ABSTRACT
Wood-decaying fungi are the major decomposers of plant litter. Heavy sequencing efforts on genomes of wood-decaying fungi have recently been made due to the interest in their lignocellulolytic enzymes; however, most parts of their proteomes remain uncharted. We hypothesized that wood-decaying fungi would possess promiscuous enzymes for detoxifying antifungal phytochemicals remaining in the dead plant bodies, which can be useful biocatalysts. We designed a computational mass spectrometry-based untargeted metabolomics pipeline for the phenotyping of biotransformation and applied it to 264 fungal cultures supplemented with antifungal plant phenolics. The analysis identified the occurrence of diverse reactivities by the tested fungal species. Among those, we focused on O-xylosylation of multiple phenolics by one of the species tested, Lentinus brumalis. By integrating the metabolic phenotyping results with publicly available genome sequences and transcriptome analysis, a UDP-glycosyltransferase designated UGT66A1 was identified and validated as an enzyme catalyzing O-xylosylation with broad substrate specificity. We anticipate that our analytical workflow will accelerate the further characterization of fungal enzymes as promising biocatalysts.
Subject(s)
Glucosyltransferases , Lentinula , Metabolomics , Metabolomics/methods , Lentinula/enzymology , Glucosyltransferases/chemistry , Glucosyltransferases/isolation & purification , Glucosyltransferases/metabolism , Phytochemicals/metabolism , Xylose/metabolism , Genome, Fungal , Liquid Chromatography-Mass SpectrometryABSTRACT
Dramatic cellular reorganization in mitosis critically depends on the timely and temporal phosphorylation of a broad range of proteins, which is mediated by the activation of the mitotic kinases and repression of counteracting phosphatases. The mitosis-to-interphase transition, which is termed mitotic exit, involves the removal of mitotic phosphorylation by protein phosphatases. Although protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) drive this reversal in animal cells, the phosphatase network associated with ordered bulk dephosphorylation in mitotic exit is not fully understood. Here, we describe a new mitotic phosphatase relay in which Wip1/PPM1D phosphatase activity is essential for chromosomal passenger complex (CPC) translocation to the anaphase central spindle after release from the chromosome via PP1-mediated dephosphorylation of histone H3T3. Depletion of endogenous Wip1 and overexpression of the phosphatase-dead mutant disturbed CPC translocation to the central spindle, leading to failure of cytokinesis. While Wip1 was degraded in early mitosis, its levels recovered in anaphase and the protein functioned as a Cdk1-counteracting phosphatase at the anaphase central spindle and midbody. Mechanistically, Wip1 dephosphorylated Thr-59 in inner centromere protein (INCENP), which, subsequently bound to MKLP2 and recruited other components to the central spindle. Furthermore, Wip1 overexpression is associated with the overall survival rate of patients with breast cancer, suggesting that Wip1 not only functions as a weak oncogene in the DNA damage network but also as a tumor suppressor in mitotic exit. Altogether, our findings reveal that sequential dephosphorylation of mitotic phosphatases provides spatiotemporal regulation of mitotic exit to prevent tumor initiation and progression.
