Establishing a structure-function relationship between biomolecular condensates and protein degradation

我们的身体含有大约10万种蛋白质，这些蛋白质基本上能够或调节我们生命所依赖的每一个生化过程。为了使这些蛋白质发挥其正常作用，绝大多数必须保持其可溶性功能状态。为了维持这种健康的平衡，细胞进化出复杂的质量控制网络，识别异常和受损的成分，并摧毁它们。为了实现这一点，细胞需要在空间上组织它们的成分来促进这些特定的反应和过程。相分离，如油与醋混合，是细胞用来将蛋白质和其他生物分子聚集在一起的一种重要的区隔化方法，具有多种功能，包括：1)增加酶反应，2)暂停过程以减轻细胞压力，或3)将成分集中在一起以供细胞废物处理机器吸收。这些液滴含有一系列不同的蛋白质和其他成分，根据它们的生物功能，它们可以是非常流动的，也可以是更像凝胶的成分。在某些疾病的情况下，从液体状态到不可逆的固体结构的进一步组成变化可能对细胞有害。细胞如何控制活细胞内的相边界，它们如何知道何时在正确的时间在正确的位置形成液滴？为了回答这些问题，有必要了解控制液滴组成和特性的因素。在这个项目中，我们建议设计和构建新的液滴形成生物分子，这些生物分子可以：(1)易于操纵，以引入影响液滴物理属性的位点导向变化（即改变液滴的流动性）；(2)以可控的方式特异性地将其他蛋白质招募到液滴中，以评估这些结合伙伴的影响。我们将利用互补的实验技术，在试管和细胞内确定新型液滴的系统变化如何影响结构的形成和溶解。通过结合自噬体形成的识别位点（自噬的关键过程-细胞的废物处理机制），我们将监测液滴结构的变化如何改变细胞处理它们的能力。随着这种结构-功能关系的建立，我们将设计人工相分离分子，可以驱动从细胞中去除任何致病蛋白质，用于治疗帕金森病等疾病。

Our bodies contain some 100,000 proteins that enable or regulate essentially every biochemical process on which our lives depend. For these proteins to perform their normal roles, the vast majority must remain in their soluble functional states. To maintain this healthy balance, cells have evolved intricate quality control networks that identify aberrant and damaged components and destroy them. For this to occur, cells need to spatially organise their components to promote these specific reactions and processes. Phase separation, such as when oil is mixed with vinegar, is an important method of compartmentalisation used by the cell to cluster together proteins and other biomolecules for a variety of functions including to: 1) increase enzyme reactions, 2) suspend processes to alleviate cellular stress, or 3) concentrate components together for uptake by the cell's waste-disposal machinery. These droplets contain an array of different proteins and other components and, depending on their biological function, they can be very fluid in nature or they can have a more gel-like composition. In the case of some diseases, further compositional changes from a liquid-like state to irreversible solid-like structures can be harmful to the cell. How does the cell control the phase boundaries within live cells and how do they know when to form droplets in the right place at the right time? To answer these questions, it is necessary to understand the factors that control the droplet composition and characteristics. In this project we propose to design and build novel liquid-droplet forming biomolecules that can: (1) be easily manipulated to introduce site-directed changes that impact on the droplet's physical attributes (i.e. changing the fluidity of the droplet) and (2) specifically recruiting other proteins to the droplets in a controllable-manner to evaluate the impact of these binding partners. We will determine how systematic changes to the novel liquid-droplets affect formation and dissolution of the structures, both in the test-tube and inside cells using complementary experimental techniques. By incorporating a recognition site for autophagosome formation (a key process in autophagy - the cell's waste-disposal mechanism), we will monitor how the changes to droplet structure change the cell's ability to dispose of them. With this structure-function relationship established, we will design artificial phase-separating molecules that can drive the removal of any disease-causing proteins from the cell for use as therapeutics to treat disorders such as Parkinson's disease.