RESUMEN
Human Cripto-1 is a member of the epidermal growth factor (EGF)-Cripto-FRL-1-Cryptic (CFC) family family and performs critical roles in cancer and various pathological and developmental processes. Recently we demonstrated that a soluble form of Cripto-1 suppresses the self-renewal and enhances the differentiation of cancer stem cells (CSCs). A functional form of soluble Cripto-1 was found to be difficult to obtain because of the 12 cysteine residues in the protein which impairs the folding process. Here, we optimized the protocol for a T7 expression system, purification from inclusion bodies under denatured conditions refolding of a His-tagged Cripto-1 protein. A concentrations of 0.2-0.4 mM isopropyl ß-D-1-thiogalactopyranoside (IPTG) at 37°C was found to be the optimal concentration for Cripto-1 expression while imidazole at 0.5 M was the optimum concentration to elute the Cripto-1 protein from a Ni-column in the smallest volume. Cation exchange column chromatography of the Cripto-1 protein in the presence of 8 M urea exhibited sufficient elution profile at pH 5, which was more efficient at recovery. The recovery of the protein reached to more than 26.6% after refolding with arginine. The purified Cripto-1 exhibited high affinity to the anti-ALK-4 antibody and suppressed sphere forming ability of CSCs at high dose and induced cell differentiation.
Asunto(s)
Neoplasias , Células Madre Neoplásicas , Diferenciación Celular , Factor de Crecimiento Epidérmico/química , Factor de Crecimiento Epidérmico/farmacología , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Humanos , Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismoRESUMEN
Telomerase is a unique ribonucleoprotein enzyme that is required for continued cell proliferation. To generate catalytically active telomerase, human telomerase reverse transcriptase (hTERT) must translocate to the nucleus and assemble with the RNA component of telomerase. The molecular chaperones heat shock protein 90 (Hsp90) and p23 maintain hTERT in a conformation that enables nuclear translocation. However, the regulatory role of chaperones in nuclear transport of hTERT remains unclear. In this work, we demonstrate that immunophilin FK506-binding protein (FKBP)52 linked the hTERT-Hsp90 complex to the dynein-dynactin motor, thereby promoting the transport of hTERT to the nucleus along microtubules. FKBP52 interacted with the hTERT-Hsp90 complex through binding of the tetratricopeptide repeat domain to Hsp90 and binding of the dynamitin (Dyt) component of the dynein-associated dynactin complex to the peptidyl prolyl isomerase domain. The depletion of FKBP52 inhibited nuclear transport of hTERT, resulting in cytoplasmic accumulation. Cytoplasmic hTERT was rapidly degraded through ubiquitin (Ub)-dependent proteolysis, thereby abrogating telomerase activity. In addition, overexpression of dynamitin, which is known to dissociate the dynein-dynactin motor from its cargoes, reduced telomerase activity. Collectively, these results provide a molecular mechanism by which FKBP52 modulates telomerase activity by promoting dynein-dynactin-dependent nuclear import of hTERT.
Asunto(s)
Citoplasma/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Unión a Tacrolimus/metabolismo , Telomerasa/metabolismo , Transporte Activo de Núcleo Celular/genética , Transporte Activo de Núcleo Celular/fisiología , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Humanos , Immunoblotting , Inmunoprecipitación , Prostaglandina-E Sintasas/genética , Prostaglandina-E Sintasas/metabolismo , Interferencia de ARN , Proteínas de Unión a Tacrolimus/genética , Telomerasa/genética , Técnicas del Sistema de Dos Híbridos , Ubiquitinación/genética , Ubiquitinación/fisiologíaRESUMEN
Human telomeres associate with shelterin, a six-protein complex that protects chromosome ends from being recognized as sites of DNA damage. The shelterin subunit TRF2 (telomeric repeat-binding factor 2) protects telomeres by facilitating their organization into the protective capping structure. We have reported previously that the DNA-PKcs (DNA-dependent protein kinase catalytic subunit)-interacting protein KIP associates with telomerase through an interaction with hTERT (human telomerase reverse transcriptase). In the present study, we identify KIP as a novel interacting partner of TRF2. KIP is able to interact with both TRF2 and DNA-PKcs at telomeres. Because KIP is required for the association between TRF2 and DNA-PKcs, the interplay of these three proteins may provide a mechanism for the recruitment of DNA-PKcs to telomeres. We also show that KIP binding to TRF2 enhances the telomere-binding activity of TRF2, suggesting that KIP acts as a positive regulator of TRF2 function. Furthermore, depletion of KIP induces DNA-damage response foci at telomeres, thereby leading to induction of growth arrest, cellular senescence and altered cell cycle distribution. Collectively, our findings suggest that KIP, in addition to its association with catalytically active telomerase, plays important roles in the maintenance of functional telomeres and the regulation of telomere-associated DNA-damage response. Thus KIP represents a new pathway for modulating telomerase and telomere function in cancer.