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1.
Mol Pharm ; 20(1): 128-135, 2023 01 02.
Article in English | MEDLINE | ID: mdl-36352823

ABSTRACT

Asymmetric small interfering RNAs (asiRNAs) that mediate RNA interference have been investigated for therapeutic use in various tissues, including skin tissue. Androgenetic alopecia (AGA) is caused by a combination of genetic factors, resulting in sensitivity to dihydrotestosterone (DHT), which binds to the androgen receptor (AR) to mediate a series of biomolecular changes leading to hair loss. This study aimed to evaluate the therapeutic potential of a cell-penetrating, AR-targeting asiRNA (cp-asiAR) for AGA treatment, which was designed to silence the AR gene. AGA mouse models were developed by stimulation with DHT, and ex vivo human scalp tissues were also used for analysis. Cp-asiAR-mediated changes in mRNA expression and protein levels of AR were assessed along with the examination of phenotypic improvements in mouse model of AGA. We also assessed downstream signaling associated with AR in primary human dermal papilla (DP) cells. Several cp-asiARs were screened for selecting the optimal sequence of AR using cell lines in vitro. A cholesterol-conjugated, chemically modified cp-asiAR candidate was optimized under passive uptake conditions in vitro. Intradermal cp-asiAR injection efficiently reduced mRNA and protein levels corresponding to AR in mouse models. Moreover, cp-asiAR injection promoted hair growth in mouse models with DHT-induced AGA. In ex vivo human hair follicle culture, the proportion of telogen hair decreased, and the mean hair bulb diameter increased in the cp-asiAR-treated group. In isolated primary human DP cells, AR expression was effectively downregulated by cp-asiAR. Furthermore, cp-asiAR attenuated DHT-mediated increases in interleukin-6, transforming growth factor-ß1, and dickkopf-1 levels. No significant toxicity was observed in DP cells after cp-asiAR treatment. Cp-asiAR treatment showed effective downregulation of AR expression and prevention of DHT-mediated alterations in the hair cycle and hair diameter, indicating its potential as a novel therapeutic option for AGA.


Subject(s)
Alopecia , Receptors, Androgen , Mice , Animals , Humans , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , RNA, Small Interfering/metabolism , Alopecia/drug therapy , Alopecia/genetics , Hair/metabolism , Hair Follicle , Disease Models, Animal , RNA, Messenger/genetics , RNA, Messenger/metabolism
2.
Nat Commun ; 13(1): 2572, 2022 05 11.
Article in English | MEDLINE | ID: mdl-35546148

ABSTRACT

Recent development of the chemical inhibitors specific to oncogenic KRAS (Kirsten Rat Sarcoma 2 Viral Oncogene Homolog) mutants revives much interest to control KRAS-driven cancers. Here, we report that AIMP2-DX2, a variant of the tumor suppressor AIMP2 (aminoacyl-tRNA synthetase-interacting multi-functional protein 2), acts as a cancer-specific regulator of KRAS stability, augmenting KRAS-driven tumorigenesis. AIMP2-DX2 specifically binds to the hypervariable region and G-domain of KRAS in the cytosol prior to farnesylation. Then, AIMP2-DX2 competitively blocks the access of Smurf2 (SMAD Ubiquitination Regulatory Factor 2) to KRAS, thus preventing ubiquitin-mediated degradation. Moreover, AIMP2-DX2 levels are positively correlated with KRAS levels in colon and lung cancer cell lines and tissues. We also identified a small molecule that specifically bound to the KRAS-binding region of AIMP2-DX2 and inhibited the interaction between these two factors. Treatment with this compound reduces the cellular levels of KRAS, leading to the suppression of KRAS-dependent cancer cell growth in vitro and in vivo. These results suggest the interface of AIMP2-DX2 and KRAS as a route to control KRAS-driven cancers.


Subject(s)
Lung Neoplasms , Proto-Oncogene Proteins p21(ras) , Cell Transformation, Neoplastic , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Nuclear Proteins/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolism
3.
Nat Commun ; 13(1): 1169, 2022 03 04.
Article in English | MEDLINE | ID: mdl-35246514

ABSTRACT

Blood-brain barrier (BBB) integrity is critical for proper function of the central nervous system (CNS). Here, we show that the endothelial Unc5B receptor controls BBB integrity by maintaining Wnt/ß-catenin signaling. Inducible endothelial-specific deletion of Unc5B in adult mice leads to BBB leak from brain capillaries that convert to a barrier-incompetent state with reduced Claudin-5 and increased PLVAP expression. Loss of Unc5B decreases BBB Wnt/ß-catenin signaling, and ß-catenin overexpression rescues Unc5B mutant BBB defects. Mechanistically, the Unc5B ligand Netrin-1 enhances Unc5B interaction with the Wnt co-receptor LRP6, induces its phosphorylation and activates Wnt/ß-catenin downstream signaling. Intravenous delivery of antibodies blocking Netrin-1 binding to Unc5B causes a transient BBB breakdown and disruption of Wnt signaling, followed by neurovascular barrier resealing. These data identify Netrin-1-Unc5B signaling as a ligand-receptor pathway that regulates BBB integrity, with implications for CNS diseases.


