A truncated analogue of CCL2 mediates anti-fibrotic effects on murine fibroblasts independently of CCR2.

The truncated [1+9-76] CCL2 analogue, also known as 7ND, has been described in numerous reports as an anti-inflammatory and anti-fibrotic agent in a wide spectrum of animal models, e.g. models of cardiovascular disease, graft versus host disease and bleomycin-induced pulmonary fibrosis. 7ND has been reported to function as a competitive inhibitor of CCL2 signaling via CCR2 in human in vitro systems. In contrast, the mechanistic basis of 7ND action in animal models has not been previously reported. Here we have studied how 7ND interacts with CCL2 and CCR2 of murine origin. Surprisingly, 7ND was shown to be a weak inhibitor of murine CCL2/CCR2 signaling and displaced murine CCL2 (JE) from the receptor with a K(i)>1 µM. Using surface plasmon resonance, we found that 7ND binds murine CCL2 with a K(d) of 670 nM, which may indicate that 7ND inhibits murine CCL2/CCR2 signaling by a dominant negative mechanism rather than by competitive binding to the CCR2 receptor. In addition we observed that sub-nanomolar levels of 7ND mediate anti-fibrotic effects in CCR2 negative fibroblasts cultured from fibrotic lung of bleomycin-induced mice. Basal levels of extracellular matrix proteins were reduced (collagen type 1 and fibronectin) as well as expression levels of α-smooth muscle actin and CCL2. Our conclusion from these data is that the previously reported effects of 7ND in murine disease models most probably are mediated via mechanisms independent of CCR2.

The crystal structure of guinea pig 11beta-hydroxysteroid dehydrogenase type 1 provides a model for enzyme-lipid bilayer interactions.

The metabolic reduction of 11-keto groups in glucocorticoid steroids such as cortisone leads to the nuclear receptor ligand cortisol. This conversion is an example of pre-receptor regulation and constitutes a novel pharmacological target for the treatment of metabolic disorders such as insulin resistance and possibly other derangements observed in the metabolic syndrome, such as hyperlipidemia, hypertension, and lowered insulin secretion. This reaction is carried out by the NADPH-dependent type 1 11beta-hydroxysteroid dehydrogenase (11beta-HSD1), an enzyme attached through an integral N-terminal transmembrane helix to the lipid bilayer and located with its active site within the lumen of the endoplasmic reticulum. Here we report the crystal structure of recombinant guinea pig 11beta-HSD1. This variant was determined in complex with NADP at 2.5 A resolution and crystallized in the presence of detergent and guanidinium hydrochloride. The overall structure of guinea pig 11beta-HSD1 shows a clear relationship to other members of the superfamily of short-chain dehydrogenases/reductases but harbors a unique C-terminal helical segment that fulfills three essential functions and accordingly is involved in subunit interactions, contributes to active site architecture, and is necessary for lipid-membrane interactions. The structure provides a model for enzyme-lipid bilayer interactions and suggests a funneling of lipophilic substrates such as steroid hormones from the hydrophobic membrane environment to the enzyme active site.

High-level production and optimization of monodispersity of 11beta-hydroxysteroid dehydrogenase type 1.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is an intraluminally oriented, endoplasmic reticulum (ER)-bound enzyme catalyzing the interconversion between inactive cortisone and hormonally active cortisol. Heterologous production of 11beta-HSD1, devoid of its N-terminal transmembrane segment, is possible but yields only small amounts of soluble protein. Here we show that the soluble portion of recombinant 11beta-HSD1 produced in E. coli is found mainly as multimeric aggregates in the absence of detergent, and to a large extent associated with the endogenous chaperonin GroEL and other E. coli proteins. By co-overexpressing GroEL/ES and adding an 11beta-HSD1 inhibitor during protein synthesis, we have increased the accumulation of soluble 11beta-HSD1 by more than one order of magnitude. Using monodispersity as a screening criterion, we have also optimized the purification process by evaluating various solubilizing systems for the chromatographic steps, finally obtaining stable monodisperse preparations of both human and guinea pig 11beta-HSD1. By analytical ultracentrifugation, we could demonstrate that 11beta-HSD1 mainly exists as a dimer in the solubilized state. Moreover, active site titration of human 11beta-HSD1 revealed that at least 75% of the protein in a typical preparation represents active enzyme. Equilibrium unfolding experiments indicate that addition of inhibitor and the cofactor NADP(H) can stabilize the conformational stability of this enzyme in an additive manner. The outlined procedure may provide a general method for preparing similar proteins to oligomeric homogeneity and with retained biological activity.

Crystal structure of the heterodimeric complex of LXRalpha and RXRbeta ligand-binding domains in a fully agonistic conformation.

The nuclear receptor heterodimers of liver X receptor (LXR) and retinoid X receptor (RXR) are key transcriptional regulators of genes involved in lipid homeostasis and inflammation. We report the crystal structure of the ligand-binding domains (LBDs) of LXRalpha and RXRbeta complexed to the synthetic LXR agonist T-0901317 and the RXR agonist methoprene acid (Protein Data Base entry 1UHL). Both LBDs are in agonist conformation with GRIP-1 peptides bound at the coactivator binding sites. T-0901317 occupies the center of the LXR ligand-binding pocket and its hydroxyl head group interacts with H421 and W443, residues identified by mutational analysis as critical for ligand-induced transcriptional activation by T-0901317 and various endogenous oxysterols. The topography of the pocket suggests a common anchoring of these oxysterols via their 22-, 24- or 27-hydroxyl group to H421 and W443. Polyunsaturated fatty acids act as LXR antagonists and an E267A mutation was found to enhance their transcriptional inhibition. The present structure provides a powerful tool for the design of novel modulators that can be used to characterize further the physiological functions of the LXR-RXR heterodimer.