A Concise Synthesis of Carolacton

A synthesis of carolacton, a myxobacterial natural product that has profound effects on Streptococcus mutans biofilms, is reported. The synthesis proceeds via a longest linear sequence of 14 steps from an Evans β-ketoimide and enabled preliminary evaluations of the effects of late-stage intermediates on S. mutans biofilms. These studies suggest that further investigations into carolacton’s structure–function relationships are warranted

Largazole (L) and largazole ester (E) inhibit ubiquitin E1 in a dose dependent manner <i>in vitro</i>.

<p>(A,C) L and E inhibit transfer of ubiquitin onto E1 in a concentration-dependent manner. Thioester assay of E1 activity using fluorescein ubiquitin (Ub-F). Thioester bond formation between E1 and Ub-F is ATP-dependent (lane 2 vs. lane 1). In addition, DMSO has no effect on the formation of the thioester linkage as seen in lane 2 of both gels. 50 nM E1 was incubated with decreasing concentrations of L (A) or E (C) for 15 minutes at room temperature followed by addition of a cocktail containing ATP and Ub-F. After 5 minutes of incubation, the reactions were resolved by SDS-PAGE under non-reducing conditions. Ub-F was used to show equal loading. (B,D) Thioester assay of the ubiquitin transfer from E1 to E2 (Cdc34). Largazole or Largazole ester, when preincubated with 50 nM E1 for 15 minutes, inhibit the transfer of ubiquitin from E1 to Cdc34 in a concentration-dependent manner. (E) Largazole selectively inhibits the activity of E1 not E2. 50 nM E1 was pre-charged with ATP and then added to Cdc34 that was previously incubated with decreasing concentrations (1 mM–16 µM) of L in thioester reaction mixture. (F) Largazole ester inhibits E2 at high concentrations. Pre-charged E1 was added to reactions that contained Cdc34 pre-incubated with E ranging from 1 mM to 16 µM and resolved by SDS-PAGE under non-reducing conditions. Complete inhibition of ubiquitin transfer to E2 was observed at 1 mM of E, with only modest inhibition at 500 µM. (G) Largazole thiol (T) has no effect on transfer of ubiquitin onto E1. The reaction was carried out as described in A,C.</p

Largazole stabilizes p27 expression in Kip16 cells and inhibits p27 ubiquitination <i>in vitro.</i>

<p>(A) Fluorescent and corresponding bright-field images of Kip16 cells treated with varying concentrations of Largazole (L). L treatment induces the expression of GFP-p27 in a dose-dependent fashion. Addition of MG132 (1 µM) prevents the degradation of GFP-p27 via the ubiquitination and subsequent proteasomal degradation pathway. The vehicle control, DMSO, has no effect on the reporter protein stabilization. (B) L fails to inhibit the phosphorylation of p27 by the Cdk2/CyclinE complex compared to the positive control. L (250 µM, lane 3, and 125 µM, lane 4) was incubated with the Cdk2/CyclinE complex prior to the autophosphorylation of Cdk2/CyclinE step. Phosphorylated-p27 was identified by protein standard. (C) L, K, and E reduce polyubiquitinated forms of p27 while M and S have no inhibitory effects. Ubiquitin-activating enzyme E1 (100 nM), UBA1, was incubated with 100 µM of each compound prior to the reaction. (D) E reduces polyubiquitinated forms of Trf1 in a dose-dependent fashion. UBA1 (100 nM) was incubated with either DMSO or various concentrations of E ranging from 250 µM to 1 µM prior to the reaction.</p

Chemical structures of Largazole, synthetic analogs, and Trichostatin A.

<p>Largazole (L) includes a substituted 4-methythiazoline linearly fused to a thiazole, a 3-hydroxy-7-mercaptohept-4-enoic acid, a thioester moiety, and a hydrocarbon tail. Analogs include a substituted ketone (K) and ester (E) in place of the thioester moiety, a macrocycle lacking the thioester moiety and hydrocarbon tail (M), an analog containing a macrocycle broken at carbon-3 of the enoic acid (S), and a thiol analog lacking the thioester moiety (T). Trichostatin A (TSA) contains a hydroxamic acid functional group.</p

Investigation into the selectivity of Largazole ketone.

