B9-17: A suitable construct for apolipoprotein B-containing lipoprotein assembly studies.

摘要

Atherosclerosis, hardening and narrowing of the arteries, is the principal underlying cause of heart attacks, strokes, and peripheral vascular disease, which kills more than 600,000 Americans each year. High plasma levels of low-density lipoproteins (LDL) are linked to the formation of atherosclerotic plaques in arteries. LDL is the last metabolic product of very low-density lipoprotein (VLDL), which is secreted from the liver along with one molecule of apolipoprotein B (apoB). Current therapies to control levels of LDL include: cholesterol synthesis inhibitors or statins, low-fat diets and antisense oligonucleotides to reduce cholesterol levels. Recent studies recommend lower clinical levels of plasma LDL to maintain an individual's health, especially of those who have already developed atheroscle- rotic plaques. However, existing therapies are often unable to achieve these aggressive limits. Furthermore, patients have shown various levels of intolerance to these treatments. In order to develop new, targeted drugs, that can control LDL levels with minimal side effects, it is imperative to understand, in detail, the process of apoB-containing lipoprotein formation.;ApoB is one of the largest human proteins known (4563 residues) and previous attempts to solve the structure have been unsuccessful, mainly due to analyzing the protein as a whole or by large sections. To advance the field we will go by a different approach. I present here a construct that represents roughly 8% of the whole protein, apoB9-17 (residues 430 to 782). This section of the protein is believed to play a pivotal role in the assembly process of LDL. My hypothesis is that this construct will be well-behaved and suitable for structural and functional analysis. The study shows that apoB9-17 can be produced in considerable quantities from bacterial cells and can be purified by means of a 6-histidine tag with a good yield. Furthermore, circular dichroism analysis shows the construct contains the expected secondary structure at room temperature and is stable at a wide temperature range (50 to 70 °C) at low concentrations.;The construct here described will be useful to test the effect of mutations such as the one found in patients with Familial hypobetalipoproteinemia (FHBL). Furthermore, this construct contains two regions believed to be of vital importance for LDL particle formation: the alpha-helical region (residues 430 to 570) is believed to associate with MTP at the initial stages of LDL formation and the c-sheet (residues 614 to 782), which may form part of the lipid recruiting process. Both essential aspects to ultimately develop therapies that can modulate VLDL particle formation.