Subject(s)
Chromosomes/metabolism , Mitosis , Protein Phosphatase 2C/metabolism , Spindle Apparatus/metabolism , Anaphase , Aurora Kinase B/metabolism , CDC2 Protein Kinase/metabolism , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes/genetics , DNA Damage , Humans , Kinesins/antagonists & inhibitors , Kinesins/genetics , Kinesins/metabolism , Phosphorylation , Protein Binding , Protein Phosphatase 1/antagonists & inhibitors , Protein Phosphatase 1/genetics , Protein Phosphatase 1/metabolism , Protein Phosphatase 2/antagonists & inhibitors , Protein Phosphatase 2/genetics , Protein Phosphatase 2/metabolism , Protein Phosphatase 2C/antagonists & inhibitors , Protein Phosphatase 2C/genetics , RNA Interference , RNA, Small Interfering/metabolism , Survivin/metabolismABSTRACT
In our previous work, a series of 2-amino-3,4-dihydroquinazoline derivativesusing an electron acceptor group was reported to be potent T-type calcium channel blockers and exhibit strong cytotoxic effects against various cancerous cell lines. To investigate the role of the guanidine moiety in the 2-amino-3,4-dihydroquinazoline scaffold as a pharmacophore for dual biological activity, a new series of 2-thio-3,4-dihydroquniazoline derivatives using an electron donor group at the C2-position was synthesized and evaluated for T-type calcium channel blocking activity and cytotoxic effects against two human cancerous cell lines (lung cancer A549 and colon cancer HCT-116). Among them, compound 6g showed potent inhibition of Cav3.2 currents (83% inhibition) at 10 µM concentrations. The compound also exhibited IC50 values of 5.0 and 6.4 µM against A549 and HCT-116 cell lines, respectively, which are comparable to the parental lead compound KYS05090. These results indicate that the isothiourea moiety similar to the guanidine moiety of 2-amino-3,4-dihydroquinazoline derivatives may be an essential pharmacophore for the desired biological activities. Therefore, our preliminary work can provide the opportunity to expand a chemical repertoire to improve affinity and selectivity for T-type calcium channels.
Subject(s)
Antineoplastic Agents/pharmacology , Calcium Channel Blockers/pharmacology , Calcium Channels, T-Type/metabolism , Quinazolines/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Calcium Channel Blockers/chemical synthesis , Calcium Channel Blockers/chemistry , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Molecular Structure , Quinazolines/chemical synthesis , Quinazolines/chemistry , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
We previously reported the strong inhibitory potency of N-phenyl-N'-(4- benzyloxyphenoxycarbonyl)-4-chlorophenylsulfonyl hydrazide (PBCH) on lipopolysaccharide (LPS)-induced prostaglandin E2 (PGE2) production in macrophages. Herein, we characterized PBCH as a microsomal prostaglandin E synthase-1 (mPGES-1) inhibitor and evaluated its anti-inflammatory effects using in vivo experimental models. PBCH inhibited PGE2 production in various activated cells in addition to inhibiting the mPGES-1 activity. In the ear edema and paw edema rat models, PBCH significantly reduced ear thickness and paw swelling, respectively. Besides, in adjuvant-induced arthritis (AIA) rat model, PBCH decreased paw swelling, plasma rheumatoid factor (RF), and receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) ratio. Furthermore, while PBCH reduced the plasma prostaglandin E metabolite (PGEM) levels, it did not affect the plasma levels of prostacyclin (PGI2) and thromboxane A2 (TXA2). Our data suggest that PBCH downregulates PGE2 production by interfering with the mPGES-1 activity, thus reducing edema and arthritis in rat models.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Dinoprostone/metabolism , Down-Regulation/drug effects , Enzyme Inhibitors/pharmacology , Hydrazines/pharmacology , Prostaglandin-E Synthases/antagonists & inhibitors , Thiazoles/pharmacology , A549 Cells , Animals , Anti-Inflammatory Agents/therapeutic use , Dinoprostone/biosynthesis , Disease Models, Animal , Enzyme Inhibitors/therapeutic use , Humans , Hydrazines/therapeutic use , Inflammation/drug therapy , Inflammation/metabolism , Male , Mice , RAW 264.7 Cells , Rats , Rats, Sprague-Dawley , Thiazoles/therapeutic useABSTRACT
Active turnover of spindle microtubules (MTs) for the formation of a bi-orientated spindle, chromosome congression and proper chromosome segregation is regulated by MT depolymerases such as the kinesin-13 family and the plus-end-tracking proteins (+TIPs). However, the control mechanisms underlying the spindle MT dynamics that are responsible for poleward flux at the minus end of MTs are poorly understood. Here, we show that Mdp3 (also known as MAP7D3) forms a complex with DDA3 (also known as PSRC1) and controls spindle dynamics at the minus end of MTs by inhibiting DDA3-mediated Kif2a recruitment to the spindle. Aberrant Kif2a activity at the minus end of spindle MTs in Mdp3-depleted cells decreased spindle stability and resulted in unaligned chromosomes in metaphase, lagging chromosomes in anaphase, and chromosome bridges in telophase and cytokinesis. Although they play opposing roles in minus-end MT dynamics, acting as an MT destabilizer and an MT stabilizer, respectively, DDA3 and Mdp3 did not affect the localization of each other. Thus, the DDA3 complex orchestrates MT dynamics at the MT minus end by fine-tuning the recruitment of Kif2a to regulate minus-end MT dynamics and poleward MT flux at the mitotic spindle.
