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NF-κB transcription factors are critical regulators of innate and adaptive immunity and major mediators of inflammatory signaling. The NF-κB signaling is dysregulated in a significant number of cancers and drives malignant transformation through maintenance of constitutive pro-survival signaling and downregulation of apoptosis. Overactive NF-κB signaling results in overexpression of pro-inflammatory cytokines, chemokines and/or growth factors leading to accumulation of proliferative signals together with activation of innate and select adaptive immune cells. This state of chronic inflammation is now thought to be linked to induction of malignant transformation, angiogenesis, metastasis, subversion of adaptive immunity, and therapy resistance. Moreover, accumulating evidence indicates the involvement of NF-κB signaling in induction and maintenance of invasive phenotypes linked to epithelial to mesenchymal transition (EMT) and metastasis. In this review we summarize reported links of NF-κB signaling to sequential steps of transition from epithelial to mesenchymal phenotypes. Understanding the involvement of NF-κB in EMT regulation may contribute to formulating optimized therapeutic strategies in cancer. Video Abstract.
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FN-kappa B , Neoplasias , Humanos , FN-kappa B/metabolismo , Transición Epitelial-Mesenquimal/genética , Transducción de Señal , Neoplasias/metabolismo , Transformación Celular Neoplásica , Fenotipo , Línea Celular TumoralRESUMEN
Dysregulation of the adaptor protein Abelson interactor 1 (ABI1) is linked to malignant transformation. To interrogate the role of ABI1 in cancer development, we mapped the ABI1 interactome using proximity-dependent labeling (PDL) with biotin followed by mass spectrometry. Using a novel PDL data filtering strategy, considering both peptide spectral matches and peak areas of detected peptides, we identified 212 ABI1 proximal interactors. These included WAVE2 complex components such as CYFIP1, NCKAP1, or WASF1, confirming the known role of ABI1 in the regulation of actin-polymerization-dependent processes. We also identified proteins associated with the TAK1-IKK pathway, including TAK1, TAB2, and RIPK1, denoting a newly identified function of ABI1 in TAK1-NF-κB inflammatory signaling. Functional assays using TNFα-stimulated, ABI1-overexpressing or ABI1-deficient cells showed effects on the TAK1-NF-kB pathway-dependent signaling to RIPK1, with ABI1-knockout cells being less susceptible to TNFα-induced, RIPK1-mediated, TAK1-dependent apoptosis. In sum, our PDL-based strategy enabled mapping of the ABI1 proximal interactome, thus revealing a previously unknown role of this adaptor protein in TAK1/RIPK1-based regulation of cell death and survival.
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Proteómica , Factor de Necrosis Tumoral alfa , Humanos , Factor de Necrosis Tumoral alfa/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Transducción de Señal , FN-kappa B/metabolismo , Apoptosis/fisiología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
While the bone marrow (BM) microenvironment is significantly remodelled in acute myeloid leukaemia (AML), molecular insight into AML-specific alterations in the microenvironment has been historically limited by the analysis of liquid marrow aspirates rather than core biopsies that contain solid-phase BM stroma. We assessed the effect of anthracycline- and cytarabine-based induction chemotherapy on both haematopoietic and non-haematopoietic cells directly in core BM biopsies using RNA-seq and histological analysis. We compared matched human core BM biopsies at diagnosis and 2 weeks after cytarabine- and anthracycline-based induction therapy in responders (<5% blasts present after treatment) and non-responders (≥5% blasts present after treatment). Our data indicated enrichment in vimentin (VIM), platelet-derived growth factor receptor beta (PDGFRB) and Snail family transcriptional repressor 2 (SNAI2) transcripts in responders, consistent with the reactivation of the mesenchymal population in the BM stroma. Enrichment of osteoblast maturation-related transcripts of biglycan (BGN), osteopontin (SPP1) and osteonectin (SPARC) was observed in non-responders. To the best of our knowledge, this is the first report demonstrating distinct osteogenic and mesenchymal transcriptome profiles specific to AML response to induction chemotherapy assessed directly in core BM biopsies. Detailing treatment response-specific alterations in the BM stroma may inform optimised therapeutic strategies for AML.
