Separation of plasma lipoproteins by density-gradient ultracentrifugation
Abstract
The separation of plasma lipoproteins into VLDL, LDL, and HDL by a single 24-hr ultracentrifugation in swinging-bucket rotors is described. The mean recovery of loaded lipoprotein cholesterol is 89.3 ± 1.3%. The method employs a discontinuous salt gradient and separates VLDL, LDL, and HDL as verified by cellulose acetate- and immuno-electrophoresis. The method offers some advantages for research applications.
References (19)
- F.T. Hatch et al.
Advan. Lipid Res
(1968) - W.J. Lossow et al.
J. Lipid Res
(1969) - A. Zlatkis et al.
Anal. Biochem
(1969) - S. Switzer et al.
J. Lipid Res
(1965) - O. Minari et al.
J. Lipid Res
(1963) - K.L. Harris et al.
J. Lipid Res
(1970) - O.F. de Lalla et al.
Methods of Biochem. Anal
(1954) - R.J. Havel et al.
J. Clin. Invest
(1955) - F.T. Lindgren et al.
J. Phys. Colloid Chem
(1951)
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Density-based gradient ultracentrifugation (G-UC) was used for lipoprotein depletion before EV isolation from plasma through size-exclusion chromatography (SEC) or serial centrifugation (SC). Recovered EVs were analyzed by size, concentration, cellular source, ultrastructure, and bottom-up proteomics.
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Combination of G-UC+SEC significantly reduced EV lipoprotein contamination without interfering in EV cellular source, gene ontology, and ultrastructure, allowing the recovery of highly pure EVs with potential implications for functional assays and proteomic and lipidomic analyses.
TMEM241 is a UDP-N-acetylglucosamine transporter required for M6P modification of NPC2 and cholesterol transport
2023, Journal of Lipid ResearchAccurate intracellular cholesterol traffic plays crucial roles. Niemann Pick type C (NPC) proteins NPC1 and NPC2, are two lysosomal cholesterol transporters that mediate the cholesterol exit from lysosomes. However, other proteins involved in this process remain poorly defined. Here, we find that the previously unannotated protein TMEM241 is required for cholesterol egressing from lysosomes through amphotericin B-based genome-wide CRISPR-Cas9 KO screening. Ablation of TMEM241 caused impaired sorting of NPC2, a protein utilizes the mannose-6-phosphate (M6P) modification for lysosomal targeting, resulting in cholesterol accumulation in the lysosomes. TMEM241 is a member of solute transporters 35 nucleotide sugar transporters family and localizes on the cis-Golgi network. Our data indicate that TMEM241 transports UDP-N-acetylglucosamine (UDP-GlcNAc) into Golgi lumen and UDP-GlcNAc is used for the M6P modification of proteins including NPC2. Furthermore, Tmem241-deficient mice display cholesterol accumulation in pulmonary cells and behave pulmonary injury and hypokinesia. Taken together, we demonstrate that TMEM241 is a Golgi-localized UDP-GlcNAc transporter and loss of TMEM241 causes cholesterol accumulation in lysosomes because of the impaired M6P-dependent lysosomal targeting of NPC2.
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Combined GIPR/GLP1R agonism attenuates atherosclerosis severity by diminishing inflammation and increasing VLDL turnover. We anticipate that combined GIPR/GLP1R agonism is a promising strategy to lower cardiometabolic risk in humans.
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