The retinoic acid-related orphan receptor ␣ (ROR␣) is an orphan member of the subfamily 1 of nuclear hormone receptors. Our recent structural and functional studies have led to the hypothesis that cholesterol or a cholesterol derivative is the natural ligand of ROR␣. We have now solved the x-ray crystal structure of the ligand binding domain of ROR␣ in complex with cholesterol-3-O-sulfate following a ligand exchange experiment. In contrast to the 3-hydroxyl of cholesterol, the 3-O-sulfate group makes additional direct hydrogen bonds with three residues of the ROR␣ ligand binding domain, namely NH-Gln 289 , NH-Tyr 290 , and NH1-Arg 370 . When compared with the complex with cholesterol, seven well ordered water molecules have been displaced, and the ligand is slightly shifted toward the hydrophilic part of the ligand binding pocket, which is ideally suited for interactions with a sulfate group. These additional ligand-protein interactions result in an increased affinity of cholesterol sulfate when compared with cholesterol, as shown by mass spectrometry analysis done under native conditions and differential scanning calorimetry. Moreover, mutational studies show that the higher binding affinity of cholesterol sulfate translates into an increased transcriptional activity of ROR␣. Our findings suggest that cholesterol sulfate could play a crucial role in the regulation of ROR␣ in vivo.The group of retinoic acid-related orphan nuclear receptors (ROR) 1 is encoded by three different genes (␣, ␤, and ␥) (1). ROR␣ has been implicated in numerous age-related phenotypes such as atherosclerosis, cerebellar atrophy, immunodeficiency, and bone metabolism (2). ROR␣ was still considered an orphan receptor until we recently reported the first crystal structure of the ROR␣ LBD. It had revealed a ligand that was unexpectedly present, namely cholesterol (3). We also had shown that the transcriptional activity of ROR␣ could be modulated by changes in intracellular cholesterol level or mutation of residues involved in cholesterol binding. This has led to the hypothesis that ROR␣ could play a key role in the regulation of cholesterol homeostasis and thus represents an important drug target in cholesterol-related diseases. Despite the relatively high homology between ROR␣ LBD and ROR␤ LBD (63%), cholesterol seems not to be a ligand for the ROR␤ isoform, as reported recently by . This indicates a possible distinct function for ROR␤ and ROR␣. An inspection of the x-ray structure of the complex between ROR␣ LBD and cholesterol had shown that in the hydrophilic part of the LBP, there is space for a substituent attached to the hydroxy group of cholesterol, if water molecules are displaced (3). The presence of three arginines (Arg 292 , Arg 370 , and Arg 367 ) and of two free backbone amide nitrogens (NH-Gln 289 and NH-Tyr 290 ) strongly suggested a negatively charged substituent with at least two hydrogen-bond acceptor functionalities. Docking studies led to the prediction that cholesterol sulfate should have higher affinity than cholest...

Separate primary and validating clinical studies concur that tumor VEGF level is the most important prognostic parameter among several markers of tumor angiogenesis and proteolysis.

