Publications by authors named "Fuchu He"

365 Publications

Posttranscriptional regulation of de novo lipogenesis by glucose-induced O-GlcNAcylation.

Mol Cell 2021 Feb 23. Epub 2021 Feb 23.

Department of Biochemistry and Molecular Medicine, The George Washington University School of Medicine and Health Science, 2300 Eye Street, N.W., Washington, DC 20037, USA; GW Cancer Center, George Washington University School of Medicine and Health Sciences, Washington, DC 20052, USA. Electronic address:

O-linked β-N-acetyl glucosamine (O-GlcNAc) is attached to proteins under glucose-replete conditions; this posttranslational modification results in molecular and physiological changes that affect cell fate. Here we show that posttranslational modification of serine/arginine-rich protein kinase 2 (SRPK2) by O-GlcNAc regulates de novo lipogenesis by regulating pre-mRNA splicing. We found that O-GlcNAc transferase O-GlcNAcylated SRPK2 at a nuclear localization signal (NLS), which triggers binding of SRPK2 to importin α. Consequently, O-GlcNAcylated SRPK2 was imported into the nucleus, where it phosphorylated serine/arginine-rich proteins and promoted splicing of lipogenic pre-mRNAs. We determined that protein nuclear import by O-GlcNAcylation-dependent binding of cargo protein to importin α might be a general mechanism in cells. This work reveals a role of O-GlcNAc in posttranscriptional regulation of de novo lipogenesis, and our findings indicate that importin α is a "reader" of an O-GlcNAcylated NLS.
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http://dx.doi.org/10.1016/j.molcel.2021.02.009DOI Listing
February 2021

Intrinsic disorder in protein domains contributes to both organism complexity and clade-specific functions.

Sci Rep 2021 Feb 4;11(1):2985. Epub 2021 Feb 4.

State Key Laboratory of Proteomics, National Center for Protein Sciences (Beijing), Beijing Proteome Research Center, Beijing Institute of Lifeomics, 38 Science Park Road, Changping District, Beijing, 102206, China.

Interestingly, some protein domains are intrinsically disordered (abbreviated as IDD), and the disorder degree of same domains may differ in different contexts. However, the evolutionary causes and biological significance of these phenomena are unclear. Here, we address these issues by genome-wide analyses of the evolutionary and functional features of IDDs in 1,870 species across the three superkingdoms. As the result, there is a significant positive correlation between the proportion of IDDs and organism complexity with some interesting exceptions. These phenomena may be due to the high disorder of clade-specific domains and the different disorder degrees of the domains shared in different clades. The functions of IDDs are clade-specific and the higher proportion of post-translational modification sites may contribute to their complex functions. Compared with metazoans, fungi have more IDDs with a consecutive disorder region but a low disorder ratio, which reflects their different functional requirements. As for disorder variation, it's greater for domains among different proteins than those within the same proteins. Some clade-specific 'no-variation' or 'high-variation' domains are involved in clade-specific functions. In sum, intrinsic domain disorder is related to both the organism complexity and clade-specific functions. These results deepen the understanding of the evolution and function of IDDs.
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http://dx.doi.org/10.1038/s41598-021-82656-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862400PMC
February 2021

Deubiquitinase Ubp3 enhances the proteasomal degradation of key enzymes in sterol homeostasis.

J Biol Chem 2021 Jan 29:100348. Epub 2021 Jan 29.

School of Basic Medical Science, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery of Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China; State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Research Unit of Proteomics & Research and Development of New Drug of Chinese Academy of Medical Sciences, Beijing Institute of Lifeomics, Beijing, 102206, China; Guizhou University School of Medicine, Guiyang 550025, China. Electronic address:

Sterol homeostasis is tightly controlled by molecules that are highly conserved from yeast to humans, the dysregulation of which plays critical roles in the development of antifungal resistance and various cardiovascular diseases. Previous studies have shown that sterol homeostasis is regulated by the ubiquitin-proteasome system. Two E3 ubiquitin ligases, Hrd1 and Doa10 are known to mediate the proteasomal degradation of HMG-CoA reductase Hmg2 and squalene epoxidase Erg1 with accumulation of the toxic sterols in cells, but the deubiquitinases (DUBs) involved are unclear. Here, we screened for DUBs responsible for sterol homeostasis using yeast strains from a DUB-deletion library. The defective growth observed in ubp3-deleted (ubp3Δ) yeast upon fluconazole treatment suggests that lack of Ubp3 disrupts sterol homeostasis. Deep-coverage quantitative proteomics reveals that ergosterol biosynthesis is rerouted into a sterol pathway that generates toxic products in the absence of Ubp3. Further genetic and biochemical analysis indicated that Ubp3 enhances the proteasome's ability to degrade the ergosterol biosynthetic enzymes Erg1 and Erg3. The retardation of ergosterol enzymes degradation in the ubp3Δ strain resulted in the severe accumulation of the intermediate lanosterol and a branched toxic sterol, and ultimately disrupted sterol homeostasis and led to the susceptibility to fluconazole. Our findings uncover a role for Ubp3 in sterol homeostasis and highlight its potential as a new antifungal target.
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http://dx.doi.org/10.1016/j.jbc.2021.100348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8027567PMC
January 2021

Author Correction: Neddylation of PTEN regulates its nuclear import and promotes tumor development.

Cell Res 2021 Mar;31(3):374

State Key Laboratory of Proteomics, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, 100850, China.

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http://dx.doi.org/10.1038/s41422-021-00470-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8027785PMC
March 2021

Conserved paradoxical relationships among the evolutionary, structural and expressional features of KRAB zinc-finger proteins reveal their special functional characteristics.

BMC Mol Cell Biol 2021 Jan 22;22(1). Epub 2021 Jan 22.

State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, 102206, China.

Background: One striking feature of the large KRAB domain-containing zinc finger protein (KZFP) family is its rapid evolution, leading to hundreds of member genes with various origination time in a certain mammalian genome. However, a comprehensive genome-wide and across-taxa analysis of the structural and expressional features of KZFPs with different origination time is lacking. This type of analysis will provide valuable clues about the functional characteristics of this special family.

