Metabolic regulation of KLHL proteins through O-glycosylation

项目摘要/摘要 Kelch样蛋白(KLHL)家族是E3泛素的关键底物适配子 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 神经退行性变。然而，大多数KLHL的上游调节和下游影响 蛋白质仍然知之甚少。特征最好的KLHL蛋白是Keap1，它与 CUL3/RBX1 E3连接酶组分介导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，阻止Keap1/CUL3/RBX1泛素化NRF2，允许NRF2 积累和激活。最近，我们证明了Keap1被O-GlcNAc，a 营养敏感、可逆的糖基化形式，这种修饰是有效的 NRF2泛素化和降解。化学抑制法阻断Keap1 O-GlcN酰化 或者，营养剥夺稳定并激活了NRF2，揭示了 营养感应和氧化还原胁迫信号。有趣的是，我们最近发现KLHL 蛋白质Gigaxin也以一种营养依赖的方式O-GlcN酰化，这表明 KLHL蛋白的糖基化可能是代谢状态和 蛋白质平衡。基于这些结果，我们假设营养敏感的O-GlcN酰化 KLHL蛋白调节蛋白平衡以响应一系列营养水平和代谢 压力。为了验证这一假设，我们将首先使用Keap1作为模型KLHL蛋白质来确定 不同的压力如何影响Keap1的糖基化和活性。接下来，我们将确定如何 Keap1糖基化影响Keap1/CUL3/RBX1复合体和Keap1与底物的相互作用， 使用有针对性的生化和无偏见的蛋白质组学方法。然后我们将确定 O-GlcN酰化对导致人类致命性疾病的巨细胞黄素的功能作用 巨大轴索神经病(GAN)。最后，我们将阐明生长素与 小鼠神经细胞和人类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和重要的 对KLHL蛋白调节和功能的洞察，并可能指出新的治疗机会 在神经退行性变、癌症和其他疾病中通过O- GlcNAc调制。

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.