Language：English   Publishing type：Research paper (scientific journal)  

Currently, the bottom-up approach, in which proteins are digested by enzymes such as trypsin prior to mass spectrometry, is the mainstream approach in mass spectrometer-based proteomics. In this approach, the enzymatic digestion process strongly affects the reproducibility of protein identification and quantification. Here, we quantitatively evaluated the enzymatic digestion of proteins under various conditions by quantitative proteomics using data-independent acquisition and found that proteins precipitated with acetone after solubilization with SDS were fully digestible without re-solubilization. This result implies that organic solvent treatment makes cells amenable to trypsin digestion. Direct trypsin digestion of methanol-fixed cells achieved the same digestion efficiency and quantitative reproducibility as the conventional method. Furthermore, this method was found to be equally applicable to mouse liver samples. The establishment of this method indicates that the sample preparation process in bottom-up proteomics can be simplified while maintaining high digestion efficiency and is expected to become a general method for sample preparation in bottom-up proteomics in the future.

DOI： 10.1093/jb/mvac101

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Publisher：Cold Spring Harbor Laboratory  

Abstract

Metabolic regulation in skeletal muscle is essential for blood glucose homeostasis. Obesity causes insulin resistance in skeletal muscle, leading to hyperglycemia and type 2 diabetes. In this study, we performed multiomic analysis of the skeletal muscle of wild-type (WT) and genetically obese (ob/ob) mice, and constructed regulatory transomic networks for metabolism after oral glucose administration. Our network revealed that metabolic regulation by glucose-responsive metabolites had a major effect on WT mice, especially carbohydrate metabolic pathways. By contrast, in ob/ob mice, much of the metabolic regulation by glucose-responsive metabolites was lost and metabolic regulation by glucose-responsive genes was largely increased, especially in carbohydrate and lipid metabolic pathways. We present some characteristic metabolic regulatory pathways found in central carbon, branched amino acids, and ketone body metabolism. Our transomic analysis will provide insights into how skeletal muscle responds to changes in blood glucose and how it fails to respond in obesity.

DOI： 10.1101/2022.03.27.486003

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Language：English   Publishing type：Research paper (scientific journal)  

Glucose homeostasis is maintained by modulation of metabolic flux. Enzymes and metabolites regulate the involved metabolic pathways. Dysregulation of glucose homeostasis is a pathological event in obesity. Analyzing metabolic pathways and the mechanisms contributing to obesity-associated dysregulation in vivo is challenging. Here, we introduce OMELET: Omics-Based Metabolic Flux Estimation without Labeling for Extended Trans-omic Analysis. OMELET uses metabolomic, proteomic, and transcriptomic data to identify relative changes in metabolic flux, and to calculate contributions of metabolites, enzymes, and transcripts to the changes in metabolic flux. By evaluating the livers of fasting ob/ob mice, we found that increased metabolic flux through gluconeogenesis resulted primarily from increased transcripts, whereas that through the pyruvate cycle resulted from both increased transcripts and changes in substrates of metabolic enzymes. With OMELET, we identified mechanisms underlying the obesity-associated dysregulation of metabolic flux in the liver.

DOI： 10.1016/j.isci.2022.103787

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.neures.2021.12.006

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Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science (PLoS)  

Although long noncoding RNAs (lncRNAs) are transcripts that do not encode proteins by definition, some lncRNAs actually contain small open reading frames that are translated. TINCR (terminal differentiation–induced ncRNA) has been recognized as a lncRNA that contributes to keratinocyte differentiation. However, we here show that TINCR encodes a ubiquitin-like protein that is well conserved among species and whose expression was confirmed by the generation of mice harboring a FLAG epitope tag sequence in the endogenous open reading frame as well as by targeted proteomics. Forced expression of this protein promoted cell cycle progression in normal human epidermal keratinocytes, and mice lacking this protein manifested a delay in skin wound healing associated with attenuated cell cycle progression in keratinocytes. We termed this protein TINCR-encoded ubiquitin-like protein (TUBL), and our results reveal a role for TINCR in the regulation of keratinocyte proliferation and skin regeneration that is dependent on TUBL.

DOI： 10.1371/journal.pgen.1009686

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.isci.2021.102217

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Language：English   Publishing type：Research paper (scientific journal)  

Impaired glucose tolerance associated with obesity causes postprandial hyperglycemia and can lead to type 2 diabetes. To study the differences in liver metabolism in healthy and obese states, we constructed and analyzed transomics glucose-responsive metabolic networks with layers for metabolites, expression data for metabolic enzyme genes, transcription factors, and insulin signaling proteins from the livers of healthy and obese mice. We integrated multiomics time course data from wild-type and leptin-deficient obese (ob/ob) mice after orally administered glucose. In wild-type mice, metabolic reactions were rapidly regulated within 10 min of oral glucose administration by glucose-responsive metabolites, which functioned as allosteric regulators and substrates of metabolic enzymes, and by Akt-induced changes in the expression of glucose-responsive genes encoding metabolic enzymes. In ob/ob mice, the majority of rapid regulation by glucose-responsive metabolites was absent. Instead, glucose administration produced slow changes in the expression of carbohydrate, lipid, and amino acid metabolic enzyme-encoding genes to alter metabolic reactions on a time scale of hours. Few regulatory events occurred in both healthy and obese mice. Thus, our transomics network analysis revealed that regulation of glucose-responsive liver metabolism is mediated through different mechanisms in healthy and obese states. Rapid changes in allosteric regulators and substrates and in gene expression dominate the healthy state, whereas slow changes in gene expression dominate the obese state.