Results: In this study, we found several conserved paradoxical phenomena about this issue. 1) Ordinary young domains/proteins tend to be disordered, but most of KRAB domains are completely structured in 64 representative species across the superclass of Sarcopterygii and most of KZFPs are also highly structured, indicating their rigid and unique structural and functional characteristics; as exceptions, old-zinc-finger-containing KZFPs have relatively disordered KRAB domains and linker regions, contributing to diverse interacting partners and functions. 2) In general, young or highly structured proteins tend to be spatiotemporal specific and have low abundance. However, by integrated analysis of 29 RNA-seq datasets, including 725 samples across early embryonic development, embryonic stem cell differentiation, embryonic and adult organs, tissues in 7 mammals, we found that KZFPs tend to express ubiquitously with medium abundance regardless of evolutionary age and structural disorder degree, indicating the wide functional requirements of KZFPs in various states. 3) Clustering and correlation analysis reveal that there are differential expression patterns across different spatiotemporal states, suggesting the specific-high-expression KZFPs may play important roles in the corresponding states. In particular, part of young-zinc-finger-containing KZFPs are highly expressed in early embryonic development and ESCs differentiation into endoderm or mesoderm. Co-expression analysis revealed that young-zinc-finger-containing KZFPs are significantly enriched in five co-expression modules. Among them, one module, including 13 young-zinc-finger-containing KZFPs, showed an 'early-high and late-low' expression pattern. Further functional analysis revealed that they may function in early embryonic development and ESC differentiation via participating in cell cycle related processes.

Conclusions: This study shows the conserved and special structural, expressional features of KZFPs, providing new clues about their functional characteristics and potential causes of their rapid evolution.
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http://dx.doi.org/10.1186/s12860-021-00346-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7821633PMC
January 2021

Histone H3K27 methyltransferase EZH2 and demethylase JMJD3 regulate hepatic stellate cells activation and liver fibrosis.

Theranostics 2021 1;11(1):361-378. Epub 2021 Jan 1.

Center for Medical Research and Innovation, Shanghai Pudong Hospital and Pudong Medical Center, Shanghai Medical College, Fudan University, Shanghai, China.

As the central hallmark of liver fibrosis, transdifferentiation of hepatic stellate cells (HSCs), the predominant contributor to fibrogenic hepatic myofibroblast responsible for extracellular matrix (ECM) deposition, is characterized with transcriptional and epigenetic remodeling. We aimed to characterize the roles of H3K27 methyltransferase EZH2 and demethylase JMJD3 and identify their effective pathways and novel target genes in HSCs activation and liver fibrosis. In primary HSCs, we analyzed effects of pharmacological inhibitions and genetic manipulations of EZH2 and JMJD3 on HSCs activation. In HSCs cell lines, we evaluated effects of EZH2 inhibition by DZNep on proliferation, cell cycling, senescence and apoptosis. In CCl and BDL murine models of liver fibrosis, we assessed effects of DZNep administration and silencing. We profiled rat primary HSCs transcriptomes with RNA-seq, screened the pathways and genes associated with DZNep treatment, analyzed EZH2 and JMJD3 regulation towards target genes by ChIP-qPCR. EZH2 inhibition by DZNep resulted in retarded growth, lowered cell viability, cell cycle arrest in S and G2 phases, strengthened senescence, and enhanced apoptosis of HSCs, decreased hepatic collagen deposition and rescued the elevated serum ALT and AST activities of diseased mice, and downregulated cellular and hepatic expressions of H3K27me3, EZH2, α-SMA and COL1A. silencing by RNA interference and showed similar effects. JMJD3 inhibition by GSK-J4 and overexpression of wild-type but not mutant enhanced or repressed HSCs activation respectively. EZH2 inhibition by DZNep transcriptionally inactivated TGF-β1 pathway, cell cycle pathways and vast ECM components in primary HSCs. EZH2 inhibition decreased H3K27me3 recruitment at target genes encoding TGF-β1 pseudoreceptor BAMBI, anti-inflammatory cytokine IL10 and cell cycle regulators CDKN1A, GADD45A and GADD45B, and increased their expressions, while overexpression manifested alike effects. EZH2 and JMJD3 antagonistically modulate HSCs activation. The therapeutic effects of DZNep as epigenetic drug in liver fibrosis are associated with the regulation of EZH2 towards direct target genes encoding TGF-β1 pseudoreceptor BAMBI, anti-inflammatory cytokine IL10 and cell cycle regulators CDKN1A, GADD45A and GADD45B, which are also regulated by JMJD3. Our present study provides new mechanistic insight into the epigenetic modulation of EZH2 and JMJD3 in HSCs biology and hepatic fibrogenesis.
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http://dx.doi.org/10.7150/thno.46360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7681085PMC
January 2021

Neddylation of PTEN regulates its nuclear import and promotes tumor development.

Cell Res 2021 Mar 9;31(3):291-311. Epub 2020 Dec 9.

State Key Laboratory of Proteomics, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, 100850, China.

PTEN tumor suppressor opposes the PI3K/Akt signaling pathway in the cytoplasm and maintains chromosomal integrity in the nucleus. Nucleus-cytoplasm shuttling of PTEN is regulated by ubiquitylation, SUMOylation and phosphorylation, and nuclear PTEN has been proposed to exhibit tumor-suppressive functions. Here we show that PTEN is conjugated by Nedd8 under high glucose conditions, which induces PTEN nuclear import without effects on PTEN stability. PTEN neddylation is promoted by the XIAP ligase and removed by the NEDP1 deneddylase. We identify Lys197 and Lys402 as major neddylation sites on PTEN. Neddylated PTEN accumulates predominantly in the nucleus and promotes rather than suppresses cell proliferation and metabolism. The nuclear neddylated PTEN dephosphorylates the fatty acid synthase (FASN) protein, inhibits the TRIM21-mediated ubiquitylation and degradation of FASN, and then promotes de novo fatty acid synthesis. In human breast cancer tissues, neddylated PTEN correlates with tumor progression and poor prognosis. Therefore, we demonstrate a previously unidentified pool of nuclear PTEN in the Nedd8-conjugated form and an unexpected tumor-promoting role of neddylated PTEN.
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http://dx.doi.org/10.1038/s41422-020-00443-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8027835PMC
March 2021

Comprehensive proteomic atlas of skin biomatrix scaffolds reveals a supportive microenvironment for epidermal development.

J Tissue Eng 2020 Jan-Dec;11:2041731420972310. Epub 2020 Nov 10.

Department of Stem Cell and Regenerative Medicine Laboratory, Institute of Health Service and Transfusion Medicine, Beijing, China.

