Microsomal triglyceride transfer-protein (MTP) and the assembly of atherogenic lipoprotein particles

This project was concerned with the biogenesis of atherogenic lipoprotein particles (VLDL), and, in particular, with molecular mechanisms leading to the development of atherosclerotic diseases. In the first part, the focus was on the function of the microsomal triglyceride transfer protein (MTP) and on processes relevant for the intracellular action of MTP. MTP activity is required for the loading of apolipoprotein B (apoB) with lipids in the endoplasmic reticulum (ER) of the liver and the intestine; mechanistically it could also be possible that MTP rather plays a role in the translocation of apoB into the ER and in its folding reactions. The functional MTP activity is mediated by a heterodimeric protein complex comprised of the MTP protein and protein disulfide isomerase (PDI). The stability of the complex is an important prerequisite for its function. The most striking results concerning MTP are listed in the following: - MTP is required for the secretion of apoB-containing lipoproteins (VLDL), but it is not the limiting component, which is rather the availability of triglycerides. - The amount and activity of MTP are not increased by estrogens, in contrast to the synthesis, assembly and secretion of VLDL; this refers to results obtained with a chicken hepatoma cell line. - The retention of MTP in the ER requires its association with PDI. - Truncated forms of MTP are not capable of forming a complex with PDI and are subject to rapid intracellular degradation. - The intracellular disposal of the truncated MTPs is mediated by the ubiquitin/proteasome-dependent ER- associated protein degradation pathway (ERAD). In the second part of the project, ER-associated protein degradation (ERAD) was analyzed in more detail, especially the role of N-linked glycosylation in this process. The most important findings are listed below: - N-linked glycosylation allows for the prolonged association of substrate proteins with the lectin-like chaperone, calnexin, in the ER, which results in temporary retention of the proteins in the ER and optimal "quality control" processes of protein folding. - Stress conditions lead to in increased binding of substrate proteins to ER chaperones and inhibition of their degradation. - The structure of the N-linked glycans on the substrate proteins plays a role in the course of glycoprotein degradation; in particular, the activity of a1,2-mannosidase(s) is essential for ERAD. These results are relevant, for instance, for the heterologous production of glycoproteins in higher eukaryotic cells.