RESUMO
ß-dystroglycan (ß-DG) assembles with lamins A/C and B1 and emerin at the nuclear envelope (NE) to maintain proper nuclear architecture and function. To provide insight into the nuclear function of ß-DG, we characterized the interaction between ß-DG and emerin at the molecular level. Emerin is a major NE protein that regulates multiple nuclear processes and whose deficiency results in Emery-Dreifuss muscular dystrophy (EDMD). Using truncated variants of ß-DG and emerin, via a series of in vitro and in vivo binding experiments and a tailored computational analysis, we determined that the ß-DG-emerin interaction is mediated at least in part by their respective transmembrane domains (TM). Using surface plasmon resonance assays we showed that emerin binds to ß-DG with high affinity (KD in the nanomolar range). Remarkably, the analysis of cells in which DG was knocked out demonstrated that loss of ß-DG resulted in a decreased emerin stability and impairment of emerin-mediated processes. ß-DG and emerin are reciprocally required for their optimal targeting within the NE, as shown by immunofluorescence, western blotting and immunoprecipitation assays using emerin variants with mutations in the TM domain and B-lymphocytes of a patient with EDMD. In summary, we demonstrated that ß-DG plays a role as an emerin interacting partner modulating its stability and function.
Assuntos
Distroglicanas/metabolismo , Proteínas de Membrana/metabolismo , Distrofia Muscular de Emery-Dreifuss/metabolismo , Proteínas Nucleares/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Linfócitos B/metabolismo , Sítios de Ligação , Linhagem Celular , Células Cultivadas , Distroglicanas/química , Distroglicanas/genética , Células HeLa , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Distrofia Muscular de Emery-Dreifuss/genética , Mutação , Membrana Nuclear/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação ProteicaRESUMO
The study of Hutchinson-Gilford progeria syndrome (HGPS) has provided important clues to decipher mechanisms underlying aging. Progerin, a mutant lamin A, disrupts nuclear envelope structure/function, with further impairment of multiple processes that culminate in senescence. Here, we demonstrate that the nuclear protein export pathway is exacerbated in HGPS, due to progerin-driven overexpression of CRM1, thereby disturbing nucleocytoplasmic partitioning of CRM1-target proteins. Enhanced nuclear export is central in HGPS, since pharmacological inhibition of CRM1 alleviates all aging hallmarks analyzed, including senescent cellular morphology, lamin B1 downregulation, loss of heterochromatin, nuclear morphology defects, and expanded nucleoli. Exogenous overexpression of CRM1 on the other hand recapitulates the HGPS cellular phenotype in normal fibroblasts. CRM1 levels/activity increases with age in fibroblasts from healthy donors, indicating that altered nuclear export is a common hallmark of pathological and physiological aging. Collectively, our findings provide novel insights into HGPS pathophysiology, identifying CRM1 as potential therapeutic target in HGPS.
Assuntos
Senilidade Prematura/metabolismo , Núcleo Celular/metabolismo , Senescência Celular , Carioferinas/metabolismo , Proteínas Nucleares/metabolismo , Progéria/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transporte Ativo do Núcleo Celular , Senilidade Prematura/patologia , Células Cultivadas , Humanos , Fenótipo , Progéria/patologia , Proteína Exportina 1RESUMO
ß-dystroglycan (ß-DG) is a key component of multiprotein complexes in the plasma membrane and nuclear envelope. In addition, ß-DG undergoes two successive proteolytic cleavages that result in the liberation of its intracellular domain (ICD) into the cytosol and nucleus. However, stimuli-inducing ICD cleavage and the physiological relevance of this proteolytic fragment are largely unknown. In this study we show for the first time that ß-DG ICD is targeted to the nucleolus where it interacts with the nuclear proteins B23 and UBF (central factor of Pol I-mediated rRNA gene transcription) and binds to rDNA promoter regions. Interestingly DG silencing results in reduced B23 and UBF levels and aberrant nucleolar morphology. Furthermore, ß-DG ICD cleavage is induced by different nucleolar stressors, including oxidative stress, acidosis, and UV irradiation, which implies its participation in the response to nucleolar stress. Consistent with this idea, overexpression of ß-DG elicited mislocalization and decreased levels of UBF and suppression of rRNA expression, which in turn provoked altered ribosome profiling and decreased cell growth. Collectively our data reveal that ß-DG ICD acts as negative regulator of rDNA transcription by impeding the transcriptional activity of UBF, as a part of the protective mechanism activated in response to nucleolar stress.