Biomaterial scaffolds are increasingly being used to drive tissue regeneration. The limited success so far in human tissues rebuilding and therapy application may be due to inadequacy of the functionality biomaterial scaffold. We developed a new decellularized method to obtain complete anatomical skin biomatrix scaffold in situ with extracellular matrix (ECM) architecture preserved, in this study. We described a skin scaffold map by integrated proteomics and systematically analyzed the interaction between ECM proteins and epidermal cells in skin microenvironment on this basis. They were used to quantify structure and function of the skin's Matrisome, comprised of core ECM components and ECM-associated soluble signals that are key regulators of epidermal development. We especially revealed that ECM played a role in determining the fate of epidermal stem cells through hemidesmosome components. These concepts not only bring us a new understanding of the role of the skin ECM niche, they also provide an attractive combinational strategy based on tissue engineering principles with skin biomatrix scaffold materials for the acceleration and enhancement of tissue regeneration.
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http://dx.doi.org/10.1177/2041731420972310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7658515PMC
November 2020

Open-pFind Verified Four Missing Proteins from Multi-Tissues.

J Proteome Res 2020 12 10;19(12):4808-4814. Epub 2020 Nov 10.

School of Basic Medical Science, Key Laboratory of Combinational Biosynthesis and Drug Discovery of Ministry of Education, School of Basic Medical Sciences, Wuhan University, Wuhan 430072, China.

The Chromosome-Centric Human Proteome Project (C-HPP) was launched in 2012 to perfect the annotation of human protein existence by identifying stronger evidence of the expression of missing proteins (MPs) at the protein level. After an 8 year effort all over the world, the number of MPs in the neXtProt database significantly decreased from 5511 (2012-02-24) to 1899 (2020-01-17). It is now more difficult to provide confident evidence of the remaining MPs because of their specific characteristics, including low abundance, low molecular weight, unexpected modifications, transmembrane structure, tissue-expression specificity, and so on. A higher resolution mass spectrometry (MS) interpretation engine might provide an opportunity to identify these buried MPs in complex samples by the combination with multi-tissue large-scale proteomics. In this study, open-pFind was used to dig MPs from 20 pairs of healthy human tissues by Wang et al. ( 2019, 15 (2), e8503) combined with our large-scale testis data set digested by three enzymes (Glu-C, Lys-C, and trypsin) with specificity for different amino acid residues ( 2019, 18 (12), 4189-4196). A total of 1 535 536 peptides with 17 283 477 peptide-spectrum matches (PSMs) were mapped to 14 279 protein entries at a false discovery rate of <1% at the PSM, peptide, and protein levels. A total of 103 MP candidates were identified, among which 86 candidates had more unique peptide numbers compared with our single testis tissue. After rigorous screening, manual checks, peptide synthesis, and matching with documented peptides from PeptideAtlas, we validated four MPs, P0C7T8 (duodenum and small intestine), Q8WWZ4 (stomach and rectum), Q8IV35 (fallopian tube), and O14921 (tonsil), at the protein level. All MS raw files have been deposited to the ProteomeXchange with identifier PXD021391.
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http://dx.doi.org/10.1021/acs.jproteome.0c00370DOI Listing
December 2020

Activation of mTORC1 by LSECtin in macrophages directs intestinal repair in inflammatory bowel disease.

Cell Death Dis 2020 10 26;11(10):918. Epub 2020 Oct 26.

State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, 102206, Beijing, China.

Damage to intestinal epithelial cells and the induction of cellular apoptosis are characteristics of inflammatory bowel disease. The C-type lectin receptor family member LSECtin promotes apoptotic cell clearance by macrophages and induces the production of anti-inflammatory/tissue growth factors, which direct intestinal repair in experimental colitis. However, the mechanisms by which the phagocytosis of apoptotic cells triggers the pro-repair function of macrophages remain largely undefined. Here, using immunoprecipitation in combination with mass spectrometry to identify LSECtin-interacting proteins, we found that LSECtin interacted with mTOR, exhibiting a role in activating mTORC1. Mechanistically, apoptotic cells enhance the interaction between LSECtin and mTOR, and increase the activation of mTORC1 induced by LSECtin in macrophages. Elevated mTORC1 signaling triggers macrophages to produce anti-inflammatory/tissue growth factors that contribute to the proliferation of epithelial cells and promote the reestablishment of tissue homeostasis. Collectively, our findings suggest that LSECtin-dependent apoptotic cell clearance by macrophages activates mTORC1, and thus contributes to intestinal regeneration and the remission of colitis.
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http://dx.doi.org/10.1038/s41419-020-03114-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589503PMC
October 2020

A high-stringency blueprint of the human proteome.

Nat Commun 2020 10 16;11(1):5301. Epub 2020 Oct 16.

Departments of Medicine-Cardiology and Biochemistry and Molecular Genetics, University of Colorado, Anschutz Medical Campus, Aurora, CO, USA.

The Human Proteome Organization (HUPO) launched the Human Proteome Project (HPP) in 2010, creating an international framework for global collaboration, data sharing, quality assurance and enhancing accurate annotation of the genome-encoded proteome. During the subsequent decade, the HPP established collaborations, developed guidelines and metrics, and undertook reanalysis of previously deposited community data, continuously increasing the coverage of the human proteome. On the occasion of the HPP's tenth anniversary, we here report a 90.4% complete high-stringency human proteome blueprint. This knowledge is essential for discerning molecular processes in health and disease, as we demonstrate by highlighting potential roles the human proteome plays in our understanding, diagnosis and treatment of cancers, cardiovascular and infectious diseases.
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http://dx.doi.org/10.1038/s41467-020-19045-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7568584PMC
October 2020

A quantitative thiol reactivity profiling platform to analyze redox and electrophile reactive cysteine proteomes.

Nat Protoc 2020 09 20;15(9):2891-2919. Epub 2020 Jul 20.

State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences • Beijing, Beijing Institute of Lifeomics, Beijing, China.

