Proteostasis Network Factors Involved in Apolipoprotein B Folding and Secretion

参与载脂蛋白 B 折叠和分泌的蛋白质稳态网络因素

• 批准号: 8145610
• 负责人: Joseph Genereux
• 金额: $ 4.84万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-15 至 2013-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8145610
• 关键词: Affect; Affinity; Antibodies; Apolipoproteins B; Binding; Biological Assay; Cell Culture Techniques; Cell Line; Cells; Chromatography; Clinical; Colorimetry; Competence; Coupled; Cytolysis; Dependence; Development; Digestion; Disease; Dyes; Endoplasmic Reticulum Degradation Pathway; Enzyme-Linked Immunosorbent Assay; Family; Fluorescence Resonance Energy Transfer; Gel Chromatography; Genetic; Hepatocyte; Homeostasis; Human; Hyperlipidemia; Immunoprecipitation; Individual; Insulin; Insulin Resistance; Intuition; Knowledge; Libraries; Lipids; Lipoproteins; Liquid Chromatography; Liver; Low-Density Lipoproteins; Mass Fragmentography; Mass Spectrum Analysis; Measurement; Measures; Mediating; Methods; Molecular Chaperones; Monoclonal Antibodies; Mus; Nature; Particle Size; Pathway interactions; Peptides; Plasma; Polyunsaturated Fatty Acids; Process; Production; Proteins; Proteomics; Proxy; Rattus; Regulation; Relative (related person); Sampling; Screening procedure; Signal Pathway; Small Interfering RNA; Stress; Structure; System; Techniques; Technology; Time; Very low density lipoprotein; Work; base; cardiovascular risk factor; hepatoma cell; high throughput analysis; high throughput screening; interest; particle; public health relevance; secretion process; tandem mass spectrometry; time use; trafficking; two-dimensional

DESCRIPTION (provided by applicant): LDL levels in the plasma are the most widely used clinical predictor for cardiovascular risk, and are absolutely determined by the extent of apoB secretion as nascent VLDL by the liver. ApoB is highly processed, and proper folding and lipidation are vital for its secretion. We will determine which proteostasis factors interact with apoB, and then evaluate these candidates by knockdown screening. This knowledge will help elucidate the complicated relationship of VLDL production to a variety of disease states, and we will use it to identify new strategies to modulate lipoprotein homeostasis. This project will involve three Specific Aims. In the first, we will apply high throughput multidimensional protein identification technology (MudPIT) to determine the lipid-dependent interactome of nascent VLDL from lysed HepG2 cells. MudPIT is an automated two-dimensional peptide separation technology combined with tandem mass spectrometry and computational fragment analysis, allowing high-throughput analysis of immunoprecipitated protein samples. By identifying the proteostasis factors that interact with apoB, we can determine which pathways are involved in apoB processing and trafficking during secretion. We will also develop assays for apoB lipidation. One will involve fusing fluorescent proteins to apoB, and using time-resolved FRET to determine particle size polydispersity. In parallel, we will develop an ELISA based colorimetric assay, whereby we will determine the effect of the extent of lipidation on the binding of various monoclonal antibodies to apoB. Finally, we will use these lipidation assays and the intuition from the proteometric results to perform knockdown screens for individual proteins whose expression is critical for proper apoB folding, processing and secretion. PUBLIC HEALTH RELEVANCE: VLDL production is misregulated in any disease states, including hyperlipidemia and insulin resistance. We will identify the factors of the proteostasis network that regulate apoB folding and processing into VLDL, and characterize the impact of these factors on VLDL secretion.

描述（由申请人提供）：血浆中的 LDL 水平是最广泛使用的心血管风险临床预测因子，并且完全由肝脏分泌的 apoB 分泌程度（作为新生 VLDL）决定。 ApoB 经过高度加工，适当的折叠和脂化对其分泌至关重要。我们将确定哪些蛋白质稳态因子与 apoB 相互作用，然后通过敲低筛选来评估这些候选因子。这些知识将有助于阐明 VLDL 产生与各种疾病状态之间的复杂关系，我们将用它来确定调节脂蛋白稳态的新策略。该项目将涉及三个具体目标。首先，我们将应用高通量多维蛋白质鉴定技术 (MudPIT) 来确定裂解的 HepG2 细胞中新生 VLDL 的脂质依赖性相互作用组。 MudPIT 是一种自动化二维肽分离技术，与串联质谱和计算片段分析相结合，可对免疫沉淀蛋白质样品进行高通量分析。通过识别与 apoB 相互作用的蛋白质稳态因子，我们可以确定哪些途径参与 apoB 分泌过程中的加工和运输。我们还将开发 apoB 脂化检测方法。其中之一涉及将荧光蛋白与 apoB 融合，并使用时间分辨 FRET 来确定粒径多分散性。与此同时，我们将开发一种基于 ELISA 的比色测定法，从而确定脂化程度对各种单克隆抗体与 apoB 结合的影响。最后，我们将利用这些脂化测定和蛋白质测定结果的直觉，对单个蛋白质进行敲低筛选，这些蛋白质的表达对于正确的 apoB 折叠、加工和分泌至关重要。 公共健康相关性：VLDL 的产生在任何疾病状态下都会受到错误调节，包括高脂血症和胰岛素抵抗。我们将确定蛋白质稳态网络中调节 apoB 折叠和加工成 VLDL 的因素，并表征这些因素对 VLDL 分泌的影响。