An Evaluation of the Crystal Structure of C-terminal Truncated Apolipoprotein A-I in Solution Reveals Structural Dynamics Related to Lipid Binding

Apolipoprotein (apo) A-I mediates many of the anti-atherogenic functions attributed to high density lipoprotein. Unfortunately, efforts toward a high resolution structure of full-length apoA-I have not been fruitful, although there have been successes with deletion mutants. Recently, a C-terminal truncation (apoA-I^Δ185–243) was crystallized as a dimer. The structure showed two helical bundles connected by a long, curved pair of swapped helical domains. To compare this structure to that existing under solution conditions, we applied small angle x-ray scattering and isotope-assisted chemical cross-linking to apoA-I^Δ185–243 in its dimeric and monomeric forms. For the dimer, we found evidence for the shared domains and aspects of the N-terminal bundles, but not the molecular curvature seen in the crystal. We also found that the N-terminal bundles equilibrate between open and closed states. Interestingly, this movement is one of the transitions proposed during lipid binding. The monomer was consistent with a model in which the long shared helix doubles back onto the helical bundle. Combined with the crystal structure, these data offer an important starting point to understand the molecular details of high density lipoprotein biogenesis.     

Cholesterol absorption and turnover in hypercholesterolemic dogs

Cholesterol absorption was measured in chronically hypercholesterolemic dogs by four methods: the fecal recovery method of Borgstr?m (1969, J. Lipid Res. 10: 331-337), the dual isotope method of Zilversmit and Hughes (1974, J. Lipid Res. 15: 465-473), the recovery of cholesterol in thoracic duct lymph collected continuously for 16 hr after a meal, and the recovery of isotopic cholesterol from the liver and plasma 24 hr after the animals consumed an isotope-containing meal. The four methods showed excellent agreement and indicated that dogs fed a cholesterol-rich synthetic diet absorb 5.2 +/- 0.5 g (mean +/- SD) of cholesterol per day and that cholesterol absorption is reasonably constant from week to week in these animals. Separate estimates of cholesterol excretion indicated that these dogs excreted 4.7 +/- 0.5 g of cholesterol per day, and thus were at or near the steady-state with regard to cholesterol input-output. These data, taken together with a previous report (1981, J. Lipid Res. 22: 598-609), indicate that the canine liver can clear up to 300 mg of chylomicron cholesterol/hr, and support the concept that chylomicron remnants do not contribute significantly to the hypercholesterolemia in these animals.     

Apo B metabolism in the cynomolgus monkey: evidence for post-transcriptional regulation.

Previous studies have shown that hepatic apo B mRNA levels do not increase in animals fed high cholesterol diets, even though plasma apo B concentrations increase markedly. As a result, it has been suggested that the diet-induced increase in plasma apo B levels was due solely to an inhibited clearance of those lipoproteins. The present study was undertaken to test that hypothesis. Hepatic apo B mRNA levels were measured in liver biopsies taken from five male cynomolgus monkeys before and twice after, they began to consume a high cholesterol diet. The diet had no effect on hepatic apo B mRNA levels, even though it caused a 7-fold increase in the plasma apo B levels. However, measurements of the apo B secretion rate in eight separate monkeys (four chow-fed and four cholesterol-fed) by isotope dilution showed that apo B secretion by the liver was increased 4-fold in the cholesterol-fed monkeys. These data, taken together, indicate that apo B secretion is not regulated by the rate at which the apo B gene is transcribed, but at some point further along in the secretion pathway.