Cysteine is unique among all protein-coding amino acids, owing to its intrinsically high nucleophilicity. The cysteinyl thiol group can be covalently modified by a broad range of redox mechanisms or by various electrophiles derived from exogenous or endogenous sources. Measuring the response of protein cysteines to redox perturbation or electrophiles is critical for understanding the underlying mechanisms involved. Activity-based protein profiling based on thiol-reactive probes has been the method of choice for such analyses. We therefore adapted this approach and developed a new chemoproteomic platform, termed 'QTRP' (quantitative thiol reactivity profiling), that relies on the ability of a commercially available thiol-reactive probe IPM (2-iodo-N-(prop-2-yn-1-yl)acetamide) to covalently label, enrich and quantify the reactive cysteinome in cells and tissues. Here, we provide a detailed and updated workflow of QTRP that includes procedures for (i) labeling of the reactive cysteinome from cell or tissue samples (e.g., control versus treatment) with IPM, (ii) processing the protein samples into tryptic peptides and tagging the probe-modified peptides with isotopically labeled azido-biotin reagents containing a photo-cleavable linker via click chemistry reaction, (iii) capturing biotin-conjugated peptides with streptavidin beads, (iv) identifying and quantifying the photo-released peptides by mass spectrometry (MS)-based shotgun proteomics and (v) interpreting MS data by a streamlined informatic pipeline using a proteomics software, pFind 3, and an automatic post-processing algorithm. We also exemplified here how to use QTRP for mining HO-sensitive cysteines and for determining the intrinsic reactivity of cysteines in a complex proteome. We anticipate that this protocol should find broad applications in redox biology, chemical biology and the pharmaceutical industry. The protocol for sample preparation takes 3 d, whereas MS measurements and data analyses require 75 min and <30 min, respectively, per sample.
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http://dx.doi.org/10.1038/s41596-020-0352-2DOI Listing
September 2020

Integrative Proteomic Characterization of Human Lung Adenocarcinoma.

Cell 2020 07;182(1):245-261.e17

Shanghai Key Laboratory of Intelligent Information Processing, School of Computer Science and Technology, Fudan University, Shanghai 200433, China.

Genomic studies of lung adenocarcinoma (LUAD) have advanced our understanding of the disease's biology and accelerated targeted therapy. However, the proteomic characteristics of LUAD remain poorly understood. We carried out a comprehensive proteomics analysis of 103 cases of LUAD in Chinese patients. Integrative analysis of proteome, phosphoproteome, transcriptome, and whole-exome sequencing data revealed cancer-associated characteristics, such as tumor-associated protein variants, distinct proteomics features, and clinical outcomes in patients at an early stage or with EGFR and TP53 mutations. Proteome-based stratification of LUAD revealed three subtypes (S-I, S-II, and S-III) related to different clinical and molecular features. Further, we nominated potential drug targets and validated the plasma protein level of HSP 90β as a potential prognostic biomarker for LUAD in an independent cohort. Our integrative proteomics analysis enables a more comprehensive understanding of the molecular landscape of LUAD and offers an opportunity for more precise diagnosis and treatment.
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http://dx.doi.org/10.1016/j.cell.2020.05.043DOI Listing
July 2020

Irradiated and CCl -treated bone marrow-derived liver macrophages exhibit different gene expression patterns and phenotypes.

Scand J Immunol 2020 Nov 2;92(5):e12916. Epub 2020 Sep 2.

State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China.

Myeloid cells infiltrate into the liver and differentiate into macrophages in different liver injury mouse models. However, the heterogeneity of bone marrow (BM)-derived LMs populations remains to be understood. To investigate this and understand the impact of the macrophage niche on the properties of recruited BM-derived macrophages, we used a non-myeloablation BM transplantation model to label and trace BM-derived LMs. Subsequently, we quantified the number of embryonic-derived liver-resident macrophages, BM-derived LMs and total LMs in CCl and irradiated acute liver injury mouse models, respectively. Finally, we compared the cell fate, gene expression patterns, chemokine signals, and surface markers of irradiated and CCl -treated BM-derived LMs. We observed that, as compared to CCl radiation generated a macrophage niche by depleting embryonic-derived liver-resident macrophages and induced the recruitment of BM-derived LMs that further settled in the liver. Irradiated and CCl -treated BM-derived LMs are different with respect to their cell fates, gene expression patterns, and chemokine expression and recruitment. They also have different surface markers shortly after differentiating from their progenitors. Our findings suggest that irradiated and CCl -treated LM populations derived from the bone marrow display different patterns of gene expression and phenotypes; these differences may be due to the availability of macrophage niche.
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http://dx.doi.org/10.1111/sji.12916DOI Listing
November 2020

Discovery of Urinary Proteomic Signature for Differential Diagnosis of Acute Appendicitis.

Biomed Res Int 2020 4;2020:3896263. Epub 2020 Apr 4.

State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing 102206, China.

Acute appendicitis is one of the most common acute abdomens, but the confident preoperative diagnosis is still a challenge. In order to profile noninvasive urinary biomarkers that could discriminate acute appendicitis from other acute abdomens, we carried out mass spectrometric experiments on urine samples from patients with different acute abdomens and evaluated diagnostic potential of urinary proteins with various machine-learning models. Firstly, outlier protein pools of acute appendicitis and controls were constructed using the discovery dataset (32 acute appendicitis and 41 control acute abdomens) against a reference set of 495 normal urine samples. Ten outlier proteins were then selected by feature selection algorithm and were applied in construction of machine-learning models using naïve Bayes, support vector machine, and random forest algorithms. The models were assessed in the discovery dataset by leave-one-out cross validation and were verified in the validation dataset (16 acute appendicitis and 45 control acute abdomens). Among the three models, random forest model achieved the best performance: the accuracy was 84.9% in the leave-one-out cross validation of discovery dataset and 83.6% (sensitivity: 81.2%, specificity: 84.4%) in the validation dataset. In conclusion, we developed a 10-protein diagnostic panel by the random forest model that was able to distinguish acute appendicitis from confusable acute abdomens with high specificity, which indicated the clinical application potential of noninvasive urinary markers in disease diagnosis.
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http://dx.doi.org/10.1155/2020/3896263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7165319PMC
January 2021

Identification of noninvasive diagnostic biomarkers for hepatocellular carcinoma by urinary proteomics.

J Proteomics 2020 08 13;225:103780. Epub 2020 Apr 13.

