Oh Kwang Kwon

Dynamic Competing Histone H4 K5K8 Acetylation and Butyrylation Are Hallmarks of Highly Active Gene Promoters

Summary Recently discovered histone lysine acylation marks increase the functional diversity of nucleosomes well beyond acetylation. Here, we focus on histone butyrylation in the context of sperm cell differentiation. Specifically, we investigate the butyrylation of histone H4 lysine 5 and 8 at gene promoters where acetylation guides the binding of Brdt, a bromodomain-containing protein, thereby mediating stage-specific gene expression programs and post-meiotic chromatin reorganization. Genome-wide mapping data show that highly active Brdt-bound gene promoters systematically harbor competing histone acetylation and butyrylation marks at H4 K5 and H4 K8. Despite acting as a direct stimulator of transcription, histone butyrylation competes with acetylation, especially at H4 K5, to prevent Brdt binding. Additionally, H4 K5K8 butyrylation also marks retarded histone removal during late spermatogenesis. Hence, alternating H4 acetylation and butyrylation, while sustaining direct gene activation and dynamic bromodomain binding, could impact the final male epigenome features.

Global Phosphoproteomic Analysis of Daphnia pulex Reveals Evolutionary Conservation of Ser/Thr/Tyr Phosphorylation

Reversible protein phosphorylations of serine, threonine, and tyrosine are critical processes in organisms ranging from prokaryotes to eukaryotes. Water fleas (Daphnids) have been used widely in ecologic and ecotoxicological studies, with more than 80% of ecotoxicological publications over the last 10 years involving planktonic genera, including Daphnia. However, the substrate proteins and the functions of phosphorylation in Daphnia remain largely unknown. Here, we report the first global screening of phosphoproteins and their sites of phosphorylation in D. pulex. We identified 103 phosphorylation sites in 91 Daphnia proteins by phosphopeptide enrichment using titanium dioxide isolation technology and an online two-dimensional liquid chromatography (2D-LC) system supported by high accuracy mass spectrometry. The identified Serine/threonine/tyrosine phosphorylation sites showed enrichment in the unstructured regions. Using Gene Ontology analysis, phosphorylated proteins were identified mainly as membrane proteins with essential biological roles such as protein binding, catalytic activity and nucleotide binding. BLASTP searching identified 21 phosphorylated sites in 20 D. pulex proteins that were evolutionally conserved between D. pulex and human. Here, we report the phosphorylation in Daphnia proteins and the predicted biological and functional roles of these phosphorylations. D. pulex might provide a promising model for examining the role of phosphorylation in biological functions.

In-depth proteomics approach of secretome to identify novel biomarker for sepsis in LPS-stimulated endothelial cells.

Sepsis and septic shock, which are conditions triggered by infection, occur with high incidence in emergency departments and are among the most common causes of death in hospitalized patients worldwide. Therefore, the identification of sepsis biomarkers for the rapid diagnosis is a major goal for researchers in the field of critical care. Endothelial cells play a pivotal role in orchestrating the inflammatory response triggered by sepsis. In this study, we used proteomics to investigate the secretome of EA.hy926 endothelial cells following lipopolysaccharide (LPS) stimulation with 1 μg/mL LPS for 12 or 24 h. SILAC in cell cultures and an online 2D‐LC‐MS/MS system were used to analyze the secretome dynamics in response to LPS. We found that 22 of the 77 secreted proteins identified in both the presence and absence of LPS and that 19 of the secreted proteins were quantified more strongly after LPS treatment for 24 h than after treatment for 12 h. By Gene Ontology and KEGG pathway analyses, we found that proteins related to the regulation of the actin cytoskeleton showed the highest secretion response to LPS stimulation. Out of the 19 candidate proteins, we focused on moesin, which is involved in the function of endothelial cells, and confirmed its amount in cellular lysates and media taken from primary human umbilical vein endothelial cells treated with LPS. To our knowledge, this study provides the first in‐depth analysis of the LPS‐induced secretome in human endothelial cells, and we propose 19 new biomarker candidates for sepsis, including moesin.