DOI： 10.1126/scisignal.aaz1236

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.isci.2020.101558

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.mito.2020.05.003

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Language：English   Publishing type：Research paper (scientific journal)  

An integrative understanding of nuclear events including transcription in normal and cancer cells requires comprehensive and quantitative measurement of protein dynamics that underlie such events. However, the low abundance of most nuclear proteins hampers their detailed functional characterization. We have now comprehensively quantified the abundance of nuclear proteins with the use of proteomics approaches in both normal and transformed human diploid fibroblasts. We found that subunits of the 26S proteasome complex were markedly down-regulated in the nuclear fraction of the transformed cells compared with that of the wild-type cells. The intranuclear proteasome abundance appeared to be inversely related to the rate of cell cycle progression, with restraint of the cell cycle being associated with an increase in the amount of proteasome subunits in the nucleus, suggesting that the nuclear proteasome content is dependent on the cell cycle. Furthermore, chromatin enrichment for proteomics (ChEP) analysis revealed enrichment of the proteasome in the chromatin fraction of quiescent cells and its apparent dissociation from chromatin in transformed cells. Our results thus suggest that translocation of the nuclear proteasome to chromatin may play an important role in control of the cell cycle and oncogenesis through regulation of chromatin-associated transcription factors.

DOI： 10.1038/s41598-020-62697-2

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Language：English   Publishing type：Research paper (scientific journal)  

Cellular signaling regulates various cellular functions via protein phosphorylation. Phosphoproteomic data potentially include information for a global regulatory network from signaling to cellular functions, but a procedure to reconstruct this network using such data has yet to be established. In this paper, we provide a procedure to reconstruct a global regulatory network from signaling to cellular functions from phosphoproteomic data by integrating prior knowledge of cellular functions and inference of the kinase-substrate relationships (KSRs). We used phosphoproteomic data from insulin-stimulated Fao hepatoma cells and identified protein phosphorylation regulated by insulin specifically over-represented in cellular functions in the KEGG database. We inferred kinases for protein phosphorylation by KSRs, and connected the kinases in the insulin signaling layer to the phosphorylated proteins in the cellular functions, revealing that the insulin signal is selectively transmitted via the Pi3k-Akt and Erk signaling pathways to cellular adhesions and RNA maturation, respectively. Thus, we provide a method to reconstruct global regulatory network from signaling to cellular functions based on phosphoproteomic data.

DOI： 10.1111/gtc.12655

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Language：English   Publishing type：Research paper (scientific journal)  

The concentrations of insulin selectively regulate multiple cellular functions. To understand how insulin concentrations are interpreted by cells, we constructed a trans-omic network of insulin action in FAO hepatoma cells using transcriptomic data, western blotting analysis of signaling proteins, and metabolomic data. By integrating sensitivity into the trans-omic network, we identified the selective trans-omic networks stimulated by high and low doses of insulin, denoted as induced and basal insulin signals, respectively. The induced insulin signal was selectively transmitted through the pathway involving Erk to an increase in the expression of immediate-early and upregulated genes, whereas the basal insulin signal was selectively transmitted through a pathway involving Akt and an increase of Foxo phosphorylation and a reduction of downregulated gene expression. We validated the selective trans-omic network in vivo by analysis of the insulin-clamped rat liver. This integrated analysis enabled molecular insight into how liver cells interpret physiological insulin signals to regulate cellular functions.

DOI： 10.1016/j.isci.2018.07.022

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC ADVANCEMENT SCIENCE  

Secretion of insulin transiently increases after eating, resulting in a high circulating concentration. Fasting limits insulin secretion, resulting in a low concentration of insulin in the circulation. We analyzed transcriptional responses to different temporal patterns and doses of insulin in the hepatoma FAO cells and identified 13 up-regulated and 16 down-regulated insulin-responsive genes (IRGs). The up-regulated IRGs responded more rapidly than did the down-regulated IRGs to transient stepwise or pulsatile increases in insulin concentration, whereas the down-regulated IRGs were repressed at lower concentrations of insulin than those required to stimulate the up-regulated IRGs. Mathematical modeling of the insulin response as two stages-(i) insulin signaling to transcription and (ii) transcription and mRNA stability-indicated that the first stage was the more rapid stage for the down-regulated IRGs, whereas the second stage of transcription was the more rapid stage for the up-regulated IRGs. A subset of the IRGs that were up-regulated or down-regulated in the FAO cells was similarly regulated in the livers of rats injected with a single dose of insulin. Thus, not only can cells respond to insulin but they can also interpret the intensity and pattern of signal to produce distinct transcriptional responses. These results provide insight that may be useful in treating obesity and type 2 diabetes associated with aberrant insulin production or tissue responsiveness.

