# Imposing order on the family of ubiquitin-conjugating (E2) enzymes through intracellular perturbation with nanobodies > **NIH NIH DP1** · BOSTON CHILDREN'S HOSPITAL · 2020 · $1,239,000 ## Abstract 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 unders... ## Key facts - **NIH application ID:** 10002176 - **Project number:** 5DP1AI150593-02 - **Recipient organization:** BOSTON CHILDREN'S HOSPITAL - **Principal Investigator:** Hidde L. Ploegh - **Activity code:** DP1 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2020 - **Award amount:** $1,239,000 - **Award type:** 5 - **Project period:** 2019-09-30 → 2024-07-31 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10002176 ## Citation > US National Institutes of Health, RePORTER application 10002176, Imposing order on the family of ubiquitin-conjugating (E2) enzymes through intracellular perturbation with nanobodies (5DP1AI150593-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10002176. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*