Subject(s)
Blood-Brain Barrier , Netrin Receptors , Animals , Blood-Brain Barrier/metabolism , Endothelial Cells/metabolism , Ligands , Mice , Netrin Receptors/genetics , Netrin Receptors/metabolism , Netrin-1/genetics , Netrin-1/metabolism , Wnt Signaling Pathway , beta Catenin/metabolism
4.
Sci Transl Med ; 12(569)2020 11 11.
Article in English | MEDLINE | ID: mdl-33177178

ABSTRACT

Lewy bodies are pathological protein inclusions present in the brain of patients with Parkinson's disease (PD). These inclusions consist mainly of α-synuclein with associated proteins, such as parkin and its substrate aminoacyl transfer RNA synthetase complex-interacting multifunctional protein-2 (AIMP2). Although AIMP2 has been suggested to be toxic to dopamine neurons, its roles in α-synuclein aggregation and PD pathogenesis are largely unknown. Here, we found that AIMP2 exhibits a self-aggregating property. The AIMP2 aggregate serves as a seed to increase α-synuclein aggregation via specific and direct binding to the α-synuclein monomer. The coexpression of AIMP2 and α-synuclein in cell cultures and in vivo resulted in the rapid formation of α-synuclein aggregates with a corresponding increase in toxicity. Moreover, accumulated AIMP2 in mouse brain was largely redistributed to insoluble fractions, correlating with the α-synuclein pathology. Last, we found that α-synuclein preformed fibril (PFF) seeding, adult Parkin deletion, or oxidative stress triggered a redistribution of both AIMP2 and α-synuclein into insoluble fraction in cells and in vivo. Supporting the pathogenic role of AIMP2, AIMP2 knockdown ameliorated the α-synuclein aggregation and dopaminergic cell death in response to PFF or 6-hydroxydopamine treatment. Together, our results suggest that AIMP2 plays a pathological role in the aggregation of α-synuclein in mice. Because AIMP2 insolubility and coaggregation with α-synuclein have been seen in the PD Lewy body, targeting pathologic AIMP2 aggregation might be useful as a therapeutic strategy for neurodegenerative α-synucleinopathies.


Subject(s)
Parkinson Disease , alpha-Synuclein , Amyloid/metabolism , Animals , Brain/metabolism , Humans , Lewy Bodies/metabolism , Mice , Nuclear Proteins , alpha-Synuclein/metabolism
5.
J Immunother Cancer ; 8(1)2020 05.
Article in English | MEDLINE | ID: mdl-32461342

ABSTRACT

BACKGROUND: The generation of antigen-specific cytotoxic T lymphocyte (CTL) responses is required for successful cancer vaccine therapy. In this regard, ligands of Toll-like receptors (TLRs) have been suggested to activate adaptive immune responses by modulating the function of antigen-presenting cells (APCs). Despite their therapeutic potential, the development of TLR ligands for immunotherapy is often hampered due to rapid systemic toxicity. Regarding the safety concerns of currently available TLR ligands, finding a new TLR agonist with potent efficacy and safety is needed. METHODS: A unique structural domain (UNE-C1) was identified as a novel TLR2/6 in the catalytic region of human cysteinyl-tRNA synthetase 1 (CARS1) using comprehensive approaches, including RNA sequencing, the human embryonic kidney (HEK)-TLR Blue system, pull-down, and ELISA. The potency of its immunoadjuvant properties was analyzed by assessing antigen-specific antibody and CTL responses. In addition, the efficacy of tumor growth inhibition and the presence of the tumor-infiltrating leukocytes were evaluated using E.G7-OVA and TC-1 mouse models. The combined effect of UNE-C1 with an immune checkpoint inhibitor, anti-CTLA-4 antibody, was also evaluated in vivo. The safety of UNE-C1 immunization was determined by monitoring splenomegaly and cytokine production in the blood. RESULTS: Here, we report that CARS1 can be secreted from cancer cells to activate immune responses via specific interactions with TLR2/6 of APCs. A unique domain (UNE-C1) inserted into the catalytic region of CARS1 was determined to activate dendritic cells, leading to the stimulation of robust humoral and cellular immune responses in vivo. UNE-C1 also showed synergistic efficacy with cancer antigens and checkpoint inhibitors against different cancer models in vivo. Further, the safety assessment of UNE-C1 showed lower systemic cytokine levels than other known TLR agonists. CONCLUSIONS: We identified the endogenous TLR2/6 activating domain from human cysteinyl-tRNA synthetase CARS1. This novel TLR2/6 ligand showed potent immune-stimulating activity with little toxicity. Thus, the UNE-C1 domain can be developed as an effective immunoadjuvant with checkpoint inhibitors or cancer antigens to boost antitumor immunity.