The crystal structure of the ligand binding domain (LBD) of the estrogen-related receptor ␣ (ERR␣, NR3B1) complexed with a coactivator peptide from peroxisome proliferator-activated receptor coactivator-1␣ (PGC-1␣) reveals a transcriptionally active conformation in the absence of a ligand. This is the first x-ray structure of ERR␣ LBD, solved to a resolution of 2.5 Å, and the first structure of a PGC-1␣ complex. The putative ligand binding pocket (LBP) of ERR␣ is almost completely occupied by side chains, in particular with the bulky side chain of Phe 328 (corresponding to Ala 272 in ERR␥ and Ala 350 in estrogen receptor ␣). Therefore, a ligand of a size equivalent to more than ϳ4 carbon atoms could only bind in the LBP, if ERR␣ would undergo a major conformational change (in particular the ligand would displace H12 from its agonist position). The x-ray structure thus provides strong evidence for ligand-independent transcriptional activation by ERR␣. The interactions of PGC-1␣ with ERR␣ also reveal for the first time the atomic details of how a coactivator peptide containing an inverted LXXLL motif (namely a LLXYL motif) binds to a LBD. In addition, we show that a PGC-1␣ peptide containing this nuclear box motif from the L3 site binds ERR␣ LBD with a higher affinity than a peptide containing a steroid receptor coactivator-1 motif and that the affinity is further enhanced when all three leucine-rich regions of PGC-1␣ are present.Nuclear hormone receptors (NRs) 1 are transcription factors that control essential developmental and physiological pathways (1). Although the transcriptional activity of NRs is often regulated by specific ligands, several members of the superfamily have no known natural ligands and are therefore referred to as orphan NRs (2). Estrogen-related receptor ␣ (ERR␣; NR3B1) was the first orphan NR to be identified on the basis of its similarity with estrogen receptor ␣ (ER␣; NR3A1) (3). ERR␣ and its relatives ERR␤ (NR3B2) and ERR␥ (NR3B3) form a small family of orphan NRs that are evolutionarily related to the estrogen receptors ER␣ and ER␤. ERRs preferentially bind to DNA sites composed of a single half-site preceded by three nucleotides with the consensus sequence TNAAGGTCA, referred to as an ERR response element. It has been shown that ERR␣ also efficiently binds to estrogen response elements and that these receptors share common target genes (4). This observation was further supported by studies demonstrating cross-talk between the ER and ERR pathways (reviewed in Ref. 5). The most striking feature observed in the phenotype of mice lacking ERR␣ is their resistance to high fat diet-induced obesity and the impaired activity of enzymes implicated in lipid metabolism. This finding led to the hypothesis that ERR␣ could be implicated in obesity or metabolic diseases (6). A function of ERR␣ on bone metabolism has also been suggested (7,8). Finally, recent publications show that ERR␣ and ERR␥ are associated with biomarkers of breast cancer and further emphasize the importance of ER-ERR cross-talk (9...

The retinoic acid-related orphan receptor alpha (RORalpha) is an orphan member of the subfamily 1 of nuclear hormone receptors. No X-ray structure of RORalpha has been described so far, and no ligand has been identified. We describe the first crystal structure of the ligand binding domain (LBD) of RORalpha, at 1.63 A resolution. This structure revealed a ligand present in the ligand binding pocket (LBP), which was identified by X-ray crystallography as cholest-5-en-3beta-ol (cholesterol). Moreover, RORalpha transcriptional activity could be modulated by changes in intracellular cholesterol level or mutation of residues involved in cholesterol binding. These findings suggest that RORalpha could play a key role in the regulation of cholesterol homeostasis and thus represents an important drug target in cholesterol-related diseases.

Deubiquitinating enzymes (DUBs) are a superfamily of thiol-and metallo proteases specialized in the processing of ubiquitin and ubiquitin-like proteins. They are responsible for the disassembly of ubiquitin chains, and for the cleavage of mono-and oligomers of this molecule, either in precursor form or attached to small Ubiquitin specific protease 7 (USP7) belongs to the family of deubiquitinating enzymes. Among other functions, USP7 is involved in the regulation of stress response pathways, epigenetic silencing and the progress of infections by DNA viruses. USP7 is a 130-kDa protein with a cysteine peptidase core, N-and C-terminal domains required for protein-protein interactions. In the present study, recombinant USP7 full length, along with several variants corresponding to domain deletions, were expressed in different hosts in order to analyze post-translational modifications, oligomerization state, enzymatic properties and subcellular localization patterns of the enzyme. USP7 is phosphorylated at S18 and S963, and ubiquitinated at K869 in mammalian cells. In in vitro activity assays, N-and C-terminal truncations affected the catalytic efficiency of the enzyme different. Both the protease core alone and in combination with the N-terminal domain are over 100-fold less active than the full length enzyme, whereas a construct including the C-terminal region displays a rather small decrease in catalytic efficiency. Limited proteolysis experiments revealed that USP7 variants containing the C-terminal domain interact more tightly with ubiquitin. Besides playing an important role in substrate recognition and processing, this region might be involved in enzyme dimerization. USP7 constructs lacking the N-terminal domain failed to localize in the cell nucleus, but no nuclear localization signal could be mapped within the enzyme's first 70 amino acids. Instead, the tumor necrosis factor receptor associated factor-like region (amino acids 70-205) was sufficient to achieve the nuclear localization of the enzyme, suggesting that interaction partners might be required for USP7 nuclear import.Abbreviations CBP, calmodulin binding protein; DUB, deubiquitinating enzyme; EGFP, enhanced green fluorescent protein; GST, glutathione S-transferase; NLS, nuclear localization signal; SUMO-1, small ubiquitin-like modifier protein 1; TAP, tandem affinity purification; TRAF, tumor necrosis factor receptor associated factor; Ub, ubiquitin; UCH, ubiquitin C-terminal hydrolase; USP, ubiquitin specific protease.

