Targeting of Histone Acetyltransferase p300 by Cyclopentenone Prostaglandin Δ12-PGJ2 through Covalent Binding to Cys1438

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Chemical Research in Toxicology, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/tx200383cInhibitors of histone acetyltransferases (HATs) are perceived to treat diseases like cancer, neurodegeneration, and AIDS. On the basis of previous studies, we hypothesized that Cys1438 in the substrate binding site could be targeted by Δ12-prostaglandin J2 (Δ12-PGJ2), a cyclopentenone prostaglandin (CyPG) derived from PGD2. We demonstrate here the ability of CyPGs to inhibit p300 HAT-dependent acetylation of histone H3. A cell-based assay system clearly showed that the α,β-unsaturation in the cyclopentenone ring of Δ12-PGJ2 was crucial for the inhibitory activity, while the 9,10-dihydro-15-deoxy- Δ12,14-PGJ2, which lacks the electrophilic carbon (at carbon 9), was ineffective. Molecular docking studies suggested that Δ12-PGJ2 places the electrophilic carbon in the cyclopentenone ring well within the vicinity of Cys1438 of p300 to form a covalent Michael adduct. Site-directed mutagenesis of the p300 HAT domain, peptide competition assay involving p300 wild type and mutant peptides, followed by mass spectrometric analysis confirmed the covalent interaction of Δ12-PGJ2 with Cys1438. Using biotinylated derivatives of Δ12-PGJ2 and 9,10-dihydro-15-deoxy- Δ12,14-PGJ2, we demonstrate the covalent interaction of Δ12-PGJ2 with the p300 HAT domain, but not the latter. In agreement with the in vitro filter binding assay, CyPGs were also found to inhibit H3 histone acetylation in cell-based assays. In addition, Δ12-PGJ2 also inhibited the acetylation of the HIV-1 Tat by recombinant p300 in in vitro assays. This study demonstrates, for the first time, that Δ12-PGJ2 inhibits p300 through Michael addition, where α,β-unsaturated carbonyl function is absolutely required for the inhibitory activity

S-nitrosation of proteins relevant to Alzheimer's disease during early stages of neurodegeneration

Protein S-nitrosation (SNO-protein), the nitric oxide-mediated posttranslational modification of cysteine thiols, is an important regulatory mechanism of protein function in both physiological and pathological pathways. A key first step toward elucidating the mechanism by which S-nitrosation modulates a protein's function is identification of the targeted cysteine residues. Here, we present a strategy for the simultaneous identification of SNO-cysteine sites and their cognate proteins to profile the brain of the CK-p25-inducible mouse model of Alzheimer's disease-like neurodegeneration. The approach-SNOTRAP (SNO trapping by triaryl phosphine)-is a direct tagging strategy that uses phosphinebased chemical probes, allowing enrichment of SNO-peptides and their identification by liquid chromatography tandem mass spectrometry. SNOTRAP identified 313 endogenous SNO-sites in 251 proteins in the mouse brain, of which 135 SNO-proteins were detected only during neurodegeneration. S-nitrosation in the brain shows regional differences and becomes elevated during early stages of neurodegeneration in the CK-p25 mouse. The SNO-proteome during early neurodegeneration identified increased S-nitrosation of proteins important for synapse function, metabolism, and Alzheimer's disease pathology. In the latter case, proteins related to amyloid precursor protein processing and secretion are S-nitrosated, correlating with increased amyloid formation. Sequence analysis of SNO-cysteine sites identified potential linear motifs that are altered under pathological conditions. Collectively, SNOTRAP is a direct tagging tool for global elucidation of the SNO-proteome, providing functional insights of endogenous SNO proteins in the brain and its dysregulation during neurodegeneration.National Institutes of Health (U.S.) (Grant CA26731)National Institutes of Health (U.S.) (Grant R01 NS051874

S-nitrosation of proteins relevant to Alzheimer’s disease during early stages of neurodegeneration

