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2.
PLoS One ; 15(4): e0232036, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32343715

RESUMO

The NUP98 and NUP214 nucleoporins (NUPs) are recurrently fused to heterologous proteins in leukemia. The resulting chimeric oncoproteins retain the phenylalanine-glycine (FG) repeat motifs of the NUP moiety that mediate interaction with the nuclear export receptor Crm1. NUP fusion leukemias are characterized by HOXA gene upregulation; however, their molecular pathogenesis remains poorly understood. To investigate the role of Crm1 in mediating the leukemogenic properties of NUP chimeric proteins, we took advantage of the Sequestosome-1 (SQSTM1)-NUP214 fusion. SQSTM1-NUP214 retains only a short C-terminal portion of NUP214 which contains FG motifs that mediate interaction with Crm1. We introduced point mutations targeting these FG motifs and found that the ability of the resulting SQSTM1-NUP214FGmut protein to interact with Crm1 was reduced by more than 50% compared with SQSTM1-NUP214. Mutation of FG motifs affected transforming potential: while SQSTM1-NUP214 impaired myeloid maturation and conferred robust colony formation to transduced hematopoietic progenitors in a serial replating assay, the effect of SQSTM1-NUP214FGmut was considerably diminished. Moreover, SQSTM1-NUP214 caused myeloid leukemia in all transplanted mice, whereas none of the SQSTM1-NUP214FGmut reconstituted mice developed leukemia. These oncogenic effects coincided with the ability of SQSTM1-NUP214 and SQSTM1-NUP214FGmut to upregulate the expression of Hoxa and Meis1 genes in hematopoietic progenitors. Indeed, chromatin immunoprecipitation assays demonstrated that impaired SQSTM1-NUP214 interaction with Crm1 correlated with impaired binding of the fusion protein to Hoxa and Meis1 genes. These findings highlight the importance of Crm1 in mediating the leukemogenic properties of SQSTM1-NUP214, and suggest a conserved role of Crm1 in recruiting oncoproteins to their effector genes.


Assuntos
Proteínas de Homeodomínio/genética , Carioferinas/metabolismo , Leucemia/metabolismo , Proteína Meis1/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína Sequestossoma-1/genética , Motivos de Aminoácidos , Animais , Linhagem Celular , Regulação Neoplásica da Expressão Gênica , Leucemia/genética , Leucemia/patologia , Camundongos , Mutagênese Sítio-Dirigida , Transplante de Neoplasias , Complexo de Proteínas Formadoras de Poros Nucleares/química , Proteínas de Fusão Oncogênica/química , Proteínas de Fusão Oncogênica/genética , Mutação Puntual , Regulação para Cima , Proteína Exportina 1
3.
PLoS One ; 10(6): e0129776, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26075887

RESUMO

PICALM (Phosphatidyl Inositol Clathrin Assembly Lymphoid Myeloid protein) is a ubiquitously expressed protein that plays a role in clathrin-mediated endocytosis. PICALM also affects the internalization and trafficking of SNAREs and modulates macroautophagy. Chromosomal translocations that result in the fusion of PICALM to heterologous proteins cause leukemias, and genome-wide association studies have linked PICALM Single Nucleotide Polymorphisms (SNPs) to Alzheimer's disease. To obtain insight into the biological role of PICALM, we performed gene expression studies of PICALM-deficient and PICALM-expressing cells. Pathway analysis demonstrated that PICALM expression influences the expression of genes that encode proteins involved in cholesterol biosynthesis and lipoprotein uptake. Gas Chromatography-Mass Spectrometry (GC-MS) studies indicated that loss of PICALM increases cellular cholesterol pool size. Isotopic labeling studies revealed that loss of PICALM alters increased net scavenging of cholesterol. Flow cytometry analyses confirmed that internalization of the LDL receptor is enhanced in PICALM-deficient cells as a result of higher levels of LDLR expression. These findings suggest that PICALM is required for cellular cholesterol homeostasis and point to a novel mechanism by which PICALM alterations may contribute to disease.


Assuntos
Colesterol/metabolismo , Homeostase , Proteínas Monoméricas de Montagem de Clatrina/metabolismo , Animais , Vias Biossintéticas/genética , Linhagem Celular , Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Camundongos , Proteínas Monoméricas de Montagem de Clatrina/genética , Especificidade de Órgãos , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores de LDL/metabolismo
4.
Nat Commun ; 5: 4998, 2014 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-25241929

RESUMO

Genome-wide association studies have identified several loci associated with Alzheimer's disease (AD), including proteins involved in endocytic trafficking such as PICALM/CALM (phosphatidylinositol binding clathrin assembly protein). It is unclear how these loci may contribute to AD pathology. Here we show that CALM modulates autophagy and alters clearance of tau, a protein which is a known autophagy substrate and which is causatively linked to AD, both in vitro and in vivo. Furthermore, altered CALM expression exacerbates tau-mediated toxicity in zebrafish transgenic models. CALM influences autophagy by regulating the endocytosis of SNAREs, such as VAMP2, VAMP3 and VAMP8, which have diverse effects on different stages of the autophagy pathway, from autophagosome formation to autophagosome degradation. This study suggests that the AD genetic risk factor CALM modulates autophagy, and this may affect disease in a number of ways including modulation of tau turnover.


