The complex of PPARalpha and PPARgamma with the dual specificity agonist AZ 242 highlights the conserved interactions required for receptor activation. Together with the NMR data, this suggests a general model for ligand activation in the PPAR family. A comparison of the ligand binding sites reveals a molecular explanation for subtype selectivity and provides a basis for rational drug design.

Transmembrane seven helix bundles form a large family of membrane inserted receptors and are responsible for a wide range of biological functions. Experimental data suggest that their overall structure is similar to bacteriorhodopsin. We describe here a new approach for the modeling of transmembrane seven helix bundles based on statistically derived environmental preference parameters combined with experimentally determined features of the receptors. The method was used to create a model for the human beta 2-adrenoreceptor. This model is physically plausible, is in reasonable agreement with experimental data and may be helpful in planning new receptor engineering experiments.

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G-protein-coupled receptors (GPCRs) form one of the largest superfamilies of membrane proteins. Obtaining high yields of GPCRs remains one of the major factors limiting a detailed understanding of their structure and function. Photoreceptor cells (PRCs) contain extensive stacks of specialized membranes where high levels of rhodopsins are naturally present, which makes them ideal for the overexpression of GPCRs. We have generated transgenic flies expressing a number of GPCRs in the PRCs. Drosophila melanogaster metabotropic glutamate receptor (DmGluRA) expressed by this novel strategy was purified to homogeneity, giving at least 3-fold higher yields than conventional baculovirus expression systems due to the higher membrane content of the PRCs. Pure DmGluRA was then reconstituted into liposomes of varying composition. Interestingly, glutamate binding was strictly dependent on the presence of ergosterol.

Procarboxypeptidase U [proCPU, thrombin‐activatable fibrinolysis inhibitor (TAFI), EC 3.4.17.20] belongs to the metallocarboxypeptidase family and is a zymogen found in human plasma. ProCPU has been proposed to be a molecular link between coagulation and fibrinolysis. Upon activation of proCPU, the active enzyme (CPU) rapidly becomes inactive due to its intrinsic instability. The inherent instability of CPU is likely to be of major importance for the in vivo down‐regulation of its activity, but the underlying structural mechanisms of this fast and spontaneous loss of activity of CPU have not yet been explained, and they severely inhibit the structural characterization of CPU. In this study, we screened for more thermostable versions of CPU to increase our understanding of the mechanism underlying the instability of CPU's activity. We have shown that single as well as a few 2–4 mutations in human CPU can prolong the half‐life of CPU's activity at 37 °C from 0.2 h of wild‐type CPU to 0.5–5.5 h for the mutants. We provide evidence that the gain in stable activity is accompanied by a gain in thermostability of the enzyme and increased resistance to proteolytic digest by trypsin. Using one of the stable mutants, we demonstrate the importance of CPU stability over proCPU concentration in down‐regulating fibrinolysis.

Objective-To study the distribution of group V secretory phospholipase A 2 (sPLA 2 ) in human and mouse lesions and compare its expression by human vascular cells, its activity toward lipoproteins, and the interaction with arterial proteoglycans (proteoglycans) with those of sPLA 2 -IIA. In addition, we also investigated the effect of a Western diet and lipopolysaccharide challenge on the aortic expression of these enzymes in mouse models. Methods and Results-Immunohistochemistry showed sPLA 2 -V in human and mouse lesions to be associated with smooth muscle cells and also surrounding foam cells in lipid core areas. mRNA of the enzyme was expressed in human lesions and human vascular cells, supporting the immunohistochemistry data. sPLA 2 -V but not sPLA 2 -IIA was active on lipoproteins in human serum. The association with proteoglycans enhanced 2-to 3-fold sPLA 2 -V activity toward low-density lipoproteins but not that of the group IIA enzyme. Experiments in mouse models showed that treatment with a Western diet induced expression of sPLA 2 -V but not that of sPLA 2 -IIA in aorta. On the other hand, lipopolysaccharide-induced acute inflammation augmented the expression of sPLA 2 -IIA but not that of sPLA 2 -V. Key Words: phospholipase Ⅲ atherogenesis Ⅲ inflammation Ⅲ lipoprotein-retention Ⅲ proteoglycans D uring atherosclerosis development, there is a progressive decrease of the lesion phospholipid content and enrichment in cholesterol. 1 Furthermore, apolipoprotein B (apoB) lipoproteins isolated from human and rabbit lesions contain less phosphatidylcholine (PC) and more sphingomyelin than circulating lipoproteins. 2,3 Therefore, lipoproteins trapped in the intima appear to be hydrolyzed by secretory phospholipases. 4 These enzymes may contribute to atherosclerosis by hydrolysis of low-density lipoprotein (LDL) phospholipids that induce fusion and increase binding of cholesterol-rich particles to intima proteoglycans, triggering further modifications. [5][6][7][8] In addition, phospholipase(s) A 2 contribute to local release of lyso-phospholipids and nonesterified fatty acids, which have proinflammatory properties in arterial cells. 9 -12 Conclusions-These See page 1421Secretory phospholipase A 2 (sPLA 2 ) group IIA is present in human atherosclerotic lesions, and experimental and clinical evidence suggest its involvement in atherosclerosis and cardiovascular disease. 13-17 sPLA 2 -IIA and the more recently cloned sPLA 2 -V are members of a family of enzymes that hydrolyze the fatty acids at the sn-2 position of glycerophospholipids. Both enzymes have low molecular weight (14 kDa), are histidine and calcium dependent, rich in disulfide bonds, are basic, and share structure similarities. 18 Several of these properties stabilize and enhance their activity in the extracellular milieu. The genes of sPLA 2 -IIA and sPLA 2 -V enzymes are located at close positions in homologous regions in mouse chromosome 4 and human chromosome 1 and share the same promoter. 19 This region was identified as an atherosclerosis ...

Although structural studies on the ligand-binding domain (LBD) have established the general mode of nuclear receptor (NR)/coactivator interaction, determinants of binding specificity are only partially understood. The LBD of estrogen receptor-␣ (ER␣), for example, interacts only with a region of peroxisome proliferator-activated receptor coactivator (PGC)-1␣, which contains the canonical LXXLL motif (NR box2), whereas the LBD of estrogen-related receptor-␣ (ERR␣) also binds efficiently an untypical, LXXYL-containing region (NR box3) of PGC-1␣. Surprisingly, in a previous structural study, the ER␣ LBD has been observed to bind NR box3 of transcriptional intermediary factor (TIF)-2 untypically via LXXYL, whereas the ERR␣ LBD binds this region of TIF-2 only poorly. Here we present a new crystal structure of the ERR␣ LBD in complex with a PGC-1␣ box3 peptide. In this structure, residues N-terminal of the PGC-1␣ LXXYL motif formed contacts with helix 4, the loop connecting helices 8 and 9, and with the C terminus of the ERR␣ LBD. Interaction studies using wild-type and mutant PGC-1␣ and ERR␣ showed that these contacts are functionally relevant and are required for efficient ERR␣/PGC-1␣ interaction. Furthermore, a structure comparison between ERR␣ and ER␣ and mutation analyses provided evidence that the helix 8 -9 loop, which differs significantly in both nuclear receptors, is a major determinant of coactivator binding specificity. Finally, our results revealed that in ERR␣ the helix 8 -9 loop allosterically links the LBD homodimer interface with the coactivator cleft, thus providing a plausible explanation for distinct PGC-1␣ binding to ERR␣ monomers and homodimers.

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