Synthesis and secretion of wild-type and mutant human plasma cholesteryl ester transfer protein in baculovirus-transfected insect cells: the carboxyl-terminal region is required for both lipoprotein binding and catalysis of transfer.

Au-Young, Janice; Fielding, Christopher J.

doi:10.1073/pnas.89.9.4094

Cited by 53 publications

(40 citation statements)

References 33 publications

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“…Sequence alignments with other neutral lipid-metabolizing and -interacting proteins led to the prediction of a putative NLBD that is involved in interactions with lipid substrates (31). We hypothesized that the NLBD in hTGH is involved in engaging hydrophobic substrates.…”

Section: Discussionmentioning

confidence: 99%

“…The conserved phenylalanine and leucine residues present within the putative NLBD (Table 2) have been proposed to provide the necessary hydrophobicity for interaction with lipids (31). In hTGH, these NLBD conserved residues are F 417 , L 418 , and L 420 and were the targets for point mutations.…”

Section: Expression Of Wt-htgh and Htgh Point Mutants In Cos-7 Cellsmentioning

confidence: 99%

“…TGH readily translocates from the aqueous milieu to diacylglycerol monolayers (13) and has been found associated with cytoplasmic lipid droplets (17) however, the regions on the protein that confer TGH affinity for lipids have not been characterized. Au-Young and Fielding (31) proposed a neutral lipid binding domain (NLBD) in human plasma cholesteryl ester transfer protein (hCETP). This putative lipid binding octapeptide forms an amphipathic a-helix with the consensus sequence FLXLXXXn (where n is a nonpolar residue and X is any amino acid except proline).…”

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confidence: 99%

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Mutation of F417 but not of L418 or L420 in the lipid binding domain decreases the activity of triacylglycerol hydrolase

Alam

Gilham

Vance

et al. 2006

Journal of Lipid Research

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Human triacylglycerol hydrolase (hTGH) has been shown to play a role in hepatic lipid metabolism. Triacylglycerol hydrolase (TGH) hydrolyzes insoluble carboxylic esters at lipid/water interfaces, although the mechanism by which the enzyme adsorbs to lipid droplets is unclear. Threedimensional modeling of hTGH predicts that catalytic residues are adjacent to an a-helix that may mediate TGH/lipid interaction. The helix contains a putative neutral lipid binding domain consisting of the octapeptide FLDLIADV (amino acid residues 417-424) with the consensus sequence FLXLXXXn (where n is a nonpolar residue and X is any amino acid except proline) identified in several other proteins that bind or metabolize neutral lipids. Deletion of this a-helix abolished the lipolytic activity of hTGH. Replacement of F 417 with alanine reduced activity by 40% toward both insoluble and soluble esters, whereas replacement of L 418 and L 420 with alanine did not. Another potential mechanism of increasing TGH affinity for lipid is via reversible acylation. Molecular modeling predicts that C 390 is available for covalent acylation. However, neither chemical modification of C 390 nor mutation to alanine affected activity. Several endoplasmic reticulum-associated carboxylesterases with broad and overlapping substrate specificities have been identified (1-5). Mammalian carboxylesterases are known to metabolize numerous analgesic and narcotic compounds, including aspirin, cocaine, heroin, procaine, and meperidine (6-9). Hepatic carboxylesterases are involved in the biotransformation of xenobiotics and natural substrates by hydrolyzing compounds containing ester, thioester, or amide bonds (10). Carboxylesterases are also used to activate anticancer prodrugs such as CPT-11 (irinotecan) (11, 12).Our laboratory has investigated the participation of hepatic carboxylesterases in lipid metabolism. We have purified a carboxylesterase that catalyzes the lipolysis of triacylglycerols and termed it triacylglycerol hydrolase (TGH) (13-15) and have isolated and expressed a cDNA for human triacylglycerol hydrolase (hTGH GenBank accession number NM_001266) (16). Hepatic TGH participates in the mobilization of stored triacylglycerol (TG) for the assembly of VLDL (14,(17)(18)(19)(20). Because VLDL is the precursor for plasma LDL and a high level of plasma LDL is a major risk factor for the development of atherosclerosis and coronary heart disease (21, 22), investigations of the mechanism of action of enzymes and proteins involved in the assembly and secretion of VLDL are of clinical importance. Hepatic VLDL assembly is predominantly regulated by the provision of lipid (23-25). It has been reported by several groups that the majority of TG in VLDL originates from cytoplasmic storage pools, and mobilization of this TG involves lipolysis followed by reesterification to reform TG before loading onto nascent apolipoprotein B-containing particles (26-30). Therefore, TGH is a pharmacological target (inhibition) to decrease plasma lipid levels, and detailed k...

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Section: Discussionmentioning

confidence: 99%

Section: Expression Of Wt-htgh and Htgh Point Mutants In Cos-7 Cellsmentioning

confidence: 99%

mentioning

confidence: 99%

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Mutation of F417 but not of L418 or L420 in the lipid binding domain decreases the activity of triacylglycerol hydrolase

Alam

Gilham

Vance

et al. 2006

Journal of Lipid Research

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“…DGAT2 also contains the consensus sequence (amino acids 80-87; FLXLXXXn, in which n is a nonpolar amino acid), for a neutral lipid binding domain found in a variety of proteins that bind to or metabolize neutral lipids, such as cholesterol ester transfer protein (CETP), hormonesensitive lipase (HSL), lecithin:cholesterol acyltransferase (LCAT), cholesterol 7a-hydroxylase, cholesterol esterase, and TG hydrolase (45,46). This domain may be involved in DG binding.…”

Section: Dgat2mentioning

confidence: 99%

Thematic Review Series: Glycerolipids. DGAT enzymes and triacylglycerol biosynthesis