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Médula Ósea , Leucemia Mieloide Aguda , Humanos , Médula Ósea/patología , Transcriptoma , Leucemia Mieloide Aguda/tratamiento farmacológico , Citarabina/uso terapéutico , Células de la Médula Ósea/patología , Antraciclinas/uso terapéutico , Biopsia , Microambiente TumoralRESUMEN
The development and use of murine myeloid progenitor cell lines that are conditionally immortalized through expression of HoxB8 has provided a valuable tool for studies of neutrophil biology. Recent work has extended the utility of HoxB8-conditional progenitors to the in vivo setting via their transplantation into irradiated mice. Here, we describe the isolation of HoxB8-conditional progenitor cell lines that are unique in their ability to engraft in the naïve host in the absence of conditioning of the hematopoietic niche. Our results indicate that HoxB8-conditional progenitors engraft in a ß1 integrin-dependent manner and transiently generate donor-derived mature neutrophils. Furthermore, we show that neutrophils derived in vivo from transplanted HoxB8-conditional progenitors are mobilized to the periphery and recruited to sites of inflammation in a manner that depends on the C-X-C chemokine receptor 2 and ß2 integrins, the same mechanisms that have been described for recruitment of endogenous primary neutrophils. Together, our studies advance the understanding of HoxB8-conditional neutrophil progenitors and describe an innovative tool that, by virtue of its ability to engraft in the naïve host, will facilitate mechanistic in vivo experimentation on neutrophils.
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The diagnosis of parenchymal central nervous system (CNS) invasion and prediction of risk for future CNS recurrence are major challenges in the management of aggressive lymphomas, and accurate biomarkers are needed to supplement clinical risk predictors. For this purpose, we studied the results of a next-generation sequencing (NGS)-based assay that detects tumor-derived DNA for clonotypic immunoglobulin gene rearrangements in the cerebrospinal fluid (CSF) of patients with lymphomas. Used as a diagnostic tool, the NGS-minimal residual disease (NGS-MRD) assay detected clonotypic DNA in 100% of CSF samples from 13 patients with known CNS involvement. They included 7 patients with parenchymal brain disease only, whose CSF tested negative by standard cytology and flow cytometry, and 6 historical DNA aliquots collected from patients at a median of 39 months before accession, which had failed to show clonal rearrangements using standard polymerase chain reaction. For risk prognostication, we prospectively collected CSF from 22 patients with newly diagnosed B-cell lymphomas at high clinical risk of CNS recurrence, of whom 8 (36%) had detectable clonotypic DNA in the CSF. Despite intrathecal prophylaxis, a positive assay of CSF was associated with a 29% cumulative risk of CNS recurrence within 12 months of diagnosis, in contrast with a 0% risk among patients with negative CSF (P = .045). These observations suggest that detection of clonotypic DNA can aid in the diagnosis of suspected parenchymal brain recurrence in aggressive lymphoma. Furthermore, the NGS-MRD assay may enhance clinical risk assessment for CNS recurrence among patients with newly diagnosed lymphomas and help select those who may benefit most from novel approaches to CNS-directed prophylaxis.
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Linfoma de Células B , Linfoma no Hodgkin , Biomarcadores , Sistema Nervioso Central , ADN , HumanosRESUMEN
BACKGROUND: Extracellular vesicles (EVs) are heterogeneous lipid bilayer particles secreted by cells. EVs contain proteins, RNA, DNA and other cargo that can have immunomodulatory effects. Cancer-derived EVs have been described as having immunomodulating effects in vivo with immunosuppressive and pro-tumor growth capabilities. However, cancer-derived EVs have also been harnessed and utilized for anti-cancer potential. METHODS: To assess the immunomodulatory effect of EVs produced by acute myeloid leukemia (AML) cells, we isolated vesicles secreted by the murine AML cell line, C1498, and investigated their effect on in vitro and in vivo immune responses. RESULTS: These leukemia-derived EVs were found to induce increased proliferation of CD3+ cells and enhanced cytolytic activity of CD3+ cells directed toward leukemic cells in vitro. Injection of leukemia-derived EVs into syngeneic naïve mice induced T cell responses in vivo and resulted in enhanced immune responses upon T cell re-stimulation in vitro. CONCLUSION: These findings indicate that C1498-derived EVs have immunomodulatory effects on cell-mediated immune responses that could potentially be utilized to facilitate anti-leukemia immune responses.
