Proteostasis Network Factors Involved in Apolipoprotein B Folding and Secretion

描述(申请人提供)：血浆中的低密度脂蛋白水平是最广泛使用的心血管风险的临床预测指标，绝对由肝脏作为新生极低密度脂蛋白分泌载脂蛋白B的程度决定。载脂蛋白B经过高度加工，适当的折叠和脂肪作用对其分泌至关重要。我们将确定哪些蛋白抑制因子与apoB相互作用，然后通过基因敲除筛选来评估这些候选因子。这一知识将有助于阐明极低密度脂蛋白的产生与各种疾病状态的复杂关系，我们将利用它来确定调节脂蛋白稳态的新策略。这个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)从裂解的HepG2细胞中确定新生极低密度脂蛋白的脂类依赖的相互作用组。MudPit是一种自动化的二维肽分离技术，结合了串联质谱仪和计算片段分析，可以高通量分析免疫沉淀蛋白质样本。通过确定与载脂蛋白B相互作用的蛋白平衡因子，我们可以确定哪些途径参与了载脂蛋白B在分泌过程中的加工和运输。我们还将开发载脂蛋白B脂化的检测方法。一种是将荧光蛋白融合到apoB上，并使用时间分辨的FRET来确定颗粒大小的多分散性。同时，我们将开发一种基于酶联免疫吸附试验的比色分析方法，由此我们将确定脂化程度对各种单抗与载脂蛋白B结合的影响。最后，我们将使用这些脂化分析和来自蛋白质计量学结果的直觉来对个别蛋白质进行击倒筛选，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。 与公共卫生相关：极低密度脂蛋白的产生在任何疾病状态下都受到错误的调节，包括高脂血症和胰岛素抵抗。我们将确定调节载脂蛋白B折叠和加工成极低密度脂蛋白的蛋白平衡网络的因素，并表征这些因素对极低密度脂蛋白分泌的影响。

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.