Protein S-nitrosation (SNO-protein), the nitric oxide-mediated posttranslational modification of cysteine thiols, is an important regulatory mechanism of protein function in both physiological and pathological pathways. A key first step toward elucidating the mechanism by which S-nitrosation modulates a protein’s function is identification of the targeted cysteine residues. Here, we present a strategy for the simultaneous identification of SNO-cysteine sites and their cognate proteins to profile the brain of the CK-p25–inducible mouse model of Alzheimer’s disease-like neurodegeneration. The approach—SNOTRAP (SNO trapping by triaryl phosphine)—is a direct tagging strategy that uses phosphine-based chemical probes, allowing enrichment of SNO-peptides and their identification by liquid chromatography tandem mass spectrometry. SNOTRAP identified 313 endogenous SNO-sites in 251 proteins in the mouse brain, of which 135 SNO-proteins were detected only during neurodegeneration. S-nitrosation in the brain shows regional differences and becomes elevated during early stages of neurodegeneration in the CK-p25 mouse. The SNO-proteome during early neurodegeneration identified increased S-nitrosation of proteins important for synapse function, metabolism, and Alzheimer’s disease pathology. In the latter case, proteins related to amyloid precursor protein processing and secretion are S-nitrosated, correlating with increased amyloid formation. Sequence analysis of SNO-cysteine sites identified potential linear motifs that are altered under pathological conditions. Collectively, SNOTRAP is a direct tagging tool for global elucidation of the SNO-proteome, providing functional insights of endogenous SNO proteins in the brain and its dysregulation during neurodegeneration.National Institutes of Health (U.S.) (NIH Grant CA26731)Massachusetts Institute of Technology. Center for Environmental Health Sciences (Grant ES002109)Simons FoundationNational Institutes of Health (U.S.) (NIH Grant R01 NS051874

Metabolite profiling and pharmacokinetic evaluation of hydrocortisone in a perfused 3D human liver bioreactor

Endotoxin lipopolysaccharide (LPS) is known to cause liver injury primarily involving inflammatory cells such as Kupffer cells, but few in vitro culture models are applicable for investigation of inflammatory effects on drug metabolism. We have developed a 3D human microphysiological hepatocyte-Kupffer-cell coculture system and evaluated the anti-inflammatory effect of glucocorticoids on liver cultures. LPS was introduced to the cultures to elicit an inflammatory response and assessed by the release of pro-inflammatory cytokines, IL6 and TNFα. A sensitive and specific RP-UHPLC-QTOF-MS method was used to evaluate hydrocortisone disappearance and metabolism at near physiological levels. For this, the systems were dosed with 100 nM hydrocortisone and circulated for two days; hydrocortisone was depleted to approximately 30 nM, with first-order kinetics. Phase I metabolites, including tetrahydrocortisone and dihydrocortisol, accounted for 8-10 % of the loss, and 45-52 % was phase II metabolites, including glucuronides of tetrahydrocortisol and tetrahydrocortisone. Pharmacokinetic parameters, i.e., half-life (t1/2), rate of elimination (kel), clearance (CL), and area under the curve (AUC), were 23.03 h, 0.03 h-1, 6.6x10-5 L. h-1 and 1.03 mg/L*h respectively. The ability of the bioreactor to predict the in vivo clearance of hydrocortisone was characterized and the obtained intrinsic clearance values correlated with human data. This system offers a physiologically-relevant tool for investigating hepatic function in an inflamed liver. Endotoxin lipopolysaccharide (LPS) is known to cause liver injury primarily involving inflammatory cells such as Kupffer cells, but few in vitro culture models are applicable for investigation of inflammatory effects on drug metabolism. We have developed a 3D human microphysiological hepatocyte-Kupffer-cell coculture system and evaluated the anti-inflammatory effect of glucocorticoids on liver cultures. LPS was introduced to the cultures to elicit an inflammatory response and assessed by the release of pro-inflammatory cytokines, IL6 and TNFα. A sensitive and specific RP-UHPLC-QTOF-MS method was used to evaluate hydrocortisone disappearance and metabolism at near physiological levels. For this, the systems were dosed with 100 nM hydrocortisone and circulated for two days; hydrocortisone was depleted to approximately 30 nM, with first-order kinetics. Phase I metabolites, including tetrahydrocortisone and dihydrocortisol, accounted for 8-10 % of the loss, and 45-52 % was phase II metabolites, including glucuronides of tetrahydrocortisol and tetrahydrocortisone. Pharmacokinetic parameters, i.e., half-life (t[subscript 1/2]), rate of elimination (k[subscript el]), clearance (CL), and area under the curve (AUC), were 23.03 h, 0.03 h[superscript -1], 6.6x10[superscript -5] L. h-1 and 1.03 mg/L*h respectively. The ability of the bioreactor to predict the in vivo clearance of hydrocortisone was characterized and the obtained intrinsic clearance values correlated with human data. This system offers a physiologically-relevant tool for investigating hepatic function in an inflamed liver.United States. Defense Advanced Research Projects Agency (DARPA-BAA-11-73 Microphysiological Systems W911NF-12-2-0039)National Institutes of Health (U.S.) (5-UH2-TR000496)Massachusetts Institute of Technology. Center for Environmental Health Sciences (P30-ES002109

