STRL33/BONZO/TYMSTR is an orphan chemokine and HIV/SIV coreceptor receptor that is expressed on activated T lymphocytes. We describe an expression cloning strategy whereby we isolated a novel chemokine, which we name CXCL16. CXCL16 is an α (CXC) chemokine but also has characteristics of CC chemokines and a structure similar to fractalkine (neurotactin) in having a transmembrane region and a chemokine domain suspended by a mucin-like stalk. A recombinant version of CXCL16 fails to mediate chemotaxis to all known chemokine receptor transfectants tested but does mediate robust chemotaxis, high affinity binding, and calcium mobilization to Bonzo receptor transfectants, indicating that this is a unique receptor ligand interaction. In vitro polarized T cell subsets including Th1, Th2, and Tr1 cells express functional Bonzo, suggesting expression of this receptor in chronic inflammation, which we further verified by demonstration of CXCL16-mediated migration of tonsil-derived CD4+ T lymphocytes. CXCL16 is expressed on the surface of APCs including subsets of CD19+ B cells and CD14+ monocyte/macrophages, and functional CXCL16 is also shed from macrophages. The combination of unique structural features of both Bonzo and CXCL16 suggest that this interaction may represent a new class of ligands for this receptor family. Additionally, this chemokine might play a unique dual role of attracting activated lymphocyte subsets during inflammation as well as facilitating immune responses via cell-cell contact.

Liver X receptors (LXRs) are ligand-activated transcription factors that belong to the nuclear receptor superfamily. LXRs activate transcription of a spectrum of genes that regulate reverse cholesterol transport, including the ATP binding cassette transporter A1 (ABCA1), and raise HDL cholesterol (HDL-C) levels. However, LXR agonists also induce genes that stimulate lipogenesis, including the sterol response element binding protein (SREBP1-c) and fatty acid synthetase (FAS). The induction of these genes in the liver cause increased hepatic triglyceride synthesis, hypertriglyceridemia, and hepatic steatosis. As LXR response elements have been identified in these promoters, it is not clear if these two processes can be separated. Herein, we demonstrate that plasma HDL-C elevation and intestinal ABCA1 induction can occur with relatively little induction of FAS and SREBP1-c in mouse liver via a selective LXR modulator GW3965. This is in contrast to the strong induction of hepatic lipogenic genes by the well-characterized LXR agonist T0901317 (T317). Consistent with the in vivo results, GW3965 is a very weak LXR activator compared with T317 in human hepatoma cells. GW3965-liganded LXR recruits selected coactivators less effectively than T317 and may explain in part the tissue selective gene induction. This demonstration that tissue and gene selective modulation is possible with selective LXR modulators has positive implications for the development of this class of antiatherosclerotic agents. -Miao, B., S. Zondlo, S. Gibbs, D. Cromley, V. P. Hosagrahara, T. G. Kirchgessner, J. Billheimer, and R. Mukherjee. Raising HDL cholesterol without inducing hepatic steatosis and hypertriglyceridemia by a selective LXR modulator.

Transcriptional activation and repression via the transcription factors p53 and p65 are mediated by hydrophobic short linear motifs (FXX phi phi) in their activation domains (ADs). To understand the molecular basis for specificity in binding to disparate biological targets, a series of chimeric peptides was synthesized, with sequences derived from the ADs of p53, which binds both the general transcriptional machinery and the repressor protein MDM2, and p65, which is reported to bind the general transcriptional machinery but not MDM2. The FXX phi phi motifs of p53 and p65 differ by only two residues, whereas the flanking sequences have no sequence identity. The affinities of the chimeric peptides to MDM2(25-117) and hTAF(II)31(1-140) were determined. Specificity for binding MDM2 via FXX phi phi motifs derives almost entirely from Trp23 of p53, with a 3.0 kcal mol(-1) loss of binding energy when Trp23 is changed to p65-derived Leu. The identity of the N-terminal flanking sequence did not significantly affect binding to MDM2. In contrast, replacement of the C-terminal sequence of p53 with that of p65 increased the affinity of the chimera for MDM2 by 1.1 kcal mol(-1), contrary to expectations. Replacement of the highly conserved residue Pro27 of p53 with Ser from p65 resulted in a 2.3 kcal mol(-1) improvement in binding to MDM2, generating a ligand (p53-P27S) (Kd = 4.7 nM) that exhibits the highest MDM2 affinity observed for a genetically encodable ligand. The basis for the increased affinity of p53-P27S over p53 was examined by circular dichroism and nuclear magnetic resonance. Pro27 disrupts the recognition alpha-helix of p53, with p53-P27S significantly more alpha-helical than p53.

