An Unbiased Chemical Proteomics Method Identifies FabI as the Primary Target of 6‑OH-BDE-47

Determination of the physical interactions of environmental chemicals with cellular proteins is important for characterizing biological and toxic mechanism of action. Yet despite the discovery of numerous bioactive natural brominated compounds, such as hydroxylated polybrominated diphenyl ethers (OH-PBDEs), their corresponding protein targets remain largely unclear. Here, we reported a systematic and unbiased chemical proteomics assay (Target Identification by Ligand Stabilization, TILS) for target identification of bioactive molecules based on monitoring ligand-induced thermal stabilization. We first validated the broad applicability of this approach by identifying both known and unexpected proteins bound by diverse compounds (anticancer drugs, antibiotics). We then applied TILS to identify the bacterial target of 6-OH-BDE-47 as enoyl-acyl carrier protein reductase (FabI), an essential and widely conserved enzyme. Using affinity pull-down and in vitro enzymatic assays, we confirmed the potent antibacterial activity of 6-OH-BDE-47 occurs via direct binding and inhibition of FabI. Conversely, overexpression of FabI rescued the growth inhibition of <i>Escherichia coli</i> by 6-OH-BDE-47, validating it as the primary in vivo target. This study documents a chemical proteomics strategy for identifying the physical and functional targets of small molecules, and its potential high-throughput application to investigate the modes-of-action of environmental compounds

An Unbiased Chemical Proteomics Method Identifies FabI as the Primary Target of 6‑OH-BDE-47

Determination of the physical interactions of environmental chemicals with cellular proteins is important for characterizing biological and toxic mechanism of action. Yet despite the discovery of numerous bioactive natural brominated compounds, such as hydroxylated polybrominated diphenyl ethers (OH-PBDEs), their corresponding protein targets remain largely unclear. Here, we reported a systematic and unbiased chemical proteomics assay (Target Identification by Ligand Stabilization, TILS) for target identification of bioactive molecules based on monitoring ligand-induced thermal stabilization. We first validated the broad applicability of this approach by identifying both known and unexpected proteins bound by diverse compounds (anticancer drugs, antibiotics). We then applied TILS to identify the bacterial target of 6-OH-BDE-47 as enoyl-acyl carrier protein reductase (FabI), an essential and widely conserved enzyme. Using affinity pull-down and in vitro enzymatic assays, we confirmed the potent antibacterial activity of 6-OH-BDE-47 occurs via direct binding and inhibition of FabI. Conversely, overexpression of FabI rescued the growth inhibition of <i>Escherichia coli</i> by 6-OH-BDE-47, validating it as the primary in vivo target. This study documents a chemical proteomics strategy for identifying the physical and functional targets of small molecules, and its potential high-throughput application to investigate the modes-of-action of environmental compounds

Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p-3

<p><b>Copyright information:</b></p><p>Taken from "Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p"</p><p>http://www.biomedcentral.com/1741-7007/5/38</p><p>BMC Biology 2007;5():38-38.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2140264.</p><p></p>) and wild-type strain (JRY2334) were shifted to the nonpermissive temperature for 0–5 h. Viability was measured as the fraction of microcolonies that formed after the incubation at 36°C within 1–2 days at permissive temperature. (B) Percentage of , , and wild-type cells that form viable microcolonies when synchronized cultures were shifted to the nonpermissive temperature. Cells were arrested in G1 (α-factor) or in S-phase (hydroxyurea) at 23°C and then maintained at restrictive temperature (36°C) for 0 to 3 h in G1 phase, in S-phase or from G1 to S-phase arrest. Averages of two independent experiments are shown. (C) FACS analysis of wild-type, (BSY539) (BSY568) and (BSY569) cells at semi-permissive temperature (26°C). Cells were arrested in α-factor (5 mg/ml) and release was performed at 26°C for 0, 10, 20, 30, 40, 50, 60, 90, 120, 150, and 190 min. (D) Cell-cycle progression of and wild-type strain at restrictive temperature for . G1-arrested cells were held at 36°C (restrictive temperature) for 1 h and then released into fresh (36°C) medium. Samples for FACS were taken at times indicated. For a detailed list of strains, see Additional file

Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p-8

<p><b>Copyright information:</b></p><p>Taken from "Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p"</p><p>http://www.biomedcentral.com/1741-7007/5/38</p><p>BMC Biology 2007;5():38-38.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2140264.</p><p></p>d from strains carrying either a TAP-tagged version of Orc1p, Orc2p, Orc4p, Orc5p, or Orc6p. For comparison, lane 2 shows marker proteins (MWs are 45.0, 66.2, and 97.4 kDa). The positions of the respective subunits of the ORC and Hat1p complex are indicated. Hat1-CBP: Hat1p with calmodulin binding protein (CBP) after TEV digestion of the TAP-tag. (B) Summary of proteins identified in purifications of TAP-tagged baits. Components of ORC and Hat1p complexes that were detected at least once with either MALDI-TOF or liquid chromatography-mass spectrometry (LC-MS) with high confidence (>90%) are indicated (M/L). (C) Architecture of the Hat1p complexes by differential tagging and subunit deletions. Strains were (BSY675), (BSY681), (BSY679), (BSY720), (BSY682). Lane 1 shows marker proteins (45.0, 66.2, and 97.4 kDa). (D) Western blot from two series of TAP purifications (Figure 1C for left panel, Additional file for right panel), probed with a-Hat1p, a-Orc2p, a-Orc3p, and a-Orc5p antibodies. (E) histone acetyltransferase activities of Hat1p complexes. Concentrated eluate (10-fold) from indicated TAP-tag purifications from Figure 1C was used for HAT-assays with C acetyl-CoA and chicken erythrocyte histones. The upper panel shows C incorporation into histones shown in the lower panel by Coomassie strain. (F) association of acetylated histone H4 with Hat1p sub-complexes from Figure 1C). Eluates from TAP-tag purifications were analyzed for Lys12 acetylated histone H4 by Western blot (a-Acetyl H4 Lys12)

Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p-1

<p><b>Copyright information:</b></p><p>Taken from "Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p"</p><p>http://www.biomedcentral.com/1741-7007/5/38</p><p>BMC Biology 2007;5():38-38.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2140264.</p><p></p>, Hat1–13myc (BSY676), and Orc5–13myc (BSY677). IPs were performed with monoclonal a-Orc3p and a-GFP antibodies. One twentieth of the extracts were loaded as whole cell extract (WCE). Immunoprecipitated Hat1–13myc was detected with mouse α-myc as a primary antibody (9E10). Note that equal amounts and concentrations of extracts were used for all IPs. (B) ORC binds Hat1p/Hat2p but not Hif1p. Strains were: untagged control, Hat1–13myc, Hat2–13myc (BSY691), and Hif1–13myc (BSY692). One twentieth of the extracts were loaded as WCE control. IPs were performed with monoclonal α-Orc3p and α-GFP control antibodies. (C) IP of Hat1p-13myc with α-Orc3p antibody in synchronized cells. Log-phase cells were either arrested in 3 μg/ml α-factor, 200 mM hydroxyurea, or 15 μg/ml nocodazole. Cells were also released from α-factor arrest and samples were taken at 20, 30, and 40 min after release (lanes 4–6). Lanes 7–12 show WCE controls. (D) FACS control of samples in panel C. Time after release is indicated (Rel)

Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p-4

<p><b>Copyright information:</b></p><p>Taken from "Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p"</p><p>http://www.biomedcentral.com/1741-7007/5/38</p><p>BMC Biology 2007;5():38-38.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2140264.</p><p></p>s either labeled with P (panels A and C) or visualized by ethidium bromide (panels B and D). (A) Binding of Hat1-TAP and Cdc45-TAP to and to control sequence at the time of origin firing and later in S-phase. Strains were (BSY679) and (BSY680). One representative experiment with input and immunoprecipitate and the percentage of precipitated DNA is shown. (B) Recruitment of Hat1p to is coincident with Cdc45p and dependent on functional ORC. Strains used for chromatin immunoprecipitation are , , and (BSY699). Strains were held in a-factor at 36°C to inactivate and then released at 23°C. (C) Recruitment of Hat1p (Hat1-TAP) to late replication origin and comparison with sequence. (D) Recruitment of Hat1p to is affected by the allele. Strains used for chromatin immunoprecipitation are , , and (BSY734)

Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p-5

<p><b>Copyright information:</b></p><p>Taken from "Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p"</p><p>http://www.biomedcentral.com/1741-7007/5/38</p><p>BMC Biology 2007;5():38-38.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2140264.</p><p></p> 10 generations. Strains were JRY2334 (wild-type control), BSY528 (), BSY535 (), BSY538 (). These strains were transformed with plasmids pDK243, pDK368-1, pDK368-2, pDK368-7, containing 1, 2, 3 or 8 sequences, respectively. Y-axis shows plasmid loss rates (loss frequency/generation). (B,C) Structural analysis of replicative intermediates by two-dimensional gel electrophoresis. Cultures of (BSY528) and wild-type (JRY2334) strains were released from G1 arrest for 15 and 30 min (30°C). The same blot was first hybridized with an specific probe (B), then stripped and rehybridized with an specific probe (C). The 15 min wild-type sample is overloaded as judged by the amount of monomers. (D) Scheme for replicative intermediates observed in two-dimensional gel electrophoresis

Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p-7

<p><b>Copyright information:</b></p><p>Taken from "Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p"</p><p>http://www.biomedcentral.com/1741-7007/5/38</p><p>BMC Biology 2007;5():38-38.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2140264.</p><p></p>erase. The upper panel shows PhosphorImage of C-labeled histones and the lower panel shows Coomassie stained gel. Protein introduced in the assay was 10, 5, 2, and 1.25 ml of the TAP-purified and concentrated product (approximately 100 ml). (B) Quantification of the results in panel A. Disintegrations/decays per min are shown on the Y-axis. (C) Equal volumes of TAP-purified proteins used for the HAT assay from (BSY700), (BSY701), and untagged control strain (JRY2334) are loaded on a silver stain gel

Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p-2

<p><b>Copyright information:</b></p><p>Taken from "Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p"</p><p>http://www.biomedcentral.com/1741-7007/5/38</p><p>BMC Biology 2007;5():38-38.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2140264.</p><p></p>t 31°C, which is the semi-permissive temperature for . Double mutants are marked with circles. (B) Plating assay of (BSY538), (BSY602), and (BSY603, BSY604) with control strains at indicated times and temperatures. Dilutions were 1:10, starting from late log-phase cultures on YPD. (C) Plating assays for mutant combinations with (BSY569) and (BSY572). (D) Plating assays as in B for mutant combinations with (BSY589-BSY591). (E) Growth comparison of (BSY595) with and at 30°C. For a detailed list of strains, see Additional file

Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p-6

<p><b>Copyright information:</b></p><p>Taken from "Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p"</p><p>http://www.biomedcentral.com/1741-7007/5/38</p><p>BMC Biology 2007;5():38-38.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2140264.</p><p></p> histones H3 and H4 with lysine to arginine substitutions at different sites in their N-terminal tails. (A) Arginine substitution in H4 lysine 5, 12 shows reduced viability in combination with on YPD at the temperature indicated (31°C). Cells were grown for 3 days. (B) The replication defect in leads to efficient loss of the marked plasmid with the covering H3/H4 wild-type genes when selection is performed on -TRP medium. When the combination of with histone mutants is nonviable, cells are less likely to lose the covering plasmid. Colonies were grown at 23°C. (C) Direct assays of transformation and loss of wild-type histone plasmid in different H3 mutant backgrounds. H3 histone mutants contain arginines substituted for lysines. (D) Plasmid loss assays with strains (AP182, AP183) containing wild-type histone H3 (pmp3), K9, 14, 18, 23, 27R substituted histone H3 (pmp8), and K14, 23R substituted histone H3 (pmp83). Loss rates were measured for (pRS316) and (YCp120). Averages and standard deviations of three independent experiments are shown