Naphthoquinone-mediated inhibition of lysine acetyltransferase KAT3B/p300, basis for non-toxic inhibitor synthesis

Hydroxynaphthoquinone-based inhibitors of the lysine acetyltransferase KAT3B (p300), such as plumbagin, are relatively toxic. Here, we report that free thiol reactivity and redox cycling properties greatly contribute to the toxicity of plumbagin. A reactive 3rd position in the naphthoquinone derivatives is essential for thiol reactivity and enhances redox cycling. Using this clue, we synthesized PTK1, harboring a methyl substitution at the 3rd position of plumbagin. This molecule loses its thiol reactivity completely and its redox cycling ability to a lesser extent. Mechanistically, non-competitive, reversible binding of the inhibitor to the lysine acetyltransferase (KAT) domain of p300 is largely responsible for the acetyltransferase inhibition. Remarkably, the modified inhibitor PTK1 was a nearly non-toxic inhibitor of p300. The present report elucidates the mechanism of acetyltransferase activity inhibition by 1,4-naphthoquinones, which involves redox cycling and nucleophilic adduct formation, and it suggests possible routes of synthesis of the non-toxic inhibitor

Tandem mass tag-based quantitative proteomic profiling identifies candidate serum biomarkers of drug-induced liver injury in humans

Diagnosis of drug-induced liver injury (DILI) and its distinction from other liver diseases are significant challenges in drug development and clinical practice. We used Tandem Mass Tag-labeled quantitative proteomics detecting 2323 proteins in a cohort comprising patients with DILI [at onset (DO) and follow-up (DF)], acute non-DILI [at onset (NDO) and follow-up (NDF)], and healthy volunteers (HV) to identify novel serum biomarkers. Thirteen candidates selected based on differential expression, liver-specific expression, and mechanistic relevance to liver pathology, were assessed in confirmatory and replication cohorts of HV (n=94), DO (n=123), DF (n=110), NDO (n=58) and NDF (n=37) using a targeted label-free SureQuant assay. Area under the receiver operating characteristic curve (AUC) ranging between 0.94 and 0.99 across cohorts for five of these biomarkers, reflected differentiation between DO and HV with high sensitivity and specificity. In addition, fructose-1,6-bisphosphatase 1 distinguished NDO from DO (AUC: 0.75 and 0.65) on its own or in combination with glutathione S-transferase A1 and leukocyte cell derived chemotaxin 2 (AUC: 0.78 and 0.68). These can potentially differentiate DILI and acute liver injury from non-drug etiologies