Subject(s)
Amino Acyl-tRNA Synthetases/metabolism , Cancer Vaccines/administration & dosage , Immunity, Cellular/immunology , Immunotherapy/methods , Neoplasms, Experimental/therapy , Toll-Like Receptor 2/immunology , Amino Acyl-tRNA Synthetases/chemistry , Amino Acyl-tRNA Synthetases/immunology , Animals , Cancer Vaccines/immunology , Catalytic Domain , Dendritic Cells/immunology , Female , Humans , Immunization , Ligands , Mice , Mice, Inbred C57BL , Neoplasms, Experimental/immunology , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , T-Lymphocytes, Cytotoxic/immunology , Toll-Like Receptor 2/chemistry , Toll-Like Receptor 2/metabolism
6.
Lab Anim Res ; 35: 2, 2019.
Article in English | MEDLINE | ID: mdl-31463221

ABSTRACT

We examined the precision, accuracy, and capability of detecting changes of Dual-Energy X-ray Absorptiometry (DXA) for the measurements of total-body weight (TBW), total-body fat weight (TBFW), and total-body lean weight (TBLW) in an 8-week follow-up study of rats. Twenty male rats (4-week) were divided into 2 diet groups. For 8 weeks, we measured body composition (TBW, TBFW, TBLW) by DXA and TBW by an electronic scale once a week. In week 8, we measured body composition 5 times by DXA and TBFW by dissecting experiment (EXP) of euthanized rats (12-week). Total-body fat ratio (TBFR) was defined as TBFW/(TBFW+TBLW). The precision of DXA was evaluated by measuring the coefficient of variation (CV) and accuracy was evaluated by comparing DXA-derived data with EXP data. The capability of detecting changes of DXA in follow-up study was verified by analyzing the trend of DXA-derived values over the 8 weeks. For TBW, TBFW, TBLW of DXA, CVs were 0.02 ± 0.01, 0.10 ± 0.05, 0.03 ± 0.02 and errors were - 6.996 ± 3.429 (r = 0.999), + 14.729 ± 3.663 (r = 0.982), - 21.725 ± 4.223 (r = 0.991), respectively. Prediction models were [EXP TBW = - 31.767 + 1.085 (DXA TBW), R2 = 0.998, root mean square error (RMSE) = 1.842] and [EXP TBFR = - 0.056 + 1.177 (DXA TBFR), R2 = 0.948, RMSE = 0.007]. Over 8 weeks, DXA TBW and DXA TBLW steadily increased, DXA TBFW steadily increased followed by saturation or declination, difference of DXA TBFW between 2 diet groups steadily increased. In conclusion, our study verified that DXA (iNSiGHT VET DXA, OsteoSys, Korea) is accurate and precise enough to measure body composition of rats. Additionally, we confirmed the possibility that DXA could be used for the long-term follow-up studies.

7.
Nat Commun ; 10(1): 1357, 2019 03 22.
Article in English | MEDLINE | ID: mdl-30902983

ABSTRACT

A fundamental question in biology is how vertebrates evolved and differ from invertebrates, and little is known about differences in the regulation of translation in the two systems. Herein, we identify a threonyl-tRNA synthetase (TRS)-mediated translation initiation machinery that specifically interacts with eIF4E homologous protein, and forms machinery that is structurally analogous to the eIF4F-mediated translation initiation machinery via the recruitment of other translation initiation components. Biochemical and RNA immunoprecipitation analyses coupled to sequencing suggest that this machinery emerged as a gain-of-function event in the vertebrate lineage, and it positively regulates the translation of mRNAs required for vertebrate development. Collectively, our findings demonstrate that TRS evolved to regulate vertebrate translation initiation via its dual role as a scaffold for the assembly of initiation components and as a selector of target mRNAs. This work highlights the functional significance of aminoacyl-tRNA synthetases in the emergence and control of higher order organisms.


