The assembly and secretion of ApoB containing lipoproteins

Abstract: Increased levels of cholesterol and triglycerides in the blood are important risk factors for the development of premature atherosclerosis and its complications such as coronary-heart diseases, stroke and peripheral vascular diseases. These diseases are the major causes of death in Europe, USA and part of Asia. The cholesterol and triglycerides are transported in the blood together with specific proteins (apolipoproteins) as lipoproteins. In particular, the low-density lipoproteins (LDL) are associated with an increased risk for atherosclerosis, and among the LDL, small cholesterol rich particles are the most aggressive (i.e. the most atherogenic). LDL is formed from very low density lipoproteins (VLDL) that is assembled in the liver and secreted into the blood. An overproduction of large VLDL particle give rise to increased levels of the small, cholesterol rich and atherogenic LDL. Increased biosynthesis of large VLDL is a major feature of metabolic diseases such as insulin resistance/ type 2 diabetes and is one of the earliest signs of these diseases. Increased biosynthesis of large VLDL is therefore a major reason why type 2 diabetes is very often diagnosed after the patient has suffered a cardiovascular event. In order to be able to prevent premature cardiovascular events in for example type 2 diabetes we need detailed information on how VLDL is assemble and how the process is regulated. The aim of this thesis is to characterize the assembly pathway that leads to the formation of VLDL. The structural apolipoprotein (apo) of VLDL (and LDL) is referred to as apo B. There exist two forms of apoB; apoB-100 and apoB-48. They are encoded by the same gene and apoB-48 corresponds to the N-terminal 48 % of apoB-100. In humans, apoB-100 is synthesized in the liver, while apoB-48 is synthesized in the intestine. In some animals, for example the rat, both proteins are expressed in the liver, where they form VLDL. This is also true for the cell line McARH7777 cells that I have used in my studies. The assembly and secretion of VLDL occurs in two steps in the endoplasmic reticulum (ER) of the cells. The first step occurs during the translation and translocation of the protein to the lumen of the rough ER and results in a pre-VLDL particle, i.e. an apoB containing particle that is smaller and less lipidated than bona fide VLDL. Pre-VLDL is loosely associated with the ER membrane and acquires the major amount of lipids (forming bona fide VLDL) in a second step in a smooth membrane compartment. The first step is dependent on the microsomal triglyceride transfer protein (MTP), a protein that transfers triglycerides from the ER membrane to the growing apoB protein. We have demonstrated that MTP is needed during the translation and translocation of apoB to the ER lumen but also for a period after apoB has been completed. This "posttranslational MTP window" is necessary for the pre-VLDL particle to be able to assemble VLDL in the second step. An important part of the first step is the folding of apoB. Certain regions of apoB have been demonstrated to be more difficult to fold. One such region occurs between apoB-48 and apoB-53. Another complex folding pattern occurs in the C-terminal 23 % of apoB-100 (where the protein makes a loop and fold back over the preceding polypeptide chain). We demonstrated that the "posttranslational MTP window " was not related to the folding of these regions but rather to the need to increase the lipid content of pre-VLDL. We could however demonstrate that a region of the last part of apoB-100 (between apoB-72 and apoB-80) specifically bound to Protein Disulfide Isomerase (PDI) and BiP (binding protein), indicating a need for chaperons during its folding. We demonstrated that the second step occurs in another compartment than Rough ER, which indicate that pre-VLDL has to be transferred to this compartment before it can be fully lipidated. The second step is important since it is likely to determine both the amount of apoB that should be secreted and the size of the VLDL particle that is secreted. Sequences in apoB, in particular the C-terminal 17 % of apoB-48 determines the efficiency of the second step. Moreover the second step seems to be supported by several chaperons such as PDI, CaBP2 (calcium binding protein 2),BiP, calreticulin and GRP94 (glucose regulatory protein 94