Yen

Stone

Koliwad

et al. 2008

Journal of Lipid Research

913

782

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Triacylglycerols (triglycerides) (TGs) are the major storage molecules of metabolic energy and FAs in most living organisms. Excessive accumulation of TGs, however, is associated with human diseases, such as obesity, diabetes mellitus, and steatohepatitis. The final and the only committed step in the biosynthesis of TGs is catalyzed by acylCoA:diacylglycerol acyltransferase (DGAT) enzymes. The genes encoding two DGAT enzymes, DGAT1 and DGAT2, were identified in the past decade, and the use of molecular tools, including mice deficient in either enzyme, has shed light on their functions. Although DGAT enzymes are involved in TG synthesis, they have distinct protein sequences and differ in their biochemical, cellular, and physiological functions. Both enzymes may be useful as therapeutic targets for diseases. Here we review the current knowledge of DGAT enzymes, focusing on new advances since the cloning of their genes, including possible roles in human health and diseases.-Yen, C-L. E., S. J. Stone, S. Koliwad, C. Harris, and R. V. Farese, Jr. DGAT enzymes and triacylglycerol biosynthesis. J. Lipid Res. 2008. 49: 2283-2301. Supplementary key words triacylglycerolsTriacylglycerols (triglycerides) (TGs), a major type of neutral lipid, are a heterogeneous group of molecules with a glycerol backbone and three FAs attached by ester bonds. The physical and chemical properties of TG differ based on chain length and the degree to which their FAs are desaturated. TGs serve multiple important functions in living organisms. Chief among these, they are the major storage molecules of FA for energy utilization and the synthesis of membrane lipids. Because they are highly reduced and anhydrous, TGs store 6-fold more energy than the same amount of hydrated glycogen (1). In plants, TGs are a major component of seed oils, which are valuable resources for dietary consumption and industrial uses. TG from plants and microorganisms can also be used for biofuels. In animals, energy stores of TG are concentrated primarily in adipocytes, although TGs are also found prominently in myocytes, hepatocytes, enterocytes, and mammary epithelial cells. In addition to energy storage, TG synthesis in cells may protect them from the potentially toxic effects of excess FA. In the enterocytes and hepatocytes of most mammals, TGs are synthesized for the assembly and secretion of lipoproteins, which transport dietary and endogenously synthesized FA between tissues. Also, TGs in secreted lipids acts as a component of the skinʼs surface water barrier, and collections of TG in adipose tissue provide insulation for organisms.Although TGs are essential for normal physiology, the excessive accumulation of TG in human adipose tissue results in obesity and, in nonadipose tissues, is associated with organ dysfunction. For example, excessive TG deposition in skeletal muscle and the liver is associated with insulin resistance, in the liver with nonalcoholic steatohepatitis, and in the heart with cardiomyopathy (2, 3). Owing to worldwide increases in the p...

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“…Most fusogenic proteins have distinct, amphipathic amino acid sequences with hydrophobicity indexes ranging from 0.5 to 0.7 (29). Candidate fusogenic regions of CETP include the putative ␣-helix spanning amino acids 454 -457 (30). These amino acids are in the highly conserved C-terminal part of CETP that contains binding sites for CE and TG (31).…”

Section: Table III Physical Properties Of (A-i)rhdl and (A-ii)rhdl Afmentioning

confidence: 99%

Evidence That Cholesteryl Ester Transfer Protein-mediated Reductions in Reconstituted High Density Lipoprotein Size Involve Particle Fusion

Rye

Hime

Barter

1997

Journal of Biological Chemistry

100

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It is well established that cholesteryl ester transfer protein (CETP) changes the size of high density lipoproteins (HDL) during incubation in vitro. It has been suggested that HDL⅐CETP⅐HDL ternary complex formation is involved in these changes. The present results, which are consistent with CETP changing the size of spherical reconstituted HDL (rHDL) by a mechanism involving fusion, support the ternary complex hypothesis. When rHDL containing a core of cholesteryl esters and either three molecules of apolipoprotein (apo) A-I/particle, (A-I)rHDL, or six molecules of apoA-II/particle, (A-II)rHDL, were incubated individually with CETP, their respective diameters decreased from 9.4 to 7.8 nm and from 9.8 to 8.8 nm. The small (A-I)rHDL and (A-II)rHDL contained, respectively, two molecules of apoA-I/particle and four molecules of apoA-II/particle. As all of the rHDL lipids and apolipoproteins were quantitatively recovered at the end of the incubations, it was apparent that there was a 50% increase in the number of particles. This increase in the number of particles can be explained as follows: (i) sequential binding of two rHDL to CETP to generate a ternary complex, (ii) fusion of the rHDL in the ternary complex, and (iii) rearrangement of the fusion product into three small particles. Various spectroscopic techniques were used to show that the small rHDL were structurally distinct from the original rHDL. These results provide the first evidence that CETP mediates the fusion of spherical rHDL.

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Synthesis and secretion of wild-type and mutant human plasma cholesteryl ester transfer protein in baculovirus-transfected insect cells: the carboxyl-terminal region is required for both lipoprotein binding and catalysis of transfer.

Cited by 53 publications

References 33 publications

Mutation of F417 but not of L418 or L420 in the lipid binding domain decreases the activity of triacylglycerol hydrolase

Mutation of F417 but not of L418 or L420 in the lipid binding domain decreases the activity of triacylglycerol hydrolase

Thematic Review Series: Glycerolipids. DGAT enzymes and triacylglycerol biosynthesis

Evidence That Cholesteryl Ester Transfer Protein-mediated Reductions in Reconstituted High Density Lipoprotein Size Involve Particle Fusion

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