Subject(s)
Peptide Chain Initiation, Translational , Threonine-tRNA Ligase/metabolism , Amino Acid Sequence , Animals , Blood Vessels/growth & development , Blood Vessels/metabolism , Eukaryotic Initiation Factor-4E , Eukaryotic Initiation Factor-4F/metabolism , Eukaryotic Initiation Factor-4G/metabolism , HEK293 Cells , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Mice, Inbred C57BL , Protein Binding , RNA Cap-Binding Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Species Specificity , Threonine-tRNA Ligase/chemistry , Vertebrates/growth & development , Vertebrates/metabolism , Zebrafish
8.
FASEB J ; 33(3): 4341-4354, 2019 03.
Article in English | MEDLINE | ID: mdl-30592630

ABSTRACT

Fibrosis is characterized by the increased accumulation of extracellular matrix (ECM), which drives abnormal cell proliferation and progressive organ dysfunction in many inflammatory and metabolic diseases. Studies have shown that halofuginone, a racemic halogenated derivative, inhibits glutamyl-prolyl-transfer RNA-synthetase (EPRS)-mediated fibrosis. However, the mechanism by which this occurs is unclear. We explored the mechanistic aspects of how EPRS could develop liver fibrotic phenotypes in cells and animal models. Treatment with TGF-ß1 up-regulated fibronectin and collagen I levels in LX2 hepatic stellate cells. This effect was inhibited in prolyl-transfer RNA synthetase (PRS)-suppressed LX2 cells. Using the promoter luciferase assay, TGF-ß1-mediated collagen I, α1 chain transcription and γ2 basal laminin transcription in LX2 cells were down-regulated by EPRS suppression, suggesting that EPRS may play roles in ECM production at transcriptional levels. Furthermore, signal transducer and activator of transcription (STAT) signaling activation was involved in the effects of TGF-ß1 on ECM expression in a PRS-dependent manner. This was mediated via a protein-protein complex formation consisting of TGF-ß1 receptor, EPRS, Janus kinases, and STAT6. Additionally, ECM expression in fibrotic livers overlapped with EPRS expression along fibrotic septa regions and was positively correlated with STAT6 activation in carbon tetrachloride-treated mice. This was less obvious in livers of Eprs-/+ mice. These findings suggest that, during fibrosis development, EPRS plays roles in nontranslational processes of ECM expression via intracellular signaling regulation upon TGF-ß1 stimulation.-Song, D.-G., Kim, D., Jung, J. W., Nam, S. H., Kim, J. E., Kim, H.-J., Kim, J. H., Lee, S.-J., Pan, C.-H., Kim, S., Lee, J. W. Glutamyl-prolyl-tRNA synthetase induces fibrotic extracellular matrix via both transcriptional and translational mechanisms.


Subject(s)
Amino Acyl-tRNA Synthetases/metabolism , Extracellular Matrix/metabolism , Protein Biosynthesis/genetics , Transcription, Genetic/genetics , Amino Acyl-tRNA Synthetases/genetics , Animals , Cell Line , Collagen Type I/genetics , Collagen Type I/metabolism , Down-Regulation/genetics , Extracellular Matrix/genetics , Fibrosis/genetics , Fibrosis/metabolism , Hepatic Stellate Cells/metabolism , Humans , Liver/metabolism , Liver Cirrhosis/genetics , Liver Cirrhosis/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Signal Transduction/genetics , Transforming Growth Factor beta1/genetics , Transforming Growth Factor beta1/metabolism , Up-Regulation/genetics
9.
Front Pharmacol ; 9: 1337, 2018.
Article in English | MEDLINE | ID: mdl-30524284

ABSTRACT

Idiopathic pulmonary fibrosis (IPF), a chronic disease of unknown cause, is characterized by abnormal accumulation of extracellular matrix (ECM) in fibrotic foci in the lung. Previous studies have shown that the transforming growth factor ß1 (TGFß1) and signal transducers and activators of transcription (STAT) pathways play roles in IPF pathogenesis. Glutamyl-prolyl-tRNA-synthetase (EPRS) has been identified as a target for anti-fibrosis therapy, but the link between EPRS and TGFß1-mediated IPF pathogenesis remains unknown. Here, we studied the role of EPRS in the development of fibrotic phenotypes in A549 alveolar epithelial cells and bleomycin-treated animal models. We found that EPRS knockdown inhibited the TGFß1-mediated upregulation of fibronectin and collagen I and the mesenchymal proteins α-smooth muscle actin (α-SMA) and snail 1. TGFß1-mediated transcription of collagen I-α1 and laminin γ2 in A549 cells was also down-regulated by EPRS suppression, indicating that EPRS is required for ECM protein transcriptions. Activation of STAT signaling in TGFß1-induced ECM expression was dependent on EPRS. TGFß1 treatment resulted in EPRS-dependent in vitro formation of a multi-protein complex consisting of the TGFß1 receptor, EPRS, Janus tyrosine kinases (JAKs), and STATs. In vivo lung tissue from bleomycin-treated mice showed EPRS-dependent STAT6 phosphorylation and ECM production. Our results suggest that epithelial EPRS regulates the expression of mesenchymal markers and ECM proteins via the TGFß1/STAT signaling pathway. Therefore, epithelial EPRS can be used as a potential target to develop anti-IPF treatments.

