CSDE1 as a Post Transcriptional Regulator of the LDLR

CSDE1 作为 LDLR 的转录后调节因子

• 批准号: 10290273
• 负责人: John S Chorba
• 金额: $ 63.46万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-15 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10290273
• 关键词: 3' Untranslated Regions; Address; Affect; Atherosclerosis; Berberine; Binding; Biological; Blood Circulation; CRISPR interference; Cardiovascular system; Cause of Death; Cell Culture Techniques; Cell membrane; Cell model; Cell surface; Cells; Cellular biology; Cholesterol; Cholesterol Homeostasis; Clinic; Coronary heart disease; Data; Development; Disease; Drug Targeting; Elements; Endotoxins; Environment; Event; Future; Gene Expression Profiling; Genes; Genetic Transcription; Goals; Health; Heart Diseases; Hepatic; Human; Immunoprecipitation; Inflammatory; Lipids; Liver; Low Density Lipoprotein Receptor; Low-Density Lipoproteins; Maintenance; Mass Spectrum Analysis; Mediating; Messenger RNA; Mission; Modeling; Molecular Chaperones; Pathway interactions; Pharmaceutical Preparations; Plasma; Play; Post-Transcriptional Regulation; Proteins; RNA; RNA Binding; Regulation; Research Design; Risk Factors; Role; Specificity; Surface; System; Therapeutic; Tissues; Transcript; Transcriptional Regulation; United States; United States National Institutes of Health; Virus; Whole Organism; Work; blood lipoprotein; crosslink; genome-wide; improved; in vivo; inhibitor/antagonist; innovation; insight; knock-down; mRNA Decay; mRNA Stability; modifiable risk; mouse model; novel; overexpression; public health relevance; receptor expression; recruit; therapeutic target; tissue culture; transcriptome; transcriptome sequencing; uptake

Low-density lipoprotein (LDL) is a key risk factor for atherosclerotic heart disease, the leading cause of death in the US. Therapies that upregulate the hepatic LDLR lower LDL and protect against cardiovascular events. Moreover, the LDLR also plays a fundamental role in maintenance of intracellular cholesterol homeostasis. Therefore, the mechanisms that regulate the LDLR are important to understand. Via an innovative genome- wide CRISPR interference screen, we identified CSDE1 as a novel post-transcriptional modulator of LDLR mRNA stability. In tissue culture, knockdown of CSDE1 is as powerful as knockdown of the targets of statins or PCSK9 inhibitors, the most effective LDL lowering therapies in the clinic. Moreover, CSDE1 also acts independently of these targets. However, both the mechanism of and specificity for CSDE1's effect on the hepatic LDLR mRNA remain unknown. Therefore, the overall goal of this proposal is to establish the role of CSDE1 as a specific regulator of the hepatic LDLR and circulating LDL. In Aim 1, we will establish the mechanism by which hepatic CSDE1 regulates LDLR mRNA stability. Because CSDE1 binds the 3' UTR of LDLR, but its effects are tissue-dependent, we hypothesize that CSDE1 recruits other factors required for LDLR mRNA decay. We will therefore establish the dynamic range of CSDE1's effect through overexpression studies, use mass spectrometry approaches to identify the additional protein components required for CSDE1- mediated LDLR mRNA decay, and place CSDE1 in the context of a natural compound, berberine, which affects LDLR mRNA stability through specific elements in the LDLR 3' UTR. In Aim 2, we will identify the role of hepatic CSDE1 in post-transcriptional regulation of the LDLR. We will use global translational profiling (RNA- seq) to evaluate the effects of CSDE1 knockdown and overexpression on the hepatic transcriptome. We will combine this with cross-linking and immunoprecipitation (CLIP) studies to identify the direct RNA interactors, and their binding motifs, of hepatic CSDE1. In Aim 3, we will establish whether the effect of hepatic CSDE1 persists in vivo. We will use a mouse model to evaluate the effect of Csde1 knockdown and overexpression on plasma lipids and in vivo hepatic Ldlr regulation. Together, this project will identify the mechanism and fidelity of a promising new regulator of the LDLR, an extremely important player in the development of atherosclerosis. The results will establish CSDE1 as a promising therapeutic target. Moreover, they will provide insight into the mechanism of specificity of a general mechanism of mRNA regulation, with implications for fundamental cell biology and other disease states.

低密度脂蛋白（LDL）是动脉粥样硬化心脏病的关键危险因素，这是美国的主要死亡原因。上调肝LDLR下LDL并预防心血管事件的疗法。此外，LDLR在维持细胞内胆固醇稳态方面也起着基本作用。因此，调节LDLR的机制对于理解很重要。通过创新的基因组CRISPR干扰屏幕，我们将CSDE1确定为LDLR mRNA稳定性的新型后转录调节剂。在组织培养中，CSDE1的敲低与他汀类药物或PCSK9抑制剂靶标一样强大，这是诊所中最有效的LDL降低疗法。此外，CSDE1还独立于这些目标。但是，CSDE1对肝LDLR mRNA的影响的机理和特异性均未清楚。因此，该提案的总体目标是确定CSDE1作为肝LDLR和循环LDL的特定调节剂的作用。在AIM 1中，我们将建立肝CSDE1调节LDLR mRNA稳定性的机制。由于CSDE1结合了LDLR的3'UTR，但其作用是组织依赖性的，因此我们假设CSDE1募集了LDLR mRNA Decay所需的其他因素。因此，我们将通过过表达研究建立CSDE1效应的动态范围，使用质谱方法来确定CSDE1-介导的LDLR mRNA衰变所需的其他蛋白质成分，并将CSDE1放置在天然化合物的背景下，berberine，Berberine，通过影响LDLR mRNA稳定性，从而影响LDLR mRNA稳定性，从而影响LDLR的特定元素。在AIM 2中，我们将确定肝CSDE1在LDLR转录后调节中的作用。我们将使用全局翻译分析（RNA-SEQ）来评估CSDE1敲低和过表达对肝转录组的影响。我们将将其与肝CSDE1的直接RNA相互作用者及其结合基序相结合（剪辑）研究（剪辑）研究。在AIM 3中，我们将确定肝CSDE1在体内的影响是否存在。我们将使用小鼠模型来评估CSDE1敲低和过表达对血浆脂质和体内肝LDLR调节的影响。该项目将共同确定LDLR新的新调节剂的机制和忠诚，这是动脉粥样硬化发展中极为重要的参与者。结果将确定CSDE1作为有前途的治疗靶点。此外，它们将洞悉mRNA调节一般机制的特异性机制，对基本细胞生物学和其他疾病状态产生影响。