記述言語：英語   掲載種別：研究論文（学術雑誌）  

Reliable secretome analysis is crucial for understanding cellular communication and developing therapeutic strategies. However, conventional protein quantification methods, such as the bicinchoninic acid (BCA) assay, can overestimate protein concentrations in concentrated culture media, leading to inconsistent protein loading and compromised quantitative accuracy in mass spectrometry-based proteomics. To address this methodological challenge, we developed an improved sample preparation method for secretome analysis. Our approach introduces a concentration rate-based normalization method that adjusts sample volumes according to the ultrafiltration concentration ratio, ensuring more consistent protein loading across samples. This method enabled reliable identification of 3468 secreted proteins with high reproducibility (r > 0.93) in a model system of nuclear DNA (nucDNA)-induced inflammation in HeLa cells. Secretome profiles were distinctly altered by nucDNA transfection, with 89 proteins showing significant differential release between control and nucDNA-transfected wild-type HeLa cells. Furthermore, we identified a subset of proteins, including chaperone and proteasome complexes, that were consistently released across all conditions, suggesting their potential utility as internal controls for secretome analysis. This study presents a practical solution to the methodological challenge in secretome analysis, enabling more reliable and reproducible secretome profiling. This improved methodology represents an important step toward establishing standardized protocols for secretome analysis, ultimately enhancing the quality and comparability of research in this rapidly growing field.

DOI： 10.1093/biomethods/bpaf068

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.isci.2024.109121

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

Abstract

Each tissue has a dominant set of functional proteins required to mediate tissue-specific functions. Epigenetic modifications, transcription, and translational efficiency control tissue-dominant protein production. However, the coordination of these regulatory mechanisms to achieve such tissue-specific protein production remains unclear. Here, we analyzed the DNA methylome, transcriptome, and proteome in mouse liver and skeletal muscle. We found that DNA hypomethylation at promoter regions is globally associated with liver-dominant or skeletal muscle-dominant functional protein production within each tissue, as well as with genes encoding proteins involved in ubiquitous functions in both tissues. Thus, genes encoding liver-dominant proteins, such as those involved in glycolysis or gluconeogenesis, the urea cycle, complement and coagulation systems, enzymes of tryptophan metabolism, and cytochrome P450-related metabolism, were hypomethylated in the liver, whereas those encoding-skeletal muscle-dominant proteins, such as those involved in sarcomere organization, were hypomethylated in the skeletal muscle. Thus, DNA hypomethylation characterizes genes encoding tissue-dominant functional proteins.

DOI： 10.1038/s41598-023-46393-5

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その他リンク： https://www.nature.com/articles/s41598-023-46393-5

掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.isci.2023.107552

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掲載種別：研究論文（学術雑誌）   出版者・発行元：EMBO  

DOI： 10.15252/embj.2022112869

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Although several ribosomal protein paralogs are expressed in a tissue-specific manner, how these proteins affect translation and why they are required only in certain tissues have remained unclear. Here we show that RPL3L, a paralog of RPL3 specifically expressed in heart and skeletal muscle, influences translation elongation dynamics. Deficiency of RPL3L-containing ribosomes in RPL3L knockout male mice resulted in impaired cardiac contractility. Ribosome occupancy at mRNA codons was found to be altered in the RPL3L-deficient heart, and the changes were negatively correlated with those observed in myoblasts overexpressing RPL3L. RPL3L-containing ribosomes were less prone to collisions compared with RPL3-containing canonical ribosomes. Although the loss of RPL3L-containing ribosomes altered translation elongation dynamics for the entire transcriptome, its effects were most pronounced for transcripts related to cardiac muscle contraction and dilated cardiomyopathy, with the abundance of the encoded proteins being correspondingly decreased. Our results provide further insight into the mechanisms and physiological relevance of tissue-specific translational regulation.

DOI： 10.1038/s41467-023-37838-6

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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出版者・発行元：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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.neures.2021.12.006

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掲載種別：研究論文（学術雑誌）   出版者・発行元：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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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.isci.2021.102217

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.isci.2020.101558

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.mito.2020.05.003

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1126/scisignal.aaf3739

Web of Science

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1111/gtc.12406

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記述言語：日本語   出版者・発行元：(一社)日本糖尿病学会  

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記述言語：英語  

DOI： 10.1016/j.tibtech.2015.12.013

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

DOI： 10.14889/jhupo.2012.0.187.0

CiNii Article

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bbrc.2010.05.074

Web of Science

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1111/j.1365-2443.2009.01353.x

Web of Science

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