Deciphering the mechanism of the human proteasome by mass photometry

在每个真核细胞中，蛋白酶体是负责多肽降解的重要分子机器。它将蛋白质循环成可重复使用的氨基酸，并防止多肽积累成有毒的聚集体。为此，蛋白酶体识别被泛素标记的蛋白质，并将它们拉入催化腔进行降解。蛋白酶体是许多调节途径的中心部分，因为它有助于调节蛋白质的相对浓度。这使得它成为一个理想的靶标，用于治疗由神经退行性疾病或癌症引起的蛋白质降解失调的细胞。对于靶向药物设计，深入了解泛素与蛋白酶体识别位点之间的相互作用以及蛋白酶体的催化作用是必不可少的。虽然人类26S蛋白酶体已经被研究了几十年，但动力学数据几乎无法获得，并且需要大量不同技术的合并结果。因此，对底物识别、结合和靶向降解的全面理解仍然难以捉摸。这是由于主要的批量实验是可用的，这不允许跟踪26S蛋白酶体的复杂催化过程。在描述的研究行动中，我将利用一种新的技术，它可以检测单分子散射的光，而不需要引入人工标记：干涉散射显微镜（iSCAT）。当与合适的校准相结合时，这种被称为质量光度法（MP）的技术能够在几秒钟内以高达数十kDa的精度测定蛋白质的质量。质谱法可以让我研究底物和26S蛋白酶体之间的相互作用。我的目标是分析底物与蛋白酶体的结合，以及基于泛素标签质量的单分子分析中去泛素化的下游处理步骤以及蛋白质水解。首先，我将通过获得泛素化底物和蛋白酶体的平衡质量分布，应用MP来确定平均结合亲和力和去泛素化/蛋白质水解速率。在第二组实验中，在快速混合后，将测量溶液中蛋白质复合物的质量分布作为时间的函数。这将为结合后的过程（去泛素化和蛋白质水解）提供单独的动力学信息。第三，我将蛋白酶体固定在一个界面上，并在上面的溶液中提供底物。这将允许在一致的实验设计中在分子水平上实时跟踪完整的催化循环。所描述的实验将对我们对蛋白酶体降解过程的理解产生直接影响：对整个催化循环的详细机制和动力学描述。这将激发神经退行性和癌症研究中药物设计的新概念。

In every eukaryotic cell, the proteasome is a vital molecular machine responsible for degradation of polypeptides. It recycles proteins into reusable amino acids and prevents accumulation of peptides into toxic aggregates. For this, the proteasome recognizes proteins flagged by a ubiquitin tag and pulls them in its catalytic lumen for degradation. The proteasome is a central part of many regulatory pathways as it facilitates adjusting the relative concentrations of proteins. This renders it an ideal target for drugs addressing cells which suffer from miss-regulated protein degradation, e.g. caused by neurodegenerative diseases or cancer.For targeted drug design, a profound understanding of the interaction between ubiquitin and the proteasomal recognition sites as well as the proteasome’s catalytic action is essential. Although the human 26S proteasome is being studied for several decades, kinetic data is barely available and requires merging results of an immense amount of different techniques. Consequently, a comprehensive understanding of substrate recognition, binding, and targeted degradation remains elusive. This is due to the fact that mainly bulk experiments are available, which do not allow tracking the complex catalytic proceedings of the 26S proteasome.In the described research action, I will make use of a novel technique, which detects light scattered by single molecules without the need for introducing artificial labels: interferometric scattering microscopy (iSCAT). When combined with a suitable calibration, this technique –termed mass photometry (MP)– enables determining the mass of proteins at a precision of up to tens of kDa and within few seconds.Mass photometry will allow me to study the interaction between substrates and the 26S proteasome. I aim to analyze binding of substrates to the proteasome and the downstream processing steps of deubiquitylation as well as proteolysis in a single-molecule-based assay depending on the quality of the ubiquitin tag. First, I will apply MP to determine the average binding affinities and deubiquitylation/proteolysis rates by acquiring equilibrium mass distributions of ubiquitylated substrates and proteasomes. In a second set of experiments, the mass distribution of the protein complexes in solution will be measured as a function of time after rapid mixing. This will yield separate kinetic information for the processes after binding (deubiquitylation and proteolysis). Thirdly, I will immobilize the proteasome on an interface and supply substrates in the solution on top. This will allow following the complete catalytic cycle in real-time on a molecular level within a consistent experimental design.The described experiments will have an immediate impact on our understanding of proteasomal degradation processes: a detailed mechanistic and kinetic description of the whole catalytic cycle. This will stimulate novel concepts for the design of drugs in neurodegenerative and cancer research.