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Although the pathogenesis of primary myelofibrosis (PMF) and other myeloproliferative neoplasms (MPNs) is linked to constitutive activation of the JAK-STAT pathway, JAK inhibitors have neither curative nor MPN-stem cell-eradicating potential, indicating that other targetable mechanisms are contributing to the pathophysiology of MPNs. We previously demonstrated that Abelson interactor 1 (Abi-1), a negative regulator of Abelson kinase 1, functions as a tumor suppressor. Here we present data showing that bone marrow-specific deletion of Abi1 in a novel mouse model leads to development of an MPN-like phenotype resembling human PMF. Abi1 loss resulted in a significant increase in the activity of the Src family kinases (SFKs), STAT3, and NF-κB signaling. We also observed impairment of hematopoietic stem cell self-renewal and fitness, as evidenced in noncompetitive and competitive bone marrow transplant experiments. CD34+ hematopoietic progenitors and granulocytes from patients with PMF showed decreased levels of ABI1 transcript as well as increased activity of SFKs, STAT3, and NF-κB. In aggregate, our data link the loss of Abi-1 function to hyperactive SFKs/STAT3/NF-κB signaling and suggest that this signaling axis may represent a regulatory module involved in the molecular pathophysiology of PMF.
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Proteínas Adaptadoras Transductoras de Señales/genética , Médula Ósea/patología , Proteínas del Citoesqueleto/genética , Eliminación de Gen , Mielofibrosis Primaria/genética , Mielofibrosis Primaria/patología , Animales , Médula Ósea/metabolismo , Autorrenovación de las Células , Células Cultivadas , Regulación hacia Abajo , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , FN-kappa B/metabolismo , Mielofibrosis Primaria/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Familia-src Quinasas/metabolismoRESUMEN
The introduction of tyrosine kinase inhibitors (TKI) has transformed chronic myeloid leukemia (CML) into a chronic disease with long-term survival exceeding 85%. However, resistance of CML stem cells to TKI may contribute to the 50% relapse rate observed after TKI discontinuation in molecular remission. We previously described a model of resistance to imatinib mesylate (IM), in which K562 cells cultured in high concentrations of imatinib mesylate showed reduced Bcr-Abl1 protein and activity levels while maintaining proliferative potential. Using quantitative phosphoproteomic analysis of these IM-resistant cells, we have now identified significant upregulation of tumor progression locus (Tpl2), also known as cancer Osaka thyroid (COT1) kinase or Map3k8. Overexpression of Tpl2 in IM-resistant cells was accompanied by elevated activities of Src family kinases (SFKs) and NF-κB, MEK-ERK signaling. CD34+ cells isolated from the bone marrow of patients with CML and exposed to IMin vitro showed increased MAP3K8 transcript levels. Dasatinib (SFK inhibitor), U0126 (MEK inhibitor), and PS-1145 (IκB kinase (IKK) inhibitor) used in combination resulted in elimination of 65% of IM-resistant cells and reduction in the colony-forming capacity of CML CD34+ cells in methylcellulose assays by 80%. In addition, CML CD34+ cells cultured with the combination of inhibitors showed reduced MAP3K8 transcript levels. Overall, our data indicate that elevated Tpl2 protein and transcript levels are associated with resistance to IM and that combined inhibition of SFK, MEK, and NF-κB signaling attenuates the survival of IM-resistant CML cells and CML CD34+ cells. Therefore, combination of SFK, MEK, and NF-κB inhibitors may offer a new therapeutic approach to overcome TKI resistance in CML patients.
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Mesilato de Imatinib/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Mesilato de Imatinib/farmacología , Modelos Biológicos , Fosfopéptidos/metabolismo , Proteómica , Transducción de Señal , Familia-src Quinasas/metabolismoRESUMEN
Despite the success of tyrosine kinase inhibitor (TKI) therapy in chronic myelogenous leukemia (CML), leukemic stem/progenitor cells remain detectable even in the state of deep molecular remission. Mechanisms that allow them to persist despite continued kinase inhibition remain unclear. We have previously shown that prolonged exposure to imatinib mesylate (IM) results in dysregulation of Akt/Erk 1/2 signaling, upregulation of miR-181a, enhanced adhesiveness, and resistance to high IM. To characterize the molecular basis and reversibility of those effects, we applied gene and protein expression analysis, quantitative phosphoproteomics, and direct miR-181a inhibition to our cellular model of CML cells subjected to prolonged exposure to IM. Those cells demonstrated upregulation of pluripotency markers (SOX2, SALL4) and adhesion receptors (CD44, VLA-4, CXCR4), as well as downregulation of Hippo signaling and upregulation of transcription coactivator YAP. Furthermore, inhibition of miR-181a using a microRNA sponge inhibitor resulted in decreased transcription of SOX2 and SALL4, decreased activation of YAP, and increased sensitivity to IM. Our findings indicate that long-term exposure to IM results in dysregulation of stem cell renewal-regulatory Hippo/YAP signaling, acquisition of expression of stem cell markers and that experimental interference with YAP activity may help to restore chemosensitivity to TKI.
