Information Processing by Post-translational Modification

描述（由申请方提供）：生物体中的大多数蛋白质通过化学基团与特定氨基酸残基的共价连接进行可逆修饰。这种“翻译后修饰”（PTM）允许在生理时间尺度上调节蛋白质功能。PTM参与调节大多数细胞过程，并且它们在诸如癌症和阿尔茨海默氏症的重大疾病中经常紊乱。我们对它们的作用的理解受到其异常复杂性的阻碍。蛋白质具有多种类型的修饰（磷酸化、甲基化、ADP-核糖基化、泛素化等）并不罕见。以及不同位点上的修饰以组合方式相互作用以影响 蛋白质功能这可以产生组合修饰状态的爆炸。例如，肿瘤抑制因子和“基因组守护者”p53，本提案将重点关注，具有超过100个修饰位点，在这个单一蛋白质上产生超过1030个组合修饰状态的潜力。当然，在任何细胞条件下，这些状态中很少会存在，但不同的条件可能会引起不同的修饰模式。这 许多研究人员认为，组合PTM为细胞信息处理提供了某种形式的“代码”，如“p53代码”，“微管蛋白代码”和“组蛋白代码”。然而，虽然修饰模式和下游反应之间的相关性已经得到证实，但因果关系尚未得到证明，代码的概念充其量仍然是一个隐喻。在以前的工作中，我们已经为解决这个问题奠定了基础。我们已经介绍了“mod-形式”的概念，用于整个蛋白质的修饰模式，以及“mod-形式分布”，用于样品中每个mod-形式的比例。我们已经开发了质谱（MS）技术，其中常规的“自下而上”基于肽的MS与较新的“自上而下”或全蛋白质MS组合，以定量地限制具有少量（<5）修饰位点的典型细胞蛋白质的模态分布。我们还开发了一个新的数学框架，它表明，在模态形式的组合爆炸数学上消失在稳态，提供了一种方法来克服复杂性障碍。在此基础上，我们汇集了一群杰出的合作者，他们的专业知识涵盖细胞生物学，质谱，数学和计算，以揭示人类细胞中内源性p53是否以及如何使用PTM编码信息。我们已经获得了完整p53的第一个自上而下的质谱。通过专注于这样一个具有挑战性的范例，我们希望了解大量关于p53本身的信息，同时开发可广泛应用于其他细胞蛋白的概念和方法，其中PTM发挥着关键作用。

DESCRIPTION (provided by applicant): Most proteins in an organism are reversibly modified by the covalent attachment of chemical groups to specific amino-acid residues. Such "post-translational modification" (PTM) allows protein function to be modulated on a physiological time-scale. PTMs are involved in regulating most cellular processes and they are frequently disordered in major diseases such as cancer and Alzheimer's. Our understanding of their role has been hampered by their extraordinary complexity. It is not uncommon for a protein to have many types of modification (phosphorylation, methylation, ADP-ribosylation, ubiquitination, etc.) on multiple sites and for modifications on different sites to interact combinatorially to influence protein function. This can create an explosion of combinatorial modification states. For instance, the tumor suppressor and "guardian of the genome" p53, on which this proposal will focus, has more than 100 sites of modification, creating the potential for more than 1030 combinatorial modification states on this single protein. Of course, very few of these states will be present in any cellular condition but different conditions may elicit different patterns of modification. This had led many researchers to suggest that combinatorial PTMs provide some form of "code" for cellular information processing, such as the "p53 code", the "tubulin code" and the "histone code". However, while correlations between patterns of modification and downstream responses have been shown, causality has not been demonstrated and the concept of code remains, at best, a metaphor. In previous work, we have laid a foundation for addressing this problem. We have introduced the concepts of "mod-form", for a pattern of modification across the whole protein, and "mod-form distribution", for the proportions of each mod-form in a sample. We have developed mass- spectrometry (MS) techniques in which conventional "bottom-up" peptide-based MS is combined with newer "top-down", or whole-protein, MS to quantitatively constrain the mod-form distribution of typical cellular proteins with small numbers (<5) of modified sites. We have also developed a new mathematical framework which shows that the combinatorial explosion in mod-forms disappears mathematically at steady-state, offering a way to overcome the complexity barrier. Here, we build on this foundation by bringing together an outstanding group of collaborators, with expertise that spans cell biology, mass spectrometry, mathematics and computation, to unravel whether and how endogenous p53 in human cells uses PTMs to encode information. We have already obtained the first top-down mass-spectrum of intact p53. By focusing on such a challenging exemplar, we expect to learn a great deal about p53 itself while developing concepts and methods that can be widely applied to other cellular proteins in which PTMs play a key role.