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Daño del ADN , Telomerasa/metabolismo , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Proteínas de Unión al Calcio/genética , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Senescencia Celular , Humanos , Telomerasa/genética , Telómero/genética , Proteína 2 de Unión a Repeticiones Teloméricas/genéticaRESUMEN
2-Cysteine (Cys) peroxiredoxins (Prxs), which include mammalian Prxs I-IV, possess two conserved Cys residues that are readily oxidized by H(2)O(2) to form a disulfide. In the case of Prx I-III, the disulfide is reduced by thioredoxin, thus enabling these proteins to function as peroxidases. Prx IV was shown previously to be synthesized as a 31-kDa polypeptide with an NH(2)-terminal signal peptide that is subsequently cleaved to generate a 27-kDa form of the protein that is localized to the endoplasmic reticulum. A form of Prx IV, larger than 27 kDa revealed by immunoblot analysis was suggested to represent the unprocessed, 31-kDa form, but this larger form was detected only in spermatids of the postpubertal testis. We now show that the larger form of Prx IV (here designated Prx IV-L) detected in the testis is actually a product of alternative transcription of the Prx IV gene that is encoded by newly identified exon 1A together with exons 2-7 that are shared with the 27-kDa form (designated Prx IV-S). Prx IV-L was detected in spermatids but not in mature sperm, it could form disulfide-linked dimers but not higher order oligomers via oxidation, and it was resistant to hyperoxidation unless additional reductant was added, suggesting that its peroxidase activity is limited in vivo. Phylogenetic analysis showed that the Prx IV-S gene is present in all vertebrates examined, whereas the Prx IV-L gene was detected only in placental mammals. We suggest that Prx IV-L functions as an H(2)O(2) sensor that mediates protein thiol oxidation required for the maturation of spermatozoa in placental mammals.
Asunto(s)
Exones/fisiología , Peroxirredoxinas/biosíntesis , Maduración Sexual/fisiología , Espermátides/enzimología , Testículo/enzimología , Transcripción Genética/fisiología , Animales , Peróxido de Hidrógeno/metabolismo , Isoenzimas/biosíntesis , Isoenzimas/genética , Masculino , Ratones , Oxidación-Reducción , Peroxirredoxinas/genética , Filogenia , Espermátides/citología , Testículo/citologíaRESUMEN
The receptor activator of nuclear factor-kappa B ligand (RANKL) mediates osteoclast differentiation and functions by inducing Ca2+ oscillations, activating mitogen-activated protein kinases (MAPKs), and activating nuclear factor of activated T-cells type c1 (NFATc1) via the RANK and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) interaction. Reactive oxygen species (ROS) also plays an important role during osteoclastogenesis and Sestrin2, an antioxidant, maintains cellular homeostasis upon stress injury via regulation of ROS, autophagy, and inflammation. However, the role of Sestrin2 in osteoclastogenesis remains unknown. In this study, we investigated the role of Sestrin2 in the RANKL-RANK-TRAF6 signaling pathway during osteoclast differentiation. Deletion of Sestrin2 (Sesn2) increased bone mass and reduced the number of multinucleated osteoclasts on bone surfaces. RANKL-induced osteoclast differentiation and function decreased in Sesn2 knockout (KO) bone marrow-derived monocytes/macrophages (BMMs) due to inhibition of NFATc1 expression, but osteoblastogenesis was not affected. mRNA expression of RANKL-induced specific osteoclastogenic genes and MAPK protein expression were lower in Sesn2 KO BMMs than wild-type (WT) BMMs after RANKL treatment. However, the Sesn2 deletion did not affect ROS generation or intracellular Ca2+ oscillations during osteoclastogenesis. In contrast, the interaction between TRAF6 and p62 was reduced during osteoclasts differentiation in Sesn2 KO BMMs. The reduction in the TRAF6/p62 interaction and TRAP activity in osteoclastogenesis in Sesn2 KO BMMs was recovered to the WT level upon expression of Flag-Sesn2 in Sesn2 KO BMMs. These results suggest that Sestrin2 has a novel role in bone homeostasis and osteoclasts differentiation through regulation of NFATc1 and the TRAF6/p62 interaction.