Gamma-glutamyltranspeptidase expression by Helicobacter saguini, an enterohepatic Helicobacter species isolated from cotton top tamarins with chronic colitis

Background: Helicobacter saguini is a novel enterohepatic Helicobacter species isolated from captive cotton top tamarins with chronic colitis and colon cancer. Monoassociated H. saguini infection in gnotobiotic IL-10 −/− mice causes typhlocolitis and dysplasia; however, the virulent mechanisms of this species are unknown. Gamma-glutamyltranspeptidase (GGT) is an enzymatic virulence factor expressed by pathogenic Helicobacter and Campylobacter species that inhibits host cellular proliferation and promotes inflammatory-mediated gastrointestinal pathology. The aim of this study was to determine if H. saguini expresses an enzymatically active GGT homologue with virulence properties. Experimental procedures: Two putative GGT paralogs (HSGGT1 and HSGGT2) identified in the H. saguini genome were bioinformatically analysed to predict enzymatic functionality and virulence potential. An isogenic knockout mutant strain and purified recombinant protein of HSGGT1 were created to study enzymatic activity and virulence properties by in vitro biochemical and cell culture experiments. Results: Bioinformatic analysis predicted that HSGGT1 has enzymatic functionality and is most similar to the virulent homologue expressed by Helicobacter bilis, whereas HSGGT2 contains putatively inactivating mutations. An isogenic knockout mutant strain and recombinant HSGGT1 protein were successfully created and demonstrated that H. saguini has GGT enzymatic activity. Recombinant HSGGT1 protein and sonicate from wild-type but not mutant H. saguini inhibited gastrointestinal epithelial and lymphocyte cell proliferation without evidence of cell death. The antiproliferative effect by H. saguini sonicate or recombinant HSGGT1 protein could be significantly prevented with glutamine supplementation or the GGT-selective inhibitor acivicin. Recombinant HSGGT1 protein also induced proinflammatory gene expression in colon epithelial cells. Conclusions: This study shows that H. saguini may express GGT as a potential virulence factor and supports further in vitro and in vitro studies into how GGT expression by enterohepatic Helicobacter species influences the pathogenesis of gastrointestinal inflammatory diseases.National Institutes of Health (U.S.) (Grant T32‐OD010978)National Institutes of Health (U.S.) (Grant P30‐ES002109)National Institutes of Health (U.S.) (Grant R01‐OD01141

Targeting of Histone Acetyltransferase p300 by Cyclopentenone Prostaglandin Δ¹²-PGJ₂ through Covalent Binding to Cys¹⁴³⁸

Inhibitors of histone acetyltransferases (HATs) are perceived to treat diseases like cancer, neurodegeneration, and AIDS. On the basis of previous studies, we hypothesized that Cys¹⁴³⁸ in the substrate binding site could be targeted by Δ¹²-prostaglandin J₂ (Δ¹²-PGJ₂), a cyclopentenone prostaglandin (CyPG) derived from PGD₂. We demonstrate here the ability of CyPGs to inhibit p300 HAT-dependent acetylation of histone H3. A cell-based assay system clearly showed that the α,β-unsaturation in the cyclopentenone ring of Δ¹²-PGJ₂ was crucial for the inhibitory activity, while the 9,10-dihydro-15-deoxy-Δ^12,14-PGJ₂, which lacks the electrophilic carbon (at carbon 9), was ineffective. Molecular docking studies suggested that Δ¹²-PGJ₂ places the electrophilic carbon in the cyclopentenone ring well within the vicinity of Cys¹⁴³⁸ of p300 to form a covalent Michael adduct. Site-directed mutagenesis of the p300 HAT domain, peptide competition assay involving p300 wild type and mutant peptides, followed by mass spectrometric analysis confirmed the covalent interaction of Δ¹²-PGJ₂ with Cys¹⁴³⁸. Using biotinylated derivatives of Δ¹²-PGJ₂ and 9,10-dihydro-15-deoxy-Δ^12,14-PGJ₂, we demonstrate the covalent interaction of Δ¹²-PGJ₂ with the p300 HAT domain, but not the latter. In agreement with the <i>in vitro</i> filter binding assay, CyPGs were also found to inhibit H3 histone acetylation in cell-based assays. In addition, Δ¹²-PGJ₂ also inhibited the acetylation of the HIV-1 Tat by recombinant p300 in <i>in vitro</i> assays. This study demonstrates, for the first time, that Δ¹²-PGJ₂ inhibits p300 through Michael addition, where α,β-unsaturated carbonyl function is absolutely required for the inhibitory activity