SummaryThe normal development of shoot structures depends on controlling the growth, proliferation and differentiation of cells derived from the shoot apical meristem. We have identi®ed the CYP78A5 gene encoding a putative cytochrome P450 monooxygenase that is the ®rst member of the CYP78 family from Arabidopsis. This gene is strongly expressed in the peripheral regions of the vegetative and reproductive shoot apical meristems, de®ning a boundary between the central meristematic zone and the developing organ primordia. In addition, CYP78A5 shows a dynamic pattern of expression durinḡ oral development. Overexpression of CYP78A5 affects multiple cell types, causing twisting and kinking of the stem and defects in¯oral development. To de®ne the relationship of CYP78A5 to genes controlling meristem function, we examined CYP78A5 expression in plants mutant for SHOOT MERISTEMLESS, ZWILLE and ARGONAUTE, and have found that CYP78A5 expression is altered in these mutant backgrounds. We propose that CYP78A5 has a role in regulating directional growth in the peripheral region of the shoot apical meristem in response to cues established by genes regulating meristem function.

Tyrosine kinases are critical mediators of intracellular signaling and of intracellular responses to extracellular signaling. Changes in tyrosine kinase activity are implicated in numerous human diseases, including cancers, diabetes, and pathogen infectivity. To address questions in tyrosine phosphorylation, we have designed a protein tyrosine kinase-inducible domain, a small, genetically encodable protein motif whose structure is dependent on its tyrosine phosphorylation state. Tyrosine kinase-inducible domain peptides are based on EF-hand loops in which a structurally critical Glu12 residue is replaced by tyrosine at residue 11 or at residue 15 of the protein. Tyrosine kinase-inducible domain peptides bind terbium(III) in a phosphorylation-dependent manner, showing strong terbium luminescence when phosphorylated but weak terbium luminescence when not phosphorylated. Lanthanide binding was confirmed by NMR. A tyrosine kinase-inducible domain peptide, pKID-Abl, was designed to incorporate a recognition sequence of the Abl kinase. Incubation of pKID-Abl with Abl kinase resulted in a large increase in terbium luminescence. This increase in luminescence was abolished when pKID-Abl and Abl kinase were incubated with the Abl kinase inhibitor Gleevec. In addition, incubation of phosphorylated pKID-Abl with the tyrosine phosphatase YOP resulted in a large reduction in terbium luminescence. pKID-Abl was employed as a fluorescent sensor of Abl tyrosine kinase activity in HeLa cell extracts, exhibiting low luminescence with extracts from serum-starved cells and increased luminescence using extracts from EGF-treated cells. These results indicate that tyrosine kinase-inducible domains may be used as sensors of tyrosine kinase and tyrosine phosphatase activity and in the detection of tyrosine kinase inhibitors.

The APETALA3 floral homeotic gene is required for petal and stamen development in Arabidopsis. APETALA3 transcripts are first detected in a meristematic region that will give rise to the petal and stamen primordia, and expression is maintained in this region during subsequent development of these organs. To dissect how the APETALA3 gene is expressed in this spatially and temporally restricted domain, various APETALA3 promoter fragments were fused to the uidA reporter gene encoding beta-glucuronidase and assayed for the resulting patterns of expression in transgenic Arabidopsis plants. Based on these promoter analyses, we defined cis-acting elements required for distinct phases of APETALA3 expression, as well as for petal-specific and stamen-specific expression. By crossing the petal-specific construct into different mutant backgrounds, we have shown that several floral genes, including APETALA3, PISTILLATA, UNUSUAL FLORAL ORGANS, and APETALA1, encode trans-acting factors required for second-whorl-specific APETALA3 expression. We have also shown that the products of the APETALA1, APETALA3, PISTILLATA and AGAMOUS genes bind to several conserved sequence motifs within the APETALA3 promoter. We present a model whereby spatially and temporally restricted APETALA3 transcription is controlled via interactions between proteins binding to different domains of the APETALA3 promoter.

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