Publications by authors named "Dae-Kyum Kim"

39 Publications

Alternative glycosylation controls endoplasmic reticulum dynamics and tubular extension in mammalian cells.

Sci Adv 2021 May 7;7(19). Epub 2021 May 7.

Department of Biomolecular Medicine and Center for Medical Genetics, Ghent University, Ghent, Belgium.

The endoplasmic reticulum (ER) is a central eukaryotic organelle with a tubular network made of hairpin proteins linked by hydrolysis of guanosine triphosphate nucleotides. Among posttranslational modifications initiated at the ER level, glycosylation is the most common reaction. However, our understanding of the impact of glycosylation on the ER structure remains unclear. Here, we show that exostosin-1 (EXT1) glycosyltransferase, an enzyme involved in -glycosylation, is a key regulator of ER morphology and dynamics. We have integrated multiomics and superresolution imaging to characterize the broad effect of inactivation, including the ER shape-dynamics-function relationships in mammalian cells. We have observed that inactivating induces cell enlargement and enhances metabolic switches such as protein secretion. In particular, suppressing in mouse thymocytes causes developmental dysfunctions associated with the ER network extension. Last, our data illuminate the physical and functional aspects of the ER proteome-glycome-lipidome structure axis, with implications in biotechnology and medicine.
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http://dx.doi.org/10.1126/sciadv.abe8349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8104865PMC
May 2021

SARS-CoV-2 Nonstructural Proteins 1 and 13 Suppress Caspase-1 and the NLRP3 Inflammasome Activation.

Microorganisms 2021 Feb 26;9(3). Epub 2021 Feb 26.

Department of Life Science, Gachon University, Seongnam-Si, Gyeonggi-do 13120, Korea.

Viral infection-induced activation of inflammasome complexes has both positive and negative effects on the host. Proper activation of inflammasome complexes induces down-stream effector mechanisms that inhibit viral replication and promote viral clearance, whereas dysregulated activation has detrimental effects on the host. Coronaviruses, including SARS-CoV and MERS-CoV, encode viroporins that activate the NLRP3 inflammasome, and the severity of coronavirus disease is associated with the inflammasome activation. Although the NLRP3 inflammasome activation is implicated in the pathogenesis of coronaviruses, these viruses must evade inflammasome-mediated antiviral immune responses to establish primary replication. Screening of a complementary DNA (cDNA) library encoding 28 SARS-CoV-2 open reading frames (ORFs) showed that two nonstructural proteins (NSPs), NSP1 and NSP13, inhibited caspase-1-mediated IL-1β activation. NSP1 amino acid residues involved in host translation shutoff and NSP13 domains responsible for helicase activity were associated with caspase-1 inhibition. In THP-1 cells, both NSP1 and NSP13 significantly reduced NLRP3-inflammasome-induced caspase-1 activity and IL-1β secretion. These findings indicate that SARS-CoV-2 NSP1 and NSP13 are potent antagonists of the NLRP3 inflammasome.
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http://dx.doi.org/10.3390/microorganisms9030494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996899PMC
February 2021

Transcriptomic Bioinformatic Analyses of Atria Uncover Involvement of Pathways Related to Strain and Post-translational Modification of Collagen in Increased Atrial Fibrillation Vulnerability in Intensely Exercised Mice.

Front Physiol 2020 23;11:605671. Epub 2020 Dec 23.

Department of Biology, York University, Toronto, ON, Canada.

Atrial Fibrillation (AF) is the most common supraventricular tachyarrhythmia that is typically associated with cardiovascular disease (CVD) and poor cardiovascular health. Paradoxically, endurance athletes are also at risk for AF. While it is well-established that persistent AF is associated with atrial fibrosis, hypertrophy and inflammation, intensely exercised mice showed similar adverse atrial changes and increased AF vulnerability, which required tumor necrosis factor (TNF) signaling, even though ventricular structure and function improved. To identify some of the molecular factors underlying the chamber-specific and TNF-dependent atrial changes induced by exercise, we performed transcriptome analyses of hearts from wild-type and TNF-knockout mice following exercise for 2 days, 2 or 6 weeks of exercise. Consistent with the central role of atrial stretch arising from elevated venous pressure in AF promotion, all 3 time points were associated with differential regulation of genes in atria linked to mechanosensing (focal adhesion kinase, integrins and cell-cell communications), extracellular matrix (ECM) and TNF pathways, with TNF appearing to play a permissive, rather than causal, role in gene changes. Importantly, mechanosensing/ECM genes were only enriched, along with tubulin- and hypertrophy-related genes after 2 days of exercise while being downregulated at 2 and 6 weeks, suggesting that early reactive strain-dependent remodeling with exercise yields to compensatory adjustments. Moreover, at the later time points, there was also downregulation of both collagen genes and genes involved in collagen turnover, a pattern mirroring aging-related fibrosis. By comparison, twofold fewer genes were differentially regulated in ventricles vs. atria, independently of TNF. Our findings reveal that exercise promotes TNF-dependent atrial transcriptome remodeling of ECM/mechanosensing pathways, consistent with increased preload and atrial stretch seen with exercise. We propose that similar preload-dependent mechanisms are responsible for atrial changes and AF in both CVD patients and athletes.
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http://dx.doi.org/10.3389/fphys.2020.605671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793719PMC
December 2020

Sequential 1,3-- to - and 1,3-- to -Migration of Sulfonyl Groups through the Synthesis of 1,4-Diazepines from the Aza-[5 + 2] Cycloaddition of Indoloazomethine Ylides.

Org Lett 2020 08 4;22(16):6562-6567. Epub 2020 Aug 4.

The Korean Academy of Science and Technology, Seongnam 13630, Republic of Korea.

