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

基本信息

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
项目类别：
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

项目摘要

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）通过结合和解偶的修饰符来调节蛋白质功能到蛋白质靶标。这些反应的不平衡导致人类疾病。泛素折叠式修饰符1（UFM1）的结合蛋白质靶标（Ufmylation）已与内质网（ER）的许多细胞过程有关。相比之下，关于UFM1 deNogation（De-Fufmylation）的作用的知之甚少，从而从中删除UFM1的过程 ufmylated蛋白。在骨骼和神经发育障碍。值得注意的是，携带UFSP2缺乏的患者衍生的成纤维细胞显示过多线粒体呼吸和核苷酸代谢中的ufmylation（高氟化）和缺陷 ufmylation在细胞代谢中的未描述的作用。这些发现表明ufmylation是一种新的调节剂线粒体功能和核苷酸代谢。长期目标是了解ufmylation如何调节细胞代谢。总体目的是了解紫外解调节线粒体的分子机制呼吸和核苷酸代谢，以及UFSP2介导的去二酰化的分子机理。这中心假设是UFSP2缺乏原因（i）线粒体核糖体（Moritoribosomes）和电子传输链（ETC）复合物I，改变蛋白质定位和/或功能，导致蛋白质降低 ETC复合物的丰度和/或功能丧失；（ii）核苷酸代谢中酶的过度氟化途径，导致酶活性的变化和核苷酸代谢的扰动。理由是探测高氧化物的定位和测量活性，ETC复合体I和丝氨酸羟甲基转移酶-2（SHMT2）将揭示ufmylation如何调节线粒体呼吸和核苷酸代谢。此外，UFSP2与ufmylated靶标的复合物的结构将揭示结构基础底物识别和脱氧反应。中央假设将通过追求三个具体目标来检验：1）确定地球体和复合物的过度氟化如何损害线粒体呼吸； 2）调查 SHMT2过度氟化对核苷酸代谢的影响； 3）确定底物的结构基础人UFSP2的识别和反应反应。对于第一个目标，蛋白质丰度，定位和酶线粒体核糖体和ETC复合物的活性将在患者衍生的UFSP2耗电中测量 WT UFSP2细胞。对于第二个目标，同位素跟踪将用于评估丝氨酸和核苷酸的代谢速率合成，而SHMT2的定位和酶活性将在患者衍生的UFSP2消耗中进行评估 WT UFSP2细胞。对于第三个目标，将及其UFM1偶联的底物中的UFSP2被困，纯化和结构确定。提出的研究具有创新性，因为它将阐明脱水的作用和紫外线化对细胞代谢的影响首次。拟议的研究很重要，因为它（1）（2）有助于ufmylome的身份，揭示了ufmylation是线粒体呼吸的新型调节剂和核苷酸代谢和（3）揭示了UFSP2的底物识别和解偶反应的结构基础。