The roles of the UFM1 post-translational modification in cellular metabolism

项目摘要 细胞使用翻译后修饰(PTM)通过结合和去结合修饰来调节蛋白质功能 以蛋白质为靶标。这些反应的不平衡会导致人类疾病。泛素折叠修饰物1(UFM1)的偶联 蛋白靶标(UFM化)与内质网(ER)的许多细胞过程有关。相比之下， 人们对UFM1去共轭(去UFM化)的作用知之甚少，即从 UFMylated蛋白质。在骨骼和骨质疏松症患者中发现了UFSP2，一种去UFM化酶的缺陷 神经发育障碍。值得注意的是，含有UFSP2缺乏症的患者来源的成纤维细胞显示出过量的 UFM化(超UFM化)与线粒体呼吸和核苷酸代谢缺陷的关系，表明迄今 未描述的UFM化在细胞代谢中的作用。这些发现表明，UFM化是一种新的调节因子 线粒体功能和核苷酸代谢。长期目标是了解UFM化如何调节细胞 新陈代谢。总体目标是了解UFM化调控线粒体的分子机制 呼吸和核苷酸代谢，以及UFSP2介导的去UFM化作用的分子机制。这个 中心假说是，UFSP2缺乏导致(I)线粒体核糖体(有丝分裂糖体)的超UFM化和 电子传输链(ETC)复合体I改变蛋白质的定位和/或功能，导致蛋白质减少 ETC复合体的丰度和/或功能丧失；以及(Ii)核苷酸代谢中酶的超UFM化 途径，导致酶活性的变化和核苷酸代谢的扰动。其基本原理是探查 ETC复合体I和丝氨酸的超超微结构定位和活性测定 羟甲基转移酶-2(SHMT2)，将揭示UFM化如何调节线粒体呼吸和核苷酸 新陈代谢。此外，UFSP2与UFMylated靶的络合物的结构将揭示UFSP2的结构基础 底物识别和去UFM化反应。核心假设将通过追求三个具体目标来检验：1) 确定线粒体和复合体I的超UFMM化如何损害线粒体的呼吸；2)研究 SHMT2超UFM化对核苷酸代谢的影响；3)确定底物的结构基础 人UFSP2的识别和去结合反应。对于第一个目标，蛋白质丰度、定位和酶 线粒体核糖体和ETC复合体I的活性将在患者来源的UFSP2耗尽与 WT UFSP2细胞。对于第二个目标，将使用同位素示踪来评估丝氨酸和核苷酸的代谢率。 合成，而SHMT2的定位和酶活性将在患者来源的UFSP2耗尽与 WT UFSP2细胞。对于第三个目的，将捕获、纯化UFSP2与其与UFM1结合的底物 和结构决定的。提出的这项研究是创新的，因为它将阐明去UFM化和 首次发现UFM化对细胞代谢的影响。拟议的研究具有重要意义，因为它(1) 有助于识别UFMylome，(2)揭示了UFM化是一种新的线粒体呼吸调节因子和 核苷酸代谢，以及(3)揭示了UFSP2底物识别和去结合反应的结构基础。

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 its UFM1-conjugated substrate will be trapped, purified and structure-determined. The research proposed is innovative because it will elucidate the roles of de-UFMylation and the effects of UFMylation on cellular metabolism for the first time. The proposed research is significant because it (1) contributes to the identity of the UFMylome, (2) reveals UFMylation as a novel regulator of mitochondrial respiration and nucleotide metabolism, and (3) reveals the structural basis for substrate recognition and deconjugation reaction of UFSP2.