Cholesteryl ester transfer protein (CETP) shuttles various lipids between lipoproteins, resulting in the net transfer of cholesteryl esters from atheroprotective, high-density lipoproteins (HDL) to atherogenic, lower-density species. Inhibition of CETP raises HDL cholesterol and may potentially be used to treat cardiovascular disease. Here we describe the structure of CETP at 2.2-A resolution, revealing a 60-A-long tunnel filled with two hydrophobic cholesteryl esters and plugged by an amphiphilic phosphatidylcholine at each end. The two tunnel openings are large enough to allow lipid access, which is aided by a flexible helix and possibly also by a mobile flap. The curvature of the concave surface of CETP matches the radius of curvature of HDL particles, and potential conformational changes may occur to accommodate larger lipoprotein particles. Point mutations blocking the middle of the tunnel abolish lipid-transfer activities, suggesting that neutral lipids pass through this continuous tunnel.

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Vertebrate cells synthesize two forms of the 82-to 90-kilodalton heat shock protein that are encoded by distinct gene families. In HeLa cells, both proteins (hsp89ai and hsp890) are abundant under normal growth conditions and are synthesized at increased rates in response to heat stress. Only the larger form, hsp89a, is induced by the adenovirus EIA gene product (M. C. Simon, K. Kitchener, H. T. Kao, E. Hickey, L. Weber, R. Voellmy, N. Heintz, and J. R. Nevins, Mol. Cell. Biol. 7:2884-2890. We have isolated a human hsp89a gene that shows complete sequence identity with heat-and ElA-inducible cDNA used as a hybridization probe. The 5'-flanking region contained overlapping and inverted consensus heat shock control elements that can confer heat-inducible expression on a 1-globin reporter gene. The gene contained 10 intervening sequences. The first intron was located adjacent to the translation start codon, an arrangement also found in the Drosophila hsp82 gene. The spliced mRNA sequence contained a single open reading frame encoding an 84,564-dalton polypeptide showing high homology with the hsp82 to hsp90 proteins of other organisms. The deduced hsp89a protein sequence differed from the human hsp890 sequence reported elsewhere (N. F. Rebbe, J. Ware, R. M. Bertina, P. Modrich, and D. W. Stafford (Gene 53:235-245, 1987) in at least 99 out of the 732 amino acids. Transcription of the hsp89a gene was induced by serum during normal cell growth, but expression did not appear to be restricted to a particular stage of the cell cycle. hsp89a mRNA was considerably more stable than the mRNA encoding hsp7O, which can account for the higher constitutive rate of hsp89 synthesis in unstressed cells.The 82-to 90-kilodalton (kDa) class of heat shock proteins (HSPs) have long been recognized as cytoplasmic proteins that are abundant in the absence of stress (40,42,78) and which are induced to higher levels of synthesis by heat shock. In avian and mammalian cells and tissues, these proteins (hereafter referred to as hsp89) have been found in association with several different regulatory and structural proteins. hsp89 has been shown to interact with several viral oncogene products that possess tyrosine kinase activity, including pp60src (10, 55), and the yes (46),fps (55),fes, and fgr (85) gene products. In rabbit reticulocytes, hsp89 has been identified as the 90-kDa component of highly purified preparations of the hemin-controlled translational repressor, an eIF-2ot-specific protein kinase (63). hsp89 appears to stimulate the activity of this enzyme. In avian (3,85) and calf (60) cells, hsp89 has been identified as the non-steroidbinding subunit of the estrogen receptor complex and has since been shown to be a common component of other steroid hormone receptors (33). The steroid-binding component of these receptors appears to be inactive with respect to DNA binding when complexed with hsp89 (30,58,66).

Variation in individual response to statin therapy has been widely studied for a potential genetic component. Multiple genes have been identified as potential modulators of statin response, but few study findings have replicated. To further examine these associations, 2735 individuals on statin therapy, half on atorvastatin and the other half divided among fluvastatin, lovastatin, pravastatin and simvastatin were genotyped for 43 SNPs in 16 genes that have been implicated in statin response. Associations with lowdensity lipoprotein cholesterol (LDL-C) lowering, total cholesterol lowering, HDL-C elevation and triglyceride lowering were examined. The only significant associations with LDL-C lowering were found with apoE2 in which carriers of the rare allele who took atorvastatin lowered their LDL-C by 3.5% more than those homozygous for the common allele and with rs2032582 (S893A in ABCB1) in which the two groups of homozygotes differed by 3% in LDL-C lowering. These genetic effects were smaller than those observed with the demographic variables of age and gender. The magnitude of all the differences found is sufficiently small that genetic data from these genes should not influence clinical decisions on statin administration.

