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.
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.
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|Shoulders, Matthew D; Ryno, Lisa M; Genereux, Joseph C et al. (2013) Stress-independent activation of XBP1s and/or ATF6 reveals three functionally diverse ER proteostasis environments. Cell Rep 3:1279-92|