Described herein is the sequential 1,3-- to - and 1,3-- to -migration of sulfonyl groups through the synthesis of 1,4-diazepines from an operationally simple thermal aza-[5 + 2] cycloaddition reaction of indoloazomethine ylides with dialkyl acetylenedicarboxylates under mild conditions, leading to the formation of -sulfonylated 1,4-diazepines.
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http://dx.doi.org/10.1021/acs.orglett.0c02333DOI Listing
August 2020

A Comprehensive, Flexible Collection of SARS-CoV-2 Coding Regions.

G3 (Bethesda) 2020 09 2;10(9):3399-3402. Epub 2020 Sep 2.

Donnelly Centre, University of Toronto, Toronto, Ontario, Canada

The world is facing a global pandemic of COVID-19 caused by the SARS-CoV-2 coronavirus. Here we describe a collection of codon-optimized coding sequences for SARS-CoV-2 cloned into Gateway-compatible entry vectors, which enable rapid transfer into a variety of expression and tagging vectors. The collection is freely available. We hope that widespread availability of this SARS-CoV-2 resource will enable many subsequent molecular studies to better understand the viral life cycle and how to block it.
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http://dx.doi.org/10.1534/g3.120.401554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7467003PMC
September 2020

Quantitative proteomic analysis of trypsin-treated extracellular vesicles to identify the real-vesicular proteins.

J Extracell Vesicles 2020 30;9(1):1757209. Epub 2020 Apr 30.

Department of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea.

Extracellular vesicles (EVs) are nano-sized vesicles surrounded by a lipid bilayer and released into the extracellular milieu by most of cells. Although various EV isolation methods have been established, most of the current methods isolate EVs with contaminated non-vesicular proteins. By applying the label-free quantitative proteomic analyses of human colon cancer cell SW480-derived EVs, we identified trypsin-sensitive and trypsin-resistant vesicular proteins. Further systems biology and protein-protein interaction network analyses based on their cellular localization, we classified the trypsin-sensitive and trypsin-resistant vesicular proteins into two subgroups: 363 candidate real-vesicular proteins and 151 contaminated non-vesicular proteins. Moreover, the protein interaction network analyses showed that candidate real-vesicular proteins are mainly derived from plasma membrane (46.8%), cytosol (36.6%), cytoskeleton (8.0%) and extracellular region (2.5%). On the other hand, most of the contaminated non-vesicular proteins derived from nucleus, Golgi apparatus, endoplasmic reticulum and mitochondria. In addition, ribosomal protein complexes and T-complex proteins were classified as the contaminated non-vesicular proteins. Taken together, our trypsin-digested proteomic approach on EVs is an important advance to identify the real-vesicular proteins that could help to understand EV biogenesis and protein cargo-sorting mechanism during EV release, to identify more reliable EV diagnostic marker proteins, and to decode pathophysiological roles of EVs.
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http://dx.doi.org/10.1080/20013078.2020.1757209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7241501PMC
April 2020

A reference map of the human binary protein interactome.

Nature 2020 04 8;580(7803):402-408. Epub 2020 Apr 8.

Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston, MA, USA.

Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype-phenotype relationships. Here we present a human 'all-by-all' reference interactome map of human binary protein interactions, or 'HuRI'. With approximately 53,000 protein-protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome, transcriptome and proteome data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein-protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes.
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http://dx.doi.org/10.1038/s41586-020-2188-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7169983PMC
April 2020

Quantifying immune-based counterselection of somatic mutations.

PLoS Genet 2019 07 25;15(7):e1008227. Epub 2019 Jul 25.

Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

Somatic mutations in protein-coding regions can generate 'neoantigens' causing developing cancers to be eliminated by the immune system. Quantitative estimates of the strength of this counterselection phenomenon have been lacking. We quantified the extent to which somatic mutations are depleted in peptides that are predicted to be displayed by major histocompatibility complex (MHC) class I proteins. The extent of this depletion depended on expression level of the neoantigenic gene, and on whether the patient had one or two MHC-encoding alleles that can display the peptide, suggesting MHC-encoding alleles are incompletely dominant. This study provides an initial quantitative understanding of counter-selection of identifiable subclasses of neoantigenic somatic variation.
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http://dx.doi.org/10.1371/journal.pgen.1008227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6657826PMC
July 2019

Network-based prediction of protein interactions.

Nat Commun 2019 03 18;10(1):1240. Epub 2019 Mar 18.

Network Science Institute and Department of Physics, Northeastern University, Boston, MA, 02115, USA.

Despite exceptional experimental efforts to map out the human interactome, the continued data incompleteness limits our ability to understand the molecular roots of human disease. Computational tools offer a promising alternative, helping identify biologically significant, yet unmapped protein-protein interactions (PPIs). While link prediction methods connect proteins on the basis of biological or network-based similarity, interacting proteins are not necessarily similar and similar proteins do not necessarily interact. Here, we offer structural and evolutionary evidence that proteins interact not if they are similar to each other, but if one of them is similar to the other's partners. This approach, that mathematically relies on network paths of length three (L3), significantly outperforms all existing link prediction methods. Given its high accuracy, we show that L3 can offer mechanistic insights into disease mechanisms and can complement future experimental efforts to complete the human interactome.
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http://dx.doi.org/10.1038/s41467-019-09177-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6423278PMC
March 2019

The Impact of Oncogenic EGFRvIII on the Proteome of Extracellular Vesicles Released from Glioblastoma Cells.

Mol Cell Proteomics 2018 10 13;17(10):1948-1964. Epub 2018 Jul 13.