10.
Sci Rep ; 8(1): 15025, 2018 10 09.
Article in English | MEDLINE | ID: mdl-30302025

ABSTRACT

Genomes are mostly protected from constant DNA-damaging threats, either internal or external, which ultimately sustain the organism. Herein, we report that AIMP3, a previously demonstrated tumour suppressor, plays an essential role in maintaining genome integrity in adult mice. Upon induction of the temporal systemic deletion of AIMP3 by tamoxifen in adult mice, the animals developed an acute radiation syndrome-like phenotype, typified by scleroderma, hypotrophy of haematopoietic cells and organs, and intestinal failure. Induction of γH2AX, an early marker of DNA double-strand breaks, was observed in the spleen, intestine, and the highly replicating embryonic cortex. In addition, sub-lethal irradiation of AIMP3 mKO mice dramatically affected organ damage and survival. Using isolated MEFs from conditional KO mice or AIMP3 knockdown cells, we confirmed the presence of spontaneously occurring DNA double-strand breaks by COMET assay and γH2AX induction. Furthermore, γH2AX removal was delayed, and homologous DNA repair activity was significantly reduced. Reduction of RPA foci formation and subsequent Rad51 foci formation probably underlie the significant reduction in homologous recombination activity in the absence of AIMP3. Together, our data demonstrate that AIMP3 plays a role in genome stability through the DNA repair process.


Subject(s)
Acute Radiation Syndrome/genetics , Histones/genetics , Peptide Elongation Factors/genetics , Rad51 Recombinase/genetics , Acute Radiation Syndrome/pathology , Animals , Comet Assay , DNA Breaks, Double-Stranded/radiation effects , DNA Damage/radiation effects , DNA Repair/genetics , DNA Repair/radiation effects , Fibroblasts/radiation effects , Genomic Instability/radiation effects , Homologous Recombination/radiation effects , Humans , Mice , Mice, Knockout , Phenotype , Radiation , Radiation, Ionizing , Tumor Suppressor Proteins/genetics
11.
J Clin Invest ; 128(11): 5034-5055, 2018 11 01.
Article in English | MEDLINE | ID: mdl-30188867

ABSTRACT

Lysyl-tRNA synthetase (KRS) functions canonically in cytosolic translational processes. However, KRS is highly expressed in colon cancer, and localizes to distinct cellular compartments upon phosphorylations (i.e., the plasma membranes after T52 phosphorylation and the nucleus after S207 phosphorylation), leading to probably alternative noncanonical functions. It is unknown how other subcellular KRSs crosstalk with environmental cues during cancer progression. Here, we demonstrate that the KRS-dependent metastatic behavior of colon cancer spheroids within 3D gels requires communication between cellular molecules and extracellular soluble factors and neighboring cells. Membranous KRS and nuclear KRS were found to participate in invasive cell dissemination of colon cancer spheroids in 3D gels. Cancer spheroids secreted GAS6 via a KRS-dependent mechanism and caused the M2 polarization of macrophages, which activated the neighboring cells via secretion of FGF2/GROα/M-CSF to promote cancer dissemination under environmental remodeling via fibroblast-mediated laminin production. Analyses of tissues from clinical colon cancer patients and Krs-/+ animal models for cancer metastasis supported the roles of KRS, GAS6, and M2 macrophages in KRS-dependent positive feedback between tumors and environmental factors. Altogether, KRS in colon cancer cells remodels the microenvironment to promote metastasis, which can thus be therapeutically targeted at these bidirectional KRS-dependent communications of cancer spheroids with environmental cues.