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Proteínas Adaptadoras Transductoras de Señales/genética , Regulación hacia Abajo/efectos de los fármacos , Mesilato de Imatinib/farmacología , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Vía de Señalización Hippo , Humanos , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , MicroARNs/genética , Fosfoproteínas/metabolismo , Complejo GPIb-IX de Glicoproteína Plaquetaria/genética , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Transducción de Señal/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAPRESUMEN
INTRODUCTION: During studies on chemotherapy-induced apoptosis in lymphoid cells, we noted that aggregation of spectrin occurred early in apoptosis, i.e. before activation of initiator caspase(s) and prior to exposure of phosphatidylserine (PS). We also found that protein kinase C theta (PKC-θ) co-localized with spectrin in these aggregates. Our previously published studies indicated that in formation of early apoptotic spectrin aggregates, either PKC-θ or other apoptosis-related proteins are involved. Taking into consideration above data, we decided to test the effect of PKC-θ and Fas-associated death domain protein (FADD) on spectrin aggregation in these cells during tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. MATERIAL AND METHODS: For PKC-θ gene (PRKCQ) or FADD gene expression silencing in Jurkat T cells we used lentiviral particles containing shRNA and scrambled shRNA, respectively. Spectrin aggregates were detected by Western blotting after Triton-X 100 extraction in pellet and soluble fractions or by confocal imaging. RESULTS: TRAIL-induced apoptosis results in spectrin aggregation and leads to translocation and aggregation of PKC-θ. We found that phorbol-myristate acetate, a PKC activator and translocation inducer, has only a small effect on spectrin aggregation. To further confirm this, we have also shown that knock down ofPRKCQin Jurkat T cells accelerates the formation of TRAIL-induced spectrin aggregates. Transient overexpression of theß-spectrin C-terminal fragment, containing multiple S/T phosphorylation sites, potential substrate sites for PKC-θ, accelerated the formation of spectrin aggregates. Silencing of downstream TRAIL receptor effector gene,FADD, delayed aggregation of spectrin, but did not reduce PKC-θ localization to the plasma membrane. CONCLUSIONS: In summary, our results show for the first time involvement of spectrin aggregation in TRAIL receptor-FADD apoptotic pathway and indicate that TRAIL-induced spectrin aggregate formation is mediated by FADD and negatively regulated by PKC-θ.
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Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Espectrina/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Caspasas/metabolismo , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Proteína de Dominio de Muerte Asociada a Fas/genética , Humanos , Isoenzimas/biosíntesis , Isoenzimas/genética , Células Jurkat , Linfocitos , Fosforilación , Proteína Quinasa C/biosíntesis , Proteína Quinasa C/genética , Proteína Quinasa C-theta , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Transducción de Señal , Linfocitos T/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
It was shown previously that an ankyrin-sensitive, phosphatidylethanolamine/phosphatidylcholine (PE/PC) binding site maps to the N-terminal part of the ankyrin-binding domain of ß-spectrin (ankBDn). Here we have identified the amino acid residues within this domain which are responsible for recognizing monolayers and bilayers composed of PE/PC mixtures. In vitro binding studies revealed that a quadruple mutant with substituted hydrophobic residues W1771, L1775, M1778 and W1779 not only failed to effectively bind PE/PC, but its residual PE/PC-binding activity was insensitive to inhibition with ankyrin. Structure prediction and analysis, supported by in vitro experiments, suggests that "opening" of the coiled-coil structure underlies the mechanism of this interaction. Experiments on red blood cells and HeLa cells supported the conclusions derived from the model and in vitro lipid-protein interaction results, and showed the potential physiological role of this binding. We postulate that direct interactions between spectrin ankBDn and PE-rich domains play an important role in stabilizing the structure of the spectrin-based membrane skeleton.