DOI： 10.1126/scisignal.aaf3739

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

A key issue in the study of signal transduction is how multiple signaling pathways are systematically integrated into the cell. We have now performed multiple phosphoproteomics analyses focused on the dynamics of the T-cell receptor (TCR) signaling network and its subsystem mediated by the Ca2+ signaling pathway. Integration of these phosphoproteomics data sets and extraction of components of the TCR signaling network dependent on Ca2+ signaling showed unexpected phosphorylation kinetics for candidate substrates of the Ca2+-dependent phosphatase calcineurin (CN) during TCR stimulation. Detailed characterization of the TCR-induced phosphorylation of a novel CN substrate, Itpkb, showed that phosphorylation of this protein is regulated by both CN and the mitogen-activated protein kinase Erk in a competitive manner. Phosphorylation of additional CN substrates was also found to be regulated by Erk and CN in a similar manner. The combination of multiple phosphoproteomics approaches thus showed two major subsystems mediated by Erk and CN in the TCR signaling network, with these subsystems regulating the phosphorylation of a group of proteins in a competitive manner.

DOI： 10.1111/gtc.12406

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Language：Japanese   Publisher：(一社)日本糖尿病学会  

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Language：English   Publisher：ELSEVIER SCIENCE LONDON  

We propose 'trans-omic' analysis for reconstructing global biochemical networks across multiple omic layers by use of both multi-omic measurements and computational data integration. We introduce technologies for connecting multi-omic data based on prior knowledge of biochemical interactions and characterize a biochemical trans-omic network by concepts of a static and dynamic nature. We introduce case studies of metabolism-centric trans-omic studies to show how to reconstruct a biochemical trans-omic network by connecting multi-omic data and how to analyze it in terms of the static and dynamic nature. We propose a trans-ome-wide association study (trans-OWAS) connecting phenotypes with trans-omic networks that reflect both genetic and environmental factors, which can characterize several complex lifestyle diseases as breakdowns in the trans-omic system.

DOI： 10.1016/j.tibtech.2015.12.013

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Language：Japanese   Publisher：Japanese Proteomics Society (Japan Human Proteome Organisation)  

DOI： 10.14889/jhupo.2012.0.187.0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The chromatin organizer modifier domain (chromodomain) is present in proteins that contribute to chromatin organization and mediates their binding to methylated histone H3. Despite a high level of sequence conservation, individual chromodomains manifest substantial differences in binding preference for methylated forms of histone H3, suggesting that posttranslational modification of the chromodomain might be an important determinant of binding specificity. We now show that mouse Cbx2 (also known as M33), a homolog of Drosophila Polycomb protein, is highly phosphorylated in some cell lines. A low-mobility band of Cbx2 observed on SDS-polyacrylamide gel electrophoresis was thus converted to a higher-mobility band by treatment with alkaline phosphatase. Mass spectrometric analysis revealed serine-42, a conserved amino acid in the chromodomain, as a phosphorylation site of Cbx2. Phosphorylation of the chromodomain of Cbx2 on this residue in vitro resulted in a reduced level of binding to an H3 peptide containing trimethylated lysine-9 as well as an increase in the extent of binding to an H3 peptide containing trimethylated lysine-27, suggesting that such phosphorylation changes the binding specificity of Cbx2 for modified histone H3. Phosphorylation of the chromodomain of Cbx2 may therefore serve as a molecular switch that affects the reading of the histone modification code and thereby controls epigenetic cellular memory. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2010.05.074

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

Left-right (LR) patterning is an essential part of the animal body plan. Primary cilia are known to play a pivotal role in this process. In humans, genetic disorders of ciliogenesis cause serious congenital diseases. A comprehensive mechanism that regulates ciliogenesis has not been proposed so far. Here, we show that EED, a core member of the Polycomb group (PcG) genes and a presumed player in many epigenetic processes, is required for ciliogenesis and subsequent LR patterning in the medaka fish, Oryzias latipes. Moderate knockdown of oleed, a medaka homolog of EED, preferentially caused situs inversus. In the affected embryo, the cilia in Kupffer's vesicle showed various defects in their structure, position and motility. Furthermore, we demonstrated that oleed maintains the expression of Noto, which, in mice, regulates ciliogenesis and LR patterning. This study provides the first evidence for the involvement of epigenetic plasticity in LR patterning through ciliogenesis.

DOI： 10.1111/j.1365-2443.2009.01353.x

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