<p>(A) Largazole ketone (K) fails to inhibit the ligation of ubiquitin onto Uba1p, a homologue of UBA1 from <i>S. pombe</i>. Formation of Uba1p-ubiquitin adducts was determined by thioester assay utilizing fluorescein-ubiquitin. Uba1p (1.03 µM) was incubated with either DMSO or various concentrations of K serially diluted from 1000 µM to 31 µM. (B) K inhibits ligation of SUMO-1 onto human SUMO E1 in a concentration-dependent fashion. Reduction of E1-SUMO adducts was determined by thioester assay utilizing fluorescein-SUMO-1. hSUMO E1 (500 nM) was incubated with either DMSO or various concentrations of K serially diluted from 1000 µM to 31 µM.</p

Solution Structure of CCL19 and Identification of Overlapping CCR7 and PSGL‑1 Binding Sites

CCL19 and CCL21 are chemokines involved in the trafficking of immune cells, particularly within the lymphatic system, through activation of CCR7. Concurrent expression of PSGL-1 and CCR7 in naive T-cells enhances recruitment of these cells to secondary lymphoid organs by CCL19 and CCL21. Here the solution structure of CCL19 is reported. It contains a canonical chemokine domain. Chemical shift mapping shows the N-termini of PSGL-1 and CCR7 have overlapping binding sites for CCL19 and binding is competitive. Implications for the mechanism of PSGL-1’s enhancement of resting T-cell recruitment are discussed

Specialized proteins in the secretome of <i>S. parasitica</i>.

<p><i>(A)</i> Distributions of major classes of specialized secreted proteins compared between animal and plant pathogenic oomycetes. <i>P. infestans</i> represents <i>Phytophthora</i> species. <i>(B) S. parasitica</i> secreted proteins that carry various lectin domain fusions are schematically drawn. Domains or domain architectures unique to <i>S. parasitica</i> are marked with an asterisk. Proteins containing single domains are also listed. <i>(C)</i> Phylogenetic relationship of lectins. The <i>S. parasitica</i> disintegrin gene (SPRG_01285 groups with bacterial homologs; gal_lectin gene (SPRG_05731)) groups with animal species. All other paralogous <i>S. parasitica</i> disintegrin and gal_lectin genes group closely with these two representatives, respectively, and are not shown.</p

Genome statistics and intron features of oomycetes.

a<p>Total contig length adjusted for the regions of haplotype assemblies.</p>b<p>Genome statistics derived from publication of these genomes.</p>c<p>Measured by repeatMasker with de novo RepeatScout.</p>d<p>The repeat content in the assembled sequence is listed in the brackets.</p

Taxonomy and ancestral genomic features in <i>S. parasitica</i>.

<p><i>(A)</i> Animal pathogenic and plant pathogenic oomycetes reside in different taxonomic units. <i>(B)</i> Comparison of intron number in phytopathogenic oomycetes (the average count from the total genes of <i>P. infestans</i>, <i>P. ramorum</i>, <i>P. sojae</i>, <i>Py. ultimum</i> and <i>H. arabidopsidis</i>) and <i>S. parasitica</i> among all genes. <i>(C)</i> Significant difference in intron number in 4008 orthologous genes shared by <i>S. parasitica</i> and <i>Phytophthora</i> species (average intron count of <i>P. infestans</i>, <i>P. sojae</i> and <i>P. ramorum</i>). (Wilcoxon test, p<0.001). <i>(D)</i> Large number of chitinase genes belonging to CAZy family GH-18 in <i>S. parasitica</i> (red) compared to other oomycetes (black; Ps = <i>P. sojae</i>, Pr = <i>P. ramorum</i>, PITG = <i>P. infestans</i>, Hp = <i>H. arabidopsidis</i>, Pyu = <i>Py. ultimum</i>, ALNC = <i>A. laibachii</i>). The phylogenetic tree was constructed with chitinase genes from oomycetes using Maximum likelihood method.</p

Molecular genetic events associated with the evolution of animal and plant pathogenesis in oomycetes.

<p>The lineages of animal pathogens are colored red and the lineages of plant pathogens are colored green. The basal lineage is colored brown. S and N pathways refer to sulfite and nitrite assimilation, respectively.</p