Human PXR Forms a Tryptophan Zipper-Mediated Homodimer †

The human nuclear receptor pregnane X receptor (PXR) responds to a wide variety of potentially harmful chemicals and coordinates the expression of genes central to xenobiotic and endobiotic metabolism. Structural studies reveal that the PXR ligand binding domain (LBD) uses a novel sequence insert to form a homodimer unique to the nuclear receptor superfamily. Terminal β-strands from each monomeric LBD interact in an ideal antiparallel fashion to bury potentially exposed surface β-strands, generating a ten-stranded intermolecular β-sheet. Conserved tryptophan and tyrosine residues lock across the dimer interface and provide the first tryptophan-zipper (Trp-Zip) interaction observed in a native protein. We show using analytical ultracentrifugation that the PXR LBD forms a homodimer in solution. We further find that removal of the interlocking aromatic residues eliminates dimer formation but does not affect PXR's ability to interact with DNA, RXRα, or ligands. Disruption of the homodimer significantly reduces receptor activity in transient transfection experiments, however, and effectively eliminates the receptor's recruitment of the transcriptional coactivator SRC-1 both in vitro and in vivo. Taken together, these results suggest that the unique Trp-Zip-mediated PXR homodimer plays a role in the function of this nuclear xenobiotic receptor

A chemical method for the sequential degradation of peptides from the carboxy terminus

A chemical method is described that will determine sequentially from the carboxy terminus the amino acid sequence of a protein or peptide fragment. The procedure involves attachment of peptide to a rigid glass support specifically at the amino terminus. Once the peptide is immobilized on the support, organic reagents and solvents may be used without difficulty. The free carboxy-terminal amino acid anion is converted to an acyl azide when allowed to react with bis(p-nitrophenyl)phosphorylazide in dimethylformamide. This acyl azide is then themolyzed to an isocyanate via the Curtius rearrangement in the presence of p-methoxybenzyl alcohol (pMBA), ultimately leading to an N-(1-aminoalkyl)peptidyl amide. This compound as the free base is coupled directly to ethyl chlorooxalate. Employing basic conditions, the N-(1-)O-ethyloxalyl)aminoalkyl)amide intermediate is cyclized internally via the amide nitrogen to yield an N-acetylated-2-alkyl-4,5-imidazolidinedione intermediate that is cleaved from the solid support most efficiently employing strong basic conditions. This procedure leaves the remaining peptide attached to the glass support, and the degraded amino acid derivatized as a 2-alkyl-4,5-imidazolidinedione (2I45D). The identity of the amino acid-2I45Ds are evaluated along with the cleavage efficiency using reverse-phase high-performance liquid chromatography. The exact conditions necessary to obtain maximum yields in all these transformations are discussed and described. Problems associated with cleavage efficiency are also discussed, in addition to experiments proposed to deal with these problems. The new carboxy-terminal amino acid of the adhered peptide continues the stepwise sequential degradation using this procedure

Inhibition of peroxisome proliferator-activated receptor (PPAR)-mediated keratinocyte differentiation by lipoxygenase inhibitors.

Lipoxygenase (LOX) metabolites from arachidonic acid and linoleic acid have been implicated in atherosclerosis, inflammation, keratinocyte differentiation and tumour progression. We previously showed that peroxisome proliferator-activated receptors (PPARs) play a role in keratinocyte differentiation and that the PPARalpha ligand 8S-hydroxyeicosatetraenoic acid is important in this process. We hypothesized that blocking LOX activity would block PPAR-mediated keratinocyte differentiation. Three LOX inhibitors, nordihydroguaiaretic acid, quercetin and morin, were studied for their effects on primary keratinocyte differentiation and PPAR activity. All three LOX inhibitors blocked calcium-induced expression of the differentiation marker keratin 1. In addition, activity of a PPAR-responsive element was inhibited in the presence of all three inhibitors, and this effect was mediated primarily through PPARalpha and PPARgamma. LOX inhibitors decreased the activity of a chimaeric PPAR-Gal4-ligand-binding domain reporter system and this effect was reversed by addition of PPAR ligands. Ligand-binding studies revealed that the LOX inhibitors bind directly to PPARs and demonstrate a novel mechanism for these inhibitors in altering PPAR-mediated gene expression

Gamma Tocopherol Upregulates the Expression of 15-S-HETE and Induces Growth Arrest Through a Ppar Gamma-Dependent Mechanism in PC-3 Human Prostate Cancer Cells

