Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones

The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology

Mitochondrial Dysfunction Leads to Deconjugation of Quercetin Glucuronides in Inflammatory Macrophages

<div><p>Dietary flavonoids, such as quercetin, have long been recognized to protect blood vessels from atherogenic inflammation by yet unknown mechanisms. We have previously discovered the specific localization of quercetin-3-<i>O</i>-glucuronide (Q3GA), a phase II metabolite of quercetin, in macrophage cells in the human atherosclerotic lesions, but the biological significance is poorly understood. We have now demonstrated the molecular basis of the interaction between quercetin glucuronides and macrophages, leading to deconjugation of the glucuronides into the active aglycone. <i>In vitro</i> experiments showed that Q3GA was bound to the cell surface proteins of macrophages through anion binding and was readily deconjugated into the aglycone. It is of interest that the macrophage-mediated deconjugation of Q3GA was significantly enhanced upon inflammatory activation by lipopolysaccharide (LPS). Zymography and immunoblotting analysis revealed that β-glucuronidase is the major enzyme responsible for the deglucuronidation, whereas the secretion rate was not affected after LPS treatment. We found that extracellular acidification, which is required for the activity of β-glucuronidase, was significantly induced upon LPS treatment and was due to the increased lactate secretion associated with mitochondrial dysfunction. In addition, the β-glucuronidase secretion, which is triggered by intracellular calcium ions, was also induced by mitochondria dysfunction characterized using antimycin-A (a mitochondrial inhibitor) and siRNA-knockdown of Atg7 (an essential gene for autophagy). The deconjugated aglycone, quercetin, acts as an anti-inflammatory agent in the stimulated macrophages by inhibiting the c-Jun N-terminal kinase activation, whereas Q3GA acts only in the presence of extracellular β-glucuronidase activity. Finally, we demonstrated the deconjugation of quercetin glucuronides including the sulfoglucuronides <i>in vivo</i> in the spleen of mice challenged with LPS. These results showed that mitochondrial dysfunction plays a crucial role in the deconjugation of quercetin glucuronides in macrophages. Collectively, this study contributes to clarifying the mechanism responsible for the anti-inflammatory activity of dietary flavonoids within the inflammation sites.</p> </div

Cell-surface binding of Q3GA.

<p>(A) Chemical structure of quercetin-3-<i>O</i>-glucuronide (Q3GA). (B) Time-dependent accumulation of Q3GA in the RAW264 cells. Cells were treated with Q3GA (50 μM) for the indicated time periods. (C) time-dependent dissociation of Q3GA bound to RAW264 cells. Cells were treated with Q3GA (50 μM) for 1 h, washed, and then incubated in the fresh medium for indicated time periods. Data points represent duplicate determinations. (D) Inhibition of Q3GA accumulation in RAW264 cells by various transporter inhibitors. Cells were pretreated with inhibitors for 15 min, followed by Q3GA (50 μM) treatment for 15 min. The concentrations of inhibitors were as follows: DIDS, taurocholic acid (TCA), p-aminohippuric acid (PAH), and estradiol-17-β-glucuronide (E17G), 500 μM; sodium azide, 5 mM. (E) Dose-dependent inhibition of Q3GA accumulation in RAW264 cells by DIDS. Data points represent duplicate determinations. (F) Inhibition of the accumulation of quercetin-3-<i>O</i>-glycosides in RAW264 cells by DIDS. The DIDS-treated cells were incubated with each quercetin-3-<i>O</i>-glycoside (20 μM) for 15 min. Q3G, quercetin-3-<i>O</i>-glucoside; Q3Gal, quercetin-3-<i>O</i>-galactoside. (G) Anti-DIDS immunoreactivity of the lysates of the DIDS-treated RAW264 cells. Cells were treated with DIDS followed by a quick trypsinization for 1 min. β-Actin was detected as a positive control for intracellular proteins. (H) Effects of quick trypsinization of RAW264 cells on the accumulation of Q3GA and quercetin. Q3GA- or quercetin-treated cells were incubated with trypsin for 1 min. (I) Effect of DIDS treatment on the accumulation of quercetin. Data in all bar graphs are presented as the average ± S.D. (n=3). Asterisks indicate a significant difference (<i>p</i> < 0.05). NS, not statistically significant.</p

Deconjugation of quercetin glucuronides <i>in</i><i>vivo</i>.

<p>(A) Immunoblot analysis for β-glucuronidase (GAPDH as control) in the various tissues in ICR mice. (B) Accumulation of the quercetin derivatives determined by HPLC-ECD in the spleen of ICR mice fed with 0.5% quercetin diet for 24 h followed by injection of LPS. <i>Left</i>, total metabolites; middle, aglycones; <i>right</i>, sulfates. (C) Accumulation of quercetin derivatives in the plasma of ICR mice injected with LPS. ND, not detected. Asterisks indicate a significant difference (<i>p</i> < 0.05). NS, not statistically significant. Data points represent the average ± S.D. (n=6). </p