Browsing by Subject "Membrane Glycoproteins"
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Item A Bacterial Cholesterol Sensor to Assess Cholesterol Accessibility in Red Blood Cells(2016-01-19) Chakrabarti, Rima Shah; Radhakrishnan, Arun; Cohen, Jonathan C.; Hobbs, Helen H.Mammals are able to gain cholesterol from two sources: diet and endogenous synthesis. However, the only means of cholesterol removal is reverse cholesterol transport (RCT), in which cholesterol is transported to the liver and exported into bile. While high density lipoprotein (HDL) is considered to be the major conduit for RCT, studies with HDL-deficient animals reveal no defect in tissue cholesterol balance. We hypothesize that red blood cells (RBCs), which contain 50% of blood cholesterol, also play a role in RCT. To measure accessible cholesterol in RBCs, we developed an assay that utilizes the cholesterol binding properties of the toxin Anthrolysin-O (ALO). We purified and fluorescently labeled domain 4 of ALO (fALOD4). We then incubated fALOD4 with RBCs from 164 subjects and measured fluorescence intensity using flow cytometry. Both intra-assay and intra-individual variability of the assay were less than 10%. In the test population, fALOD4 binding varied 10-fold. fALOD4 binding did not correlate with total RBC cholesterol but did correlate with RBC phosphatidylcholine (PC) (-0.42, p=6e-7) and lyso-phosphatidylcholine (LPC) (0.40, p=6e-6). Increasing the LPC:PC ratio in RBCs with phospholipase A2 (PLA2) increased fALOD4 binding by 3-fold. fALOD4 binding also correlated with plasma HDL (0.30, p=6e-4) and triglycerides (-0.57, p=2e-12). These data suggest that RBC accessible cholesterol varies in a population, is driven by intrinsic RBC phospholipid composition and interacts with known cholesterol transporters in the blood. Future studies will determine if variability in fALOD4 binding is driven by non-lipid RBC membrane components, is genetically determined, or contributes to atherosclerosis.Item On Cholesterol Transport Between Membranes(August 2021) Trinh, Michael Nguyen; Mendell, Joshua T.; Chen, Zhijian J.; Abrams, John M.; Brown, Michael S.; Goldstein, Joseph L.The studies described in this dissertation focus on investigation of the pathways for transport of cholesterol from one organelle to another in animal cells. Cells have evolved elaborate transport mechanisms to assure an optimum cholesterol content within their membranes. Dysregulation of cholesterol transport causes common diseases, including atherosclerosis. The major source of cellular cholesterol comes from Low Density Lipoprotein (LDL). When plasma membranes are low in cholesterol, cells produce LDL receptors which bind LDL and mediate its uptake by endocytosis and its delivery to lysosomes. Within lysosomes the cholesteryl esters of LDL are hydrolyzed. The free cholesterol binds to a soluble lysosomal protein called Niemann Pick C2 (NPC2) which delivers it to a membrane-embedded protein called NPC1 which inserts the cholesterol into the lysosome membrane. From there the cholesterol moves to the plasma membrane (PM) through a pathway that is unknown. When the PM becomes saturated with cholesterol, any excess is transported to the endoplasmic reticulum (ER) to repress production of LDL receptors and to be stored in lipid droplets. The work described here 1) showed that triazole antifungal drugs inhibit lysosomal cholesterol export by binding to the membrane domain of NPC1 2) used itraconazole to solve the crystal structure of NPC1 at 3.3Å, 3) revealed that cholesterol is transported out of lysosomes through interactions between two or more NPC1 molecules, and 4) utilized CRISPR-Cas9 whole-genome knockout screens to identify all the genes involved in the transport and uptake of LDL cholesterol. From these screens in the latter study, we discovered that a specific phospholipid, phosphatidylserine (PS), is required for PM-to-ER cholesterol transport. These studies provide supporting evidence towards a vision of one-way directional transport of LDL-derived cholesterol from lysosomes to the PM to the ER.Item Variability of Cholesterol Accessibility in Human Red Blood Cells Measured Using a Bacterial Cholesterol Binding Toxin(2017-03-31) Chakrabarti, Rima Shah; Radhakrishnan, Arun; Horton, Jay D.; Hofmann, Sandra L.Cholesterol partitions into accessible and sequestered pools in cell membranes. Here, we describe a new assay using fluorescently-tagged anthrolysin O, a cholesterol-binding bacterial toxin, to measure accessible cholesterol in human red blood cells (RBCs). Accessible cholesterol levels were stable within individuals, but varied >10 fold among individuals. Significant variation was observed among ethnic groups (Blacks>Hispanics>Whites). Variation in accessibility of RBC cholesterol was unrelated to the cholesterol content of RBCs or plasma, but was associated with the phospholipid composition of the RBC membranes and with plasma triglyceride levels. Pronase treatment of RBCs only modestly altered cholesterol accessibility. Plasma from various healthy individuals differentially modulated RBC cholesterol accessibility. Individuals on hemodialysis, who have an unexplained increase in atherosclerotic risk, had significantly higher RBC cholesterol accessibility. Our data indicate that RBC accessible cholesterol is a stable phenotype with significant inter-individual variability. Factors both intrinsic and extrinsic to the RBC contribute to variation in its accessibility. This assay provides a new tool to assess cholesterol homeostasis among tissues in humans.