Subject(s)
Kinesins/metabolism , Microtubule-Associated Proteins/metabolism , Mitosis , Phosphoproteins/metabolism , Spindle Apparatus/metabolism , Chromosomes, Human/metabolism , HeLa Cells , Humans , Kinetochores/metabolism , Microtubules/metabolism , Polymerization , Protein BindingABSTRACT
Sakuranetin (SKN), found in cherry trees and rice, is a flavanone with various pharmacological activities. It is biosynthesized from naringenin in rice or cherry trees, and the metabolism of SKN has been studied in non-human species. The present study aimed to investigate the metabolic pathways of SKN in human liver microsomes and identify the phase I and phase II metabolites, as well as evaluate the potential for drugâ»herb interactions through the modulation of drug metabolizing enzymes (DMEs). HPLC-DAD and HPLC-electrospray mass spectrometry were used to study the metabolic stability and identify the metabolites from human liver microsomes incubated with SKN. The potential of SKN to inhibit the DMEs was evaluated by monitoring the formation of a DME-specific product. The cytochrome P450 2B6 and 3A4-inductive effects were studied using promoter reporter assays in human hepatocarcinoma cells. The major pathways for SKN metabolism include B-ring hydroxylation, 5-O-demethylation, and conjugation with glutathione or glucuronic acid. The phase I metabolites were identified as naringenin and eriodictyol. SKN was found to be a UDP-glucuronosyltransferases (UGT) 1A9 inhibitor, whereas it induced transactivation of the human pregnane X receptor-mediated cytochrome P450 (CYP) 3A4 gene.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Flavonoids/metabolism , Glucuronosyltransferase/metabolism , Liver/metabolism , Animals , Chromatography, High Pressure Liquid , Cytochrome P-450 Enzyme Inhibitors/pharmacology , Cytochrome P-450 Enzyme System/genetics , Hep G2 Cells , Humans , Liver/drug effects , Metabolic Detoxication, Phase I , Metabolic Detoxication, Phase II , Metabolome , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , NADP/metabolism , Pregnane X Receptor , Promoter Regions, Genetic/genetics , Receptors, Steroid/metabolism , Transcriptional Activation/genetics , Uridine Diphosphate Glucuronic Acid/metabolismABSTRACT
The error-free segregation of chromosomes, which requires the precisely timed search and capture of chromosomes by spindles during early mitotic and meiotic cell division, is responsible for genomic stability and is achieved by the spindle assembly checkpoint in the metaphase-anaphase transition. Mitotic kinases orchestrate M phase events, such as the reorganization of cell architecture and kinetochore (KT) composition with the exquisite phosphorylation of mitotic regulators, to ensure timely and temporal progression. However, the molecular mechanisms underlying the changes of KT composition for stable spindle attachment during mitosis are poorly understood. Here, we show that the sequential action of the kinase Cdk1 and the phosphatase Cdc14A control spindle attachment to KTs. During prophase, the mitotic spindle protein Spag5/Astrin is transported into centrosomes by Kinastrin and phosphorylated at Ser-135 and Ser-249 by Cdk1, which, in prometaphase, is loaded onto the spindle and targeted to KTs. We also demonstrate that Cdc14A dephosphorylates Astrin, and therefore the overexpression of Cdc14A sequesters Astrin in the centrosome and results in aberrant chromosome alignment. Mechanistically, Plk1 acts as an upstream kinase for Astrin phosphorylation by Cdk1 and targeting phospho-Astrin to KTs, leading to the recruitment of outer KT components, such as Cenp-E, and the stable attachment of spindles to KTs. These comprehensive findings reveal a regulatory circuit for protein targeting to KTs that controls the KT composition change of stable spindle attachment and chromosome integrity.