MS/MS of Synthetic Peptide Is Not Sufficient to Confirm New Types of Protein Modifications

Protein post-translational modification (PTM) is one of the major regulatory mechanisms that fine-tune protein functions. Undescribed mass shifts, which may suggest novel types of PTMs, continue to be discovered because of the availabilities of more sensitive mass spectrometry technologies and more powerful sequence alignment algorithms. In this study, the histone extracted from HeLa cells was analyzed using an approach that takes advantages of in vitro propionylation, efficient peptide separation using isoelectric focusing fractionation, and the high sensitivity of the linear ion trap coupled with hybrid FT mass spectrometer. One modified peptide was identified with a new type of protein modification (+42 Da), which was assigned to acetylation of threonine 15 in histone2A. The modified peptide was verified by careful manual evaluation of the tandem mass spectrum and confirmed by high-resolution MS/MS analysis of the corresponding synthetic peptide. However, HPLC coelution and MS/MS/MS of key ions showed that the +42 Da mass shifts at threonine residue did not correspond to acetylation. The key fragment ion, y4, in the MS/MS/MS spectra (indicative of the modification site) differed between the in vivo and synthetic peptide. We showed that the misidentification was originated from sequence homologues and chemical derivitization during sample preparation. This result indicated that a more stringent procedure that includes MS/MS, MS/MS/MS, and HPLC coelution of synthetic peptides is required to identify a new PTM.

Global analysis of phosphoproteome dynamics in embryonic development of zebrafish (Danio rerio).

The zebrafish (Danio rerio) is a popular animal model used for studies on vertebrate development and organogenesis. Recent research has shown a similarity of approximately 70% between the human and zebrafish genomes and about 84% of human disease‐causing genes have common ancestry with that of the zebrafish genes. Zebrafish embryos have a number of desirable features, including transparency, a large size, and rapid embryogenesis. Protein phosphorylation is a well‐known PTM, which can carry out various biological functions. Recent MS developments have enabled the study of global phosphorylation patterns by using MS‐based proteomics coupled with titanium dioxide phosphopeptide enrichment. In the present study, we identified 3500 nonredundant phosphorylation sites on 2166 phosphoproteins and quantified 1564 phosphoproteins in developing embryos of zebrafish. The distribution of Ser/Thr/Tyr phosphorylation sites in zebrafish embryos was found to be 88.9, 10.2, and 0.9%, respectively. A potential kinase motif was predicted using Motif‐X analysis, for 80% of the identified phosphorylation sites, with the proline‐directed motif appearing most frequently, and 35 phosphorylation sites having the pSF motif. In addition, we created six phosphoprotein clusters based on their dynamic pattern during the development of zebrafish embryos. Here, we report the largest dataset of phosphoproteins in zebrafish embryos and our results can be used for further studies on phosphorylation sites or phosphoprotein dynamics in zebrafish embryos.

A Comparison of the In Vitro Inhibitory Effects of Thelephoric Acid and SKF-525A on Human Cytochrome P450 Activity

Thelephoric acid is an antioxidant produced by the hydrolysis of polyozellin, which is isolated from Polyozellus multiplex. In the present study, the inhibitory effects of polyozellin and thelephoric acid on 9 cytochrome P450 (CYP) family members (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) were examined in pooled human liver microsomes (HLMs) using a cocktail probe assay. Polyozellin exhibited weak inhibitory effects on the activities of all 9 CYPs examined, whereas thelephoric acid exhibited dose- and time-dependent inhibition of all 9 CYP isoforms (IC50 values, 3.2–33.7 μM). Dixon plots of CYP inhibition indicated that thelephoric acid was a competitive inhibitor of CYP1A2 and CYP3A4. In contrast, thelephoric acid was a noncompetitive inhibitor of CYP2D6. Our findings indicate that thelephoric acid may be a novel, non-specific CYP inhibitor, suggesting that it could replace SKF-525A in inhibitory studies designed to investigate the effects of CYP enzymes on the metabolism of given compounds.

In vitro inhibitory effect of luotonin A on human CYP1A

Luotonin A, a pyrroloquinolinequinoline alkaloid, is a natural inhibitor of topoisomerase I. In the present study, cytochrome P450 (CYP) inhibition by luotonin A was examined in pooled human liver microsomes (HLMs) and human recombinant cDNA-expressed human CYPs using a cocktail probe assay to investigate potential drug-drug interactions. Luotonin A selectively inhibited CYP1A2-catalyzed phenacetin O-deethylation with an IC50 of 6.3 μM in HLMs, and strongly decreased CYP1A2-catalyzed phenacetin O-deethylation dose-dependently in HLMs, but did not inhibit it time-dependently. Furthermore, the Lineweaver-Burk and secondary plots for the inhibition of CYP1A2 in HLMs well fitted competitive inhibition mode. Luotonin A showed the selectivity of inhibitory effects on CYP1A1 and CYP1A2 in human recombinant cDNA-expressed CYP 1A1 and 1A2, respectively. Luotonin A was found to be a potent CYP1A inhibitor that might cause drug-drug interactions when co-administrated with CYP1A substrates.

