Imposing order on the family of ubiquitin-conjugating (E2) enzymes through intracellular perturbation with nanobodies

摘要 泛素-蛋白酶体系统影响着真核细胞生物学的几乎每一个方面。泛素缀合是 以高度层次化的方式组织，其中插入了约40个E2型泛素结合酶家族 一个或两个E1型泛素激活酶和数以百计的E3泛素连接酶之间，后者 负责基板改性。E2的多样性和它们所服务的许多E3使其具有挑战性 来整理它们对活细胞中整个过程的贡献：大多数已知的 E2s的特性是基于体外实验。因此，在本建议中，我将填补我们知识上的这一空白 并研究E2家族的体内特性。我将应用新的方法来递送E2s-in硫酯键， 一个标记的泛素-进入活细胞，一个目标，以前没有达到，以探测他们的贡献，整体 泛素化的过程。以转基因小鼠为目的，我将进一步开发一套抗体 这些片段将用于控制和指导细胞内不同E2的相互作用， 以前可能的。因此，我的建议的重点是在体内分析泛素化，使用新的 开发的工具包将在生物学的其他领域找到应用。单结构域抗体片段（VHH或 纳米抗体）是保留抗原结合特性的最小免疫球蛋白衍生片段。目前 它们的生产围绕着使用骆驼科动物作为仅有重链的抗体的唯一天然来源， 然后从中推导出VHH。VHH是不寻常的，因为它们可以在胞质溶胶中表达为单一的VHH。 保留抗原结合特性的链。因此，它们可用于破坏细胞内蛋白质- 蛋白质相互作用，或加强蛋白质之间不会自行雅阁发生的相互作用。这些 我将利用这些性质来操纵上述E2和它们参与的反应，特别是在 炎症反应的背景。我建议改造老鼠，使它们能够产生重 链抗体作为VHH的来源，从而消除了对较大和较大的免疫的需要。 笨重的美洲驼骆驼或羊驼筛选具有合适性质的抗体片段将采用 最大化获得单结构域抗体的可能性的新型细菌双杂交筛选方法 能够识别和调节活细胞中的E2。 虽然我的方法是以E2为中心的，但它将作为标准遗传方法的一个例子， （诱变;缺失; shRNA; Cas9/CRISPR）可以通过留下靶蛋白质组的方法来补充。 完整的，而不是依赖于外源引入的生物制剂（VHH或纳米抗体）作为分子扰动剂。 VHH作为结晶伴侣的能力确保了这种扰动的后果 即使不是原子级的，也可以用分子级的分辨率来理解。这个概念显然可以转置到任何复形 兴趣之路。这就是这项建议的另一个开拓性因素。

Abstract The ubiquitin-proteasome system affects almost every aspect of eukaryotic cell biology. Ubiquitin conjugation is organized in a highly hierarchical manner, with a family of ~40 E2-type ubiquitin conjugating enzymes interposed between one or two E1-type ubiquitin-activating enzymes and literally hundreds of E3 ubiquitin ligases, the latter charged with substrate modification. The multiplicity of E2s and the many E3s they serve has made it challenging to sort out their individual contributions to the overall process in living cells: most of what is known for the properties of E2s is based on in vitro experiments. In this proposal I will therefore fill this gap in our knowledge and study the in vivo properties of the E2 family. I will apply new methods to deliver E2s -in thioester linkage with a tagged ubiquitin- into living cells, a goal not previously within reach, to probe their contributions to the overall process of ubiquitylation. Using mice genetically modified for the purpose, I will further develop a suite of antibody fragments that will be used to control and direct interactions of the different E2s intracellularly in ways not previously possible. The focus of my proposal is thus on an in vivo analysis of ubiquitylation, using a newly developed toolkit that will find application in other areas of biology. Single domain antibody fragments (VHHs or nanobodies) are the smallest immunoglobulin-derived fragments that retain antigen binding properties. Currently their production revolves around the use of camelids as the only natural source of heavy chain-only antibodies, from which VHHs are then derived. VHHs are unusual, in that they can be expressed in the cytosol as single chains with retention of antigen binding properties. Consequently, they can be used to disrupt intracellular protein- protein interactions, or to enforce interactions between proteins that would not occur on their own accord. These properties I will exploit to manipulate the above E2s and the reactions in which they participate, especially in the context of the inflammatory response. I propose to engineer mice such that they are capable of producing heavy chain only antibodies as a source of VHHs, thus eliminating the need for immunization of the larger and more cumbersome llamas, camels or alpacas. Screening for antibody fragments with suitable properties will employ a novel bacterial two-hybrid screening procedure to maximize the likelihood of obtaining single domain antibodies that can recognize and modulate E2s in living cells. While my approach is centered on E2s, it will serve as an example of how standard genetic approaches (mutagenesis; deletion; shRNA; Cas9/CRISPR) can be complemented by methods that leave the target proteome intact, instead relying on exogenously introduced biologicals (VHHs or nanobodies) as molecular perturbants. The ability of VHHs to serve as crystallization chaperones ensures that the consequences of such perturbations can be understood at molecular, if not atomic resolution. This concept is obviously transposable to any complex pathway of interest. Therein lies an additional pioneering element of this proposal.