Subject(s)
Anaphase/physiology , Cell Cycle Proteins/metabolism , Kinetochores/metabolism , Metaphase/physiology , CDC2 Protein Kinase , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , HeLa Cells , Humans , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation/physiology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Tyrosine Phosphatases , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Polo-Like Kinase 1ABSTRACT
Aurora B activation is triggered at the mitotic entry and required for proper microtubule-kinetochore attachment at mitotic phase. Therefore, Aurora B should be in inactive form in interphase to prevent aberrant cell cycle progression. However, it is unclear how the inactivation of Aurora B is sustained during interphase. In this study, we find that IK depletion-induced mitotic arrest leads to G2 arrest by Aurora B inhibition, indicating that IK depletion enhances Aurora B activation before mitotic entry. IK binds to Aurora B, and colocalizes on the nuclear foci during interphase. Our data further show that IK inhibits Aurora B activation through recruiting PP2A into IK and Aurora B complex. It is thus believed that IK, as a scaffold protein, guides PP2A into Aurora B to suppress its activity in interphase until mitotic entry.
Subject(s)
Aurora Kinase B/metabolism , Cytokines/metabolism , Protein Phosphatase 2/metabolism , Aurora Kinase B/antagonists & inhibitors , Benzamides/pharmacology , Cytokines/antagonists & inhibitors , Cytokines/genetics , Enzyme Activation/drug effects , G2 Phase Cell Cycle Checkpoints/drug effects , HEK293 Cells , HeLa Cells , Humans , Interphase , M Phase Cell Cycle Checkpoints , Phosphorylation/drug effects , Protein Binding , Protein Kinase Inhibitors/pharmacology , Quinazolines/pharmacology , RNA Interference , RNA, Small Interfering/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Tubulin/metabolismABSTRACT
Larrea nitida Cav. (LNC), which belongs to the family Zygophyllaceae, is widely indigenous and used in South America to treat various pathological conditions. It contains the antioxidant and antiinflammatory but toxic nordihydroguaiaretic acid (NDGA) as well as O-methylated metabolite of NDGA (MNDGA) as bioactive compounds. The hepatic metabolism-based toxicological potential of extracts of LNC (LNE), NDGA, and MNDGA has not previously been reported. The present study aimed to characterize the phase I and phase II hepatic metabolism and reactive intermediates of LNE, NDGA, and MNDGA and their effects on the major drug-metabolizing enzymes in vitro and ex vivo. A methanol extract of LNC collected from Chile as well as NDGA and MNDGA isolated from LNE were subjected to metabolic stability assays in liver microsomes in the presence of the cofactors reduced nicotinamide dinucleotide phosphate (NADPH) and/or uridine 5'-diphosphoglucuronic acid (UDPGA). Cytochrome P450 (CYP) inhibition assays were performed using CYP isozyme-specific model substrates to examine the inhibitory activities of LNE, NDGA, and MNDGA, which were expressed as % inhibition and IC50 values. Ex vivo CYP induction potential was investigated in the liver microsomes prepared from the rats intraperitoneally administered with LNE. Glutathione (GSH) adduct formation was monitored by LC-MS3 analysis of the microsomal incubation samples with either NDGA or MNDGA and an excess of GSH to determine the formation of electrophilic reactive intermediates. Both NDGA and MNDGA were stable to NADPH-dependent phase I metabolism, but labile to glucuronide conjugation. LNE, NDGA, and MNDGA showed significant inhibitory effects on CYP1A2, 2C9, 2D6, and/or 3A4, with IC50 values in the micromolar range. LNE was found to be a CYP1A2 inducer in ex vivo rat experiments, and mono- and di-GSH adducts of both NDGA and MNDGA were identified by LC-MS3 analysis. Our study suggests that hepatic clearance is the major elimination route for the lignans NDGA and MNDGA present in LNE. These lignans may possess the ability to modify biomacromolecules via producing reactive intermediates. In addition, LNE, NDGA, and MNDGA are found to be inhibitors for various CYP isozymes such as CYP2C9 and 3A4. Thus, the consumption of LNC as an herbal preparation or NDGA may cause metabolism-driven herb-drug interactions. Copyright © 2016 John Wiley & Sons, Ltd.