Subject(s)
Colonic Neoplasms/enzymology , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Lysine-tRNA Ligase/biosynthesis , Macrophages/enzymology , Neoplasm Proteins/biosynthesis , Spheroids, Cellular/enzymology , Tumor Microenvironment , Animals , Chemokine CXCL1/genetics , Chemokine CXCL1/metabolism , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Fibroblast Growth Factor 2/genetics , Fibroblast Growth Factor 2/metabolism , Fibroblasts/enzymology , Fibroblasts/pathology , HCT116 Cells , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Lysine-tRNA Ligase/genetics , Macrophages/pathology , Mice , Mice, Inbred BALB C , Mice, Knockout , Neoplasm Metastasis , Neoplasm Proteins/genetics , Spheroids, Cellular/pathology
12.
Cell Death Dis ; 9(10): 972, 2018 09 24.
Article in English | MEDLINE | ID: mdl-30250065

ABSTRACT

Aminoacyl-tRNA synthetase-interacting multifunctional protein-3 (AIMP3) is a component of the multi-aminoacyl-tRNA synthetase complex and is involved in diverse cellular processes. Given that AIMP3 deficiency causes early embryonic lethality in mice, AIMP3 is expected to play a critical role in early mouse development. To elucidate a functional role of AIMP3 in early mouse development, we induced AIMP3 depletion in mouse embryonic stem cells (mESCs) derived from blastocysts of AIMP3f/f; CreERT2 mice. In the present study, AIMP3 depletion resulted in loss of self-renewal and ability to differentiate to three germ layers in mESCs. AIMP3 depletion led to accumulation of DNA damage by blocking double-strand break repair, in particular homologous recombination. Through microarray analysis, the p53 signaling pathway was identified as being activated in AIMP3-depleted mESCs. Knockdown of p53 rescued loss of stem cell characteristics by AIMP3 depletion in mESCs. These results imply that AIMP3 depletion in mESCs leads to accumulation of DNA damage and p53 transactivation, resulting in loss of stemness. We propose that AIMP3 is involved in maintenance of genome stability and stemness in mESCs.


Subject(s)
Genomic Instability/physiology , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/metabolism , Tumor Suppressor Proteins/metabolism , Amino Acyl-tRNA Synthetases/genetics , Amino Acyl-tRNA Synthetases/metabolism , Animals , Blotting, Western , Cell Cycle/genetics , Cell Cycle/physiology , Cells, Cultured , Cellular Reprogramming/genetics , Cellular Reprogramming/physiology , Computational Biology , DNA Damage/genetics , DNA Damage/physiology , Genomic Instability/genetics , Mice , Protein Binding , Signal Transduction/genetics , Signal Transduction/physiology , Tumor Suppressor Proteins/genetics
13.
Exp Mol Med ; 50(1): e424, 2018 01 12.
Article in English | MEDLINE | ID: mdl-29328069

ABSTRACT

Mucin1 (MUC1), a heterodimeric oncoprotein, containing tandem repeat structures with a high proportion of threonine, is aberrantly overexpressed in many human cancers including pancreatic cancer. Since the overall survival rate of pancreatic cancer patients has remained low for several decades, novel therapeutic approaches are highly needed. Intestinal mucin has been known to be affected by dietary threonine supply since de novo synthesis of mucin proteins is sensitive to luminal threonine concentration. However, it is unknown whether biosynthesis of MUC1 is regulated by threonine in human cancers. In this study, data provided suggests that threonine starvation reduces the level of MUC1 and inhibits the migration of MUC1-expressing pancreatic cancer cells. Interestingly, knockdown of threonyl-tRNA synthetase (TRS), an enzyme that catalyzes the ligation of threonine to its cognate tRNA, also suppresses MUC1 levels but not mRNA levels. The inhibitors of TRS decrease the level of MUC1 protein and prohibit the migration of MUC1-expressing pancreatic cancer cells. In addition, a positive correlation between TRS and MUC1 levels is observed in human pancreatic cancer cells. Concurrent with these results, the bioinformatics data indicate that co-expression of both TRS and MUC1 is correlated with the poor survival of pancreatic cancer patients. Taken together, these findings suggest a role for TRS in controlling MUC1-mediated cancer cell migration and provide insight into targeting TRS as a novel therapeutic approach to pancreatic cancer treatment.


Subject(s)
Mucin-1/biosynthesis , Pancreatic Neoplasms/pathology , Threonine-tRNA Ligase/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Enzyme Inhibitors/pharmacology , Fatty Alcohols/pharmacology , Gene Expression Regulation, Neoplastic , Humans , Mucin-1/metabolism , Pancreatic Neoplasms/mortality , Survival Analysis , Threonine/metabolism , Threonine/pharmacology , Threonine-tRNA Ligase/antagonists & inhibitors , Threonine-tRNA Ligase/genetics , Tissue Array Analysis
14.
ACS Pharmacol Transl Sci ; 1(1): 21-31, 2018 Sep 14.
Article in English | MEDLINE | ID: mdl-32219202

ABSTRACT

Although abnormal increases in the level or activity of cyclin-dependent kinase 4 (CDK4) occur frequently in cancer, the underlying mechanism is not fully understood. Here, we show that methionyl-tRNA synthetase (MRS) specifically stabilizes CDK4 by enhancing the formation of the complex between CDK4 and a chaperone protein. Knockdown of MRS reduced the CDK4 level, resulting in G0/G1 cell cycle arrest. The effects of MRS on CDK4 stability were more prominent in the tumor suppressor p16INK4a-negative cancer cells because of the competitive relationship of the two proteins for binding to CDK4. Suppression of MRS reduced cell transformation and the tumorigenic ability of a p16INK4a-negative breast cancer cell line in vivo. Further, the MRS levels showed a positive correlation with those of CDK4 and the downstream signals at high frequency in p16INK4a-negative human breast cancer tissues. This work revealed an unexpected functional connection between the two enzymes involving protein synthesis and the cell cycle.