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Ancirinas/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Espectrina/química , Espectrina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión/genética , Sitios de Unión/fisiología , Western Blotting , Dicroismo Circular , Transferencia Resonante de Energía de Fluorescencia , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación , Unión Proteica/genética , Unión Proteica/fisiología , Espectrina/genéticaRESUMEN
Abl interactor 1 (Abi1) plays a critical function in actin cytoskeleton dynamics through participation in the WAVE2 complex. To gain a better understanding of the specific role of Abi1, we generated a conditional Abi1-KO mouse model and MEFs lacking Abi1 expression. Abi1-KO cells displayed defective regulation of the actin cytoskeleton, and this dysregulation was ascribed to altered activity of the WAVE2 complex. Changes in motility of Abi1-KO cells were manifested by a decreased migration rate and distance but increased directional persistence. Although these phenotypes did not correlate with peripheral ruffling, which was unaffected, Abi1-KO cells exhibited decreased dorsal ruffling. Western blotting analysis of Abi1-KO cell lysates indicated reduced levels of the WAVE complex components WAVE1 and WAVE2, Nap1, and Sra-1/PIR121. Although relative Abi2 levels were more than doubled in Abi1-KO cells, the absolute Abi2 expression in these cells amounted only to a fifth of Abi1 levels in the control cell line. This finding suggests that the presence of Abi1 is critical for the integrity and stability of WAVE complex and that Abi2 levels are not sufficiently increased to compensate fully for the loss of Abi1 in KO cells and to restore the integrity and function of the WAVE complex. The essential function of Abi1 in WAVE complexes and their regulation might explain the observed embryonic lethality of Abi1-deficient embryos, which survived until approximately embryonic day 11.5 and displayed malformations in the developing heart and brain. Cells lacking Abi1 and the conditional Abi1-KO mouse will serve as critical models for defining Abi1 function.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Encéfalo/embriología , Proteínas del Citoesqueleto/metabolismo , Embrión de Mamíferos/embriología , Corazón/embriología , Complejos Multiproteicos/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Línea Celular , Movimiento Celular/fisiología , Proteínas del Citoesqueleto/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Complejos Multiproteicos/genética , Familia de Proteínas del Síndrome de Wiskott-Aldrich/genéticaRESUMEN
Fatty acyl-CoA esters are extremely important in cellular homeostasis. They are intermediates in both lipid metabolism and post-translational protein modifications. Among these modification events, protein palmitoylation seems to be unique by its reversibility which allows dynamic regulation of the protein hydrophobicity. The recent discovery of an enzyme family that catalyze protein palmitoylation has increased the understanding of the enzymology of the covalent attachment of fatty acids to proteins. Despite that, the molecular mechanism of supplying acyl-CoA esters to this reaction is yet to be established. Acyl-coenzyme A-binding proteins are known to bind long-chain acyl-CoA esters with very high affinity. Therefore, they play a significant role in intracellular acyl-CoA transport and pool formation. The purpose of this work is to explore the potential of one of the acyl-CoA-binding proteins to participate in the protein palmitoylation. In this study, a recombinant form of ACBP derived from human erythroid cells was expressed in E. coli, purified, and functionally characterized. We demonstrate that recombinant hACBP effectively binds palmitoyl-CoA in vitro, undergoing a shift from a monomeric to a dimeric state, and that this ligand-binding ability is involved in erythrocytic membrane phosphatidylcholine (PC) remodeling but not in protein acylation.
