The overall objectives of the proposed research are to study the molecular details of the mechanisms regulating the folding and secretion of apolipoprotein B (apo B). Apo B is a large hydrophobic glycoprotein with unique structural properties that are essential for the assembly and secretion of triglyceride rich lipoproteins (very low density lipoproteins and chylomicrons) and for the receptor-mediated catabolism of low density lipoproteins (LDL), the major carriers of plasma cholesterol. Plasma levels of apo B are positively correlated with an increased risk of the premature development of coronary heart disease. The detailed mechanisms regulating the secretion of apo B are incompletely understood. Secretion of proteins in general involve the assistance of helper proteins: """"""""receptors,"""""""" folding enzymes and molecular chaperones residing in the cytosol and the endoplasmic reticulum. The hypothesis is that helper proteins that assist apo B during various stages of its biogenesis play a crucial role in the regulation of its secretion and are therefore relevant to the pathogenesis of the premature development of coronary heart disease. It is further expected that one or more helper proteins will be unique to apo B and could be targeted as a means to modulate the secretion of apo B on VLDL.
The aims of the present proposal are to detect, identify, and characterize helper proteins mediating translocation, folding and assembly of apo B with lipids. To that end, cells that normally secrete lipoproteins (HepG-2), as well as non-lipoprotein secreting cells derived from mammary tissue (C127), stably transfected to express C-terminally truncated human apo B forms, will be utilized. The methodology to be used involves pulse-chase protocols followed by immunoprecipitation of cell lysates under low stringency conditions combined with the use of cross-linking reagents. Helper proteins will be identified using available antibodies and their mode of interaction with apo B will be studied. Helper proteins that are unique to apo B and that have not been identified thus far will be identified by micro sequencing followed by searches of databases. Two categories will be defined: a) known protein/s with a novel function or b) novel protein/s of unknown sequence. The mode of their interaction with apo B will be studied.
