Project Summary Cells use post-translational modifications (PTM) to modulate protein function by conjugating and deconjugating modifiers to protein targets. Imbalance in these reactions leads to human disease. Conjugation of ubiquitin-fold modifier 1 (UFM1) to protein targets (UFMylation) has been linked to many cellular processes at the endoplasmic reticulum (ER). In contrast, much less is known about the roles of UFM1 deconjugation (de-UFMylation), the process of removing UFM1 from UFMylated proteins. Defects in UFSP2, a de-UFMylation enzyme, were identified in patients with skeletal and neurodevelopmental disorders. Notably, patient-derived fibroblasts harboring UFSP2 deficiency show excessive amounts of UFMylation (hyper-UFMylation) and defects in mitochondrial respiration and nucleotide metabolism, indicating as-yet undescribed roles of UFMylation in cellular metabolism. These findings suggest UFMylation as a novel regulator of mitochondrial function and nucleotide metabolism. The long-term goal is to understand how UFMylation regulates cellular metabolism. The overall objective is to understand the molecular mechanism by which UFMylation regulates mitochondrial respiration and nucleotide metabolism, and the molecular mechanism of action of UFSP2-mediated de-UFMylation. The central hypothesis is that UFSP2 deficiency causes (i) hyper-UFMylation of mitochondrial ribosomes (mitoribosomes) and the electron transport chain (ETC) complex I which change protein localization and/or function, leading to decreased protein abundance and/or loss-of-function of ETC complexes; and (ii) hyper-UFMylation of enzymes in nucleotide metabolic pathways, leading to changes in enzyme activity and perturbation of nucleotide metabolism. The rationale is that probing localization and measuring activity of the hyper-UFMylated mitoribosomes, the ETC Complex I and serine hydroxymethyltransferase-2 (SHMT2), will reveal how UFMylation regulates mitochondrial respiration and nucleotide metabolism. In addition, the structure of UFSP2 in complex with UFMylated targets will reveal the structural basis for substrate recognition and de-UFMylation reaction. The central hypothesis will be tested by pursuing three specific aims: 1) Determine how hyper-UFMylation of mitoribosomes and Complex I impairs mitochondrial respiration; 2) Investigate the effect of hyper-UFMylation of SHMT2 on nucleotide metabolism; and 3) Determine the structural basis for substrate recognition and deconjugation reaction of human UFSP2. For the first aim, protein abundance, localization and enzyme activity of mitochondrial ribosomes and the ETC Complex I will be measured in patient-derived UFSP2-depleted versus WT UFSP2 cells. For the second aim, isotope tracing will be used to evaluate metabolic rates of serine and nucleotide synthesis, while the localization and enzyme activity of SHMT2 will be assessed in patient-derived UFSP2-depleted versus WT UFSP2 cells. For the third aim, the UFSP2 in complex with...