Subject(s)
Larrea/chemistry , Lignans/chemistry , Liver/metabolism , Microsomes, Liver/drug effects , Animals , Female , Herb-Drug Interactions , Humans , Lignans/pharmacology , RatsABSTRACT
Spindle dynamics drives chromosome movement and mitotic progression during mitosis. Microtubule (MT)-associated proteins (MAPs) regulate MT stabilization/destabilization and MT polymerization/depolymerization for congression of sister chromatids at the mitotic equator and subsequent segregation toward the spindle poles. Here, we identified ANKRD53 as a novel DDA3-interacting protein through proteomic analysis. Based on expression profiles, ANKRD53 is phosphorylated by mitotic kinases during mitosis. In ANKRD53-depleted HeLa cells, the progression of mitosis was delayed and the number of unaligned chromosomes increased substantially. In addition, spindle MT polymerization decreased and the spindle assembly checkpoint (SAC) was concomitantly activated by the decreased spindle dynamics in ANKRD53-depleted cells. Although ANKRD53 is recruited to the mitotic spindle by DDA3, it counteracts the activity of DDA3 for spindle MT polymerization. Furthermore, ANKRD53 depletion increased the number of bi-nuclei and polylobed nuclei. Thus, ANKRD53 is recruited to the mitotic spindle by DDA3 and acts as a regulator of spindle dynamics and cytokinesis.
Subject(s)
Carrier Proteins/metabolism , Cell Nucleus/physiology , Chromosomes/physiology , Mitosis/physiology , Phosphoproteins/metabolism , Spindle Apparatus/physiology , Cell Nucleus/ultrastructure , Chromosomes/ultrastructure , Gene Expression Regulation/physiology , HeLa Cells , Humans , Spindle Apparatus/ultrastructureABSTRACT
Spindle microtubules (MTs) capture kinetochores (KTs) on the centromere sequence of sister chromatids to align at the mitotic equator and segregate toward spindle poles during mitosis. For efficient chromosome capture, KTs initially attach to the lateral surface of a MT, providing a considerably larger contact surface than the MT tip. A sequential change of KT composition upon spindle attachment enables a conversion from lateral to stable end-on attachment. However, the molecular link between spindle dynamics and KT composition is not fully understood. Here, we report that Ska1 and DDA3 act as molecular linkers in the interplay between KTs and spindle dynamics. After recruitment of Kif2a onto the mitotic spindle by DDA3, Ska1 targets Kif2a to the minus-end of spindle MTs and facilitates spindle dynamics. Furthermore, DDA3 targets Ska1 to KTs to stabilize end-on attachment. Thus, our findings identified a definite regulatory mechanism of the search and capture process for stable spindle attachment through cross-talk between spindle dynamics and KT composition mediated by DDA3 and Ska1.
Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , Kinetochores/metabolism , Mitosis/physiology , Molecular Motor Proteins/metabolism , Phosphoproteins/metabolism , Spindle Apparatus/physiology , HeLa Cells , Humans , Protein BindingABSTRACT
Cancerous inhibitor of protein phosphatase 2A (CIP2A) is overexpressed in most human cancers and has been described as being involved in the progression of several human malignancies via the inhibition of protein phosphatase 2A (PP2A) activity toward c-Myc. However, with the exception of this role, the cellular function of CIP2A remains poorly understood. On the basis of yeast two-hybrid and coimmunoprecipitation assays, we demonstrate here that NIMA (never in mitosis gene A)-related kinase 2 (NEK2) is a binding partner for CIP2A. CIP2A exhibited dynamic changes in distribution, including the cytoplasm and centrosome, depending on the cell cycle stage. When CIP2A was depleted, centrosome separation and the mitotic spindle dynamics were impaired, resulting in the activation of spindle assembly checkpoint signaling and, ultimately, extension of the cell division time. Our data imply that CIP2A strongly interacts with NEK2 during G2/M phase, thereby enhancing NEK2 kinase activity to facilitate centrosome separation in a PP1- and PP2A-independent manner. In conclusion, CIP2A is involved in cell cycle progression through centrosome separation and mitotic spindle dynamics.