State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China. Electronic address:

Hepatocellular carcinoma (HCC) ranks fourth in cancer mortality worldwide, and third in China. Hepatitis B virus (HBV) infection is a main risk factor for HCC in China, and the early diagnosis of HCC in high-risk population is very important. However, the commonly used diagnostic biomarker alpha-fetoprotein has limitations in clinical practice. In order to identify reliable and noninvasive HCC urinary biomarkers, a high-throughput proteomics streamline was applied in the analysis of urine samples from 74 HCC and 82 high-risk patients with chronic HBV infected liver diseases. Candidate diagnostic markers were screened by feature selection algorithm, and were combined with random forest or simple voting algorithms in the training dataset. Then the multiple feature models were validated in an independent test dataset. The selected features were further verified by Multiple Reaction Monitoring (MRM) in another independent dataset. By integrating 7 features screened in the discovery phase, random forest model achieved AUC of 0.92 and 0.87 in training and test datasets, respectively, while voting model performed better with AUC of 0.94 and 0.90, respectively. In the MRM dataset, the 7 features were targeted quantified, and voting model integrating the 7 features achieved AUC of 0.95. Our work highlights the potential of noninvasive urinary protein biomarkers in HCC diagnosis with high-risk population, which will be beneficial to HCC auxiliary diagnosis and HCC surveillance. SIGNIFICANCE: A high throughput urinary proteome analysis platform was committed into the discovery of noninvasive HCC biomarkers in high-risk patients with chronic HBV infected liver diseases. The combination of 7 urinary features achieved good performance in distinguishing HCC from high-risk population. The expression of the 7 features was validated by targeted MRM, and the integration of the features also worked well in the MRM dataset. This is the first time that urinary proteomic strategy was applied in discovering HCC biomarkers from high-risk population. This result will be helpful for HCC auxiliary diagnosis and surveillance in a noninvasive way.
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http://dx.doi.org/10.1016/j.jprot.2020.103780DOI Listing
August 2020

Ubiquitin Linkage Specificity of Deubiquitinases Determines Cyclophilin Nuclear Localization and Degradation.

iScience 2020 Apr 13;23(4):100984. Epub 2020 Mar 13.

State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Research Unit of Proteomics & Research and Development of New Drug of Chinese Academy of Medical Sciences, Beijing Institute of Lifeomics, 38 Science Park Road, Beijing 102206, China; School of Basic Medical Science, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery of Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China; Guizhou University School of Medicine, Guiyang 550025, China; Second Clinical Medicine Collage, Guangzhou University Chinese Medicine, Guangzhou 510006, China. Electronic address:

Ubiquitin chain specificity has been described for some deubiquitinases (DUBs) but lacks a comprehensive profiling in vivo. We used quantitative proteomics to compare the seven lysine-linked ubiquitin chains between wild-type yeast and its 20 DUB-deletion strains, which may reflect the linkage specificity of DUBs in vivo. Utilizing the specificity and ubiquitination heterogeneity, we developed a method termed DUB-mediated identification of linkage-specific ubiquitinated substrates (DILUS) to screen the ubiquitinated lysine residues on substrates modified with certain chains and regulated by specific DUB. Then we were able to identify 166 Ubp2-regulating substrates with 244 sites potentially modified with K63-linked chains. Among these substrates, we further demonstrated that cyclophilin A (Cpr1) modified with K63-linked chain on K151 site was regulated by Ubp2 and mediated the nuclear translocation of zinc finger protein Zpr1. The K48-linked chains at non-K151 sites of Cpr1 were mainly regulated by Ubp3 and served as canonical signals for proteasome-mediated degradation.
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http://dx.doi.org/10.1016/j.isci.2020.100984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115106PMC
April 2020

Expression of human Krüppel-like factor 3 in peripheral blood as a promising biomarker for acute leukemia.

Cancer Med 2020 04 26;9(8):2803-2811. Epub 2020 Feb 26.

Department of Hematology, Peking University First Hospital, Beijing, China.

Background: Universal gene targets are in persistent demand by real-time quantitative polymerase chain reaction (RT-qPCR)-based methods in acute leukemia (AL) diagnosis and monitoring. Human Krüppel-like factor 3 (hKLF3), a newly cloned human transcription factor, has proved to be a regulator of hematopoiesis.

Methods: Sanger sequencing was performed in bone marrow (BM) samples from 17 AL patients for mutations in hKLF3 coding exons. hKLF3 expression in peripheral blood (PB) and BM samples from 45 AL patients was dynamically detected by RT-qPCR. PB samples from 31 healthy donors were tested as normal controls.

Results: No mutation was sequenced in hKLF3 coding exons. hKLF3 expression in PB of AL was significantly lower than that in healthy donors [0.30 (0.02-1.07) vs 1.18 (0.62-3.37), P < .0001]. Primary acute myeloid leukemia (AML) exhibited the least expression values compared with secondary AML and acute lymphoblastic leukemia. Receiver operating characteristic (ROC) analyses suggested that hKLF3 expression in PB was a good marker for AML diagnosis with an AUC of 0.99 (95% CI 0.98-1.00) and an optimum cutoff value of 0.67 (sensitivity 93.94% and specificity 93.55%). hKLF3 expression was upregulated significantly when AML patients acquired morphological complete remission (CR), and the level of hKLF3 seemed to be higher in patients with deeper CR than in patients with minimal residual disease (MRD). Paired PB and BM samples showed highly consistent alteration in hKLF3 expression (r = .6533, P = .001). Besides, a significantly converse correlation between decreased hKLF3 expression in PB and markers for leukemic load was observed.

Conclusions: hKLF3 expression in PB may act as a potential marker for AL diagnosis and monitoring.
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http://dx.doi.org/10.1002/cam4.2911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7163096PMC
April 2020

FibroAtlas: A Database for the Exploration of Fibrotic Diseases and Their Genes.

Cardiol Res Pract 2019 30;2019:4237285. Epub 2019 Dec 30.

School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China.

Background: Fibrosis is a highly dynamic process caused by prolonged injury, deregulation of the normal processes of wound healing, and extensive deposition of extracellular matrix (ECM) proteins. During fibrosis process, multiple genes interact with environmental factors. Over recent decades, tons of fibrosis-related genes have been identified to shed light on the particular clinical manifestations of this complex process. However, the genetics information about fibrosis is dispersed in lots of extensive literature.

Methods: We extracted data from literature abstracts in PubMed by text mining, and manually curated the literature and identified the evidence sentences.