Assuntos
Nucléolo Celular/metabolismo , Distroglicanas/metabolismo , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , RNA Ribossômico/biossíntese , Animais , Proliferação de Células/genética , Citoplasma/metabolismo , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Distroglicanas/antagonistas & inibidores , Distroglicanas/genética , Camundongos , Mioblastos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleofosmina , Estresse Oxidativo , Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , Domínios Proteicos/genética , RNA Ribossômico/genética , Ribossomos/metabolismo , Transcrição Gênica , Regulação para Cima/genéticaRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
RESUMO
ß-Dystroglycan (ß-DG) is a plasma membrane protein that has ability to target to the nuclear envelope (NE) to maintain nuclear architecture. Nevertheless, mechanisms controlling ß-DG nuclear localization and the physiological consequences of a failure of trafficking are largely unknown. We show that ß-DG has a nuclear export pathway in myoblasts that depends on the recognition of a nuclear export signal located in its transmembrane domain, by CRM1. Remarkably, NES mutations forced ß-DG nuclear accumulation resulting in mislocalization and decreased levels of emerin and lamin B1 and disruption of various nuclear processes in which emerin (centrosome-nucleus linkage and ß-catenin transcriptional activity) and lamin B1 (cell cycle progression and nucleoli structure) are critically involved. In addition to nuclear export, the lifespan of nuclear ß-DG is restricted by its nuclear proteasomal degradation. Collectively our data show that control of nuclear ß-DG content by the combination of CRM1 nuclear export and nuclear proteasome pathways is physiologically relevant to preserve proper NE structure and activity.
Assuntos
Distroglicanas/metabolismo , Carioferinas/metabolismo , Laminina/metabolismo , Membrana Nuclear/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Linhagem Celular , Distroglicanas/genética , Carioferinas/genética , Laminina/genética , Camundongos , Membrana Nuclear/genética , Complexo de Endopeptidases do Proteassoma/genética , Receptores Citoplasmáticos e Nucleares/genética , Proteína Exportina 1RESUMO
ß-Dystroglycan (ß-DG) is a transmembrane protein with critical roles in cell adhesion, cytoskeleton remodeling and nuclear architecture. This functional diversity is attributed to the ability of ß-DG to target to, and conform specific protein assemblies at the plasma membrane (PM) and nuclear envelope (NE). Although a classical NLS and importin α/ß mediated nuclear import pathway has already been described for ß-DG, the intracellular trafficking route by which ß-DG reaches the nucleus is unknown. In this study, we demonstrated that ß-DG undergoes retrograde intracellular trafficking from the PM to the nucleus via the endosome-ER network. Furthermore, we provided evidence indicating that the translocon complex Sec61 mediates the release of ß-DG from the ER membrane, making it accessible for importins and nuclear import. Finally, we show that phosphorylation of ß-DG at Tyr890 is a key stimulus for ß-DG nuclear translocation. Collectively our data describe the retrograde intracellular trafficking route that ß-DG follows from PM to the nucleus. This dual role for a cell adhesion receptor permits the cell to functionally connect the PM with the nucleus and represents to our knowledge the first example of a cell adhesion receptor exhibiting retrograde nuclear trafficking and having dual roles in PM and NE.
RESUMO
AIMS: Previous reports have demonstrated that alterations or reduced expression of Dystroglycan (Dg) complex (αDg and ßDg subunits) are related to progression and severity of neoplastic solid tissues. Therefore we determined the expression pattern and subcellular distribution of Dg complex in Acute Myeloid Leukemia (AML) primary blasts (M1, M2, and M3 phenotypes), as well as HL-60 and Kasumi-1 leukemia cell lines. Additionally, we evaluated the relative expression of the main enzymes controlling α-Dg glycosylation to ascertain the post-translational modifications in the leukemia cell phenotype. MAIN METHODS: Primary leukemia blasts and leukemia cell lines were processed by confocal analysis to determine the subcellular distribution of α-Dg, ß-Dg, and phosphorylated ß-Dg (Y892), to evaluate the expression pattern of the different Dg species we performed Western Blot (WB) assays, while the messenger RNA (mRNA) expression of enzymes involved in α-Dg glycosylation, such as POMGnT1, POMT1, POMT2, LARGE, FKTN, and FKRP, were evaluated by qualitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Finally, in an attempt to ameliorate the leukemia cell phenotype, we transfected leukemia cells with a plasmid expressing the Dg complex. KEY FINDINGS: The Dg complex was altered in leukemia cells, including decreased mRNA, protein, and α-Dg glycosylated levels, mislocalization of ß-Dg, and a diminution of mRNA expression of LARGE in patients leukemia blasts and in cell lines. Interestingly, the exogenous expression of Dg complex promoted filopodial formation, differentiation, and diminished proliferation, attenuating some HL-60 and Kasumi cells characteristics. SIGNIFICANCE: Dg complex integrity and balance are required for a proper hematopoietic cell function, in that its disruption might contribute to leukemia pathophysiology.
