Anti-UFM1 monoclonal antibody Made to order

The covalent conjugation of ubiquitin-fold modifier 1 (UFM1) to proteins generates a signal that regulates transcription, response to cell stress, and differentiation. Ufmylation is initiated by ubiquitin-like modifier activating enzyme 5 (UBA5), which activates and transfers UFM1 to ubiquitin-fold modifier-conjugating enzyme 1 (UFC1). The details of the interaction between UFM1 and UBA5 required for UFM1 activation and its downstream transfer are however unclear. In this study, we described and characterized a combined linear LC3-interacting region/UFM1-interacting motif (LIR/UFIM) within the C terminus of UBA5. This single motif ensures that UBA5 binds both UFM1 and light chain 3/gamma-aminobutyric acid receptor-associated proteins (LC3/GABARAP), two ubiquitin (Ub)-like proteins. We demonstrated that LIR/UFIM is required for the full biological activity of UBA5 and for the effective transfer of UFM1 onto UFC1 and a downstream protein substrate both in vitro and in cells. Taken together, our study provides important structural and functional insights into the interaction between UBA5 and Ub-like modifiers, improving the understanding of the biology of the ufmylation pathway.

Ubiquitin-like proteins are conserved in eukaryotes and involved in numerous cellular processes. Ufm1 is proved to play important roles in endoplasmic reticulum homeostasis, vesicle transportation and embryonic development. Enzyme cascade of Ufm1 is similar to that of ubiquitin. Mature Ufm1 is activated and conjugated to substrates by assistance of Ufm1 activating enzyme Uba5 (E1), Ufm1 conjugating enzyme Ufc1 (E2), and Ufm1 ligating enzyme Ufl1 (E3). Here, we determined the solution structure of TbUfm1 from Trypanosoma brucei (T. brucei) by NMR spectroscopy and explored the interactions between TbUfm1 and TbUba5/TbUfc1/TbUfl1. TbUfm1 adopts a typical beta-grasp fold, which partially wraps a central alpha-helix and the other two helixes. NMR chemical shift perturbation indicated that TbUfm1 interacts with TbUba5 via a hydrophobic pocket formed by alpha 1 alpha 2 beta 1 beta 2. Although the structure and Uba5-interaction mode of TbUfm1 are conserved in Ufm1 proteins, there are also some differences, which might reflect the potential diversity of Ufm1 in evolution and biological functions.