From the ‡Research Institute of the McGill University Health Centre, Glen Site, McGill University, Montreal, Quebec, H4A 3J1, Canada;

Glioblastoma multiforme (GBM) is a highly aggressive and heterogeneous form of primary brain tumors, driven by a complex repertoire of oncogenic alterations, including the constitutively active epidermal growth factor receptor (EGFRvIII). EGFRvIII impacts both cell-intrinsic and non-cell autonomous aspects of GBM progression, including cell invasion, angiogenesis and modulation of the tumor microenvironment. This is, at least in part, attributable to the release and intercellular trafficking of extracellular vesicles (EVs), heterogeneous membrane structures containing multiple bioactive macromolecules. Here we analyzed the impact of EGFRvIII on the profile of glioma EVs using isogenic tumor cell lines, in which this oncogene exhibits a strong transforming activity. We observed that EGFRvIII expression alters the expression of EV-regulating genes (vesiculome) and EV properties, including their protein composition. Using mass spectrometry, quantitative proteomic analysis and Gene Ontology terms filters, we observed that EVs released by EGFRvIII-transformed cells were enriched for extracellular exosome and focal adhesion related proteins. Among them, we validated the association of pro-invasive proteins (CD44, BSG, CD151) with EVs of EGFRvIII expressing glioma cells, and downregulation of exosomal markers (CD81 and CD82) relative to EVs of EGFRvIII-negative cells. Nano-flow cytometry revealed that the EV output from individual glioma cell lines was highly heterogeneous, such that only a fraction of vesicles contained specific proteins (including EGFRvIII). Notably, cells expressing EGFRvIII released EVs double positive for CD44/BSG, and these proteins also colocalized in cellular filopodia. We also detected the expression of homophilic adhesion molecules and increased homologous EV uptake by EGFRvIII-positive glioma cells. These results suggest that oncogenic EGFRvIII reprograms the proteome and uptake of GBM-related EVs, a notion with considerable implications for their biological activity and properties relevant for the development of EV-based cancer biomarkers.
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http://dx.doi.org/10.1074/mcp.RA118.000644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166673PMC
October 2018

Akkermansia muciniphila-derived extracellular vesicles influence gut permeability through the regulation of tight junctions.

Exp Mol Med 2018 02 23;50(2):e450. Epub 2018 Feb 23.

Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, Republic of Korea.

The gut microbiota has an important role in the gut barrier, inflammation and metabolic functions. Studies have identified a close association between the intestinal barrier and metabolic diseases, including obesity and type 2 diabetes (T2D). Recently, Akkermansia muciniphila has been reported as a beneficial bacterium that reduces gut barrier disruption and insulin resistance. Here we evaluated the role of A. muciniphila-derived extracellular vesicles (AmEVs) in the regulation of gut permeability. We found that there are more AmEVs in the fecal samples of healthy controls compared with those of patients with T2D. In addition, AmEV administration enhanced tight junction function, reduced body weight gain and improved glucose tolerance in high-fat diet (HFD)-induced diabetic mice. To test the direct effect of AmEVs on human epithelial cells, cultured Caco-2 cells were treated with these vesicles. AmEVs decreased the gut permeability of lipopolysaccharide-treated Caco-2 cells, whereas Escherichia coli-derived EVs had no significant effect. Interestingly, the expression of occludin was increased by AmEV treatment. Overall, these results imply that AmEVs may act as a functional moiety for controlling gut permeability and that the regulation of intestinal barrier integrity can improve metabolic functions in HFD-fed mice.
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http://dx.doi.org/10.1038/emm.2017.282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5903829PMC
February 2018

TNF-α and IFN-γ Together Up-Regulates Par-4 Expression and Induce Apoptosis in Human Neuroblastomas.

Biomedicines 2017 12 26;6(1). Epub 2017 Dec 26.

National Centre for Cell Science, Savitribai Phule Pune University, Ganeshkhind, Pune 411007, India.

The objective of this study was to examine the combined effect of Interferon-gamma (IFN-γ) and Tumor Necrosis factor-alpha (TNF-α) on cytotoxicity and expression of prostate apoptosis response-4 (Par-4) and Par-4 interacting proteins B-cell lymphoma (Bcl-2), nuclear factor kappa-light-chain-enhancer of activated B cells/p65 subunit (NF-κB/p65), Ak mouse strain thymoma (Akt) in human neuroblastoma (NB) cells. Materials and methods included human neuroblastoma cell lines-SK-N-MC, SK-N-SH, and SH-SY5Y, which were treated with IFN-γ and TNF-α individually, or in combination, and were assessed for viability by tetrazolium (MTT) assay. Apoptosis was monitored by hypodiploid population (by flow cytometry), DNA fragmentation, Poly (ADP-ribose) polymerase (PARP) cleavage, and caspase-8 activity. Transcript level of Par-4 was measured by RT-PCR. Protein levels of Par-4 and suppressor of cytokine signaling 3 (SOCS-3) were assessed by immunoblotting. Cellular localization of Par-4 and p65 was examined by immunofluorescence. Unbiased transcript analysis for IFN-γ, TNF-α, and Par-4 were analyzed from three independent clinical datasets from neuroblastoma patients. In terms of results, SK-N-MC cells treated with a combination of, but not individually with, IFN-γ and TNF-α induced apoptosis characterized by hypodiploidy, DNA fragmentation, PARP cleavage, and increased caspase-8 activity. Apoptosis was associated with up-regulation of Par-4 mRNA and protein expression. Immunofluorescence studies revealed that Par-4 was localized exclusively in cytoplasm in SK-N-MC cells cultured for 24 h. but showed nuclear localization at 48 h. Treatment with IFN-γ and TNF-α together enhanced the intensity of nuclear Par-4. In gene expression, data from human neuroblastoma patients, levels of IFN-γ, and TNF-α have strong synergy with Par-4 expression and provide good survival advantage. The findings also demonstrated that apoptosis was associated with reduced level of pro-survival proteins-Bcl-2 and Akt and NF-κB/p65. Furthermore, the apoptotic effect induced by IFN-γ-induced Signal Transducer and Activator of Transcription-1(STAT-1), and could be due to down-regulation of suppressor of cytokine signaling-3 (SOCS3). The study concludes that a combinatorial approach using IFN-γ and TNF-α can be explored to maximize the effect in chemotherapy in neuroblastoma, and implies a role for Par-4 in the process.
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http://dx.doi.org/10.3390/biomedicines6010004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874661PMC
December 2017

Comparative Interactomes of VRK1 and VRK3 with Their Distinct Roles in the Cell Cycle of Liver Cancer.

