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

UFM1翻译后修饰在细胞代谢中的作用

• 批准号: 10722954
• 负责人: Phong Thanh Nguyen
• 金额: $ 12.5万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10722954
• 关键词: Biological; Biological Process; C-terminal; Cell Respiration; Cell physiology; Cells; Child; Complex; Defect; Disease; Electron Transport; Endoplasmic Reticulum; Enzymes; Fibroblasts; Foundations; Genetic; Glucose; Glycine Hydroxymethyltransferase; Goals; Homeostasis; Human; Impairment; Isotope Labeling; Isotopes; Link; Measures; Mediating; Metabolic Diseases; Metabolic Pathway; Metabolism; Mission; Mitochondria; Mitochondrial Diseases; Mitochondrial Proteins; Modification; Molecular; Molecular Mechanisms of Action; N-terminal; National Institute of General Medical Sciences; Neurodevelopmental Disability; Neurodevelopmental Disorder; Nucleotide Biosynthesis; Nucleotides; Oxidative Phosphorylation; Oxidative Phosphorylation Deficiency; Pathway interactions; Patients; Post-Translational Protein Processing; Process; Protein Biosynthesis; Proteins; Public Health; Reaction; Reporting; Research; Research Personnel; Respiration; Ribosomes; Role; Serine; Structure; Testing; Time; Ubiquitin; Ubiquitination; Variant; Work; career; enzyme activity; human disease; innovation; loss of function; metabolic rate; metabolomics; mitochondrial dysfunction; novel; nucleotide metabolism; protein complex; protein function; respiratory; skeletal disorder; skills

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.

项目摘要 细胞使用翻译后修饰（PTM）通过缀合和解缀合修饰剂来调节蛋白质功能 蛋白质靶点。这些反应的不平衡导致人类疾病。泛素折叠修饰物1（UFM 1）的缀合 与蛋白质靶点的结合（UFM化）与内质网（ER）的许多细胞过程有关。与此相反， 关于UFM 1去缀合（de-UFMylation）的作用知之甚少， UFMylated蛋白质。UFSP 2缺陷，一种去UFM化酶，在骨骼和骨骼肌疾病患者中被发现。 神经发育障碍值得注意的是，携带UFSP 2缺陷的患者来源的成纤维细胞显示出过量的UFSP 2。 UFMylation（hyper-UFMylation）和线粒体呼吸和核苷酸代谢缺陷，表明迄今为止， UFM化在细胞代谢中的未描述的作用。这些发现表明UFM化作为一种新的调节剂， 线粒体功能和核苷酸代谢。长期的目标是了解UFM化如何调节细胞内 新陈代谢.总体目标是了解UFM化调节线粒体的分子机制， 呼吸和核苷酸代谢，以及UFSP 2介导的去UFM作用的分子机制。的 中心假设是UFSP 2缺乏导致（i）线粒体核糖体（mitoribosomes）的超UFM化， 电子传递链（ETC）复合物I改变蛋白定位和/或功能，导致蛋白质减少 ETC复合物的丰度和/或功能丧失;和（ii）核苷酸代谢中的酶的超UFM化 途径，导致酶活性的变化和核苷酸代谢的扰动。理由是， 定位和测量超UFMylated线粒体、ETC复合物I和丝氨酸蛋白酶的活性 羟甲基转移酶-2（SHMT 2），将揭示UFM化如何调节线粒体呼吸和核苷酸 新陈代谢.此外，UFSP 2与UFMylated靶标复合的结构将揭示UFSP 2的结构基础。 底物识别和去UFM化反应。中心假设将通过追求三个具体目标来检验：1） 确定线粒体和复合物I的超UFM化如何损害线粒体呼吸; 2）研究线粒体呼吸的机制。 SHMT 2的超UFM化对核苷酸代谢的影响;和3）确定底物的结构基础 人UFSP 2的识别和解缀合反应。对于第一个目标，蛋白质丰度，定位和酶 线粒体核糖体和ETC复合物I的活性将在患者来源的UFSP 2缺失的对照组中测量。 WT UFSP 2细胞。第二个目标是利用同位素示踪技术来评价丝氨酸和核苷酸的代谢率 SHMT 2的定位和酶活性将在患者来源的UFSP 2缺失与UFSP 2合成中进行评估。 WT UFSP 2细胞。对于第三个目的，将捕获、纯化与其UFM 1缀合的底物复合的UFSP 2 并且由结构决定。这项研究是创新的，因为它将阐明去UFM化和 UFMylation对细胞代谢的影响。这项研究的意义在于：（1） 有助于UFMylome的身份，（2）揭示UFMylation作为线粒体呼吸的新调节因子， 核苷酸代谢，和（3）揭示了底物识别和解偶联反应的结构基础。