Subject(s)
Autoantigens/metabolism , Cell Division/physiology , Centrosome/metabolism , G2 Phase/physiology , Membrane Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Autoantigens/genetics , Cell Cycle Checkpoints/physiology , Cytoplasm/genetics , Cytoplasm/metabolism , HEK293 Cells , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins , Membrane Proteins/genetics , Mice , NIMA-Related Kinases , Protein Phosphatase 1/genetics , Protein Phosphatase 1/metabolism , Protein Phosphatase 2/genetics , Protein Phosphatase 2/metabolism , Protein Serine-Threonine Kinases/genetics , Signal Transduction/physiology , Two-Hybrid System TechniquesABSTRACT
Although loss of Sirt1 leads to chromosome aneuploidy, which accounts for higher tumor susceptibility, the molecular mechanisms remain unclear. Herein, we demonstrate that Sirt1 directly regulates Plk1, of which activity is critical for mitotic progression and spindle dynamics. Depletion or inhibition of Sirt1 significantly perturbs the formation of the mitotic spindle, leading to defective chromosome segregation. Elevated depolymerization of the mitotic spindle following loss of Sirt1 was associated with the deregulation of Plk1 activity. Thus, we conclude that Sirt1 may contribute to a mitotic regulator that controls spindle dynamics through Plk1 activity, resulting in fine-tuning of Plk1 dependent microtubule dynamics.
Subject(s)
Cell Cycle Proteins/metabolism , Mitosis , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Sirtuin 1/metabolism , Spindle Apparatus/metabolism , Chromosome Segregation , HEK293 Cells , HeLa Cells , Humans , Phosphorylation , Sirtuin 1/genetics , Polo-Like Kinase 1ABSTRACT
The centrosome is an important cellular organelle which nucleates microtubules (MTs) to form the cytoskeleton during interphase and the mitotic spindle during mitosis. The Cep290 is one of the centrosomal proteins and functions in cilia formation. Even-though it is in the centrosome, the function of Cep290 in mitosis had not yet been evaluated. In this study, we report a novel function of Cep290 that is involved in spindle positioning. Cep290 was identified as an interacting partner of DDA3, and we confirmed that Cep290 specifically localizes in the mitotic centrosome. Depletion of Cep290 caused a reduction of the astral spindle, leading to misorientation of the mitotic spindle. MT polymerization also decreased in Cep290-depleted cells, suggesting that Cep290 is involved in spindle nucleation. Furthermore, DDA3 stabilizes and transports Cep290 to the centrosome. Therefore, we concluded that DDA3 controls astral spindle formation and spindle positioning by targeting Cep290 to the centrosome.
Subject(s)
Antigens, Neoplasm/metabolism , Centrosome/metabolism , Neoplasm Proteins/metabolism , Phosphoproteins/metabolism , Spindle Apparatus/metabolism , Antigens, Neoplasm/genetics , Cell Cycle Proteins , Cytoskeletal Proteins , Gene Knockdown Techniques , HeLa Cells , Humans , Microtubules/metabolism , Mitosis/physiology , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/genetics , Protein Stability , Protein Transport , RNA, Small Interfering/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Estrogen receptors are activated by the hormone estrogen and they control cell growth by altering gene expression as a transcription factor. So far two estrogen receptors have been found: ERα and ERß. Estrogen receptors are also implicated in the development and progression of breast cancer. Here, we found that ERα localized on the spindle and spindle poles at the metaphase during mitosis. Depletion of ERα generated unaligned chromosomes in metaphase cells and lagging chromosomes in anaphase cells in a transcription-independent manner. Furthermore, the levels of ß-tubulin and γ-tubulin were reduced in ERα-depleted cells. Consistent with this, polymerization of microtubules in ERα-depleted cells and turnover rate of α/ß-tubulin were decreased than in control cells. We suggest that ERα regulates chromosome alignment and spindle dynamics by stabilizing microtubules during mitosis.