Results: We presented FibroAtlas, which included 1,439 well-annotated fibrosis-associated genes. FibroAtlas 1.0 is the first attempt to build a nonredundant and comprehensive catalog of fibrosis-related genes with supporting evidence derived from curated published literature and allows us to have an overview of human fibrosis-related genes.
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http://dx.doi.org/10.1155/2019/4237285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012261PMC
December 2019

Phosphoproteomics Enables Molecular Subtyping and Nomination of Kinase Candidates for Individual Patients of Diffuse-Type Gastric Cancer.

iScience 2019 Dec 6;22:44-57. Epub 2019 Nov 6.

State Key Laboratory of Proteomics, Joint Laboratory of Gastrointestinal Oncology, Beijing Proteome Research Center, National Center for Protein Sciences (The PHOENIX Center, Beijing), Beijing Institute of Lifeomics, Beijing 102206, China; State Key Laboratory of Genetic Engineering and Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Institute of Biomedical Sciences, Fudan University, Shanghai 200433, China. Electronic address:

The diffuse-type gastric cancer (DGC) constitutes a subgroup of gastric cancer with poor prognosis and no effective molecular therapies. Here, we report a phosphoproteomic landscape of DGC derived from 83 tumors together with their nearby tissues. Based on phosphorylation, DGC could be classified into three molecular subtypes with distinct overall survival (OS) and chemosensitivity. We identified 16 kinases whose activities were associated with poor OS. These activated kinases covered several cancer hallmark pathways, with the MTOR signaling network being the most frequently activated. We proposed a patient-specific strategy based on the hierarchy of clinically actionable kinases for prioritization of kinases for further clinical evaluation. Our global data analysis indicates that in addition to finding activated kinase pathways in DGC, large-scale phosphoproteomics could be used to classify DGCs into subtypes that are associated with distinct clinical outcomes as well as nomination of kinase targets that may be inhibited for cancer treatments.
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http://dx.doi.org/10.1016/j.isci.2019.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6931223PMC
December 2019

Open-pFind Enhances the Identification of Missing Proteins from Human Testis Tissue.

J Proteome Res 2019 12 7;18(12):4189-4196. Epub 2019 Nov 7.

State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing) , Beijing Institute of Lifeomics , Beijing 102206 , China.

In recent years, high-throughput technologies have contributed to the development of a more precise picture of the human proteome. However, 2129 proteins remain listed as missing proteins (MPs) in the newest neXtProt release (2019-02). The main reasons for MPs are a low abundance, a low molecular weight, unexpected modifications, membrane characteristics, and so on. Moreover, >50% of the MS/MS data have not been successfully identified in shotgun proteomics. Open-pFind, an efficient open search engine, recently released by the pFind group in China, might provide an opportunity to identify these buried MPs in complex samples. In this study, proteins and potential MPs were identified using Open-pFind and three other search engines to compare their performance and efficiency with three large-scale data sets digested by three enzymes (Glu-C, Lys-C, and trypsin) with specificity on different amino acid (AA) residues. Our results demonstrated that Open-pFind identified 44.7-93.1% more peptide-spectrum matches and 21.3-61.6% more peptide sequences than the second-best search engine. As a result, Open-pFind detected 53.1% more MP candidates than MaxQuant and 8.8% more candidate MPs than Proteome Discoverer. In total, 5 (PE2) of the 124 MP candidates identified by Open-pFind were verified with 2 or 3 unique peptides containing more than 9 AAs by using a spectrum theoretical prediction with pDeep and synthesized peptide matching with pBuild after spectrum quality analysis, isobaric post-translational modification, and single amino acid variant filtering. These five verified MPs can be saved as PE1 proteins. In addition, three other MP candidates were verified with two unique peptides (one peptide containing more than 9 AAs and the other containing only 8 AAs), which was slightly lower than the criteria listed by C-HPP and required additional verification information. More importantly, unexpected modifications were detected in these MPs. All MS data sets have been deposited into ProteomeXchange with the identifier PXD015759.
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http://dx.doi.org/10.1021/acs.jproteome.9b00376DOI Listing
December 2019

The Role of Kupffer Cells as Mediators of Adipose Tissue Lipolysis.

J Immunol 2019 11 4;203(10):2689-2700. Epub 2019 Oct 4.

State Key Laboratory of Proteomics, National Center for Protein Sciences, Beijing, Beijing Proteome Research Center, Beijing Institute of Lifeomics, 102206 Beijing, China

Kupffer cells (KCs) are the resident macrophages of the liver, and they respond to and counteract metabolic stresses, such as those imposed by high-fat diet feeding in mouse models. However, little is known regarding the role of these cells in maintaining metabolic homeostasis under metabolically normal conditions. In this study, we found that depletion of KCs in vivo led to enhanced lipolysis in adipose tissue by increasing the expression of FGF21, a metabolic regulator, in hepatocytes. IL-1β secreted from KCs contributed to the suppression of FGF21 expression in hepatocytes. FGF21 overexpression led to a lean phenotype and enhanced lipolysis in mice. KC depletion resulted in a lack of IL-1β signaling in the liver, leading to elevated expression of FGF21 in hepatocytes. FGF21 promoted lipolysis in adipose tissue and led to hyperlipidemia and decreased body weight. The secretion of IL-1β in KCs was mediated by bacterial products. Antibiotic treatment also led to enhanced lipolysis. Therefore, the current study identified a physiological role of KCs in the regulation of adipose lipolysis.
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http://dx.doi.org/10.4049/jimmunol.1900366DOI Listing
November 2019

[Mouse liver proteome database].

Sheng Wu Gong Cheng Xue Bao 2019 Sep;35(9):1715-1722

National Center for Protein Sciences·Beijing, Beijing 102206, China.

The liver is the metabolic center of mammalian body. Systematic study on liver's proteome expression under different physiological and pathological conditions helps us understand the functional mechanisms of the liver. With the rapid development of liquid chromatography tandem mass spectrometry technique, numerous studies on liver physiology and pathology features produced a large number of proteomics data. In this paper, 834 proteomics experiments of mouse liver were systematically collected and the mouse liver proteome database (Mouse Liver Portal, http://mouseliver.com) was established. The Mouse Liver Portal contains the liver's proteomics data under different physiology and pathology conditions, such as different gender, age, circadian rhythm, cell type and different phase of partial hepatectomy, non-alcoholic fatty liver. This portal provides the changes in proteins' expression in different conditions of the liver, differently expressed proteins and the biological processes which they are involved in, potential signal transduction and regulatory networks. As the most comprehensive mouse liver proteome database, it can provide important resources and clues for liver biology research.
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http://dx.doi.org/10.13345/j.cjb.190166DOI Listing
September 2019

Proteomics Links Ubiquitin Chain Topology Change to Transcription Factor Activation.

