Optimization of protein UFMylation modification method and its application in substrate identification in human cells

UFMylation plays an essential role in regulating intracellular physiological and pathological processes. Accumulating evidence has demonstrated that dysregulation of UFMylation is closely associated with the progression of various diseases, including cancers and developmental disorders. However, efficient and specific methods for detecting UFMylated substrate proteins remain challenging. In this study, we generated the UFM1-specific protease 1/2 knockout (UFSP1^KO/UFSP2^KO) human embryonic kidney 293T cell line and validated it as the most suitable cellular model for the screening, identification, and confirmation of UFMylated substrate proteins. Furthermore, exogenous expression of the E3 ligase components, UFM1-specific ligase 1 (UFL1) and DDRGK domain-containing protein 1 (DDRGK1), significantly enhanced protein UFMylation levels, enabling the large-scale enrichment of UFMylated substrates. Collectively, we have established a more efficient, specific, and reliable method for UFMylation substrate research, facilitating in-depth investigation into its biological functions and regulatory mechanisms.