Proteostasis Network Factors Involved in Apolipoprotein B Folding and 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指标，并且绝对由肝脏作为新生VLDL的载脂蛋白ob分泌程度决定。载脂蛋白b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抑制因子与载脂蛋白ob相互作用，然后通过敲低筛选评估这些候选因子。这一知识将有助于阐明VLDL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鉴定技术（MudPIT）来确定裂解HepG2细胞中新生VLDL的脂质依赖性相互作用组。MudPIT是一种自动的二维肽分离技术，结合串联质谱和计算片段分析，可以对免疫沉淀蛋白样品进行高通量分析。通过鉴定与载脂蛋白ob相互作用的蛋白抑制因子，我们可以确定在分泌过程中哪些途径参与了载脂蛋白ob的加工和运输。我们还将开发载脂蛋白脂化试验。其中一种方法是将荧光蛋白与载脂蛋白b融合，并使用时间分辨FRET来确定颗粒大小的多分散性。同时，我们将开发一种基于ELISA的比色测定法，由此我们将确定脂化程度对各种单克隆抗体与载脂蛋白ob结合的影响。最后，我们将使用这些脂化实验和蛋白质测定结果的直觉来对单个蛋白质进行敲低筛选，这些蛋白质的表达对载脂蛋白的正确折叠、加工和分泌至关重要。

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.