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Inhibidor de la Unión a Diazepam/química , Inhibidor de la Unión a Diazepam/metabolismo , Células Eritroides/metabolismo , Regulación de la Expresión Génica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Inhibidor de la Unión a Diazepam/aislamiento & purificación , Inhibidor de la Unión a Diazepam/farmacología , Células Eritroides/química , Escherichia coli/genética , Humanos , Lipoilación/efectos de los fármacos , Datos de Secuencia Molecular , Unión Proteica , Isoformas de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacologíaRESUMEN
It was previously shown that the beta-spectrin ankyrin-binding domain binds lipid domains rich in PE in an ankyrin-dependent manner, and that its N-terminal sequence is crucial in interactions with phospholipids. In this study, the effect of the full-length ankyrin-binding domain of beta-spectrin on natural erythrocyte and HeLa cell membranes was tested. It was found that, when encapsulated in resealed erythrocyte ghosts, the protein representing the full-length ankyrin-binding domain strongly affected the shape and barrier properties of the erythrocyte membrane, and induced partial spectrin release from the membrane, while truncated mutants had no effect. As found previously (Bok et al. Cell Biol. Int. 31 (2007) 1482-94), overexpression of the full-length GFP-tagged ankyrin-binding domain aggregated and induced aggregation of endogenous spectrin, but this was not the case with overexpression of proteins truncated at their N-terminus. Here, we show that the aggregation of spectrin was accompanied by the aggregation of integral membrane proteins that are known to be connected to spectrin via ankyrin, i.e. Na(+)K(+)ATP-ase, IP3 receptor protein and L1 CAM. By contrast, the morphology of the actin cytoskeleton remained unchanged and aggregation of cadherin E or N did not occur upon the overexpression of either full-length or truncated ankyrin-binding domain proteins. The obtained results indicate a substantial role of the lipid-binding part of the beta-spectrin ankyrin-binding domain in the determination of the membrane and spectrin-based skeleton functional properties.
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Ancirinas/metabolismo , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Espectrina/metabolismo , Citoesqueleto de Actina/ultraestructura , Actinas/metabolismo , Ancirinas/química , Sitios de Unión , Cadherinas/metabolismo , Citoesqueleto , Eritrocitos/citología , Células HeLa , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Espectrina/química , Espectrina/genéticaRESUMEN
The major component of the cell membrane skeleton, spectrin, is anchored in the cell membrane via interactions with membrane proteins. It has been previously shown that both erythroid and non-erythroid spectrin interact directly with membrane phospholipids (mainly aminophospholipids). One of the binding sites responsible for these interactions is located in the ankyrin-binding domain. In the present study, in order to better understand the character of binding, a more detailed investigation of the interactions between the beta-spectrin fragments corresponding to the truncated mutants of the ankyrin-binding domain (Frag1 and Frag3) and liposomes of different compositions were carried out. The obtained results suggest that the binding of both spectrin fragments with liposomes induces conformational changes within the protein. Analysis of the changes in intrinsic tryptophan fluorescence spectra upon binding with liposomes, together with quenching studies (from the water and membrane hydrocarbon environment), allows for qualitative description of changes in proteins conformation. Our results suggest that the largest conformational changes occur for Frag1 bound to PC : PE (2 : 3) liposomes what is consistent with previous studies on monolayers. They are also in good agreement with those obtained previously for native erythroid and nonerythroid spectrin molecules.
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Sitios de Unión/genética , Membrana Dobles de Lípidos/química , Espectrina/química , Espectrina/genética , Algoritmos , Animales , Ancirinas/química , Fluorescencia , Liposomas/química , Potenciales de la Membrana , Modelos Químicos , Modelos Moleculares , Mutación , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Análisis Espectral , Triptófano/química , Agua/químicaRESUMEN
It was previously shown that ankyrins play a crucial role in the membrane skeleton arrangement. Purifying ankyrinR obtained from erythrocytes is a time-consuming process. Therefore, cloned and bacterially expressed ankyrinR-spectrin-binding domain (AnkSBD) is a demanded tool for studying spectrin-ankyrin interactions. In this communication, we report on the cloning and purification of AnkSBD and describe the results of binding experiments, in which we showed high-affinity interactions between the AnkSBD construct and isolated erythrocyte or non-erythroid spectrins. pEGFP-AnkSBD-transfected cells co-localised with non-erythroid spectrin in HeLa cells. The functional interactions of the AnkSBD construct in vivo and in vitro open many possibilities to study the structure and function of this domain, which has not yet been as extensively studied when compared to the aminoterminal domain of this protein.