Mol Cell 2019 10 20;76(1):126-137.e7. Epub 2019 Aug 20.

State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Research Unit of Proteomics & Research and Development of New Drug of Chinese Academy of Medical Sciences, Institute of Lifeomics, Beijing 102206, P.R. China; Key Laboratory of Combinatorial Biosynthesis and Drug Discovery of Ministry of Education, School of Pharmaceutical Sciences, School of Medicine, Wuhan University, Wuhan 430072, P.R. China; Guizhou University School of Medicine, Guiyang 550025, P.R. China. Electronic address:

A surprising complexity of ubiquitin signaling has emerged with identification of different ubiquitin chain topologies. However, mechanisms of how the diverse ubiquitin codes control biological processes remain poorly understood. Here, we use quantitative whole-proteome mass spectrometry to identify yeast proteins that are regulated by lysine 11 (K11)-linked ubiquitin chains. The entire Met4 pathway, which links cell proliferation with sulfur amino acid metabolism, was significantly affected by K11 chains and selected for mechanistic studies. Previously, we demonstrated that a K48-linked ubiquitin chain represses the transcription factor Met4. Here, we show that efficient Met4 activation requires a K11-linked topology. Mechanistically, our results propose that the K48 chain binds to a topology-selective tandem ubiquitin binding region in Met4 and competes with binding of the basal transcription machinery to the same region. The change to K11-enriched chain architecture releases this competition and permits binding of the basal transcription complex to activate transcription.
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http://dx.doi.org/10.1016/j.molcel.2019.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7751889PMC
October 2019

Trans-acting non-synonymous variant of FOXA1 predisposes to hepatocellular carcinoma through modulating FOXA1-ERα transcriptional program and may have undergone natural selection.

Carcinogenesis 2020 04;41(2):146-158

State Key Laboratory of Genetic Engineering and Collaborative Innovation Center for Genetics and Development, School of Life Sciences; Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China.

Interplay of pioneer transcription factor forkhead box A1 (FOXA1) and estrogen receptor has been implicated in sexual dimorphism in hepatocellular carcinoma (HCC), but etiological relevance of its polymorphism was unknown. In the case control study (1152 patients versus1242 controls), we observed significant increase in HCC susceptibility in hepatitis B virus carriers associated with a non-synonymous Thr83Ala variant of FOXA1 (odds ratio [OR], 1.28; 95% confidence interval [CI], 1.11-1.48, for Ala83-containing genotype, after validation in an independent population with 933 patients versus 1030 controls), a tightly linked (CGC)5/6or7 repeat polymorphism at its promoter (OR 1.32; 95% CI 1.10-1.60, for (CGC)6or7-repeat-containing genotype), and their combined haplotype (OR 1.50; 95% CI 1.24-1.81, for (CGC)6or7-Ala83 haplotype). The susceptible FOXA1-Ala83 impairs its interaction with ERα, attenuates transactivation toward some of their dual target genes, such as type 1 iodothyronine deiodinase, UDP glucuronosyltransferase 2 family, polypeptide B17 and sodium/taurocholate cotransporting polypeptide, but correlates with strengthened cellular expression of α-fetoprotein (AFP) and elevated AFP serum concentration in HCC patients (n = 1096). The susceptible FOXA1 cis-variant with (CGC)6or7 repeat strengthens the binding to transcription factor early growth response 1 and enhances promoter activity and gene expression. Evolutionary population genetics analyses with public datasets reveal significant population differentiation and unique haplotype structure of the derived protective FOXA1-Thr83 and suggest that it may have undergone positive natural selection in Chinese population. These findings epidemiologically highlight the functional significance of FOXA1-ERα transcriptional program and regulatory network in liver cancer development.
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http://dx.doi.org/10.1093/carcin/bgz136DOI Listing
April 2020

Long Noncoding RNA p53-Stabilizing and Activating RNA Promotes p53 Signaling by Inhibiting Heterogeneous Nuclear Ribonucleoprotein K deSUMOylation and Suppresses Hepatocellular Carcinoma.

Hepatology 2020 01 12;71(1):112-129. Epub 2019 Aug 12.

State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, China.

To identify hepatocellular carcinoma (HCC)-implicated long noncoding RNAs (lncRNAs), we performed an integrative omics analysis by integrating mRNA and lncRNA expression profiles in HCC tissues. We identified a collection of candidate HCC-implicated lncRNAs. Among them, we demonstrated that an lncRNA, which is named as p53-stabilizing and activating RNA (PSTAR), inhibits HCC cell proliferation and tumorigenicity through inducing p53-mediated cell cycle arrest. We further revealed that PSTAR can bind to heterogeneous nuclear ribonucleoprotein K (hnRNP K) and enhance its SUMOylation and thereby strengthen the interaction between hnRNP K and p53, which ultimately leads to the accumulation and transactivation of p53. PSTAR is down-regulated in HCC tissues, and the low PSTAR expression predicts poor prognosis in patients with HCC, especially those with wild-type p53. Conclusion: This study sheds light on the tumor suppressor role of lncRNA PSTAR, a modulator of the p53 pathway, in HCC.
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http://dx.doi.org/10.1002/hep.30793DOI Listing
January 2020

OsteoporosAtlas: a human osteoporosis-related gene database.

PeerJ 2019 26;7:e6778. Epub 2019 Apr 26.

State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Lifeomics, Beijing, China.

Background: Osteoporosis is a common, complex disease of bone with a strong heritable component, characterized by low bone mineral density, microarchitectural deterioration of bone tissue and an increased risk of fracture. Due to limited drug selection for osteoporosis and increasing morbidity, mortality of osteoporotic fractures, osteoporosis has become a major health burden in aging societies. Current researches for identifying specific loci or genes involved in osteoporosis contribute to a greater understanding of the pathogenesis of osteoporosis and the development of better diagnosis, prevention and treatment strategies. However, little is known about how most causal genes work and interact to influence osteoporosis. Therefore, it is greatly significant to collect and analyze the studies involved in osteoporosis-related genes. Unfortunately, the information about all these osteoporosis-related genes is scattered in a large amount of extensive literature. Currently, there is no specialized database for easily accessing relevant information about osteoporosis-related genes and miRNAs.

