides an important way of intracellular communication. The Hay laboratory elaborated important molecular details pertaining to basic biochemical reactions that are needed for SUMO modification. SUMO by itself is a sizable protein, that is attached to certain residues of its target proteins and several proteins are needed to confer sumoylation. So called 'systems approaches' aim to provide a comprehensive analysis and understanding of the 'SUMO language'. Such analysis requires the identification, of all SUMO modified proteins, and a detailed analysis of how and to which extend SUMO modification changes in response to various physiological and pathophysiological conditions. First experiments to assess the global role of SUMO modification were done in with cell cultures. While those systems have obvious experimental advantages, these cells do not necessarily reflect the behaviour of cells in intact organisms. This is why we plan to use the small roundworm C. elegans as a model system to globally asses the extend, and importance of sumoylation in a living organism. C. elegans was chosen as the simplest possible animal system. Studies are facilitated by the simplicity of the organism at the developmental and anatomical level, by the ease of its maintenance, as well as by the availability of methods that allow for manipulating its genetic information procedures necessary to study the importance of SUMO modification. Despite of its simplicity, C. elegans is a multicellular organism that shares many fundamental genetic programs with humans. Thus results obtained in the C. elegans system are likely to be applicable to humans and working with worms does not raise ethical concerns. As part of our studies we want to express modified versions of SUMO in the worm, such that we can best apply the advanced methods we have developed to isolate all SUMO modified proteins. To identify those proteins, but also to assess the extent of SUMO modification we will use a method we recently developed allowing for the identification and quantification of thousands of worm proteins. We not only want to find all SUMO modified proteins, but also want to assess the importance of SUMO modification. To start with this, we chose an important biological processes extensively studied in our labs. 1) Meiosis is a specialised cell division, needed for the generation of germ cells. During this division the duplicated set of chromosomes occurring in normal cells is reduced to just one copy. The subsequent fusion of germ cell restores a duplicated set of chromosomes one coming from the father, another one coming from the mother. Not only this, during meiosis a process referred to as recombination also mixes individual chromosomes such that chromosomes contain elements from both grandparents. Recombination ensures genetic diversity. 2) Recombination pertains to the breaking and linking DNA molecules. Recombination is used when DNA damaging agents such as ionizing irradiation breaks chromosomes. We already have know that SUMO modification is important for meiosis and recombination and we now want perform a systematic analysis of this. Our studies are important to understand how the genetic information is passed on from one generation to the next.