Mol Cells 2017 Sep 20;40(9):621-631. Epub 2017 Sep 20.

Department of Life Science, Pohang University of Science and Technology, Pohang 37673, Korea.

Vaccinia-related kinase 1 (VRK1) and VRK3 are members of the VRK family of serine/threonine kinases and are principally localized in the nucleus. Despite the crucial roles of VRK1/VRK3 in physiology and disease, the molecular and functional interactions of VRK1/VRK3 are poorly understood. Here, we identified over 200 unreported VRK1/VRK3-interacting candidate proteins by affinity purification and LC-MS/MS. The networks of VRK1 and VRK3 interactomes were found to be associated with important biological processes such as the cell cycle, DNA repair, chromatin assembly, and RNA processing. Interactions of interacting proteins with VRK1/VRK3 were confirmed by biochemical assays. We also found that phosphorylations of XRCC5 were regulated by both VRK1/VRK3, and that of CCNB1 was regulated by VRK3. In liver cancer cells and tissues, VRK1/VRK3 were highly upregulated and its depletion affected cell cycle progression in the different phases. VRK3 seemed to affect S phase progression and G2 or M phase entry and exit, whereas VRK1 affects G1/S transition in the liver cancer, which could be explained by different interacting candidate proteins. Thus, this study not only provides a resource for investigating the unidentified functions of VRK1/VRK3, but also an insight into the regulatory roles of VRK1/VRK3 in biological processes.
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http://dx.doi.org/10.14348/molcells.2017.0108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5638770PMC
September 2017

Interactomic analysis of REST/NRSF and implications of its functional links with the transcription suppressor TRIM28 during neuronal differentiation.

Sci Rep 2016 12 15;6:39049. Epub 2016 Dec 15.

Department of Life Sciences, Pohang University of Science and Technology, Pohang, Gyeongbuk, Korea.

RE-1 silencing transcription factor (REST) is a transcriptional repressor that regulates gene expression by binding to repressor element 1. However, despite its critical function in physiology, little is known about its interaction proteins. Here we identified 204 REST-interacting proteins using affinity purification and mass spectrometry. The interactome included proteins associated with mRNA processing/splicing, chromatin organization, and transcription. The interactions of these REST-interacting proteins, which included TRIM28, were confirmed by co-immunoprecipitation and immunocytochemistry, respectively. Gene Ontology (GO) analysis revealed that neuronal differentiation-related GO terms were enriched among target genes that were co-regulated by REST and TRIM28, while the level of CTNND2 was increased by the knockdown of REST and TRIM28. Consistently, the level of CTNND2 increased while those of REST and TRIM28 decreased during neuronal differentiation in the primary neurons, suggesting that CTNND2 expression may be co-regulated by both. Furthermore, neurite outgrowth was increased by depletion of REST or TRIM28, implying that reduction of both REST and TRIM28 could promote neuronal differentiation via induction of CTNND2 expression. In conclusion, our study of REST reveals novel interacting proteins which could be a valuable resource for investigating unidentified functions of REST and also suggested functional links between REST and TRIM28 during neuronal development.
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http://dx.doi.org/10.1038/srep39049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5157023PMC
December 2016

An Acrodermatitis Enteropathica-Associated Zn Transporter, ZIP4, Regulates Human Epidermal Homeostasis.

J Invest Dermatol 2017 04 7;137(4):874-883. Epub 2016 Dec 7.

Basic Research & Innovation Division, R&D Unit, AmorePacific Corporation, Yongin, Republic of Korea. Electronic address:

Acrodermatitis enteropathica is an autosomal recessive disorder characterized by scaly eczematous dermatosis accompanied by alopecia and diarrhea. Various mutations in the SLC39A4 gene (ZIP4), which encodes a zinc transporter, are responsible for this disorder. However, the molecular mechanism underlying the involvement of ZIP4 in the pathogenesis of this condition has yet to be established. In this study, we report the role of ZIP4 in human epidermis. ZIP4 is predominantly expressed in human keratinocytes, and its expression is dramatically reduced on epidermal differentiation. ZIP4 knockdown in human keratinocytes down-regulates zinc (Zn) levels and the transcriptional activity of a key epidermal Zn-binding protein, ΔNp63, and dysregulates epidermal differentiation in a reconstituted human skin model, resulting in the appearance of proliferating keratinocytes even in the uppermost layers of the skin. We verified that, among the amino acid residues in its Zn-binding motif, Cys205 is critical for the processing and nuclear distribution of ΔNp63 and, therefore, Zn-dependent transcriptional activity. Our results suggest that ZIP4 is essential for maintaining human epidermal homeostasis through the regulation of Zn-dependent ΔNp63 activity and can provide insight into the molecular mechanisms responsible for the cutaneous symptoms observed in Acrodermatitis enteropathica patients.
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http://dx.doi.org/10.1016/j.jid.2016.11.028DOI Listing
April 2017

SIRT6 Depletion Suppresses Tumor Growth by Promoting Cellular Senescence Induced by DNA Damage in HCC.

PLoS One 2016 8;11(11):e0165835. Epub 2016 Nov 8.

Department of Life Sciences, Pohang University of Science and Technology, Pohang, Gyeongbuk, Korea.

