HypE-specific Nanobodies as Tools to Modulate HypE-mediated Target AMPylation |
| |
Authors: | Matthias C. Truttmann Qin Wu Sarah Stiegeler Joao N. Duarte Jessica Ingram Hidde L. Ploegh |
| |
Affiliation: | From the ‡Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142 and ;the §Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 |
| |
Abstract: | The covalent addition of mono-AMP to target proteins (AMPylation) by Fic domain-containing proteins is a poorly understood, yet highly conserved post-translational modification. Here, we describe the generation, evaluation, and application of four HypE-specific nanobodies: three that inhibit HypE-mediated target AMPylation in vitro and one that acts as an activator. All heavy chain-only antibody variable domains bind HypE when expressed as GFP fusions in intact cells. We observed localization of HypE at the nuclear envelope and further identified histones H2–H4, but not H1, as novel in vitro targets of the human Fic protein. Its role in histone modification provides a possible link between AMPylation and regulation of gene expression. |
| |
Keywords: | Histone Modification Nuclear Membrane Phage Display Post-translational Modification (PTM) Recombinant Protein Expression AMPylation Fic Proteins HypE VHHs |
|
|