Reference no: EM133543234
Assignment:
Michael Brown and Joseph Goldstein are physician-scientists. They jointly won the Nobel Prize in Physiology or Medicine in 1985 because of their contributions to the regulation of cholesterol metabolism. They found that receptors on cell surfaces mediate the uptake of the cholesterol-containing lipoprotein particles - low-density lipoproteins (LDL) that circulate in the bloodstream. This led to a great improvement in our understanding of the pathogenesis of cardiovascular disease.
In the 1970s, they set out to identify the molecular mechanisms of a human genetic disease called familial hypercholesterolemia (FH). At the time, they knew the following about FH: 1) the levels of cholesterol in the blood are many times above normal in FH patients; 2) FH patients can develop heart attack early in life; 3) the excess circulating cholesterol in FH patients is contained in LDL lipoprotein particles; 4) FH is inherited as an autosomal dominant trait (certain mutations in FH patient can cause disease in the heterozygous state); and 5) HMG CoA reductase (3-hydroxy-3-methyl-glutaryl-CoA reductase) catalyzes a rate-limiting step in cholesterol production.
They hypothesized that there are receptors of LDL at the cell surface in human bodies that are important for the uptake of LDL and the function of these receptors is impaired in FH patients. To test the hypothesis, they did a series of experiments to examine the regulation of HMG-CoA reductase by cholesterol-carrying lipoproteins using human fibroblasts. We know now that hepatocytes (the parenchymal cells of the liver play very important roles in controlling cholesterol homeostasis, and the regulation of cholesterol metabolism is well-conserved between human fibroblasts and hepatocytes.
Question 1: They cultured fibroblasts that were isolated from donors who were healthy subjects and homozygous FH patients. Cells were cultured in a medium containing LDL cholesterol. They examined the activities of HMG-CoA reductase in these fibroblasts. Were the HMG-CoA reductase activities expected to be higher or lower in normal fibroblasts compared with that in fibroblasts isolated from FH patients?
Q 2: They also examined the activities of HMG-CoA reductase after the cholesterol-carrying lipoproteins were removed from the culture medium. Compared with the HMG-CoA reductase activities in the cells before the cholesterol-carrying lipoproteins were removed, what changes in the HMG-CoA reductase activities did they expect to see in the normal and diseased fibroblasts, respectively?
Q 3: In a different experiment, they cultured cells in an LDL-cholesterol-free medium and examined the HMG-CoA reductase activities after sufficient LDL was added back into the culture medium. What changes in the HMG-CoA reductase activities did they expect to see in the normal and diseased fibroblasts, respectively?
Q 4: They mixed cholesterol with ethanol and albumin to form a quasi-soluble emulsion that can enter the normal or diseased fibroblasts independent of receptor uptake. The mixture was added to fibroblasts that were cultured in the medium without cholesterol-carrying lipoprotein. What changes of HMG-CoA reductase activities would they expect to observe in normal and diseased fibroblasts, respectively?
With the results from additional experiments, they hypothesized that a cell surface receptor was involved. They purified the LDL receptor in 1982 and cloned its human gene shortly thereafter. They sought to determine how the LDL receptor pathway regulates cholesterol homeostasis.
Q 5: Can you describe the effects of the LDL receptor pathway on the levels of intracellular and circulating cholesterol in humans? Can you explain how low levels of intracellular cholesterol regulate the LDL receptor pathway and cholesterol synthesis?
Q 6: If you treated normal fibroblasts and diseased fibroblasts with statin when cells are cultured in the medium with cholesterol-carrying lipoprotein, what do you expect to see about LDL receptor expression in normal and diseased fibroblasts respectively?
In a few multigenerational heterozygous FH families, no mutations in LDL receptor or APOB (another known mutation that can cause hypercholesterolemia) were detected. Genetic analysis in these families revealed a region on chromosome 1p34.1-p32 that cosegregated with elevated LDL cholesterol levels. This region contained the gene for a new serine protease called PCSK9 (proprotein convertase subtilisin/kexin type 9). Mechanistic studies revealed that PCSK9 binds to the LDL receptor at the cell surface of hepatocytes and promotes LDL receptor lysosomal degradation.
Q 7: In humans, both gain-of-function and loss-of-function mutations of PCSK9 were identified. The gain-of-function of PCSK9 promotes LDL receptor degradation, while the loss-of-function of PCSK9 inhibits LDL receptor degradation. Do you expect to see an increase or a decrease of circulating LDL cholesterol levels in humans expressing gain-of-function or loss-of-function PCSK9, respectively, compared with that in humans expressing a normal PCSK9?