RESUMEN
This study was undertaken to compare the labeling efficiencies of three iron-oxide based MRI contrast agents [Feridex, Resovist and monocrystalline iron oxide (MION)] and to evaluate their effects on the biological properties of human mesenchymal stem cells (hMSCs). The hMSCs were cultivated for 1 and 7 days after 24-h labeling with iron oxide nanoparticles (12.5 microg Fe/mL) in the presence of poly-L-lysine (0.75 microg/mL). The hMSCs were labeled more efficiently with use of Feridex, Resovist as compared to MION. No significant differences were observed in terms of viability and proliferation of labeled hMSCs. The level of Oct-4 mRNA increased in labeled hMSCs at day 1 and the cellular phenotype changed from CD45-/CD44+/CD29+ to CD45low/CD44+/CD29+ at day 7, which closely resembles the phenotype of fresh bone marrow-derived hMSCs. Our study has demonstrated that the Feridex or Resovist is the preferred labeling agent for hMSCs. There was a change in Oct-4 and CD45 expression after labeling.
Asunto(s)
Medios de Contraste/farmacología , Imagen por Resonancia Magnética , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Biomarcadores/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Compuestos Férricos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hierro/metabolismo , Células Madre Mesenquimatosas/ultraestructura , Nanopartículas/ultraestructura , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Coloración y EtiquetadoRESUMEN
The Drosophila lymph gland, the larval hematopoietic organ comprised of prohemocytes and mature hemocytes, has been a valuable model for understanding mechanisms underlying hematopoiesis and immunity. Three types of mature hemocytes have been characterized in the lymph gland: plasmatocytes, lamellocytes, and crystal cells, which are analogous to vertebrate myeloid cells, yet molecular underpinnings of the lymph gland hemocytes have been less investigated. Here, we use single-cell RNA sequencing to comprehensively analyze heterogeneity of developing hemocytes in the lymph gland, and discover previously undescribed hemocyte types including adipohemocytes, stem-like prohemocytes, and intermediate prohemocytes. Additionally, we identify the developmental trajectory of hemocytes during normal development as well as the emergence of the lamellocyte lineage following active cellular immunity caused by wasp infestation. Finally, we establish similarities and differences between embryonically derived- and larval lymph gland hemocytes. Altogether, our study provides detailed insights into the hemocyte development and cellular immune responses at single-cell resolution.
Asunto(s)
Drosophila melanogaster/citología , Drosophila melanogaster/genética , Hemocitos/citología , Hemocitos/metabolismo , Transcriptoma , Animales , Animales Modificados Genéticamente , Diferenciación Celular/genética , Linaje de la Célula/genética , Drosophila melanogaster/metabolismo , Infestaciones Ectoparasitarias/genética , Infestaciones Ectoparasitarias/metabolismo , Infestaciones Ectoparasitarias/patología , Perfilación de la Expresión Génica , Hematopoyesis/genética , Interacciones Huésped-Parásitos/genética , Interacciones Huésped-Parásitos/fisiología , Tejido Linfoide/citología , Tejido Linfoide/metabolismo , Tejido Linfoide/parasitología , RNA-Seq , Análisis de la Célula Individual , Avispas/patogenicidadRESUMEN
The receptor activator of nuclear factor-kappa B ligand (RANKL) induces osteoclastogenesis by induction of Ca2+ oscillation, calcineurin activation and translocation into the nucleus of nuclear factor of activated T cells type c1 (NFATc1). Homer proteins are scaffold proteins. They regulate Ca2+ signaling by modulating the activity of multiple Ca2+ signaling proteins. Homers 2 and 3, but not Homer1, also independently affect the interaction between NFATc1 and calcineurin. However, to date, whether and how the Homers are involved in osteoclastogenesis remains unknown. In the present study, we investigated Homer2 and Homer3 roles in Ca2+ signaling and NFATc1 function during osteoclast differentiation. Deletion of Homer2/Homer3 (Homer2/3) markedly decreased the bone density of the tibia, resulting in bone erosion. RANKL-induced osteoclast differentiation is greatly facilitated in Homer2/3 DKO bone marrow-derived monocytes/macrophages (BMMs) due to increased NFATc1 expression and nuclear translocation. However, these findings did not alter RANKL-induced Ca2+ oscillations. Of note, RANKL treatment inhibited Homer proteins interaction with NFATc1, but it was restored by cyclosporine A treatment to inhibit calcineurin. Finally, RANKL treatment of Homer2/3 DKO BMMs significantly increased the formation of multinucleated cells. These findings suggest a novel potent mode of bone homeostasis regulation through osteoclasts differentiation. Specifically, we found that Homer2 and Homer3 regulate NFATc1 function through its interaction with calcineurin to regulate RANKL-induced osteoclastogenesis and bone metabolism.
