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水平是心血管风险最广泛使用的临床预测因子，并且绝对取决于APOB分泌为肝脏新生VLDL的程度。 APOB经过高度处理，适当的折叠和脂质对其分泌至关重要。我们将确定哪些蛋白抑制因子与APOB相互作用，然后通过敲低筛选评估这些候选物。这些知识将有助于阐明VLDL生产与各种疾病状态的复杂关系，我们将使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，我们将应用高吞吐量多维蛋白识别技术（MUDPIT）来确定来自裂解的HEPG2细胞的新生VLDL的脂质依赖性相互作用。 Mudpit是一种自动化的二维肽分离技术，结合了串联质谱和计算片段分析，从而可以对免疫沉淀的蛋白质样品进行高通量分析。通过识别与APOB相互作用的蛋白质抑制因子，我们可以确定分泌过程中与APOB处理和运输中涉及哪些途径。我们还将开发APOB脂质的测定法。一种将涉及将荧光蛋白融合到APOB，并使用时间分辨的FRET来确定粒径多分散性。同时，我们将开发一种基于ELISA的比色测定法，从而确定脂质程度对各种单克隆抗体与APOB的结合的影响。最后，我们将使用这些脂化测定法和蛋白质计量结果的直觉来执行单个蛋白质的敲低筛选，这些蛋白对于适当的APOB折叠，加工和分泌至关重要。 公共卫生相关性：在包括高脂血症和胰岛素抵抗在内的任何疾病状态下，VLDL的产生都误导。我们将确定调节APOB折叠和处理为VLDL的Proteostasis网络的因素，并表征这些因素对VLDL分泌的影响。