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

摘要 泛素-蛋白酶体系统几乎影响真核细胞生物学的各个方面。泛素结合是 以高度分层的方式组织，其中插入了约 40 个 E2 型泛素结合酶家族 一种或两种 E1 型泛素激活酶和数百种 E3 泛素连接酶之间，后者 负责底物修饰。 E2 的多样性以及它们所服务的众多 E3 使其具有挑战性 整理出它们对活细胞整个过程的个人贡献：大部分已知的 E2s 的特性基于体外实验。因此，在本提案中，我将填补我们的知识空白 并研究E2家族的体内特性。我将应用新方法来传递 E2s -硫酯键 标记的泛素-进入活细胞，这是以前无法实现的目标，以探究它们对整体的贡献 泛素化过程。为此目的，我将使用经过基因改造的小鼠，进一步开发一套抗体 将用于控制和指导细胞内不同 E2 相互作用的片段 以前可能。因此，我建议的重点是使用新的方法对泛素化进行体内分析。 开发的工具包将在生物学的其他领域得到应用。单域抗体片段（VHH 或 纳米抗体）是保留抗原结合特性的最小的免疫球蛋白衍生片段。目前 他们的生产围绕使用骆驼科动物作为纯重链抗体的唯一天然来源， 然后从中导出 VHH。 VHH 很不寻常，因为它们可以在细胞质中以单个形式表达 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发生的相互作用。这些 我将利用这些属性来操纵上述 E2 及其参与的反应，特别是在 炎症反应的背景。我建议对小鼠进行基因改造，使其能够产生重质 仅链抗体作为 VHH 的来源，从而消除了对更大和更多的免疫的需要 笨重的美洲驼、骆驼或羊驼。筛选具有合适特性的抗体片段将采用 新型细菌双杂交筛选程序，最大限度地提高获得单域抗体的可能性 可以识别和调节活细胞中的 E2。 虽然我的方法以 E2 为中心，但它将作为标准遗传方法如何进行的示例 （诱变；缺失；shRNA；Cas9/CRISPR）可以通过留下目标蛋白质组的方法进行补充 完整的，而不是依赖外源引入的生物制品（VHH 或纳米抗体）作为分子扰动物。 VHH 作为结晶伴侣的能力确保了这种扰动的后果 即使不是原子分辨率，也可以在分子分辨率上进行理解。这个概念显然可以移植到任何复杂的 兴趣途径。该提案还有一个开创性的元素。

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.