Unlocking the therapeutic potential of E3 ubiquitin ligases through structure-function studies and Cryo-EM

我们体内的所有细胞都需要丢弃废物，如受损或不再需要的蛋白质，以确保细胞保持健康和功能。小分子蛋白泛素在此过程中起着识别标签的作用。泛素是最通用的后修饰，它可以作为蛋白质上的单个部分存在，这在蛋白质运输和胞内吞作用中很重要，但也作为泛素链存在。直到最近十年，我们才开始认识到泛素系统的复杂性。例如，我们现在知道，泛素可以利用它的7个赖氨酸残基中的任何一个，或者它的N-末端Met，将两条泛素链组装在一起形成链。因此，在酵母和人类细胞中发现了多达8种蛋白质连接类型（Met 1、K6、K11、K27、K29、K33、K48和K63），其中E3泛素连接酶通过介导泛素转移到蛋白质底物上，在蛋白质泛素化过程中起着关键作用。根据加入的泛素信号的类型，这可以触发泛素-蛋白酶体系统的抑制。因此，在理论上可以通过特异性E3的连接酶活性的靶向抑制来实现预防与年龄相关的疾病中的重要蛋白质的降解，而不是抑制蛋白酶体的当前和一般/非特异性方法。E3 HECT连接酶是用于药物发现的非常好的候选者，因为它们具有基于Cys的酶活性。尽管有越来越多的证据表明这些大酶对人类健康的重要性，但这些大酶有限的结构信息和生物化学知识阻碍了它们作为治疗靶点的潜力。学生将进行E3 HECT泛素连接酶的结构-功能研究。学生将接受蛋白质表达和纯化、泛素化分析、生物制药技术、蛋白质晶体学和最先进的Cryo-EM的培训。一个重要的目标将是建立在真核系统中表达和纯化全长HECT E3酶的策略，用于Cryo-EM研究和未来的药物发现项目。该项目将受益于与蛋白质组学和化学生物学专家的持续国际合作，以及巴斯和布里斯托的支持和动态研究环境。由于我们在蛋白质泛素化，结构-功能研究和Cryo-EM的综合专业知识，学生将处于理想的位置，对泛素领域产生影响。

All cells within our body need to discard waste material such as damaged or no longer wanted proteinsto ensure that cells remain healthy and functional. The small protein ubiquitin is essential in thisprocess by functioning as an identification tag. Ubiquitin is the most versatile post-translationalmodification, it can exist as a single moiety on proteins, and this is important in protein trafficking andendocytosis, but also as a ubiquitin chain. It is only in the last decade that we have come to appreciatethe complexity of the ubiquitin system. We now know for instance that ubiquitin can use any of itsseven lysine residues, or its N-terminal Met, to assemble two ubiquitin chains together in order toform chains. Therefore, as many as eight linkage types have been found on proteins (i.e. Met1, K6,K11, K27, K29, K33, K48 and K63) in yeast and human cells.E3 ubiquitin ligases plays a key role in protein ubiquitination, by mediating the transfer of ubiquitinonto protein substrates. Depending on the type of ubiquitin signal added, this can trigger recognitionby the Ubiquitin-Proteasome System. Therefore, preventing the degradation of important proteins inage-related diseases could in theory be achieved through the targeted inhibition of the ligase activityof specific E3s, rather than the current and general/unspecific approach of inhibiting the proteasome.E3 HECT ligases are very good candidates for drug discovery given their Cys-based enzyme activity.However, the limited structural information and biochemical knowledge of these large enzymes hashindered their potential as therapeutic targets, despite mounting evidence for their importance inhuman health.In this project, the student will undertake structure-function studies of E3 HECT ubiquitin ligases. Thestudent will be trained in protein expression and purification, ubiquitination assays, biophysicaltechniques, protein crystallography and state-of-the-art Cryo-EM. An important goal will be to alsoestablish strategies for the expression and purification of full-length HECT E3 enzymes in eukaryoticsystems, for Cryo-EM studies and future drug discovery projects. This project will benefit from ongoinginternational collaboration with experts in proteomics and chemical biology as well as supportive anddynamic research environments at Bath and Bristol. Because of our combined expertise in proteinubiquitination, structure-function studies and Cryo-EM, the student will be in an ideal position to makean impact on the ubiquitin field.