Novel Roles of Cullin-RING E3 Ligases in Liver Pathophysiology

项目摘要 NAFLD与肥胖和2型糖尿病密切相关。都提高了对这种疾病的认识 因此，迫切需要研究NAFLD的病理生理学和发现新的治疗靶点。库林- RING E3连接酶（CRL）是泛素连接酶的一个独特的亚类，其在泛素连接酶的neddylation后被激活。 临时脚手架哺乳动物细胞含有识别特定蛋白质的功能不同的CRL复合物 泛素化和随后的蛋白酶体降解的底物。CRL被认为是有前途的药物 由于它们调节疾病相关的细胞途径，底物选择性较高， 对抑制的广泛细胞影响。最近，CRLs已成为营养信号的新型调节剂 以及不同器官系统的炎症反应。然而，肝脏CRL在NAFLD中的作用 病理生理学仍然是未知的。该项目的目标是描述功能影响， CRLs调节肝脏脂质和葡萄糖代谢的潜在机制，并建立CRLs 作为开发NAFLD和糖尿病治疗的有效治疗靶点。该项目是根据 我们的关键发现是肝脏CRL在人类NASH肝脏和小鼠NAFLD中异常激活， 通过靶向Cullin neddylation对肝CRL活性的药理学抑制改善了胰岛素敏感性， 降低高血糖症和肝脏脂肪变性，通过对抗异常升高的促血管生成和脂肪生成 活动推动NAFLD进展。我们将进行机制研究，以建立一个工作模型 由此药理学CRL抑制防止异常的肝胰岛素受体底物降解， NAFLD中肝脏胰岛素抵抗的关键潜在原因，并减弱ChREBP驱动的从头 脂肪生成，因此同时实现葡萄糖和降脂作用。设计了具体目标 剖析特定的下游CRL复合物，它介导cullin neddylation的有益作用， 抑制剂，并确定和功能验证CRL底物受体，可以作为新的 未来药物开发的治疗目标。这项研究采用了新的组织特异性基因敲除小鼠， AAV 8介导的肝脏特异性基因递送和药理学治疗模型。这些模型将在 结合分子和细胞生物学技术，代谢表型分析， 高胰岛素-正葡萄糖钳夹以及无偏转录组学和蛋白质组学方法。通过采用 这些国家的最先进的模型和技术，我们的工作假设可以严格测试和新的 将获得机械的见解。

Project Summary NAFLD is closely associated with obesity and type-2 diabetes. Both improved understanding of the disease pathophysiology and discovery of novel therapeutic targets for NAFLD treatment are urgently needed. Cullin- RING E3 ligases (CRLs) are a unique sub-class of ubiquitin ligases that are activated upon neddylation of the cullin scaffold. Mammalian cells contain functionally distinct CRL complexes that recognize specific protein substrates for ubiquitination and subsequent proteasomal degradation. CRLs are considered promising drug targets due to their regulation of disease relevant cellular pathways, higher substrate selectivity and lack of broad cellular impact upon inhibition. Recently, CRLs have emerged as novel regulators of nutrient signaling and inflammatory response in different organ systems. However, the roles of hepatic CRLs in NAFLD pathophysiology is still largely unknown. The goals of this project are to delineate the functional impact and underlying mechanisms by which CRLs regulate hepatic lipid and glucose metabolism and to establish CRLs as valid therapeutic targets for developing NAFLD and diabetes treatment. This project is developed based on our key findings that hepatic CRLs are abnormally activated in human NASH livers and murine NAFLD, and pharmacological inhibition of hepatic CRL activity by targeting cullin neddylation improved insulin sensitivity, reduced hyperglycemia and liver steatosis by countering the abnormally elevated gluconeogenic and lipogenic activity that drives NAFLD progression. We will perform mechanistic studies to establish a working model whereby pharmacological CRL inhibition prevents abnormal liver insulin receptor substrate degradation, the key underlying cause of hepatic insulin resistance in NAFLD, and attenuates ChREBP-driven de novo lipogenesis, therefore simultaneously achieves glucose and lipid-lowering effects. Specific aims are designed to dissect the specific downstream CRL complex that mediates the beneficial effects of cullin neddylation inhibitors and to identify and functionally validate the CRL substrate receptors that can serve as new therapeutic targets for future drug development. This study employs new tissue-specific genetic knockout mice, AAV8-mediated liver-specific gene delivery, and pharmacological treatment models. These models will be investigated with a combination of molecular and cell biology techniques, metabolic phenotyping, hyperinsulinemic-euglycemic clamp, and unbiased transcriptomics and proteomics approaches. By employing these state-of-the-art models and techniques, our working hypothesis can be rigorously tested and new mechanistic insights will be obtained.