Metabolic regulation of KLHL proteins through O-glycosylation

通过 O-糖基化调节 KLHL 蛋白的代谢

• 批准号: 10380171
• 负责人: MICHAEL S BOYCE
• 金额: $ 52.7万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-15 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10380171
• 关键词: Address; Affect; Antioxidants; Binding; Biochemical; CUL3 gene; Cell Nucleus; Cells; Characteristics; Chemicals; Complex; Cysteine; Cytoplasmic Protein; Data; Disease; Family; Family member; Fibroblasts; Genes; Genetic Transcription; Health; Homeostasis; Human; Impairment; Intermediate Filament Proteins; Intermediate Filaments; Lead; Link; Lung; Malignant Neoplasms; Mammals; Maps; Mass Spectrum Analysis; Mediating; Metabolic; Metabolic stress; Metabolism; Methods; Modeling; Modification; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nuclear Proteins; Nutrient; Ovarian; Oxidants; Oxidation-Reduction; Oxidative Stress; Pathway interactions; Patients; Phenotype; Physiology; Post-Translational Protein Processing; Protein Family; Protein Glycosylation; Proteins; Proteolysis; Proteomics; RBX1 gene; Regulation; Resistance; Role; Signal Transduction; Site; Starvation; Stress; Substrate Interaction; Substrate Specificity; Testing; Tissues; Ubiquitination; Work; base; detection of nutrient; disability; disease phenotype; experimental study; giant axonal neuropathy; gigaxonin; glycosylation; human disease; human tissue; in vivo; insight; loss of function; member; mortality; novel; novel strategies; novel therapeutics; nutrient deprivation; prevent; protein degradation; proteostasis; response; sensor; sugar; toxicant; ubiquitin-protein ligase

Project Summary/Abstract The Kelch-like (KLHL) family of proteins are critical substrate adaptors for E3 ubiquitin ligases and mutations in KLHL genes underlie a range of human diseases, including cancer and neurodegeneration. However, the upstream regulation and downstream effects of most KLHL proteins remain poorly understood. The best-characterized KLHL protein is KEAP1, which binds to the CUL3/RBX1 E3 ligase components to mediate the ubiquitination and destruction of NRF2, a master transcriptional regulator of redox stress signaling. Redox-active compounds disable KEAP1, preventing the ubiquitination of NRF2 by KEAP1/CUL3/RBX1 and allowing NRF2 accumulation and activation. Recently, we demonstrated that KEAP1 is modified by O-GlcNAc, a nutrient-sensitive, reversible form of glycosylation, and this modification is required for efficient NRF2 ubiquitination and degradation. Blockade of KEAP1 O-GlcNAcylation by chemical inhibition or nutrient deprivation stabilizes and activates NRF2, revealing a new regulatory link between nutrient sensing and redox stress signaling. Interestingly, we recently discovered that the KLHL protein gigaxonin is also O-GlcNAcylated in a nutrient-dependent manner, indicating that glycosylation of KLHL proteins may be a general regulatory link between metabolic status and proteostasis. Based on these results, we hypothesize that nutrient-sensitive O-GlcNAcylation of KLHL proteins regulates proteostasis in response to a range of nutrient levels and metabolic stresses. To test this hypothesis, we will first exploit KEAP1 as a model KLHL protein to determine how various stresses impact KEAP1 glycosylation and activity. Next, we will determine how KEAP1 glycosylation affects the KEAP1/CUL3/RBX1 complex and KEAP1-substrate interactions, using both targeted biochemical and unbiased proteomic approaches. We will then determine the functional role of O-GlcNAcylation on gigaxonin, whose mutations cause the fatal human disease giant axon neuropathy (GAN). Finally, we will elucidate the relationship between gigaxonin glycosylation and GAN disease phenotypes using murine neuronal and human GAN patient fibroblast models. The successful completion of our project will provide broad and significant insights into KLHL protein regulation and function, and may point to new therapeutic opportunities in neurodegeneration, cancer and other diseases by manipulating KLHL proteins through O- GlcNAc modulation.

项目概要/摘要 Kelch 样 (KLHL) 蛋白家族是 E3 泛素的关键底物接头 KLHL 基因的连接酶和突变是一系列人类疾病的基础，包括癌症和 神经变性。然而，大多数KLHL的上游监管和下游影响 蛋白质仍然知之甚少。最具特征的 KLHL 蛋白是 KEAP1，它结合 与 CUL3/RBX1 E3 连接酶成分介导 NRF2 的泛素化和破坏， 氧化还原应激信号的主要转录调节因子。氧化还原活性化合物禁用 KEAP1，防止 KEAP1/CUL3/RBX1 对 NRF2 泛素化并允许 NRF2 积累和激活。最近，我们证明了 KEAP1 是由 O-GlcNAc 修饰的，O-GlcNAc 是一种 营养敏感的、可逆的糖基化形式，并且这种修饰是有效的 NRF2 泛素化和降解。通过化学抑制阻断 KEAP1 O-GlcNAc 酰化 或营养剥夺稳定并激活 NRF2，揭示了 NRF2 之间新的调控联系 营养传感和氧化还原应激信号。有趣的是，我们最近发现 KLHL 蛋白质 gigaxonin 也以营养依赖性方式被 O-GlcNA 酰化，表明 KLHL 蛋白的糖基化可能是代谢状态和代谢状态之间的一般调节联系。 蛋白质稳态。基于这些结果，我们假设营养敏感的 O-GlcNAc 酰化 KLHL 蛋白根据一系列营养水平和代谢调节蛋白质稳态 压力。为了检验这个假设，我们首先利用 KEAP1 作为 KLHL 蛋白模型来确定 各种应激如何影响 KEAP1 糖基化和活性。接下来，我们将确定如何 KEAP1 糖基化影响 KEAP1/CUL3/RBX1 复合物和 KEAP1-底物相互作用， 使用靶向生化和无偏见的蛋白质组学方法。然后我们将确定 O-GlcNAcylation 对 gigaxonin 的功能作用，其突变导致致命的人类疾病 巨轴突神经病（GAN）。最后，我们来阐明一下gigaxonin之间的关系 使用鼠神经元和人类 GAN 患者的糖基化和 GAN 疾病表型 成纤维细胞模型。我们项目的成功完成将为我们提供广泛而重要的 对 KLHL 蛋白调节和功能的见解，可能会带来新的治疗机会 通过 O- 操纵 KLHL 蛋白来治疗神经退行性疾病、癌症和其他疾病 GlcNAc 调节。