Identification of UFM1 candidate targets from mouse brain

Genetic variants that hinder post-translational protein modifications by the ubiquitin-like modifier UFM1 (UFMylation) cause encephalopathies. UFMylation regulates endoplasmic reticulum (ER) homeostasis, but how UFMylation-deficiencies cause selective neurological defects is unknown. In the framework of our studies on two types of UFMylation pathologies, UFM1 loss and expression of a pathogenic UFM1-R81C variant, we conducted a pilot screen for neuronal UFM1 candidate targets in the mouse brain. We used anti-UFM1 antibodies to affinity-purify potentially UFM1-modified proteins from mouse brain and identified them via a gel-based proteomic approach. Apart from free UFM1, UBA5 and UFC1 as major members of the UFMylation machinery were found to be enriched, providing proof-of-concept to our approach. Among the ~300 potentially UFMylated proteins exclusively identified in the anti-UFM1 antibody sample, we found several guanyl-nucleotide exchange factors, including DOCK4 as the most prominent candidate target. While the UFMylation of DOCK4 remains to be established, this candidate is particularly interesting as it is associated with neurodevelopmental delay and microcephaly. Thus, our findings may open a new area of future investigation toward the role UFMylation in neuronal biology and pathology.