Quantitation of Lipid Transfer Activity

Wetterau, John R.; Zilversmit, D. B.

doi:10.1002/9780470110515.ch4

Cited by 20 publications

(11 citation statements)

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“…The lipid transfer activities of purified proteins were also measured employing a classical radiolabeled vesicle transfer assay (11,12). hMTP-FLAG showed a robust triacylglycerol transfer activity (19.2 Ϯ 1.1%), whereas only minimal transfer was detected using purified dMTP-FLAG (0.6 Ϯ 0.2%) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In the absence of MTP, nascent apoB folds incorrectly and undergoes proteasomal degradation. Although mammalian MTP has been shown to transfer triacylglycerols, cholesteryl esters, and phospholipids between membrane vesicles in vitro (11)(12)(13)(14), its predominant activity is toward triacylglycerols (12,14). Several antagonists of the lipid transfer activity of MTP are potent inhibitors of the triacylglycerol transfer activity (14,30).…”

Section: Discussionmentioning

confidence: 99%

“…Lipid Transfer Assays-Radiolabeled lipid transfer assays were performed according to the method described by Wetterau and Zilversmit (11,12 . After 48 h, the cells received 1 ml of media (ϪOA) or media containing 0.4 mM oleic acid, 1.5% BSA, and 1 mM glycerol (ϩOA).…”

Section: Methodsmentioning

confidence: 99%

“…More recent evidence suggests that MTP lipid transfer activity is also responsible for lipid accretion within the secretory pathway and that MTP potentially stabilizes lipid vesicles in the endoplasmic reticulum (reviewed in Refs. 2 and 3).MTP transfers several lipids including triacylglycerols, cholesterol esters, and phospholipids between vesicles in vitro (11)(12)(13)(14). It has been suggested that MTP transiently interacts with a membrane, extracts lipids, and then delivers them to another lipid acceptor or to nascent apoB lipoproteins (15).…”

mentioning

confidence: 99%

“…MTP transfers several lipids including triacylglycerols, cholesterol esters, and phospholipids between vesicles in vitro (11)(12)(13)(14). It has been suggested that MTP transiently interacts with a membrane, extracts lipids, and then delivers them to another lipid acceptor or to nascent apoB lipoproteins (15).…”

mentioning

confidence: 99%

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Phospholipid Transfer Activity of Microsomal Triacylglycerol Transfer Protein Is Sufficient for the Assembly and Secretion of Apolipoprotein B Lipoproteins

Rava

Ojakian

Shelness

et al. 2006

Journal of Biological Chemistry

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Human microsomal triacylglycerol transfer protein (hMTP) is essential for apolipoprotein B (apoB)-lipoprotein assembly and secretion and is known to transfer triacylglycerols, cholesterol esters, and phospholipids. To understand the relative importance of each lipid transfer activity, we compared the ability of hMTP and its Drosophila ortholog (dMTP) to assemble apoB lipoproteins and to transfer various lipids. apoB48 secretion was induced when co-expressed with either hMTP or dMTP in COS cells, and oleic acid supplementation further augmented secretion without altering particle density. C-terminal epitope-tagged dMTP (dMTP-FLAG) facilitated the secretion of apoB polypeptides in the range of apoB48 to apoB72 but was ϳ50% as efficient as hMTP-FLAG. Comparison of lipid transfer activities revealed that although phospholipid transfer was similar in both orthologs, dMTP was unable to transfer neutral lipids. We conclude that the phospholipid transfer activity of MTP is sufficient for the assembly and secretion of primordial apoB lipoproteins and may represent its earliest function evolved for the mobilization of lipid in invertebrates. Identification of MTP inhibitors, which selectively affect transfer of a specific lipid class, may have therapeutic potential.Lipoproteins are lipid-protein complexes that transport lipids, fatsoluble vitamins, and other hydrophobic molecules in the plasma. Apolipoprotein B (apoB) 2 is a structural protein embedded in the phospholipid monolayer on the surface of triglyceride-rich lipoproteins. It has been hypothesized that apoB contains amphipathic ␣-helical and ␤-sheet domains (1). Lipidation of the ␤-sheets is necessary for the assembly of larger apoB polypeptides into lipoprotein emulsion particles. Lipoprotein assembly begins co-translationally and microsomal triglyceride transfer protein (MTP) plays a pivotal role in this process (for reviews, see Refs. 2-6). MTP is absent in abetalipoproteinemia, a disease characterized by the deficit of plasma apoB lipoproteins and low plasma cholesterol levels (7,8). Reconstitution of MTP in heterologous systems rescues apoB secretion, whereas tissue-specific liver knock-out models recreate the lipoprotein deficiency present in abetalipoproteinemia (9, 10). The signature activity of MTP is its ability to transfer lipids between membranes. It has been demonstrated that MTP lipid transfer activity is necessary for apoB lipoprotein assembly and secretion (for reviews, see Refs. 2-6). More recent evidence suggests that MTP lipid transfer activity is also responsible for lipid accretion within the secretory pathway and that MTP potentially stabilizes lipid vesicles in the endoplasmic reticulum (reviewed in Refs. 2 and 3).MTP transfers several lipids including triacylglycerols, cholesterol esters, and phospholipids between vesicles in vitro (11)(12)(13)(14). It has been suggested that MTP transiently interacts with a membrane, extracts lipids, and then delivers them to another lipid acceptor or to nascent apoB lipoproteins (15). Kinetic studies in...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Phospholipid Transfer Activity of Microsomal Triacylglycerol Transfer Protein Is Sufficient for the Assembly and Secretion of Apolipoprotein B Lipoproteins

Rava

Ojakian

Shelness

et al. 2006

Journal of Biological Chemistry

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Liposomal FRET Assay Identifies Potent Drug‐Like Inhibitors of the Ceramide Transport Protein (CERT)

Samaha

Hamdo

Cong

et al. 2020

Chemistry A European J

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Ceramide transfer protein (CERT) mediates non‐vesicular transfer of ceramide from endoplasmic reticulum to Golgi apparatus and thus catalyzes the rate‐limiting step of sphingomyelin biosynthesis. Usually, CERT ligands are evaluated in tedious binding assays or non‐homogenous transfer assays using radiolabeled ceramides. Herein, a facile and sensitive assay for CERT, based on Förster resonance energy transfer (FRET), is presented. To this end, we mixed donor and acceptor vesicles, each containing a different fluorescent ceramide species. By CERT‐mediated transfer of fluorescent ceramide, a FRET system was established, which allows readout in 96‐well plate format, despite the high hydrophobicity of the components. Screening of a 2 000 compound library resulted in two new potent CERT inhibitors. One is approved for use in humans and one is approved for use in animals. Evaluation of cellular activity by quantitative mass spectrometry and confocal microscopy showed inhibition of ceramide trafficking and sphingomyelin biosynthesis.

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Phospholipid Transfer Proteins as Probes of Membrane Structure and Function

Crain

1990

Subcellular Biochemistry

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Quantitation of Lipid Transfer Activity

Cited by 20 publications

References 83 publications

Phospholipid Transfer Activity of Microsomal Triacylglycerol Transfer Protein Is Sufficient for the Assembly and Secretion of Apolipoprotein B Lipoproteins

Phospholipid Transfer Activity of Microsomal Triacylglycerol Transfer Protein Is Sufficient for the Assembly and Secretion of Apolipoprotein B Lipoproteins

Liposomal FRET Assay Identifies Potent Drug‐Like Inhibitors of the Ceramide Transport Protein (CERT)

Phospholipid Transfer Proteins as Probes of Membrane Structure and Function

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