15.
Front Immunol ; 8: 1801, 2017.
Article in English | MEDLINE | ID: mdl-29379495

ABSTRACT

Dendritic cells (DCs) must integrate a broad array of environmental cues to exact control over downstream immune responses including TH polarization. The multienzyme aminoacyl-tRNA synthetase complex component AIMp1/p43 responds to cellular stress and exerts pro-inflammatory functions; however, a role for DC-expressed AIMp1 in TH polarization has not previously been shown. Here, we demonstrate that the absence of AIMp1 in bone marrow-derived DC (BMDC) significantly impairs cytokine and costimulatory molecule expression, p38 MAPK signaling, and TH1 polarization of cocultured T-cells while significantly dysregulating immune-related gene expression. These deficits resulted in significantly compromised BMDC vaccine-mediated protection against melanoma. AIMp1 within the host was also critical for innate and adaptive antiviral immunity against influenza virus infection in vivo. Cancer patients with AIMp1 expression levels in the highest tertiles exhibited a 70% survival advantage at 15-year postdiagnosis as determined by bioinformatics analysis of nearly 9,000 primary human tumor samples in The Cancer Genome Atlas database. These data establish the importance of AIMp1 for the effective governance of antitumor and antiviral immune responses.

17.
Int J Oncol ; 48(4): 1553-60, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26891990

ABSTRACT

The cell-adhesion properties of cancer cells can be targeted to block cancer metastasis. Although cytosolic lysyl-tRNA synthetase (KRS) functions in protein synthesis, KRS on the plasma membrane is involved in cancer metastasis. We hypothesized that KRS is involved in cell adhesion-related signal transduction for cellular migration. To test this hypothesis, colon cancer cells with modulated KRS protein levels were analyzed for cell-cell contact and cell-substrate adhesion properties and cellular behavior. Although KRS suppression decreased expression of cell-cell adhesion molecules, cells still formed colonies without being scattered, supporting an incomplete epithelial mesenchymal transition. Noteworthy, KRS-suppressed cells still exhibited focal adhesions on laminin, with Tyr397-phopshorylated focal adhesion kinase (FAK), but they lacked laminin-adhesion-mediated extracellular signal-regulated kinase (ERK) and paxillin activation. KRS, p67LR and integrin α6ß1 were found to interact, presumably to activate ERK for paxillin expression and Tyr118 phosphorylation even without involvement of FAK, so that specific inhibition of ERK or KRS in parental HCT116 cells blocked cell-cell adhesion and cell-substrate properties for focal adhesion formation and signaling activity. Together, these results indicate that KRS can promote cell-cell and cell-ECM adhesion for migration.


Subject(s)
Epithelial-Mesenchymal Transition , Extracellular Matrix/genetics , Lysine-tRNA Ligase/genetics , Neoplasms/pathology , Cell Adhesion , Cell Movement , Focal Adhesion Protein-Tyrosine Kinases/genetics , HCT116 Cells , Humans , Lysine-tRNA Ligase/metabolism , Neoplasms/genetics , Signal Transduction
18.
Oncotarget ; 6(25): 21655-74, 2015 Aug 28.
Article in English | MEDLINE | ID: mdl-26091349

ABSTRACT

The adhesion properties of cells are involved in tumor metastasis. Although KRS at the plasma membrane is shown important for cancer metastasis, additionally to canonical roles of cytosolic KRS in protein translation, how KRS and its downstream effectors promote the metastatic migration remains unexplored. Disseminative behaviors (an earlier metastatic process) of colon cancer cell spheroids embedded in 3D collagen gels were studied with regards to cell adhesion properties, and relevance in KRS(-/+) knocked-down animal and clinical colon cancer tissues. Time-lapse imaging revealed KRS-dependent cell dissemination from the spheroids, whereas KRS-suppressed spheroids remained static due to the absence of outbound movements supported by cell-extracellular matrix (ECM) adhesion. While keeping E-cadherin at the outward disseminative cells, KRS caused integrin-involved intracellular signaling for ERK/c-Jun, paxillin, and cell-ECM adhesion-mediated signaling to modulate traction force for crawling movement. KRS-suppressed spheroids became disseminative following ERK or paxillin re-expression. The KRS-dependent intracellular signaling activities correlated with the invasiveness in clinical colon tumor tissues and in KRS(-/+) knocked-down mice tissues. Collectively, these observations indicate that KRS at the plasma membrane plays new roles in metastatic migration as a signaling inducer, and causes intracellular signaling for cancer dissemination, involving cell-cell and cell-ECM adhesion, during KRS-mediated metastasis.