Subject(s)
Chromosomes, Mammalian/metabolism , Estrogen Receptor alpha/metabolism , Mitosis , Spindle Apparatus/metabolism , Cell Survival , Chromosome Segregation , HeLa Cells , Humans , Metaphase , Protein Transport , Transcription, GeneticABSTRACT
Sirt3, one of mammalian sirtuins is a prominent mitochondrial deacetylase that controls mitochondrial oxidative pathways and the rate of reactive oxygen species. Sirt3 also regulates energy metabolism by deacetylating enzymes involved in the metabolic pathway related with lifespan. We report here a novel function of Sirt3 which was found to be involved in mitosis. Depletion of the Sirt3 protein generated unaligned chromosomes in metaphase which caused mitotic arrest by activating spindle assembly checkpoint (SAC). Furthermore, the shape and the amount of the spindles in Sirt3 depleted cells were abnormal. Microtubule (MT) polymerization also increased in Sirt3 depleted cells, suggesting that Sirt3 is involved in spindle dynamics. However, the level of acetylated tubulin was not increased significantly in Sirt3 depleted cells. The findings collectively suggest that Sirt3 is not a tubulin deacetylase but regulates the attachment of spindle MTs to the kinetochore and the subsequent chromosome alignment by increasing spindle dynamics.
Subject(s)
Chromosomes/metabolism , Mitosis , Sirtuin 3/metabolism , Spindle Apparatus/metabolism , Chromosomes/ultrastructure , HeLa Cells , Humans , RNA Interference , RNA, Small Interfering/genetics , Sirtuin 3/genetics , Spindle Apparatus/ultrastructureABSTRACT
Abnormal activation of the Wnt/ß-catenin signaling pathway frequently induces colon cancer progression. In the present study, we identified tussilagone (TSL), a compound isolated from the flower buds of Tussilago farfara, as an inhibitor on ß-catenin dependent Wnt pathway. TSL suppressed ß-catenin/T-cell factor transcriptional activity and down-regulated ß-catenin level both in cytoplasm and nuclei of HEK293 reporter cells when they were stimulated by Wnt3a or activated by an inhibitor of glycogen synthase kinase-3ß. Since the mRNA level was not changed by TSL, proteasomal degradation might be responsible for the decreased level of ß-catenin. In SW480 and HCT116 colon cancer cell lines, TSL suppressed the ß-catenin activity and also decreased the expression of cyclin D1 and c-myc, representative target genes of the Wnt/ß-catenin signaling pathway, and consequently inhibited the proliferation of colon cancer cells. Taken together, TSL might be a potential chemotherapeutic agent for the prevention and treatment of human colon cancer.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Proliferation/drug effects , Colonic Neoplasms/pathology , Sesquiterpenes/pharmacology , beta Catenin/metabolism , Colonic Neoplasms/metabolism , HEK293 Cells , Humans , Metabolic Networks and Pathways/drug effects , Proteolysis/drug effects , Wnt Proteins/antagonists & inhibitorsABSTRACT
5-Lipoxygenase (5-LO) catalyzes the formation of two major groups of leukotrienes, leukotriene B4 and cysteinyl leukotrienes (CysLTs), and it has been implicated as a promising drug target to treat various inflammatory diseases. However, its role in osteoclastogenesis has not been investigated. In this study, we used mouse bone marrow-derived macrophages (BMMs) to show that 5-LO inhibitor suppresses RANKL-induced osteoclast formation. Inhibition of 5-LO was associated with impaired activation of multiple signaling events downstream of RANK, including ERK and p38 phosphorylation, and IκB degradation, followed by a decrease in NFATc1 expression. Ectopic overexpression of a constitutively active form of NFATc1 partly rescued the antiosteoclastogenic effect of 5-LO inhibitor. The knockdown of 5-LO in BMMs also resulted in a significant reduction in RANKL-induced osteoclast formation, accompanied by decreased expression of NFATc1. Similar effects were shown with CysLT receptor (CysLTR)1/2 antagonist and small RNA for CysLTR1 in BMMs, indicating the involvement of CysLT and CysLTR1 in 5-LO-mediated osteoclastogenesis. Finally, 5-LO inhibitor suppressed LPS-induced osteoclast formation and bone loss in the in vivo mouse experiments, suggesting a potential therapeutic strategy for treating diseases involving bone destruction. Taken together, the results of this study demonstrate that 5-LO is a key mediator of RANKL-induced osteoclast formation and possibly a novel therapeutic target for bone-resorption diseases.
