The X gene product encoded by the hepatitis B virus, termed pX, is a promiscuous transactivator of a variety of viral and cellular genes under the control of diverse cis-acting elements. Although pX does not appear to directly bind DNA, pX-responsive elements include the NF-cB, AP-1, and CRE (cAMP response element) sites. Direct proteinprotein interactions occur between viral pX and the CREbinding transcription factors CREB and ATF. Here we examine the mechanism of the protein-protein interactions occurring between CREB and pX by using recombinant proteins and in vitro DNA-binding assays. We demonstrate that pX interacts with the basic region-leucine zipper domain of CREB but not with the DNA-binding domain of the yeast transactivator protein Gal4. The interaction between CREB and pX increases the affinity of CREB for the CRE site by an order of magnitude, although pX does not alter the rate of CREB dimerization. Methylation interference footprinting reveals differences between the CREB DNA and CREB-pX DNA complexes. These experiments demonstrate that pX alters the way CREB interacts with the CRE DNA and suggest that the basic, DNA-binding region of CREB is the target of pX. Transfection assays in PC12 cells with the CREBdependent somatostatin promoter demonstrate a nearly 15-fold transcriptional induction after forskolin stimulation in the presence of pX. These results support the significance of the CREB-pX protein-protein interactions in vivo.The 3.2-kb hepatitis B virus (HBV) genome contains four recognized open reading frames, three of which encode virion structural proteins. These include the surface and core antigens and viral polymerase (1). The fourth frame, conserved among all mammalian hepadnaviruses, encodes a 16.5-kDa protein, termed X antigen (1). The X gene is expressed during viral infection, producing a 1-kb mRNA (2). In transgenic mice, the HBV X protein (pX) induces liver cancer (3).pX is a transactivator of transcriptional elements, such as those present within the HBV enhancer (4, 5), simian virus 40 enhancer (6), human immunodeficiency virus long terminal repeat (7-9), and the human interferon gene (10). X-responsive cis-acting elements include NF-KB (9), AP-1 (11), and cAMP response element (CRE) (12) sites. pX does not appear to bind DNA directly. Having multiple X-responsive cis-acting elements suggests that the mechanism of pX transactivation is pleiotropic (13). Activation by pX may involve targeting different regulatory steps in these signal transduction pathways or direct protein-protein interactions with diverse cellular transcription factors and the transcriptional machinery. Recent studies (14) with inhibitors of protein kinase C reported that pX acts indirectly by activating the protein kinase C transduction pathway, thereby increasing the activity of the cognate transcription factors. A more direct mechanism of pX transactivation is supported by earlier studies (11,15) showing that pX possesses an activation domain. pX stimulates tran-The publication costs of this articl...
SUMMARY The serine esterase monoacylglycerol lipase (MGL) is primarily responsible for deactivating the signaling lipid 2-arachidonoylglycerol (2-AG), an endocannabinoid with full agonist activity at both principal cannabinoid receptors. Although MGL is recognized as a potential therapeutic target, the paucity of structural information on this enzyme has hindered development of MGL-selective inhibitors. Previously, we overexpressed and purified human MGL as the hexa-histidine-tagged recombinant protein (hMGL) and showed that it catalyzed the hydrolysis of both 2-AG and novel fluorogenic reporters. We now characterize by mass spectroscopy the hMGL active site using two chemically distinct inhibitors as direct probes: 5-((biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701) and N-arachidonylmaleimide (NAM). Suitable conditions were established for hMGL inhibition by AM6701, and the inhibitor-treated enzyme was subjected to trypsin digestion. The tryptic digest of AM6701-inhibited hMGL was analyzed by MALDI-TOF and tandem MS, which showed that AM6701 had carbamylated the serine in a GXSXG motif of the putative MGL catalytic triad. These results provide the first direct confirmation of the essential role of this serine residue for catalysis and establish the mechanism of AM6701 as a high-affinity, covalent hMGL inhibitor. When applied to NAM-treated hMGL, our direct, ligand-assisted approach revealed that partial alkylation of cysteine residues 215 and/or 249 was sufficient to achieve ~ 80% hMGL inhibition. Further alkylation at cysteine 39 did not increase the extent of enzyme inhibition. Although Cys215 and/or Cys249 mutations to alanine(s) did not affect hMGL’s ability to hydrolyze reporter substrate, as compared to nonmutated hMGL the C215A mutant was more sensitive to NAM, whereas the C249A mutation reduced the enzyme’s sensitivity to NAM. These data conclusively demonstrate a sulfhydryl-based mechanism underlying MGL inhibition by this fatty alkyl-maleimide substrate analog in which Cys249 is of paramount importance. Identification of amino acids critical to catalysis by and pharmacological modulation of hMGL provides information useful in the design of selective MGL inhibitors as potential drugs.