Subject(s)
Collagen Type I/metabolism , Colonic Neoplasms/enzymology , Lysine-tRNA Ligase/metabolism , Animals , Cadherins/metabolism , Cell Adhesion , Cell Line, Tumor , Colonic Neoplasms/pathology , Cytosol/metabolism , Extracellular Matrix/metabolism , Female , Fluorescence Resonance Energy Transfer , Fluorescent Antibody Technique, Indirect , HCT116 Cells , Humans , Mice , Neoplasm Metastasis , Paxillin/metabolism , Phosphorylation , Protein Biosynthesis , Signal Transduction
19.
Proc Natl Acad Sci U S A ; 111(42): 15084-9, 2014 Oct 21.
Article in English | MEDLINE | ID: mdl-25288775

ABSTRACT

In higher eukaryotes, one of the two arginyl-tRNA synthetases (ArgRSs) has evolved to have an extended N-terminal domain that plays a crucial role in protein synthesis and cell growth and in integration into the multisynthetase complex (MSC). Here, we report a crystal structure of the MSC subcomplex comprising ArgRS, glutaminyl-tRNA synthetase (GlnRS), and the auxiliary factor aminoacyl tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1)/p43. In this complex, the N-terminal domain of ArgRS forms a long coiled-coil structure with the N-terminal helix of AIMP1 and anchors the C-terminal core of GlnRS, thereby playing a central role in assembly of the three components. Mutation of AIMP1 destabilized the N-terminal helix of ArgRS and abrogated its catalytic activity. Mutation of the N-terminal helix of ArgRS liberated GlnRS, which is known to control cell death. This ternary complex was further anchored to AIMP2/p38 through interaction with AIMP1. These findings demonstrate the importance of interactions between the N-terminal domains of ArgRS and AIMP1 for the catalytic and noncatalytic activities of ArgRS and for the assembly of the higher-order MSC protein complex.


Subject(s)
Amino Acyl-tRNA Synthetases/chemistry , Arginine-tRNA Ligase/chemistry , Cytokines/chemistry , Neoplasm Proteins/chemistry , RNA-Binding Proteins/chemistry , Binding Sites , Chromatography, Gel , Circular Dichroism , Crystallography, X-Ray , Escherichia coli/metabolism , Glutathione Transferase/chemistry , Humans , Models, Molecular , Multiprotein Complexes , Mutagenesis , Mutation , Protein Biosynthesis , Protein Structure, Secondary , Protein Structure, Tertiary , Scattering, Radiation
20.
J Mol Histol ; 45(2): 121-8, 2014 Apr.
Article in English | MEDLINE | ID: mdl-23990368

ABSTRACT

Charcot-Marie-Tooth disease type 2D is a hereditary axonal and glycyl-tRNA synthetase (GARS)-associated neuropathy that is caused by a mutation in GARS. Here, we report a novel GARS-associated mouse neuropathy model using an adenoviral vector system that contains a neuronal-specific promoter. In this model, we found that wild-type GARS is distributed to peripheral axons, dorsal root ganglion (DRG) cell bodies, central axon terminals, and motor neuron cell bodies. In contrast, GARS containing a G240R mutation was localized in DRG and motor neuron cell bodies, but not axonal regions, in vivo. Thus, our data suggest that the disease-causing G240R mutation may result in a distribution defect of GARS in peripheral nerves in vivo. Furthermore, a distributional defect may be associated with axonal degradation in GARS-associated neuropathies.


Subject(s)
Adenoviridae/genetics , Charcot-Marie-Tooth Disease/enzymology , Animals , Axons/enzymology , Charcot-Marie-Tooth Disease/genetics , Charcot-Marie-Tooth Disease/pathology , Disease Models, Animal , Ganglia, Spinal/enzymology , Ganglia, Spinal/pathology , Genetic Vectors , Glycine-tRNA Ligase/genetics , Glycine-tRNA Ligase/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Motor Neurons/enzymology , Mutation, Missense , Nerve Fibers, Myelinated/enzymology , Organ Specificity , Peripheral Nerves/enzymology , Peripheral Nerves/pathology
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