Methods: We extracted data from literature abstracts in PubMed by text-mining and manual curation. Moreover, a local MySQL database containing all the data was developed with PHP on a Windows server.

Results: OsteoporosAtlas (http://biokb.ncpsb.org/osteoporosis/), the first specialized database for easily accessing relevant information such as osteoporosis-related genes and miRNAs, was constructed and served for researchers. OsteoporosAtlas enables users to retrieve, browse and download osteoporosis-related genes and miRNAs. Gene ontology and pathway analyses were integrated into OsteoporosAtlas. It currently includes 617 human encoding genes, 131 human non-coding miRNAs, and 128 functional roles. We think that OsteoporosAtlas will be an important bioinformatics resource to facilitate a better understanding of the pathogenesis of osteoporosis and developing better diagnosis, prevention and treatment strategies.
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http://dx.doi.org/10.7717/peerj.6778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6487800PMC
April 2019

UVGD 1.0: a gene-centric database bridging ultraviolet radiation and molecular biology effects in organisms.

Int J Radiat Biol 2019 08 13;95(8):1172-1177. Epub 2019 May 13.

a State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences-Beijing (PHOENIX Center), Beijing Institute of Lifeomics , Beijing , China.

Exposing to ultraviolet for a certain time will trigger some significant molecular biology effects in an organism. In the past few decades, varied ultraviolet-associated biological effects as well as their related genes, have been discovered under biologists' efforts. However, information about ultraviolet-related genes is dispersed in thousands of scientific papers, and there is still no study emphasizing on the systematic collection of ultraviolet-related genes. We collected ultraviolet-related genes and built this gene-centric database UVGD based on literature mining and manual curation. Literature mining was based on the ultraviolet-related abstracts downloaded from PubMed, and we obtained sentences in which ultraviolet keywords and genes co-occur at single-sentence level by using bio-entity recognizer. After that, manual curation was implemented in order to identify whether the genes are related to ultraviolet or not. We built the ultraviolet-related knowledge base UVGD 1.0 (URL: http://biokb.ncpsb.org/UVGD/ ), which contains 663 ultraviolet-related genes, together with 17 associated biological processes, 117 associated phenotypes, and 2628 MeSH terms. UVGD is helpful to understand the ultraviolet-related biological processes in organisms and we believe it would be useful for biologists to study the responding mechanisms to ultraviolet.
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http://dx.doi.org/10.1080/09553002.2019.1609127DOI Listing
August 2019

Transketolase (TKT) activity and nuclear localization promote hepatocellular carcinoma in a metabolic and a non-metabolic manner.

J Exp Clin Cancer Res 2019 Apr 11;38(1):154. Epub 2019 Apr 11.

Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China.

Background: Metabolic reprogramming is one of the hallmarks of cancer cells. The pentose phosphate pathway (PPP), a branch of glycolysis, is an important metabolic pathway for the survival and biosynthesis of cancer cells. Transketolase (TKT) is a key enzyme in the non-oxidative phase of PPP. The mechanistic details of TKT in hepatocellular carcinoma (HCC) development remain unclear.

Methods: TKT level and subcellular location were examined in HCC cell lines and tissue samples. We established the TKT overexpression and knocking-down stable cells in HCC cell lines. Proliferation, migration, viability and enzyme activity assays in vitro, tumor growth and metastasis assays in vivo were employed to test the effects of TKT on HCC development. GFP-tagged TKT truncations and mutants were used to locate the nuclear localization sequence (NLSs) of TKT. Cross-linking co-IP/MS was applied to identify the interaction proteins of nuclear TKT.

Results: We showed that TKT increased the proliferation and migration of HCC cells, as well as the viability under oxidative stress in vitro and accelerated the growth and metastasis of HCC cells in vivo. We found as a key enzyme of PPP, TKT could promote the proliferation, cell cycle, migration and viability by regulating the metabolic flux. Moreover, it was firstly reported that unlike other key enzymes in PPP, TKT showed a strong nuclear localization in HCC cells. We found not only high TKT expression, but also its nuclear localization was a prediction for poor prognosis of HCC patients. We further identified the nuclear localization sequences (NLS) for TKT and demonstrated the NLS mutations decreased the pro-tumor function of TKT independent of the enzyme activity. Cross-linking Co-IP/MS showed that nuclear TKT interacted with kinases and transcriptional coregulators such as EGFR and MAPK3, which are associated with cell activation or stress response processes. EGF treatment significantly increased the viability and proliferation of HCC cells in the enzyme-inactivating mutation TKT-D155A overexpression cells but not in the NLS-D155A double mutant group, which could be blocked by EGFR inhibitor erlotinib treatment.

Conclusions: Our research suggests that in addition to the metabolic manner, TKT can promote the development of HCC in a non-metabolic manner via its nuclear localization and EGFR pathway.
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http://dx.doi.org/10.1186/s13046-019-1131-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6458711PMC
April 2019

The deubiquitylating enzyme USP15 regulates homologous recombination repair and cancer cell response to PARP inhibitors.

Nat Commun 2019 03 15;10(1):1224. Epub 2019 Mar 15.

State Key Laboratory of Proteomics, National Center for Protein Sciences (Beijing), Beijing Proteome Research Center, Beijing Institute of Lifeomics, Beijing, 102206, China.

Poly-(ADP-ribose) polymerase inhibitors (PARPi) selectively kill breast and ovarian cancers with defects in homologous recombination (HR) caused by BRCA1/2 mutations. There is also clinical evidence for the utility of PARPi in breast and ovarian cancers without BRCA mutations, but the underlying mechanism is not clear. Here, we report that the deubiquitylating enzyme USP15 affects cancer cell response to PARPi by regulating HR. Mechanistically, USP15 is recruited to DNA double-strand breaks (DSBs) by MDC1, which requires the FHA domain of MDC1 and phosphorylated Ser678 of USP15. Subsequently, USP15 deubiquitinates BARD1 BRCT domain, and promotes BARD1-HP1γ interaction, resulting in BRCA1/BARD1 retention at DSBs. USP15 knockout mice exhibit genomic instability in vivo. Furthermore, cancer-associated USP15 mutations, with decreased USP15-BARD1 interaction, increases PARP inhibitor sensitivity in cancer cells. Thus, our results identify a novel regulator of HR, which is a potential biomarker for therapeutic treatment using PARP inhibitors in cancers.
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http://dx.doi.org/10.1038/s41467-019-09232-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6420636PMC
March 2019