The role of Sirtuin 6 (SIRT6) as a tumor suppressor or oncogene in liver cancer remains controversial. Thus, we identified the specific role of SIRT6 in the progression of hepatocellular carcinoma (HCC). SIRT6 expression was significantly higher in HCC cell lines and HCC tissues from 138 patients than in an immortalized hepatocyte cell line, THLE-2 and non-tumor tissues, respectively. SIRT6 knockdown by shRNA suppressed the growth of HCC cells and inhibited HCC tumor growth in vivo. In addition, SIRT6 silencing significantly prevented the growth of HCC cell lines by inducing cellular senescence in the p16/Rb- and p53/p21-pathway independent manners. Microarray analysis revealed that the expression of genes involved in nucleosome assembly was apparently altered in SIRT6-depleted Hep3B cells. SIRT6 knockdown promoted G2/M phase arrest and downregulation of genes encoding histone variants associated with nucleosome assembly, which could be attributed to DNA damage. Taken together, our findings suggest that SIRT6 acts as a tumor promoter by preventing DNA damage and cellular senescence, indicating that SIRT6 represents a potential therapeutic target for the treatment of HCC.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0165835PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5100879PMC
June 2017

Two distinct extracellular RNA signatures released by a single cell type identified by microarray and next-generation sequencing.

RNA Biol 2017 01 28;14(1):58-72. Epub 2016 Oct 28.

a Krefting Research Center, Department of Internal Medicine and Clinical Nutrition , University of Gothenburg , Gothenburg , Sweden.

Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in many body fluids such as blood, breast milk and cerebrospinal fluid. However, there are conflicting results regarding the nature of exRNA. Here, we have separated 2 distinct exRNA profiles released by mast cells, here termed high-density (HD) and low-density (LD) exRNA. The exRNA in both fractions was characterized by microarray and next-generation sequencing. Both exRNA fractions contained mRNA and miRNA, and the mRNAs in the LD exRNA correlated closely with the cellular mRNA, whereas the HD mRNA did not. Furthermore, the HD exRNA was enriched in lincRNA, antisense RNA, vault RNA, snoRNA, and snRNA with little or no evidence of full-length 18S and 28S rRNA. The LD exRNA was enriched in mitochondrial rRNA, mitochondrial tRNA, tRNA, piRNA, Y RNA, and full-length 18S and 28S rRNA. The proteomes of the HD and LD exRNA-containing fractions were determined with LC-MS/MS and analyzed with Gene Ontology term finder, which showed that both proteomes were associated with the term extracellular vesicles and electron microscopy suggests that at least a part of the exRNA is associated with exosome-like extracellular vesicles. Additionally, the proteins in the HD fractions tended to be associated with the nucleus and ribosomes, whereas the LD fraction proteome tended to be associated with the mitochondrion. We show that the 2 exRNA signatures released by a single cell type can be separated by floatation on a density gradient. These results show that cells can release multiple types of exRNA with substantial differences in RNA species content. This is important for any future studies determining the nature and function of exRNA released from different cells under different conditions.
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http://dx.doi.org/10.1080/15476286.2016.1249092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5270547PMC
January 2017

Fibronectin-Containing Extracellular Vesicles Protect Melanocytes against Ultraviolet Radiation-Induced Cytotoxicity.

J Invest Dermatol 2016 05 15;136(5):957-966. Epub 2016 Feb 15.

Bioscience Research Division, R&D Unit, AmorePacific Corporation, Yongin, Republic of Korea. Electronic address:

Skin melanocytes are activated by exposure to UV radiation to secrete melanin-containing melanosomes to protect the skin from UV-induced damage. Despite the continuous renewal of the epidermis, the turnover rate of melanocytes is very slow, and they survive for long periods. However, the mechanisms underlying the survival of melanocytes exposed to UV radiation are not known. Here, we investigated the role of melanocyte-derived extracellular vesicles in melanocyte survival. Network analysis of the melanocyte extracellular vesicle proteome identified the extracellular matrix component fibronectin at a central node, and the release of fibronectin-containing extracellular vesicles was increased after exposure of melanocytes to UVB radiation. Using an anti-fibronectin neutralizing antibody and specific inhibitors of extracellular vesicle secretion, we demonstrated that extracellular vesicles enriched in fibronectin were involved in melanocyte survival after UVB radiation. Furthermore, we observed that in the hyperpigmented lesions of patients with melasma, the extracellular space around melanocytes contained more fibronectin compared with normal skin, suggesting that fibronectin is involved in maintaining melanocytes in pathological conditions. Collectively, our findings suggest that melanocytes secrete fibronectin-containing extracellular vesicles to increase their survival after UVB radiation. These data provide important insight into how constantly stimulated melanocytes can be maintained in pathological conditions such as melasma.
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http://dx.doi.org/10.1016/j.jid.2015.08.001DOI Listing
May 2016

In Vivo Differentiation of Therapeutic Insulin-Producing Cells from Bone Marrow Cells via Extracellular Vesicle-Mimetic Nanovesicles.

ACS Nano 2015 Dec 3;9(12):11718-27. Epub 2015 Nov 3.

Biomedical Research Institute, Seoul National University Hospital , Seoul 110-744, Korea.