Asunto(s)
Huesos/efectos de los fármacos , Proteínas de Andamiaje Homer/metabolismo , Macrófagos/efectos de los fármacos , Factores de Transcripción NFATC/metabolismo , Osteoclastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Ligando RANK/farmacología , Animales , Densidad Ósea/efectos de los fármacos , Huesos/metabolismo , Calcineurina/metabolismo , Señalización del Calcio/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Proteínas de Andamiaje Homer/genética , Macrófagos/citología , Macrófagos/metabolismo , Ratones Noqueados , Osteoclastos/citología , Osteoclastos/metabolismo , Unión Proteica/efectos de los fármacosRESUMEN
Endoplasmic reticulum (ER) stress is triggered by various cellular stresses that disturb protein folding or calcium homeostasis in the ER. To cope with these stresses, ER stress activates the unfolded protein response (UPR) pathway, but unresolved ER stress induces reactive oxygen species (ROS) accumulation leading to apoptotic cell death. However, the mechanisms that underlie protection from ER stress-induced cell death are not clearly defined. The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway plays a crucial role in the protection of cells against ROS-mediated oxidative damage. Keap1 acts as a negative regulator of Nrf2 activation. In this study, we investigated the role of the Nrf2-Keap1 pathway in protection from ER stress-induced cell death using tunicamycin (TM) as an ER stress inducer. We found that Nrf2 is an essential protein for the prevention from TM-induced apoptotic cell death and its activation is driven by autophagic Keap1 degradation. Furthermore, ablation of p62, an adapter protein in the autophagy process, attenuates the Keap1 degradation and Nrf2 activation that was induced by TM treatment, and thereby increases susceptibility to apoptotic cell death. Conversely, reinforcement of p62 alleviated TM-induced cell death in p62-deficient cells. Taken together, these results demonstrate that p62 plays an important role in protecting cells from TM-induced cell death through Nrf2 activation.
Asunto(s)
Proteínas de Unión al ARN/metabolismo , Proteína Sequestosoma-1/metabolismo , Animales , Apoptosis/fisiología , Autofagia/efectos de los fármacos , Autofagia/fisiología , Muerte Celular/fisiología , Estrés del Retículo Endoplásmico/fisiología , Células HEK293 , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Ratones , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/fisiología , Tunicamicina/farmacologíaRESUMEN
Low density lipoproteins (LDL) play important roles in the pathogenesis of atherosclerosis. Diabetes is associated with accelerated atherosclerosis leading to cardiovascular disease in diabetic patients. Although LDL stimulates the proliferation of arterial smooth muscle cells (SMC), the mechanisms are not fully understood. We examined the effects of native LDL and glycated LDL on the extracellular signal-regulated kinase (ERK) pathway. Addition of native and glycated LDL to rat aorta SMCs (RASMCs) stimulated ERK phosphorylation. ERK phosphorylation was not affected by exposure to the Ca2+ chelator BAPTA-AM but inhibition of protein kinase C (PKC) with GF109203X, inhibition of Src kinase with PP1 (5 microM) and inhibition of phospholipase C (PLC) with U73122/U73343 (5 microM) all reduced ERK phosphorylation in response to glycated LDL. In addition, pretreatment of the RASMCs with a cell-permeable mitogen-activated protein kinase kinase (MEK) inhibitor (PD98059, 5 microM) markedly decreased ERK phosphorylation in response to native and glycated LDL. These findings indicate that ERK phosphorylation in response to glycated LDL involves the activation of PKC, PLC, and MEK, but is independent of intracellular Ca2+.