Subject(s)
Arachidonate 5-Lipoxygenase/metabolism , Bone Resorption/prevention & control , Indoles/pharmacology , Lipoxygenase Inhibitors/pharmacology , Osteoclasts/drug effects , RANK Ligand/antagonists & inhibitors , Receptors, Leukotriene/metabolism , Animals , Arachidonate 5-Lipoxygenase/genetics , Bone Marrow/drug effects , Bone Marrow/metabolism , Bone Marrow/pathology , Bone Resorption/genetics , Bone Resorption/metabolism , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation/drug effects , Gene Knockdown Techniques , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Macrophages/pathology , Male , Mice , Mice, Inbred ICR , NFATC Transcription Factors/genetics , NFATC Transcription Factors/metabolism , Osteoclasts/metabolism , Osteoclasts/pathology , Phosphorylation/drug effects , RANK Ligand/genetics , RANK Ligand/metabolism , RNA, Small Interfering/genetics , Receptors, Leukotriene/genetics , Signal Transduction/drug effects , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
The centrosome assembles a bipolar spindle for faithful chromosome segregation during mitosis. To prevent the inheritance of DNA damage, the DNA damage response (DDR) triggers programmed spindle multipolarity and concomitant death in mitosis through a poorly understood mechanism. We identified hornerin, which forms a complex with checkpoint kinase 1 (Chk1) and polo-like kinase 1 (Plk1) to mediate phosphorylation at the polo-box domain (PBD) of Plk1, as the link between the DDR and death in mitosis. We demonstrate that hornerin mediates DDR-induced precocious centriole disengagement through a dichotomous mechanism that includes sequestration of Sgo1 and Plk1 in the cytoplasm through phosphorylation of the PBD in Plk1 by Chk1. Phosphorylation of the PBD in Plk1 abolishes the interaction with Sgo1 and phosphorylation-dependent Sgo1 translocation to the centrosome, leading to precocious centriole disengagement and spindle multipolarity. Mechanistically, hornerin traps phosphorylated Plk1 in the cytoplasm. Furthermore, PBD phosphorylation inactivates Plk1 and disrupts Cep192::Aurora A::Plk1 complex translocation to the centrosome and concurrent centrosome maturation. Remarkably, hornerin depletion leads to chemoresistance against DNA damaging agents by attenuating DDR-induced death in mitosis. These results reveal how the DDR eradicates mitotic cells harboring DNA damage to ensure genome integrity during cell division.
Subject(s)
Centrosome , Mitosis , Checkpoint Kinase 1 , Phosphorylation , Polo-Like Kinase 1ABSTRACT
BACKGROUND: Despite highly effective machinery for the maintenance of genome integrity in human embryonic stem cells (hESCs), the frequency of genetic aberrations during in-vitro culture has been a serious issue for future clinical applications. METHOD: By passaging hESCs over a broad range of timepoints (up to 6 years), the isogenic hESC lines with different passage numbers with distinct cellular characteristics, were established. RESULT: We found that mitotic aberrations, such as the delay of mitosis, multipolar centrosomes, and chromosome mis-segregation, were increased in parallel with polyploidy compared to early-passaged hESCs (EP-hESCs) with normal copy number. Through high-resolution genome-wide approaches and transcriptome analysis, we found that culture adapted-hESCs with a minimal amplicon in chromosome 20q11.21 highly expressed TPX2, a key protein for governing spindle assembly and cancer malignancy. Consistent with these findings, the inducible expression of TPX2 in EP-hESCs reproduced aberrant mitotic events, such as the delay of mitotic progression, spindle stabilization, misaligned chromosomes, and polyploidy. CONCLUSION: These studies suggest that the increased transcription of TPX2 in culture adapted hESCs could contribute to an increase in aberrant mitosis due to altered spindle dynamics.