Assuntos
Distroglicanas/genética , Regulação Neoplásica da Expressão Gênica , Leucemia Mieloide Aguda/patologia , Processamento de Proteína Pós-Traducional , Western Blotting , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Células HL-60 , Humanos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Dystroglycan has recently been characterised in blood tissue cells, as part of the dystrophin glycoprotein complex involved in the differentiation process of neutrophils. PURPOSE: In the present study we have investigated the role of dystroglycan in the human promyelocytic leukemic cell line Kasumi-1 differentiated to macrophage-like cells. METHODS: We characterised the pattern expression and subcellular distribution of dystroglycans in non-differentiated and differentiated Kasumi-1 cells. RESULTS: Our results demonstrated by WB and flow cytometer assays that during the differentiation process to macrophages, dystroglycans were down-regulated; these results were confirmed with qRT-PCR assays. Additionally, depletion of dystroglycan by RNAi resulted in altered morphology and reduced properties of differentiated Kasumi-1 cells, including morphology, migration and phagocytic activities although secretion of IL-1ß and expression of markers of differentiation are not altered. CONCLUSION: Our findings strongly implicate dystroglycan as a key membrane adhesion protein involved in actin-based structures during the differentiation process in Kasumi-1 cells.
Assuntos
Actinas/metabolismo , Diferenciação Celular/fisiologia , Distroglicanas/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Regulação para Baixo/fisiologia , Distrofina/metabolismo , Humanos , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Neutrófilos/metabolismo , Interferência de RNA/fisiologiaRESUMO
Platelets are the most prominent elements of blood tissue involved in hemostasis at sites of blood vessel injury. Platelet cytoskeleton is responsible for their shape modifications observed during activation and adhesion to the substratum; therefore the interactions between cytoskeleton and plasma membrane are critical to modulate blood platelet functions. Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to membrane/lipid rafts (MLR) and regulate lateral diffusion of membrane proteins and lipids. Resting, thrombin-activated, and adherent human platelets were processed for biochemical studies including western-blot and immunprecipitation assays and confocal analysis were performed to characterize the interaction of MLR with the main cytoskeleton elements and ß-dystroglycan as well as with the association of caveolin-1 PY14 with focal adhesion proteins. We transfected a megakaryoblast cell line (Meg-01) to deplete ß-dystroglycan, subsequent to their differentiation to the platelet progenitors. Our data showed a direct interaction of the MLR with cytoskeleton to regulate platelet shape, while an association of caveolin-1 PY14 with vinculin is needed to establish focal adhesions, which are modulated for ß-dystroglycan. In conclusion, caveolin-1 PY14 in association with platelet cytoskeleton participate in focal adhesions dynamics.
Assuntos
Plaquetas/citologia , Caveolina 1/metabolismo , Citoesqueleto/metabolismo , Microdomínios da Membrana/metabolismo , Vinculina/metabolismo , Plaquetas/metabolismo , Adesão Celular , Diferenciação Celular , Linhagem Celular , Distroglicanas/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Células Progenitoras de Megacariócitos/citologia , Trombina/metabolismoRESUMO
Dystroglycan has recently been characterized in blood tissue cells, as part of the dystrophin glycoprotein complex but to date nothing is known of its role in the differentiation process of neutrophils. We have investigated the role of dystroglycan in the human promyelocytic leukemic cell line HL-60 differentiated to neutrophils. Depletion of dystroglycan by RNAi resulted in altered morphology and reduced properties of differentiated HL-60 cells, including chemotaxis, respiratory burst, phagocytic activities and expression of markers of differentiation. These findings strongly implicate dystroglycan as a key membrane adhesion protein involved in the differentiation process in HL-60 cells.