The current diabetes mellitus pandemic constitutes an important global health problem. Reductions in the mass and function of β-cells contribute to most of the pathophysiology underlying diabetes. Thus, physiological control of blood glucose levels can be adequately restored by replacing functioning β-cell mass. Sources of functional islets for transplantation are limited, resulting in great interest in the development of alternate sources, and recent progress regarding cell fate change via utilization of extracellular vesicles, also known as exosomes and microvesicles, is notable. Thus, this study investigated the therapeutic capacity of extracellular vesicle-mimetic nanovesicles (NVs) derived from a murine pancreatic β-cell line. To differentiate insulin-producing cells effectively, a three-dimensional in vivo microenvironment was constructed in which extracellular vesicle-mimetic NVs were applied to subcutaneous Matrigel platforms containing bone marrow (BM) cells in diabetic immunocompromised mice. Long-term control of glucose levels was achieved over 60 days, and differentiation of donor BM cells into insulin-producing cells in the subcutaneous Matrigel platforms, which were composed of islet-like cell clusters with extensive capillary networks, was confirmed along with the expression of key pancreatic β-cell markers. The resectioning of the subcutaneous Matrigel platforms caused a rebound in blood glucose levels and confirmed the source of functioning β-cells. Thus, efficient differentiation of therapeutic insulin-producing cells was attained in vivo through the use of extracellular vesicle-mimetic NVs, which maintained physiological glucose levels.
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http://dx.doi.org/10.1021/acsnano.5b02997DOI Listing
December 2015

Gut microbe-derived extracellular vesicles induce insulin resistance, thereby impairing glucose metabolism in skeletal muscle.

Sci Rep 2015 Oct 29;5:15878. Epub 2015 Oct 29.

Department of Medicine, Ewha Womans University School of Medicine and Ewha Institute of Convergence Medicine, Seoul, Republic of Korea.

Gut microbes might influence host metabolic homeostasis and contribute to the pathogenesis of type 2 diabetes (T2D), which is characterized by insulin resistance. Bacteria-derived extracellular vesicles (EVs) have been suggested to be important in the pathogenesis of diseases once believed to be non-infectious. Here, we hypothesize that gut microbe-derived EVs are important in the pathogenesis of T2D. In vivo administration of stool EVs from high fat diet (HFD)-fed mice induced insulin resistance and glucose intolerance compared to regular diet (RD)-fed mice. Metagenomic profiling of stool EVs by 16S ribosomal DNA sequencing revealed an increased amount of EVs derived from Pseudomonas panacis (phylum Proteobacteria) in HFD mice compared to RD mice. Interestingly, P. panacis EVs blocked the insulin signaling pathway in both skeletal muscle and adipose tissue. Moreover, isolated P. panacis EVs induced typical diabetic phenotypes, such as glucose intolerance after glucose administration or systemic insulin injection. Thus, gut microbe-derived EVs might be key players in the development of insulin resistance and impairment of glucose metabolism promoted by HFD.
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http://dx.doi.org/10.1038/srep15878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4625370PMC
October 2015

Proteomic analysis of extracellular vesicles derived from Mycobacterium tuberculosis.

Proteomics 2015 Oct 19;15(19):3331-7. Epub 2015 Aug 19.

Department of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea.

The release of extracellular vesicles, also known as outer membrane vesicles, membrane vesicles, exosomes, and microvesicles, is an evolutionarily conserved phenomenon from bacteria to eukaryotes. It has been reported that Mycobacterium tuberculosis releases extracellular vesicles harboring immunologically active molecules, and these extracellular vesicles have been suggested to be applicable in vaccine development and biomarker discovery. However, the comprehensive proteomic analysis has not been performed for M. tuberculosis extracellular vesicles. In this study, we identified a total of 287 vesicular proteins by four LC-MS/MS analyses with high confidence. In addition, we identified several vesicular proteins associated with the virulence of M. tuberculosis. This comprehensive proteome profile will help elucidate the pathogenic mechanism of M. tuberculosis. The data have been deposited to the ProteomeXchange with identifier PXD001160 (http://proteomecentral.proteomexchange.org/dataset/PXD001160).
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http://dx.doi.org/10.1002/pmic.201500037DOI Listing
October 2015

Small RNA deep sequencing discriminates subsets of extracellular vesicles released by melanoma cells--Evidence of unique microRNA cargos.

RNA Biol 2015 ;12(8):810-23

a Krefting Research Center ; Institute of Medicine; University of Gothenburg ; Gothenburg , Sweden.

Melanoma cells release different types of extracellular vesicles (EVs) into the extracellular milieu that are involved with communication and signaling in the tumor microenvironment. Subsets of EVs include exosomes, microvesicles, and apoptotic bodies that carry protein and genetic (RNA) cargos. To define the contribution of the RNA cargo of melanoma cell derived EVs we performed small RNA sequencing to identify different small RNAs in the EV subsets. Using validated centrifugation protocols, we separated these EV subsets released by the melanoma cell line MML-1, and performed RNA sequencing with the Ion Torrent platform. Various, but different, non-coding RNAs were detected in the EV subsets, including microRNA, mitochondrial associated tRNA, small nucleolar RNA, small nuclear RNA, Ro associated Y-RNA, vault RNA and Y-RNA. We identified in total 1041 miRNAs in cells and EV subsets. Hierarchical clustering showed enrichment of specific miRNAs in exosomes, including hsa-miR-214-3p, hsa-miR-199a-3p and hsa-miR-155-5p, all being associated with melanoma progression. Comparison of exosomal miRNAs with miRNAs in clinical melanoma samples indicate that multiple miRNAs in exosomes also are expressed specifically in melanoma tissues, but not in benign naevi. This study shows for the first time the presence of distinct small RNAs in subsets of EVs released by melanoma cells, with significant similarities to clinical melanoma tissue, and provides unique insights into the contribution of EV associated extracellular RNA in cancer.
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http://dx.doi.org/10.1080/15476286.2015.1056975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4615768PMC
May 2016

Large oncosomes contain distinct protein cargo and represent a separate functional class of tumor-derived extracellular vesicles.

Oncotarget 2015 May;6(13):11327-41

Division of Cancer Biology and Therapeutics, Departments of Surgery, Biomedical Sciences and Pathology and Laboratory Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA.