1 Pentamidine, an antiprotozoal agent, has been traditionally known to cause QT prolongation and arrhythmias; however, its ionic mechanism has not been illustrated. 2 In a stable HEK-293 cell line, we observed a concentration-dependent inhibition of the hERG current with an IC 50 of 252 mM. 3 In freshly isolated guinea-pig ventricular myocytes, pentamidine showed no effect on the L-type calcium current at concentrations up to 300 mM, with a slight prolongation of the action potential duration at this concentration. 4 Since the effective concentrations of pentamidine on the hERG channel and APD were much higher than clinically relevant exposures (B1 mM free or lower), we speculated that this drug might not prolong the QT interval through direct inhibition of I Kr channel. We therefore incubated hERG-HEK cells in 1 and 10 mM pentamidine-containing media (supplemented with 10% serum) for 48 h, and examined the hERG current densities in the vehicle control and pentamidine-treated cells. 5 In all, 36 and 85% reductions of the current densities were caused by 1-and 10-mM pentamidine treatment (Po0.001 vs control), respectively. A similar level of reduction of the hERG polypeptides and a reduced intensity of the hERG protein on the surface membrane in treated cells were observed by Western blot analysis and laser-scanning confocal microscopy, respectively. 6 Taken together, our data imply that chronic administration of pentamidine at clinically relevant exposure reduces the membrane expression of the hERG channel, which may most likely be the major mechanism of QT prolongation and torsade de pointes reported in man.

Background: Human cholesteryl ester transfer protein (CETP) transfers cholesteryl esters from high-density to low-density lipoprotein particles. Results: Crystallographic, mutagenesis, and biochemical studies illuminated inhibition mechanisms of CETP by torcetrapib and a structurally distinct compound, ((2R)-3-{[4-(4-chloro-3-ethylphenoxy)pyrimidin-2-yl][3-(1,1,2,2-tetrafluoroethoxy)benzyl]-amino}-1,1,1-trifluoropropan-2-ol. Conclusion: These small molecules inhibit CETP through blocking its lipid tunnel. Significance: Potential polar interactions at compound binding site may be utilized in design of inhibitors with improved physical properties.

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Luciferase (EC 1.13.12.7) from the North American firefly, Photinus pyralis, is widely used as a reporter enzyme in cell biology. One of its distinctive properties is a pronounced susceptibility to proteolytic degradation that causes luciferase to have a very short intracellular half-life. To define the structural basis for this behavior and possibly facilitate the design of more stable forms of luciferase, limited proteolysis studies were undertaken using trypsin and chymotrypsin to identify regions of the protein whose accessible and flexible character rendered them especially sensitive to cleavage. Regions of amino acid sequence 206 -220 and 329 -341 were found to be sensitive, and because the region around 206 -220 had high homology with other luciferases, CoA ligases, and peptidyl synthetases, this region was selected for mutagenesis experiments intended to determine which of its amino acids were essential for activity. Surprisingly, many highly conserved residues including Ser 198 , Ser 201 , Thr 202 , and Gly 203 could be mutated with little effect on the luminescent activity of P. pyralis luciferase. One mutation, however, S198T, caused several alterations in enzymatic properties including shifting the pH optimum from 8.1 to 8.7, lowering the K m for Mg-ATP by a factor of 4 and increasing the half-time for light emission decay by a factor of up to 150. While the S198T luciferase was less active than wild type, activity could be restored by the introduction of the additional L194F and N197Y mutations. In addition to indicating the involvement of this region in ATP binding, these results provide a new form of the enzyme that affords a more versatile reporter system. Luciferase (EC 1.13.12.7) from the North American firefly, Photinus pyralis, is widely used as a reporter enzyme in studies of gene expression (1). Firefly luciferase first catalyzes the formation of a bound luciferin-AMP, which then reacts with O 2 to generate oxyluciferin-AMP in an excited state. Breakdown of this intermediate to oxyluciferin, CO 2 , and AMP is accompanied by the emission of a photon. The high quantum yield for this process, nearly a single photon emitted per reacted luciferin molecule (2), reflects not only an efficient catalytic machinery but also a highly favorable environment for radiative decay of an excited state.The kinetics of light emission can be modulated by coenzyme A, but this is not a required substrate (3, 4). Luciferase can also function as a ligase (5) as the AMP group of the enzyme⅐luciferyl-AMP complex can be transferred to ATP to produce diadenosine tetraphosphate.Despite many studies, relatively little was known about the structure of luciferase or the nature of its active site until recently. Cloning and sequencing of luciferase from the firefly (6) and its homologues from several beetles (reviewed in Ref . 7) have shown that these enzymes are related to certain acyltransferases (8, 9). Alignment of these related sequences reveals conserved residues that may be important for enzymatic activity. For exam...