Chronic inflammation and dietary fat consumption correlates with an increase in prostate cancer. Our previous studies in the colon have demonstrated that -tocopherol treatment could upregulate the expression of peroxisome proliferator-activated preceptors (PPAR) , a nuclear receptor involved in fatty acid metabolism as well modulation of cell proliferation and differentiation. In this study, we explored the possibility that -tocopherol could induce growth arrest in PC-3 prostate cancer cells through the regulation of fatty acid metabolism. Growth arrest (40%) and PPAR mRNA and protein upregulation was achieved with -tocopherol within 6 h. -Tocopherol-mediated growth arrest was demonstrated to be PPAR dependent using the agonist GW9662 and a PPAR dominant negative vector. -tocopherol was shown not to be a direct PPAR ligand, but rather 15-S-HETE (an endogenous PPAR ligand) was upregulated by -tocopherol treatment. 15-Lipoxygenase-2, a tumor suppressor and the enzyme that converts arachidonic acid to 15-S-HETE, was upregulated at 3 h following -tocopherol treatment. Expression of proteins downstream of the PPAR pathway were examined. Cyclin D1, cyclin D3, bcl-2, and NF B proteins were found to be downregulated following -tocopherol treatment. These data demonstrate that the growth arrest mediated by -tocopherol follows a PPAR - dependent mechanism

1,550 nm Erbium-Doped and 1,927 nm Thulium Nonablative Fractional Laser System: Best Practices and Treatment Setting Recommendations

BACKGROUND: The Fraxel Dual laser system (Solta Medical, Inc., Bothell, WA) contains a 1,550 and 1,927 nm wavelength single handpiece with different indications for each wavelength. OBJECTIVE: To discuss treatment setting recommendations and best practices for select on-label and investigational applications of the 1,550 and 1,927 nm dual laser system. MATERIALS AND METHODS: Eight board-certified dermatologists with 10 or more years of experience with the 1,550 and 1,927 nm laser system completed an online survey about their clinical experience with the system and then participated in a roundtable to share clinical perspectives and best practices for using the laser system. RESULTS: For all Fitzpatrick skin types, treatment recommendations were described for selected approved indications for the 1,550 and 1,927 nm laser system, including both lasers in combination. Treatment recommendations were also reached for investigational applications with the 1,550 nm laser and 1,927 nm laser. Best practices for using the lasers during the treatment session to achieve optimal outcomes and decrease the post-treatment recovery time were compiled. CONCLUSION: The 1,550 and 1,927 nm dual laser system is effective for a wide range of aesthetic and therapeutic applications, on and off the face and across all Fitzpatrick skin types

Lysophosphatidic acid activates peroxisome proliferator activated receptor-γ in CHO cells that over-express glycerol 3-phosphate acyltransferase-1.

Lysophosphatidic acid (LPA) is an agonist for peroxisome proliferator activated receptor-γ (PPARγ). Although glycerol-3-phosphate acyltransferase-1 (GPAT1) esterifies glycerol-3-phosphate to form LPA, an intermediate in the de novo synthesis of glycerolipids, it has been assumed that LPA synthesized by this route does not have a signaling role. The availability of Chinese Hamster Ovary (CHO) cells that stably overexpress GPAT1, allowed us to analyze PPARγ activation in the presence of LPA produced as an intracellular intermediate. LPA levels in CHO-GPAT1 cells were 6-fold higher than in wild-type CHO cells, and the mRNA abundance of CD36, a PPARγ target, was 2-fold higher. Transactivation assays showed that PPARγ activity was higher in the cells that overexpressed GPAT1. PPARγ activity was enhanced further in CHO-GPAT1 cells treated with the PPARγ ligand troglitazone. Extracellular LPA, phosphatidic acid (PA) or a membrane-permeable diacylglycerol had no effect, showing that PPARγ had been activated by LPA generated intracellularly. Transient transfection of a vector expressing 1-acylglycerol-3-phosphate acyltransferase-2, which converts endogenous LPA to PA, markedly reduced PPARγ activity, as did over-expressing diacylglycerol kinase, which converts DAG to PA, indicating that PA could be a potent inhibitor of PPARγ. These data suggest that LPA synthesized via the glycerol-3-phosphate pathway can activate PPARγ and that intermediates of de novo glycerolipid synthesis regulate gene expression

γ-Tocotrienol Induces Growth Arrest Through a Novel Pathway With TGFβ2 in Prostate Cancer

Regions along the Mediterranean and in southern Asia have lower prostate cancer incidence compared to the rest of the world. It has been hypothesized that one of the potential contributing factors for this low incidence includes a higher intake of tocotrienols. Here we examine the potential of γ-tocotrienol (GT3) to reduce prostate cancer proliferation and focus on elucidating pathways by which GT3 could exert a growth-inhibitory effect on prostate cancer cells. We find that the γ and δ isoforms of tocotrienol are more effective at inhibiting the growth of prostate cancer cell lines (PC-3 and LNCaP) compared with the γ and δ forms of tocopherol. Knockout of PPAR-γ and GT3 treatment show inhibition of prostate cancer cell growth, through a partially PPAR-γ-dependent mechanism. GT3 treatment increases the levels of the 15-lipoxygenase-2 enzyme, which is responsible for the conversion of arachidonic acid to the PPAR-γ-activating ligand 15-S-hydroxyeicosatrienoic acid. In addition, the latent precursor and the mature forms of TGFβ2 are down-regulated after treatment with GT3, with concomitant disruptions in TGFβ receptor I, SMAD-2, p38, and NF-κB signaling