S

Protein S-nitrosation (SNO-protein), the nitric oxide-mediated posttranslational modification of cysteine thiols, is an important regulatory mechanism of protein function in both physiological and pathological pathways. A key first step toward elucidating the mechanism by which S-nitrosation modulates a protein's function is identification of the targeted cysteine residues. Here, we present a strategy for the simultaneous identification of SNO-cysteine sites and their cognate proteins to profile the brain of the CK-p25-inducible mouse model of Alzheimer's disease-like neurodegeneration. The approach-SNOTRAP (SNO trapping by triaryl phosphine)-is a direct tagging strategy that uses phosphinebased chemical probes, allowing enrichment of SNO-peptides and their identification by liquid chromatography tandem mass spectrometry. SNOTRAP identified 313 endogenous SNO-sites in 251 proteins in the mouse brain, of which 135 SNO-proteins were detected only during neurodegeneration. S-nitrosation in the brain shows regional differences and becomes elevated during early stages of neurodegeneration in the CK-p25 mouse. The SNO-proteome during early neurodegeneration identified increased S-nitrosation of proteins important for synapse function, metabolism, and Alzheimer's disease pathology. In the latter case, proteins related to amyloid precursor protein processing and secretion are S-nitrosated, correlating with increased amyloid formation. Sequence analysis of SNO-cysteine sites identified potential linear motifs that are altered under pathological conditions. Collectively, SNOTRAP is a direct tagging tool for global elucidation of the SNO-proteome, providing functional insights of endogenous SNO proteins in the brain and its dysregulation during neurodegeneration.National Institutes of Health (U.S.) (Grant CA26731)National Institutes of Health (U.S.) (Grant R01 NS051874

Development and Application of the Metalloprotease Activity Multiplexed Bead-Based Immunoassay (MAMBI)

Metalloproteinases (MMPs) are zinc-dependent endopeptidases that cleave various proteins to regulate normal and diseased cellular functions, and as such, they play significant roles in human tissue development, homeostasis, and the pathogenesis of many diseases, including cancers, endometriosis, arthritis, etc. Most MMPs are produced as zymogenic latent enzymes that must be cleaved to activate their catalytic regions, and localized endogenous protein inhibitors further regulate activity. Accordingly, they operate within recursive networks to degrade extracellular matrix proteins and regulate cell signaling by cleaving growth factors and receptors at the cell surface and in the local pericellular environment. Thus, high-resolution information about the concentrations of specific active MMPs, revealing their intricate regulatory networks, may improve disease diagnosis and treatment. Here, we introduce a new and readily mastered method for measuring MMP activities in a multiplex fashion. We integrate aspects of activity-based enzyme labeling with commercial high-throughput, multiplexed protein quantification to yield the metalloproteinase activity multiplexed bead-based immunoassay (MAMBI). Assays of recombinant active MMP-1, -2, -3, -7, -8, -9, -12, and -13 establish the sensitivity and selectivity of MAMBI detection. Levels of active native MMPs are similarly characterized in conditioned cell culture medium, menstrual effluent, and uterine tissue. In a single MAMBI (5 μL), we achieve sensitivities equal to those from leading single-plex MMP activity detection strategies (e.g., 10-15 M for MMP-1). We also demonstrate high-throughput inhibitor screening via the MAMBI approach in complex, patient-derived samples.National Institutes of Health (Grant R01 EB010246