Large oncosomes (LO) are atypically large (1-10 µm diameter) cancer-derived extracellular vesicles (EVs), originating from the shedding of membrane blebs and associated with advanced disease. We report that 25% of the proteins, identified by a quantitative proteomics analysis, are differentially represented in large and nano-sized EVs from prostate cancer cells. Proteins enriched in large EVs included enzymes involved in glucose, glutamine and amino acid metabolism, all metabolic processes relevant to cancer. Glutamine metabolism was altered in cancer cells exposed to large EVs, an effect that was not observed upon treatment with exosomes. Large EVs exhibited discrete buoyant densities in iodixanol (OptiPrep(TM)) gradients. Fluorescent microscopy of large EVs revealed an appearance consistent with LO morphology, indicating that these structures can be categorized as LO. Among the proteins enriched in LO, cytokeratin 18 (CK18) was one of the most abundant (within the top 5th percentile) and was used to develop an assay to detect LO in the circulation and tissues of mice and patients with prostate cancer. These observations indicate that LO represent a discrete EV type that may play a distinct role in tumor progression and that may be a source of cancer-specific markers.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4484459PMC
http://dx.doi.org/10.18632/oncotarget.3598DOI Listing
May 2015

EVpedia: A community web resource for prokaryotic and eukaryotic extracellular vesicles research.

Semin Cell Dev Biol 2015 Apr 19;40:4-7. Epub 2015 Feb 19.

Department of Life Sciences, Pohang University of Science and Technology, Pohang, Gyeongbuk 790-784, Republic of Korea. Electronic address:

For cell-to-cell communication, all living cells including archaea, bacteria, and eukaryotes secrete nano-sized membrane vesicles into the extracellular space. These extracellular vesicles harbor specific subsets of proteins, mRNAs, miRNAs, lipids, and metabolites that represent their cellular status. These vesicle-specific cargos are considered as novel diagnostic biomarkers as well as therapeutic targets. With the advancement in high-throughput technologies on multiomics studies and improvements in bioinformatics approaches, a huge number of vesicular proteins, mRNAs, miRNAs, lipids, and metabolites have been identified, and our understanding of these complex extracellular organelles has considerably increased during these past years. In this review, we highlight EVpedia (http://evpedia.info), a community web portal for systematic analyses of prokaryotic and eukaryotic extracellular vesicles research.
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http://dx.doi.org/10.1016/j.semcdb.2015.02.005DOI Listing
April 2015

Egr-1 activation by cancer-derived extracellular vesicles promotes endothelial cell migration via ERK1/2 and JNK signaling pathways.

PLoS One 2014 12;9(12):e115170. Epub 2014 Dec 12.

Department of Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea.

Various mammalian cells, including cancer cells, shed extracellular vesicles (EVs), also known as exosomes and microvesicles, into surrounding tissues. These EVs play roles in tumor growth and metastasis by promoting angiogenesis. However, the detailed mechanism of how cancer-derived EVs elicit endothelial cell activation remains unknown. Here, we provide evidence that early growth response-1 (Egr-1) activation in endothelial cells is involved in the angiogenic activity of colorectal cancer cell-derived EVs. Both RNA interference-mediated downregulation of Egr-1 and ERK1/2 or JNK inhibitor significantly blocked EV-mediated Egr-1 activation and endothelial cell migration. Furthermore, lipid raft-mediated endocytosis inhibitor effectively blocked endothelial Egr-1 activation and migration induced by cancer-derived EVs. Our results suggest that Egr-1 activation in endothelial cells may be a key mechanism involved in the angiogenic activity of cancer-derived EVs. These findings will improve our understanding regarding the proangiogenic activities of EVs in diverse pathological conditions including cancer, cardiovascular diseases, and neurodegenerative diseases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115170PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4264882PMC
March 2016

EVpedia: a community web portal for extracellular vesicles research.

Bioinformatics 2015 Mar 10;31(6):933-9. Epub 2014 Nov 10.

Department of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea, Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, Republic of Korea, Cardiovascular Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA, Department of Clinical Immunology, Polish-American Institute of Paediatrics, Jagiellonian University Medical College, Cracow, Poland, Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands, Section of Oncology, Department of Clinical Sciences, Lund University, Lund, Sweden, The Feinstein Institute for Medical Research, Manhasset, NY, USA, Department of Microbiology, Biochemistry, and Immunology, Morehouse School of Medicine, Atlanta, GA, USA, Institute of Biomedicine and Molecular Immunology (IBIM), National Research Council (CNR), Palermo, Italy, Innovation in Vesicles and Cells for Application in Therapy, Germans Trias i Pujol Research Institute, Germans Trias i Pujol University Hospital, Badalona, Spain, INSERM, UMR837 JEAN-PIERRE Aubert Research Centre, Lille, France, Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary, Department of Biochemistry and Molecular Biology, BIO21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC, Australia, Institute of Cancer & Genetics, School of Medicine, Velindre Cancer Centre, Cardiff University, Cardiff, UK, Program in Cellular and Molecular Medicine at Boston Children's Hospital and Department of Cell Biology, Harvard Medical School, Boston, MA, USA, Section of Pulmonary, Critical Care and Sleep Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA, Department of Neurology, College of Medicine, University of Tennessee Health Science Center, Memphis, TN, USA, Cancer Biology Program, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai M

Motivation: Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging.

Results: We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research.

Availability And Implementation: The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info.
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http://dx.doi.org/10.1093/bioinformatics/btu741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4375401PMC
March 2015

In vivo kinetic biodistribution of nano-sized outer membrane vesicles derived from bacteria.

Small 2015 Jan 5;11(4):456-61. Epub 2014 Sep 5.

Department of Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, Republic of Korea.