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Ancirinas/metabolismo , Espectrina/metabolismo , Ancirinas/genética , Ancirinas/aislamiento & purificación , Secuencia de Bases , Cromatografía de Afinidad , Clonación Molecular , Cartilla de ADN , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Células HeLa , Humanos , Microscopía Fluorescente , Plásmidos , Reacción en Cadena de la Polimerasa , Unión Proteica , Espectrina/genética , Espectrina/aislamiento & purificaciónRESUMEN
It is known that erythroid and non-erythroid spectrins binding of vesicles and monolayers containing PE proved sensitive to inhibition by red blood cell ankyrin. We now show that the bacterially-expressed recombinant peptides representing betaII(brain)-spectrin's ankyrin-binding domain and its truncated mutants showed lipid-binding activity, although only those containing a full-length amino terminal fragment showed high to moderate affinity towards phospholipid mono- and bilayers and a substantial sensitivity of this binding to inhibition by ankyrin. These results are in accordance with our published data on betaI-spectrin's ankyrin-binding domain [Hryniewicz-Jankowska A, et al. Mapping of ankyrin-sensitive, PE/PC mono- and bilayer binding site in erythroid beta-spectrin. Biochem J 2004;382:677-85]. Moreover, we tested also the effect of transient transfection of living cells of several cell-lines with vectors coding for GFP-conjugates including betaII and also betaI full-length ankyrin-binding domain and their truncated fragments on the membrane skeleton organization. The transfection with constructs encoding full-length ankyrin-binding domain of betaII and betaI spectrin resulted in increased aggregation of membrane skeleton and its punctate appearance in contrast to near normal appearance of membrane skeleton of cells transiently transfected with GFP control or construct encoding ankyrin-binding domain truncated at their N-terminal region. Our results therefore indicate the importance of N-terminal region for lipid-binding activity of the beta-spectrin ankyrin-binding domain and its substantial role in maintaining the spectrin-based skeleton distribution.
Asunto(s)
Ancirinas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Lípidos de la Membrana/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Algoritmos , Animales , Ancirinas/genética , Sitios de Unión , Línea Celular , Dicroismo Circular/métodos , Escherichia coli/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa/metabolismo , Humanos , Liposomas/metabolismo , Melanoma Experimental/metabolismo , Ratones , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Pliegue de Proteína , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Alineación de Secuencia , Eliminación de Secuencia , Relación Estructura-Actividad , Resonancia por Plasmón de SuperficieRESUMEN
Understanding drug-membrane and drug-membrane protein interactions would be a crucial step towards understanding the action and biological properties of anthracyclines, as the cell membrane with its integral and peripheral proteins is the first barrier encountered by these drugs. In this paper, we briefly describe mitoxantrone-monolayer and mitoxantrone-bilayer interactions, focusing on the effect of mitoxantrone on the interactions between erythroid or nonerythroid spectrin with phosphatidylethanolamine-enriched mono- and bilayers. We found that mitoxantrone markedly modifies the interaction of erythroid and nonerythroid spectrins with phosphatidylethanolamine/phosphatidylcholine (PE/PC) monolayers. The change in delta pi induced by spectrins is several-fold larger in the presence of 72 nM mitoxantrone than in its absence: spectrin/mitoxantrone complexes induced a strong compression of the monolayer. Spin-labelling experiments showed that spectrin/mitoxantrone complexes caused significant changes in the order parameter measured using a 5'-doxyl stearate probe in the bilayer, but they practically did not affect the mobility of 16'-doxyl stearate. These results indicate close-to-surface interactions/penetrations without significant effect on the mid-region of the hydrophobic core of the bilayer. The obtained apparent equilibrium dissociation constants indicated relatively similar mitoxantrone-phospholipid and mitoxantrone-spectrin (erythroid and nonerythroid) binding affinities. These results might in part, explain the effect of mitoxantrone on spectrin distribution in the living cells.
Asunto(s)
Mitoxantrona/farmacología , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Espectrina/metabolismo , Animales , Sitios de Unión , Bovinos , Dicroismo Circular , Membrana Eritrocítica/metabolismo , Células Eritroides/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Liposomas/metabolismo , Fluidez de la Membrana/efectos de los fármacos , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Marcadores de SpinRESUMEN
The object of this paper is to review briefly the studies on the interactions of erythroid and non-erythroid spectrins with lipids in model and natural membranes. An important progress on the identification of lipid-binding sites has recently been made although many questions remain still unanswered. In particular, our understanding of the physiological role of such interactions is still limited. Another important issue is the occurrence of spectrins in membrane rafts, how they are attached to the raft and what is their function in rafts.