Application of mass spectrometry approaches to the study of the function assembly associated factors and architecture of the proteasome

细胞令人惊叹的特性之一是它能够再生和自我更新。旧的和受损的蛋白质不断被降解，新的蛋白质不断制备。降解的主要途径是蛋白酶体途径。蛋白酶体是一个由至少46种蛋白质组成的非常大的复合体，这些蛋白质组成了细胞的降解机制。这是一台以特定方式分解蛋白质的关键机器。为了防止蛋白酶体破坏细胞或自身，它只破坏标记为要破坏的蛋白质。一种常用的标签是一种小蛋白，泛素。蛋白酶体的调节颗粒(19S)识别标记的蛋白质，并充当只允许目标蛋白质进入催化颗粒(20S)的“把关人”，催化颗粒(20S)迅速分解它们。多年来，关于这一重要途径积累了大量信息，从而产生了2004年诺贝尔化学奖。然而，还有很多东西需要学习，因为研究这样一个复杂的系统并不是一项微不足道的任务。海量、异质和动态的组装给现有的结构生物学方法带来了困难。在我们的实验室中，我们使用独特的质谱学方法来研究非常大的蛋白质组件。虽然复合体中的蛋白质通过非常微妙的相互作用结合在一起，但我们能够将这些复合体保持在气相中，并检查极大的组装，如分子质量远远超过200万的蛋白酶体。最近，我们记录了结合到靶蛋白上的蛋白酶体催化颗粒的光谱并测量了其质量。要解释这些数据是非常具有挑战性的，不仅因为自由形式和蛋白质结合形式的络合物之间的质量差异相对较小，而且在理解光谱中可用的信息方面也是如此。我们惊讶地发现，我们不仅可以确定被占据的蛋白质的准确数量，还可以确定它们在蛋白酶体内腔中的位置(图2)。我们研究的另一个具有挑战性的方面是获得关于调节颗粒的结构信息。使用我们的质谱学方法，我们能够确定LID颗粒中蛋白质的空间组织，LID颗粒是形成调节颗粒的两个亚复合体之一。这些发现推动了我们未来的大部分研究。我们的目标是通过实时记录质谱图，监测单个亚基形成更大的复合体，来跟踪蛋白酶体构建块是如何聚集在一起形成这个大型组装的。其中一个悬而未决的谜团是一种被认为与20世纪90年代的盖子密切相关的复杂的生物作用，被称为信号体。我们的目标是研究这些蛋白质是如何在信号体组装的结构中组织起来的。一旦我们有了这些信息，我们就会比较Sigalosome和LID复合体的整体形状，因为最近的发现表明，它们可以相互替代。为了解决这个问题，我们正在开发一种方法，可以直接在质谱仪中测量离子的截面。我们还计划扩大我们的初步研究，了解19S调控粒子(共18个亚基)的完整亚基安排。揭示组成蛋白质的结构组织也将有助于了解它们的生物学功能。这项研究的主要吸引力之一是我们可以分析直接从细胞中取出的蛋白酶体复合体。我们将使用特定的蛋白质作为‘诱饵’来分离蛋白酶体复合体和瞬时结合的蛋白质。然后，我们将使用质谱仪来鉴定这些相关蛋白质的组成和拷贝数。这些信息将极大地促进了解降解机制是如何受到监管的。

One of the amazing properties of the cell is its ability to regenerate and renew itself. Old and damaged proteins are constantly being degraded and new proteins are continuously prepared. The major pathway for degradation is the proteasome pathway. The proteasome is a very large complex of at least 46 proteins that compose the cellular degradation machinery. It is a crucial machine that breaks down proteins in a specific manner. To prevent the proteasome from destroying the cell or itself, it destroys only proteins that have been tagged for destruction. One tag commonly used is a small protein, ubiquitin. The regulatory particle (19S) of the proteasome recognizes the labelled proteins and acts as a 'gatekeeper' only allowing targeted proteins into the catalytic particle (20S), which rapidly breaks them down. A great level of information has been accumulated over the years about this important pathway, resulting in the 2004 Nobel prize for chemistry. However, much remains to be learned, as studying such a complex system is not a trivial task. Massive heterogeneous and dynamic assemblies pose difficulties for current structural biology methods. In our laboratory, we use unique mass spectrometry approaches to investigate very large protein assemblies. Although proteins within the complex are bound by very delicate interactions, we are able to maintain these complexes in the gas phase and examine extremely large assemblies such as the proteasome with molecular masses well over two million. Recently we have recorded spectra and measured the masses of the proteasome catalytic particle bound to target proteins. To interpret the data is very challenging not only because the mass difference between free and protein bound forms of the complex are relatively small, but also in understanding the information available from the spectra. We were surprised to find that we could define not only the precise number of occupied proteins but also their location within the proteasome inner cavities (figure 2). Another challenging aspect of our research was to gain structural information regarding the regulatory particle. Using our mass spectrometry approach we were able to determine the spatial organization of proteins in the lid particle, one of the two subcomplexes that form the regulatory particle These findings prompt much of our future research. We aim to follow how the proteasome building-blocks come together to form this large assembly by recording mass spectra in real-time, monitoring the formation of larger complexes from the individual subunits. One of the unresolved mysteries is the specific biological role of a complex thought to be closely related to the 19S lid, known as the signalosome. We aim to investigate how the proteins are organized within the structure of the signalosome assembly. Once we have this information we would like to compare the overall shape of the siganalosome and lid complexes, as recent findings suggest that they can substitute for one another. To address this question we are developing methods that can measure the cross section of ions directly in the mass spectrometer. We also plan to extend our initial study and understand the full subunit arrangement of the 19S regulatory particle (a total of 18 subunits). Revealing the structural organization of the constituent proteins will also shed light on their biological functions. One of the major attractions of this research is that we can analyze proteasome complexes that have been taken directly out of the cell. We will use specific proteins as 'baits' to isolate proteasome complexes and transiently associated proteins. We will then use mass spectrometry to identify the composition and number of copies of these associated proteins. This information will greatly facilitate the understanding of how the degradation machinery is regulated.