Evaluation of kinetic distribution and behaviors of nanoparticles in vivo provides crucial clues into their roles in living organisms. Extracellular vesicles are evolutionary conserved nanoparticles, known to play important biological functions in intercellular, inter-species, and inter-kingdom communication. In this study, the first kinetic analysis of the biodistribution of outer membrane vesicles (OMVs)-bacterial extracellular vesicles-with immune-modulatory functions is performed. OMVs, injected intraperitoneally, spread to the whole mouse body and accumulate in the liver, lung, spleen, and kidney within 3 h of administration. As an early systemic inflammation response, increased levels of TNF-α and IL-6 are observed in serum and bronchoalveolar lavage fluid. In addition, the number of leukocytes and platelets in the blood is decreased. OMVs and cytokine concentrations, as well as body temperature are gradually decreased 6 h after OMV injection, in concomitance with the formation of eye exudates, and of an increase in ICAM-1 levels in the lung. Following OMV elimination, most of the inflammatory signs are reverted, 12 h post-injection. However, leukocytes in bronchoalveolar lavage fluid are increased as a late reaction. Taken together, these results suggest that OMVs are effective mediators of long distance communication in vivo.
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http://dx.doi.org/10.1002/smll.201401803DOI Listing
January 2015

Comparative interactomes of SIRT6 and SIRT7: Implication of functional links to aging.

Proteomics 2014 Jul 28;14(13-14):1610-22. Epub 2014 May 28.

Department of Life Sciences, Pohang University of Science and Technology, Pohang, Gyeongbuk, Korea.

Sirtuins are NAD(+) -dependent deacetylases that regulate a range of cellular processes. Although diverse functions of sirtuins have been proposed, those functions of SIRT6 and SIRT7 that are mediated by their interacting proteins remain elusive. In the present study, we identified SIRT6- and SIRT7-interacting proteins, and compared their interactomes to investigate functional links. Our interactomes revealed 136 interacting proteins for SIRT6 and 233 for SIRT7 while confirming seven and 111 proteins identified previously for SIRT6 and SIRT7, respectively. Comparison of SIRT6 and SIRT7 interactomes under the same experimental conditions disclosed 111 shared proteins, implying related functional links. The interaction networks of interactomes indicated biological processes associated with DNA repair, chromatin assembly, and aging. Interactions of two highly acetylated proteins, nucleophosmin (NPM1) and nucleolin, with SIRT6 and SIRT7 were confirmed by co-immunoprecipitation. NPM1 was found to be deacetylated by both SIRT6 and SIRT7. In senescent cells, the acetylation level of NPM1 was increased in conjunction with decreased levels of SIRT6 and SIRT7, suggesting that the acetylation of NPM1 could be regulated by SIRT6 and SIRT7 in the aging process. Our comparative interactomic study of SIRT6 and SIRT7 implies important functional links to aging by their associations with interacting proteins. All MS data have been deposited in the ProteomeXchange with identifiers PXD000159 and PXD000850 (http://proteomecentral.proteomexchange.org/dataset/PXD000159, http://proteomecentral.proteomexchange.org/dataset/PXD000850).
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http://dx.doi.org/10.1002/pmic.201400001DOI Listing
July 2014

Proteomics of extracellular vesicles: Exosomes and ectosomes.

Mass Spectrom Rev 2015 Jul-Aug;34(4):474-90. Epub 2014 Jan 14.

Department of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea.

Almost all bacteria, archaea, and eukaryotic cells shed extracellular vesicles either constitutively or in a regulated manner. These nanosized membrane vesicles are spherical, bilayered proteolipids that harbor specific subsets of proteins, DNAs, RNAs, and lipids. Recent research has facilitated conceptual advancements in this emerging field that indicate that extracellular vesicles act as intercellular communicasomes by transferring signals to their target cell via surface ligands and delivering receptors and functional molecules. Recent progress in mass spectrometry-based proteomic analyses of mammalian extracellular vesicles derived from diverse cell types and body fluids has resulted in the identification of several thousand vesicular proteins that provide us with essential clues to the molecular mechanisms involved in vesicle cargo sorting and biogenesis. Furthermore, cell-type- or disease-specific vesicular proteins help us to understand the pathophysiological functions of extracellular vesicles and contribute to the discovery of diagnostic and therapeutic target proteins. This review focuses on the high-throughput mass spectrometry-based proteomic analyses of mammalian extracellular vesicles (i.e., exosomes and ectosomes), EVpedia (a free web-based integrated database of high-throughput data for systematic analyses of extracellular vesicles; http://evpedia.info), and the intravesicular protein-protein interaction network analyses of mammalian extracellular vesicles. The goal of this article is to encourage further studies to construct a comprehensive proteome database for extracellular vesicles that will help us to not only decode the biogenesis and cargo-sorting mechanisms during vesicle formation but also elucidate the pathophysiological roles of these complex extracellular organelles.
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http://dx.doi.org/10.1002/mas.21420DOI Listing
April 2016

Cdk5 phosphorylates dopamine D2 receptor and attenuates downstream signaling.

PLoS One 2013 31;8(12):e84482. Epub 2013 Dec 31.

Department of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea.

The dopamine D2 receptor (DRD2) is a key receptor that mediates dopamine-associated brain functions such as mood, reward, and emotion. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase whose function has been implicated in the brain reward circuit. In this study, we revealed that the serine 321 residue (S321) in the third intracellular loop of DRD2 (D2i3) is a novel regulatory site of Cdk5. Cdk5-dependent phosphorylation of S321 in the D2i3 was observed in in vitro and cell culture systems. We further observed that the phosphorylation of S321 impaired the agonist-stimulated surface expression of DRD2 and decreased G protein coupling to DRD2. Moreover, the downstream cAMP pathway was affected in the heterologous system and in primary neuronal cultures from p35 knockout embryos likely due to the reduced inhibitory activity of DRD2. These results indicate that Cdk5-mediated phosphorylation of S321 inhibits DRD2 function, providing a novel regulatory mechanism for dopamine signaling.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0084482PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3877277PMC
September 2014
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