Intracellular cholesterol transport

Maxfield, Frederick R.; Wüstner, Daniel

doi:10.1172/jci0216500

Cited by 277 publications

(183 citation statements)

References 67 publications

(97 reference statements)

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“…Increased bulk as well as where the fluorophore is positioned could strongly influence a lipid's binding properties. As recently pointed out for cholesterol [40,41], NBD derivatization could also affect how a lipid analyte is distributed within cells. Based on these considerations, the desirability of employing unmodified lipids for binding or transfer-distribution studies is obvious.…”

Section: Discussionmentioning

confidence: 92%

Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology

Kernstock

Girotti

2007

Analytical Biochemistry

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A new approach for analyzing lipid-lipid transfer protein interactions is described. The transfer protein is genetically engineered for expression with a C-terminal biotinylated peptide extension (AviTag ® ). This allows protein anchoring to a streptavidin-coated chip for surface plasmon resonance (SPR)-based assessment of lipid binding. Sterol carrier protein-2 (SCP-2), involved in the intracellular trafficking of cholesterol, fatty acids and other lipids, was selected as the prototype. Biotinylated SCP-2 (bSCP-2) was expressed in E. coli, purified to homogeneity by mutated streptavidin (SoftLink ® ) affinity chromatography, and confirmed by mass spectrometry to contain one biotin group at the expected position. Intermembrane [ 14 C]cholesterol transfer was strongly enhanced by bSCP-2, demonstrating that it was functional. Using bSCP-2 immobilized on a Biacore streptavidin chip, we determined on-and off-rate constants along with equilibrium dissociation constants for the following analytes: oleic acid, linoleic acid, cholesterol, and fluorophore (NBD)-derivatized cholesterol. The dissociation constant for NBD-cholesterol was similar to that determined by fluorescence titration for SCP-2 in solution, thus validating the SPR approach. This method can be readily adapted to other transfer proteins and has several advantages over existing techniques for measuring lipid binding, including (i) ability to study lipids in their natural states, i.e. without relatively large reporter groups; and (ii) ability to measure on-and off-rate constants as well as equilibrium constants.

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Section: Discussionmentioning

confidence: 92%

Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology

Kernstock

Girotti

2007

Analytical Biochemistry

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“…Immunoisolated recycling endosomes from polarized MDCK cells were also found to be enriched in SM and the raft-associated proteins caveolin and flotillin (Gagescu et al, 2000). Late endosomes and lysosomes also have been shown to contain cholesterol, but to much lesser extents (Maxfield and Wustner, 2002).…”

Section: Cholesterol and Glycosphingolipids Are Specifically Requiredmentioning

confidence: 97%

Transcytotic Efflux from Early Endosomes Is Dependent on Cholesterol and Glycosphingolipids in Polarized Hepatic Cells

Nyasae

Hubbard

Tuma

2003

MBoC

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We examined the role that lipid rafts play in regulating apical protein trafficking in polarized hepatic cells. Rafts are postulated to form in the trans-Golgi network where they recruit newly synthesized apical residents and mediate their direct transport to the apical plasma membrane. In hepatocytes, single transmembrane and glycolipid-anchored apical proteins take the "indirect" route. They are transported from the trans-Golgi to the basolateral plasma membrane where they are endocytosed and transcytosed to the apical surface. Do rafts sort hepatic apical proteins along this circuitous pathway? We took two approaches to answer this question. First, we determined the detergent solubility of selected apical proteins and where in the biosynthetic pathway insolubility was acquired. Second, we used pharmacological agents to deplete raft components and assessed their effects on basolateral-to-apical transcytosis. We found that cholesterol and glycosphingolipids are required for delivery from basolateral early endosomes to the subapical compartment. In contrast, fluid phase uptake and clathrin-mediated internalization of recycling receptors were only mildly impaired. Apical protein solubility did not correlate with raft depletion or impaired transcytosis, suggesting other factors contribute to apical protein insolubility. Examination of apical proteins in Fao cells also revealed that raft-dependent sorting does not require the polarized cell context.

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“…11,18,26 As described above, the steroidogenic metabolic pathway spans two cell types; theca cells and granulosa cells (Fig. 1).…”

Section: Secretion Of Steroidsmentioning

confidence: 98%

“…7,11,14 Second, cholesterol is transferred to the inner mitochondrial membrane by the intracellular sterol carrier protein-2, steroidogenic acute regulatory protein, and peripheral benzodiazepine receptor. 11,14,18 We model the rate of cholesterol uptake from the medium as a zero-order process (Fig. 2) since the large concentration of cholesterol added to the medium for our in vitro steroidogenesis assay is probably sufficient to saturate the cholesterol transport process for the duration of the experiments.…”

Section: Ovary Uptake Of Cholesterolmentioning

confidence: 99%

Mechanistic Computational Model of Ovarian Steroidogenesis to Predict Biochemical Responses to Endocrine Active Compounds

et al. 2007

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Abstract-Sex steroids, which have an important role in a wide range of physiological and pathological processes, are synthesized primarily in the gonads and adrenal glands through a series of enzyme-mediated reactions. The activity of steroidogenic enzymes can be altered by a variety of endocrine active compounds (EAC), some of which are therapeutics and others that are environmental contaminants. A steady-state computational model of the intraovarian metabolic network was developed to predict the synthesis and secretion of testosterone (T) and estradiol (E2), and their responses to EAC. Model predictions were compared to data from an in vitro steroidogenesis assay with ovary explants from a small fish model, the fathead minnow. Model parameters were estimated using an iterative optimization algorithm. Model-predicted concentrations of T and E2 closely correspond to the time-course data from baseline (control) experiments, and dose-response data from experiments with the EAC, fadrozole (FAD). A sensitivity analysis of the model parameters identified specific transport and metabolic processes that most influence the concentrations of T and E2, which included uptake of cholesterol into the ovary, secretion of androstenedione (AD) from the ovary, and conversions of AD to T, and AD to estrone (E1). The sensitivity analysis also indicated the E1 pathway as the preferred pathway for E2 synthesis, as compared to the T pathway. Our study demonstrates the feasibility of using the steroidogenesis model to predict T and E2 concentrations, in vitro, while reducing model complexity with a steady-state assumption. This capability could be useful for pharmaceutical development and environmental health assessments with EAC.

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Intracellular cholesterol transport

Cited by 277 publications

References 67 publications

Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology

Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology

Transcytotic Efflux from Early Endosomes Is Dependent on Cholesterol and Glycosphingolipids in Polarized Hepatic Cells

Mechanistic Computational Model of Ovarian Steroidogenesis to Predict Biochemical Responses to Endocrine Active Compounds

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