The endocannabinoid system includes two G-protein coupled cannabinoid receptors (CB1 and CB2), their endogenous lipid ligands (endocannabinoids), and the enzymes and transporters that help regulate cannabinergic tone ( 1 ). Endocannabinoid signaling is ubiquitous in mammals, being particularly critical in brain , where it modulates neurotransmitter release and exhibits neuroprotective effects ( 2, 3 ). Although the fi rst two endocannabinoids identifi ed, N -arachidonoylethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (AG), have been extensively characterized, the (patho)physiological impact of endocannabinoid signaling has prompted the search for additional endocannabinoids and their related metabolites/ biosynthetic precursors, creating an evolving network of chemical species collectively termed the endocannabinoid metabolome, few of which have been functionally annotated ( 1,(3)(4)(5)(6)(7)(8)(9). Although the translational and diagnostic aspects of endocannabinoid signaling are well appreciated ( 10, 11 ), factors that infl uence and regulate the endocannabinoid metabolome profi le among various tissues and compartments remains incompletely described and understood ( 1, 10 ), as are the metabolome's relationship to lipid pathways outside of the endocannabinoid signaling system ( 3, 9 ). Abstract
1] The ChemCam instrument package on the Mars rover, Curiosity, provides new capabilities to probe the abundances of certain trace elements in the rocks and soils on Mars using the laser-induced breakdown spectroscopy technique. We focus on detecting and quantifying Li, Ba, Rb, and Sr in targets analyzed during the first 100 sols, from Bradbury Landing Site to Rocknest. Univariate peak area models and multivariate partial least squares models are presented. Li, detected for the first time directly on Mars, is generally low (<15 ppm). The lack of soil enrichment in Li, which is highly fluid mobile, is consistent with limited influx of subsurface waters contributing to the upper soils. Localized enrichments of up to~60 ppm Li have been observed in several rocks but the host mineral for Li is unclear. Bathurst_Inlet is a fine-grained bedrock unit in which several analysis locations show a decrease in Li and other alkalis with depth, which may imply that the unit has undergone low-level aqueous alteration that has preferentially drawn the alkalis to the surface. Ba (~1000 ppm) was detected in a buried pebble in the Akaitcho sand ripple and it appears to correlate with Si, Al, Na, and K, indicating a possible feldspathic composition. Rb and Sr are in the conglomerate Link at abundances >100 ppm and >1000 ppm, respectively. These analysis locations tend to have high Si and alkali abundances, consistent with a feldspar composition. Together, these trace element observations provide possible evidence of magma differentiation and aqueous alteration. Citation: Ollila, A. M., et al. (2014), Trace element geochemistry (Li, Ba, Sr, and Rb) using Curiosity's ChemCam: Early results for Gale crater from Bradbury Landing Site to Rocknest,
The endocannabinoid system's biological significance continues to grow as novel endocannabinoid metabolites are discovered. Accordingly, a myopic view of the system that focuses solely on one or two endocannabinoids, such as anandamide or 2-arachidonoyl glycerol, is insufficient to describe the biological responses to perturbations of the system. Rather, the endocannabinoid metabolome as a whole must be analyzed. The work described here is based on liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry. This method has been validated to quantify, in a single chromatographic run, the levels of 15 known or suspected metabolites of the endocannabinoid system in the rat brain and is applicable to other biological matrixes. We have obtained an endocannabinoid profile specifically for the frontal cortex of the rat brain and have determined anandamide level differences following the administration of the fatty acid amide hydrolase inhibitor AM374.
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