Global proteomic analysis of lysine acetylation in zebrafish (Danio rerio) embryos

Lysine acetylation is an important post‐translational modification (PTM). Since the development of MS‐based proteomics technology, important roles of lysine acetylation beyond histones have focused on chromatin remodeling during the cell cycle and regulation of nuclear transport, metabolism, and translation. Zebrafish (Danio rerio) is a widely used vertebrate model in genetics and biologic studies. Although studies in several mammalian species have been performed, the mechanism of lysine acetylation in D. rerio embryos is incompletely understood. Here, we investigated the global acetylome in D. rerio embryos by using an MS‐based proteomics approach. We identified 351 acetylated peptides and 377 nonredundant acetylation sites on 189 lysine‐acetylated proteins in 5‐day postfertilization (hpf) embryos of D. rerio. Among lysine‐acetylated peptides, 40.2% indicated three motifs: (ac)KxxxK, (ac)KxxxxK, and Lx(ac)K. Of 190 acetylated proteins, 81 (42.6%) were mainly distributed in the cytoplasm. Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that lysine acetylation in D. rerio was enriched in metabolic pathways. Additionally, 17 of 30 acetylated ribosomal proteins were evolutionarily conserved between zebrafish and humans. Our results indicate that acetyllysine might have regulatory effects on ribosomal proteins involved in protein biosynthesis.

The inhibitory effects of the ethanolic extract of Pimpinella brachycarpa on cytochrome P450 enzymes in humans

Pimpinella brachycarp is a widely distributed vegetable with known antibacterial, antitumor, antioxidant, antithrombotic, and anti-inflammatory effects. In the present study, a cocktail probe assay and LC-MS/MS were used to investigate the modulating effect of the ethanolic extract of P. brachycarp on CYP enzymes in human liver microsomes. The extract significantly inhibited CYP 1A2, 2B6, and 3A4 by mix-mode inhibition and CYP2C19 and 2D6 by competitive inhibition.

Investigation of nonalcoholic fatty liver disease-induced drug metabolism by comparative global toxicoproteomics

&NA; Non‐alcoholic fatty liver disease (NAFLD) includes conditions such as steatosis, non‐alcoholic steatohepatitis, and ultimately hepatocellular carcinoma. Although the pathology of NAFLD is well‐established, NAFLD‐induced drug metabolism mediated by cytochrome P450 (CYP) in the liver has remained largely unexplored. Therefore, we investigated NAFLD‐induced drug metabolism mediated by CYP by quantitative toxicoproteomics analysis. After administration of a methionine‐choline deficient (MCD) diet to induce development of NAFLD, tandem mass tags‐based liquid chromatography‐tandem mass spectrometry analysis was conducted to investigate the dynamics of hepatic proteins. A total of 1295 proteins were identified, of which 934 were quantified by proteomic analysis. Among these proteins, 21 proteins were up‐regulated and 51 proteins were down‐regulated by the MCD diet. Notably, domain annotation enrichment using InterPro indicated that proteins related to CYPs were significantly decreased. When we investigated CYP activity using in vivo and in vitro CYP cocktail assays, most CYPs were significantly decreased, whereas CYP2D was not changed after administration of the MCD diet. In conclusion, we identified significantly altered levels of CYPs and their activities induced by the MCD diet and confirmed the NAFLD‐induced drug metabolism by pharmacokinetic analysis. Graphical abstract Figure. No caption available. HighlightsNAFLD‐induced drug metabolism was investigated by comparative toxicoproteomics.Among 934 quantified proteins, 21 were up‐regulated, and 51 were down‐regulated.Domain annotation enrichment indicated CYPs were significantly decreased.The decreased CYP activities were confirmed by in vivo and in vitro assays.Our data provides understanding of the drug‐interaction in NAFLD patients.