(H12) has to move away, and thus the activation helix H12 is displaced from its agonist position. This is a novel mechanism of H12 inactivation, different from ERR␥, estrogen receptor (ER) ␣, and ER␤. H12 binds (with a surprising binding mode) in the coactivator groove of its ligand binding domain, at a similar place as a coactivator peptide. This is in contrast to ERR␥ but resembles the situation for ER␣ (raloxifene or 4-hydroxytamoxifen complexes). Our results explain the novel molecular mechanism of an inverse agonist for ERR␣ and provide the basis for rational drug design to obtain isotype-specific inverse agonists of this potential new drug target. Despite a practically filled LBP, the finding that a suitable ligand can induce an opening of the cavity also has broad implications for other orphan nuclear hormone receptors (e.g. the NGFI-B subfamily).

As part of a program aimed at the development of selective estrogen receptor modulators (SERMs), tetrahydroisoquinoline derivative 27 was discovered by high throughput screening. Successive replacements of the p-F substituent of 27 by an aminoethoxy side chain and of the 1-H of the tetrahydroisoquinoline core by a 1-Me group provided analogues 19 and 20. These compounds showed potencies in a cell-based reporter gene assay (ERE assay) varying between 0.6 and 20 nM and displayed antagonist behaviors in the MCF-7 human breast adenocarcinoma cell line with IC(50)s in the range of 2-36 nM. The effect of N-phenyl substituents on the activity and pharmacokinetic properties of tetrahydroisoquinoline analogues was explored. As a result of this investigation, two potent derivatives bearing a p-F N-aryl group, 19c and 20c, were discovered as candidates suitable for further profiling. To gain insight into the ligand-receptor interaction, the X-ray crystallographic structure of the 1-H tetrahydroisoquinoline derivative (R)-18a in complex with ERalpha-ligand binding domain (LBD)(301)(-)(553)/C-->S triple mutant was solved to 2.28 A. An overlay of this X-ray crystal structure with that reported for the complex of ERalpha-LBD(301)(-)(553)/carboxymethylated C and raloxifene (5) shows that both compounds bind to the same cleft of the receptor and display comparable binding modes, with differences being observed in the conformation of their "D-ring" phenyl groups.

SummaryCellular sites of coagulation activation within complex, intact tissues have been studied by immunohistochemical techniques. Hirudin, a specific and high affinity inihibitor of the active site of thrombin, together with antibody to hirudin were applied to sections of AMeX-fixed specimens of normal lung, kidney, placenta, freshly incised skin and unperturbed skin obtained at fresh autopsy; to rheumatoid synovial tissue; and to malignant tissue from a variety of tumor types. Staining for thrombin was observed selectively on pulmonary alveolar, rheumatoid synovial, and placental macrophages that express an intact extrinsic coagulation pathway. Staining was also observed restricted to the endothelium of capillaries in freshly incised skin but not in either unperturbed skin or in aged incisions. Staining of tumor cell bodies was observed in small cell carcinoma of the lung, renal cell carcinoma, and malignant melanoma tissues that we found previously to show tumor cell-associated procoagulant activity. This staining occurred commonly on cells within the tumor mass that were distant from stromal fibrinogen/fibrin. By contrast, tumor-associated macrophage but not tumor cell staining was seen in adenocarcinoma and squamous cell carcinoma of the lung, and little or no staining was seen in colon cancer tissue. Negative controls in which either the hirudin probe or its antibody were omitted failed to show staining. These results are in accord with previous findings and suggest that such techniques may be useful for studying the cellular sites of thrombin generation in intact tissues. We postulate that administration of potent and specific thrombin antagonists, such as hirudin, to patients with relevant tumor types might be followed by homing of hirudin to tumor cells in vivo so that effects of local thrombin generation on malignant progression can be determined.