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方方面面。泛素偶联是 以高度层次化的方式组织，插入了一个约40个E2型泛素结合酶家族 在一个或两个E1型泛素激活酶和数百个E3泛素连接酶之间，后者 负责基片修饰。E2和它们所服务的众多E3的多样性使其具有挑战性 为了找出它们在活细胞中对整个过程的单独贡献：大多数已知的 E2s的特性是基于体外实验。因此，在这项提议中，我将填补我们在知识上的这一空白 并研究E2家族的体内特性。我将应用新的方法来提供E2S-in硫酯连接 标记泛素-进入活细胞，这是一个以前无法实现的目标，以探索它们对整体的贡献 泛素化过程。以转基因小鼠为目的，我将进一步开发一套抗体 将用于控制和指导不同E2在细胞内相互作用的片段，其方式不是 以前是有可能的。因此，我的建议的重点是体内泛素化分析，使用一种新的 开发的工具包将在生物学的其他领域找到应用。单域抗体片段(VHHs或 纳米体)是最小的免疫球蛋白衍生片段，保留了抗原结合特性。目前 他们的生产围绕着使用骆驼作为唯一的重链抗体的自然来源， 然后从那里衍生出VHH。VHH是不寻常的，因为它们可以在细胞质中以单一形式表达 具有保留抗原结合特性的链。因此，它们可以用来扰乱细胞内的蛋白质- 蛋白质之间的相互作用，或强制蛋白质之间的相互作用，而这些相互作用不会自行发生。这些 属性，我将利用这些属性来操纵上面的E2及其参与的反应，特别是在 炎症反应的背景。我建议改造小鼠，使它们能够产生重的 只需链抗体作为VHH的来源，从而消除了免疫的需要，更大和更多 笨重的骆驼、骆驼或羊驼。筛选具有合适特性的抗体片段将使用 最大限度获得单域抗体的细菌双杂交筛选新方法 它可以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如何 (突变；缺失；shRNA；Cas9/CRISPR)可以通过离开目标蛋白质组的方法来补充 原封不动，取而代之的是依赖外来引入的生物制品(VHH或纳米体)作为分子扰动。 VHH作为结晶伴侣的能力确保了这种扰动的后果 可以在分子分辨率上理解，如果不是原子分辨率的话。这个概念显然可以转置到任何复合体中。 感兴趣的路径。这项提议的另一个开创性元素就在这里。

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.