Drosophila KdelR is conserved.

<p>(A) Drosophila KdelR is highly conserved with respect to its vertebrate and <i>C. elegans</i> counterparts (A) and is homologous to <i>S. cerevisiae</i> Erd2. (Mm-mouse, Hs-human , Gg-chicken, Xl-<i>Xenopus</i>, Dr-zebrafish, Dm-Drosophila, Ce- C<i>. elegans</i> and Sc- <i>S. cerevisiae</i>). Vertebrates encode two-three Kdel Receptors, whereas only a single gene is found in flies, worms and yeast. Black bars over the sequences indicate membrane spanning regions [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077618#B3" target="_blank">3</a>]. Red dots denote residues involved in ligand (KDEL) binding [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077618#B4" target="_blank">4</a>]. (Green= completely conserved residues, yellow= identical residues, purple=similar residues). (B) A Phylip unrooted tree analysis of the KDEL Receptors from the major model organisms reveals that the Drosophila Kdel Receptor is slightly more related to the vertebrate proteins than are the worm and yeast receptors.</p

<i>KdelR</i> expression profile.

<p><i>KdelR</i> is detected early in embryogenesis at the cellular blastoderm stage (A,E) and to high levels in the salivary gland beginning at embryonic stage 10 and continuing throughout embryogenesis (b-d, f-h, black arrows). <i>KdelR</i> is also expressed to elevated levels in the epidermis (cells on the embryo surface), fat body (clusters of staining in each segment in stage 11 embryos, indicated by black stars), proventriculus (white star) and a subset of gut endoderm cells (arrowheads). b-gal and RNA staining in the neural tube (nt) is also observed at late stages, although the RNA expression in the nt is not in the plane of focus of the embryos shown. Left column shows mRNA expression detected with a probe made from the <i>KdelR</i> cDNA clone CK00230 and the right column shows β-gal staining of <i>l</i>(<i>2</i>)<i>k00311</i>embryos. </p

<i>KdelR</i> mutant cuticles are smaller than wild-type cuticles and are grossly underdeveloped.

<p>Dark field (ventral) images of wild-type, <i>l</i>(<i>2</i>)<i>k00311</i> and <i>31Em</i>^<i>2</i> larvae (A-C). Note that <i>l</i>(<i>2</i>)<i>k00311</i> and <i>31Em</i>^<i>2</i> mutants are approximately 60% the length of wild type larvae and the ventral denticles are not as prominent as in their wild-type siblings (A-C). Mouthparts (MP) of <i>l</i>(<i>2</i>)<i>k00311</i> (G) and <i>31Em</i>^<i>2</i> (J) are underdeveloped and less pigmented than corresponding wild-type mouth parts (D). The filtzkörper (FK) of <i>l</i>(<i>2</i>)<i>k00311</i> (I) and <i>31Em</i>^<i>2</i> (L) are underdeveloped and do not protrude from the larval body as in wild-type (F). </p

<i>KdelR</i> and the retention of PH4αSG1 and other soluble resident proteins in the ER.

<p>(A) Staining with antibodies to the resident ER protein PH4αSG1 revealed cellular expression in WT SGs beginning at embryonic stage 11 and continuing through embryogenesis (data not shown; left panels). By embryonic stage 13, PH4αSG1 staining was observed at low levels in the salivary lumens of <i>l</i>(<i>2</i>)<i>k00311</i> mutants (middle top panel) and at high levels in the salivary lumens of <i>l</i>(<i>2</i>)<i>31Em</i>^<i>1</i> and <i>l</i>(<i>2</i>)<i>31Em</i>^<i>2</i> mutants (top right panel and data not shown). By stage 17, almost all detectable PH4αSG1 was lumenal in both <i>l</i>(<i>2</i>)<i>k00311</i> and <i>l</i>(<i>2</i>)<i>31Em</i>^<i>1</i> salivary glands. The same staining patterns were observed in all embryos examined of each genotype. (B) Stage 15 embryonic salivary glands were co-stained with antibodies to PH4αSG1, Boca, and Windbeutel. PH4αSG1 protein was observed entirely in the ER in wild-type salivary glands, in both the ER and lumen in <i>l</i>(<i>2</i>)<i>k00311</i> salivary glands, and predominantly in the lumen in <i>31Em</i>^<i>2</i> mutant salivary glands (left panels). Boca protein was barely detected in the salivary glands of <i>31Em</i>^<i>2</i> mutants, compared to the WT and <i>l</i>(<i>2</i>)<i>k00311</i> mutants, although some Boca protein can be detected in the lumens of <i>31Em</i>^<i>2</i> mutants when the image is overexposed (second column, last row, inset). Wbl localization was largely unaffected in <i>l</i>(<i>2</i>)<i>k00311</i> and <i>31Em</i>^<i>2</i> mutant salivary glands; only minimal lumenal staining of Wbl protein was observed, even when the cellular staining was at high levels (third column, second row). The changes in PH4αSG1 and Boca protein localization are more apparent in the merged images (last column). Again, the same patterns of accumulation were observed in all of the stage 15 embryos examined for each genotype.</p

Identification of <i>KdelR</i> alleles.

<p><i>KdelR</i> maps to region 31E in the Drosophila genome and <i>l</i>(<i>2</i>)<i>k00311</i> is inserted in the 5’ UTR of the <i>KdelR</i> transcript. <i>l</i>(<i>2</i>)<i>k00311</i> fails to complement deficiencies <i>Df</i>(<i>2L</i>)<i>J3, Df</i>(<i>2L</i>)<i>J106</i> and <i>Df</i>(<i>2L</i>)<i>J27</i>, but complements deficiencies <i>Df</i>(<i>2L</i>)<i>J17</i> and <i>Df</i>(<i>2L</i>)<i>J16</i>. Both EMS alleles of <i>KdelR</i> (<i>31Em1</i> and <i>31Em</i>^<i>2</i>) encode ORFs with premature stop codons. </p

Pyridinylquinazolines Selectively Inhibit Human Methionine Aminopeptidase‑1 in Cells

Methionine aminopeptidases (MetAPs), which remove the initiator methionine from nascent peptides, are essential in all organisms. While MetAP2 has been demonstrated to be a therapeutic target for inhibiting angiogenesis in mammals, MetAP1 seems to be vital for cell proliferation. Our earlier efforts identified two structural classes of human MetAP1 (<i>Hs</i>MetAP1)-selective inhibitors (<b>1</b>–<b>4</b>), but all of them failed to inhibit cellular <i>Hs</i>MetAP1. Using Mn­(II) or Zn­(II) to activate <i>Hs</i>MetAP1, we found that <b>1</b>–<b>4</b> could only effectively inhibit purified <i>Hs</i>MetAP1 in the presence of physiologically unachievable concentrations of Co­(II). In an effort to seek Co­(II)-independent inhibitors, a novel structural class containing a 2-(pyridin-2-yl)­quinazoline core has been discovered. Many compounds in this class potently and selectively inhibited <i>Hs</i>MetAP1 without Co­(II). Subsequently, we demonstrated that <b>11j</b>, an auxiliary metal-dependent inhibitor, effectively inhibited <i>Hs</i>MetAP1 in primary cells. This is the first report that an <i>Hs</i>MetAP1-selective inhibitor is effective against its target in cells