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

通过纳米抗体的细胞内扰动对泛素结合 (E2) 酶家族施加顺序

• 批准号: 10208670
• 负责人: Hidde L. Ploegh
• 金额: $ 123.9万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-30 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10208670
• 关键词: Affect; Alpaca; Antibodies; Area; Biological; Biology; CRISPR/Cas technology; Camels; Cells; Cellular biology; Charge; Complement; Complex; Crystallization; Cytosol; Elements; Engineering; Ensure; Enzymes; Eukaryotic Cell; Family; Goals; Immunization; Immunoglobulin Fragments; Immunoglobulins; In Vitro; Individual; Inflammatory Response; Internet; Knowledge; Llama; Methods; Modification; Molecular; Molecular Chaperones; Mus; Mutagenesis; Pathway interactions; Process; Production; Property; Proteins; Proteome; Reaction; Resolution; Screening procedure; Source; System; Ubiquitin; Ubiquitin family; Ubiquitin-Activating Enzymes; Ubiquitin-Conjugating Enzymes; Ubiquitination; antigen binding; base; experimental study; genetic approach; in vivo; interest; mouse model; multicatalytic endopeptidase complex; nanobodies; novel; protein protein interaction; screening; small hairpin RNA; thioester; tool; ubiquitin ligase; ubiquitin-protein ligase; yeast two hybrid system

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.

抽象的 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，一个约40 E2型泛素偶联酶的家族 在一到两个E1型泛素激活酶和数百个E3泛素连接酶之间，后者 负责底物修改。 E2的多重性和他们所提供的许多E3使它具有挑战性 弄清他们对活细胞整体过程的个人贡献：大多数已知的 E2S的性质基于体外实验。因此，在这个建议中，我将在我们的知识中填补这一空白 并研究E2家族的体内特性。我将采用新方法来交付e2s -in thioester连接 标记的泛素进入活细胞，这是一个以前不在触及的目标，以探究其对整体的贡献 泛素化过程。使用基因修饰的小鼠为此目的，我将进一步开发一套抗体 片段将用于控制和直接以细胞内的方式控制和直接相互作用 以前可能。因此，我的建议的重点是使用新的体内泛素化分析 开发的工具包将在其他生物学领域找到应用。单域抗体片段（VHHS或 纳米型）是保留抗原结合特性的最小免疫球蛋白衍生的片段。现在 他们的生产围绕使用骆驼作为重链抗体的唯一天然来源， 然后从中得出VHH。 VHHS是不寻常的，因为它们可以在细胞质中以单个 具有抗原结合特性的链。因此，它们可用于破坏细胞内蛋白质 蛋白质相互作用，或者是为了实现不会自行发生的蛋白质之间的相互作用。这些 我将利用属性来操纵上述E2及其参与的反应，尤其是在 炎症反应的背景。我建议对老鼠进行工程，使它们能够产生重量 仅链抗体作为VHH的来源，从而消除了较大和更多的免疫的需求 繁琐的美洲驼，骆驼或羊驼。筛选具有合适特性的抗体片段将采用 新型细菌两杂化筛选程序，以最大程度地获得单域抗体的可能性 可以识别和调节活细胞中的E2。 尽管我的方法以E2为中心，但它将作为标准遗传方法的一个例子 （诱变；删除； shrna; cas9/crispr）可以通过离开靶蛋白质组的方法来补充 完整地依靠外源引入的生物学（VHHS或纳米化）作为分子扰动。 VHHs充当结晶伴侣的能力确保了这种扰动的后果 可以在分子（即使不是原子分辨率）上理解。这个概念显然可以转座到任何复杂 感兴趣的途径。这是该提案的附加开创性要素。