Asunto(s)
Lipoproteínas LDL/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Músculo Liso Vascular/citología , Animales , Aorta/citología , Línea Celular , Proliferación Celular , Flavonoides/farmacología , Productos Finales de Glicación Avanzada , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Pirazoles/farmacología , Pirimidinas/farmacología , Ratas , Ratas Sprague-Dawley , Fosfolipasas de Tipo C/metabolismo , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/metabolismoRESUMEN
Sestrins (Sesns) protect cells from oxidative stress. The mechanism underlying the antioxidant effect of Sesns has remained unknown, however. The Nrf2-Keap1 pathway provides cellular defense against oxidative stress by controlling the expression of antioxidant enzymes. We now show that Sesn1 and Sesn2 interact with the Nrf2 suppressor Keap1, the autophagy substrate p62, and the ubiquitin ligase Rbx1 and that the antioxidant function of Sesns is mediated through activation of Nrf2 in a manner reliant on p62-dependent autophagic degradation of Keap1. Sesn2 was upregulated in the liver of mice subjected to fasting or subsequent refeeding with a high-carbohydrate, fat-free diet, whereas only refeeding promoted Keap1 degradation and Nrf2 activation, because only refeeding induced p62 expression. Ablation of Sesn2 blocked Keap1 degradation and Nrf2 activation induced by refeeding and thereby increased the susceptibility of the liver to oxidative damage resulting from the acute stimulation of lipogenesis associated with refeeding.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/metabolismo , Hígado/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas/metabolismo , Factores de Transcripción/metabolismo , Animales , Autofagia , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Proteína 1 Asociada A ECH Tipo Kelch , Hígado/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares , Estrés Oxidativo , Peroxidasas , Unión Proteica , Factor de Transcripción TFIIH , Transfección , Regulación hacia ArribaRESUMEN
AIMS: To define the mechanisms underlying pyrazole-induced oxidative stress and the protective role of peroxiredoxins (Prxs) and sulfiredoxin (Srx) against such stress. RESULTS: Pyrazole increased Srx expression in the liver of mice in a nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent manner and induced Srx translocation from the cytosol to the endoplasmic reticulum (ER) and mitochondria. Pyrazole also induced the expression of CYP2E1, a primary reactive oxygen species (ROS) source for ethanol-induced liver injury, in ER and mitochondria. However, increased CYP2E1 levels only partially accounted for the pyrazole-mediated induction of Srx, prompting the investigation of CYP2E1-independent ROS generation downstream of pyrazole. Indeed, pyrazole increased ER stress, which is known to elevate mitochondrial ROS. In addition, pyrazole up-regulated CYP2E1 to a greater extent in mitochondria than in ER. Accordingly, among Prxs I to IV, PrxIII, which is localized to mitochondria, was preferentially hyperoxidized in the liver of pyrazole-treated mice. Pyrazole-induced oxidative damage to the liver was greater in PrxIII(-/-) mice than in wild-type mice. Such damage was also increased in Srx(-/-) mice treated with pyrazole, underscoring the role of Srx as the guardian of PrxIII. INNOVATION: The roles of Prxs, Srx, and ER stress have not been previously studied in relation to pyrazole toxicity. CONCLUSION: The concerted action of PrxIII and Srx is important for protection against pyrazole-induced oxidative stress arising from the convergent induction of CYP2E1-derived and ER stress-derived ROS in mitochondria.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Peroxiredoxina III/metabolismo , Pirazoles/toxicidad , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Peroxiredoxina III/genéticaRESUMEN
Telomere homeostasis is regulated by telomerase and a collection of associated proteins. Telomerase is, in turn, regulated by post-translational modifications of the rate-limiting catalytic subunit hTERT. Here we show that disruption of Hsp90 by geldanamycin promotes efficient ubiquitination and proteasome-mediated degradation of hTERT. Furthermore, we have used the yeast two-hybrid method to identify a novel RING finger gene (MKRN1) encoding an E3 ligase that mediates ubiquitination of hTERT. Overexpression of MKRN1 in telomerase-positive cells promotes the degradation of hTERT and decreases telomerase activity and subsequently telomere length. Our data suggest that MKRN1 plays an important role in modulating telomere length homeostasis through a dynamic balance involving hTERT protein stability.