Assuntos
Autofagia , Proteínas Monoméricas de Montagem de Clatrina/metabolismo , Proteínas tau/metabolismo , Animais , Proteína 12 Relacionada à Autofagia , Linhagem Celular , Drosophila , Endocitose , Feminino , Fibroblastos/metabolismo , Estudo de Associação Genômica Ampla , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Fagossomos , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Fatores de Risco , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Transfecção , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Peixe-Zebra
5.
Exp Hematol ; 42(12): 1022-1030.e1, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25193880

RESUMO

Iron, an essential nutrient for cellular growth and proliferation, enters cells via clathrin-mediated endocytosis. The clathrin assembly lymphoid myeloid (CALM) protein plays an essential role in the cellular import of iron by clathrin-mediated endocytosis. CALM-AF10 leukemias harbor a single copy of the normal CALM gene and therefore may be more sensitive to the growth-inhibitory effect of iron restriction compared with normal hematopoietic cells. We found that CALM heterozygous (CALM(HET)) murine fibroblasts exhibit signs of iron deficiency, with increased surface transferrin receptor levels and reduced growth rates. CALM(HET) hematopoietic cells are more sensitive in vitro to iron chelators than their wild type counterparts. Iron chelation also displayed toxicity toward cultured CALM(HET)CALM-AF10 leukemia cells, and this effect was additive to that of chemotherapy. In mice transplanted with CALM(HET)CALM-AF10 leukemia, we found that dietary iron restriction reduced tumor burden in the spleen. However, dietary iron restriction, used alone or in conjunction with chemotherapy, did not increase survival of mice with CALM(HET)CALM-AF10 leukemia. In summary, although CALM heterozygosity results in iron deficiency and increased sensitivity to iron chelation in vitro, our data in mice do not suggest that iron depletion strategies would be beneficial for the therapy of CALM-AF10 leukemia patients.


Assuntos
Ferro da Dieta/administração & dosagem , Ferro/metabolismo , Leucemia Experimental/terapia , Proteínas de Fusão Oncogênica/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Benzoatos/farmacologia , Benzoatos/uso terapêutico , Transporte Biológico , Linhagem Celular Tumoral , Terapia por Quelação , Terapia Combinada , Deferasirox , Desferroxamina/farmacologia , Desferroxamina/uso terapêutico , Fibroblastos/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Heterozigoto , Humanos , Quelantes de Ferro/farmacologia , Quelantes de Ferro/uso terapêutico , Ferro da Dieta/farmacocinética , Leucemia Experimental/metabolismo , Camundongos , Camundongos Knockout , Proteínas Monoméricas de Montagem de Clatrina/deficiência , Proteínas Monoméricas de Montagem de Clatrina/genética , Quimera por Radiação , Baço/patologia , Triazóis/farmacologia , Triazóis/uso terapêutico , Carga Tumoral
6.
Nutrients ; 5(8): 2836-59, 2013 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-23887041

RESUMO

Iron is essential for normal cellular function. It participates in a wide variety of cellular processes, including cellular respiration, DNA synthesis, and macromolecule biosynthesis. Iron is required for cell growth and proliferation, and changes in intracellular iron availability can have significant effects on cell cycle regulation, cellular metabolism, and cell division. Perhaps not surprisingly then, neoplastic cells have been found to have higher iron requirements than normal, non-malignant cells. Iron depletion through chelation has been explored as a possible therapeutic intervention in a variety of cancers. Here, we will review iron homeostasis in non-malignant and malignant cells, the widespread effects of iron depletion on the cell, the various iron chelators that have been explored in the treatment of cancer, and the tumor types that have been most commonly studied in the context of iron chelation.


Assuntos
Anemia Ferropriva/tratamento farmacológico , Ferro da Dieta/uso terapêutico , Neoplasias/tratamento farmacológico , Anemia Ferropriva/complicações , Pontos de Checagem do Ciclo Celular , Divisão Celular , Proliferação de Células/efeitos dos fármacos , Quelantes/química , Quelantes/farmacologia , Homeostase/efeitos dos fármacos , Humanos , Neoplasias/complicações
7.
Blood ; 121(23): 4758-68, 2013 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-23487024

RESUMO

The t(10;11) chromosomal translocation gives rise to the CALM-AF10 fusion gene and is found in patients with aggressive and difficult-to-treat hematopoietic malignancies. CALM-AF10-driven leukemias are characterized by HOXA gene up-regulation and a global reduction in H3K79 methylation. DOT1L, the H3K79 methyltransferase, interacts with the octapeptide/leucine zipper domain of AF10, and this region has been shown to be necessary and sufficient for CALM-AF10-mediated transformation. However, the precise role of CALM in leukemogenesis remains unclear. Here, we show that CALM contains a nuclear export signal (NES) that mediates cytoplasmic localization of CALM-AF10 and is necessary for CALM-AF10-dependent transformation. Fusions of the CALM NES (NES(CALM)-AF10) or NES motifs from heterologous proteins (ABL1, Rev, PKIA, APC) in-frame with AF10 are sufficient to immortalize murine hematopoietic progenitors in vitro. The CALM NES is essential for CALM-AF10-dependent Hoxa gene up-regulation and aberrant H3K79 methylation, possibly by mislocalization of DOT1L. Finally, we observed that CALM-AF10 leukemia cells are selectively sensitive to inhibition of nuclear export by Leptomycin B. These findings uncover a novel mechanism of leukemogenesis mediated by the nuclear export pathway and support further investigation of the utility of nuclear export inhibitors as therapeutic agents for patients with CALM-AF10 leukemias.


Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Transformação Celular Neoplásica/patologia , Regulação da Expressão Gênica , Leucemia Experimental/etiologia , Proteínas Monoméricas de Montagem de Clatrina/fisiologia , Sinais de Exportação Nuclear/genética , Proteínas de Fusão Oncogênica/metabolismo , Sequência de Aminoácidos , Animais , Antibióticos Antineoplásicos/farmacologia , Transplante de Medula Óssea , Células Cultivadas , Ácidos Graxos Insaturados/farmacologia , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Leucemia Experimental/metabolismo , Leucemia Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas de Fusão Oncogênica/genética , Transporte Proteico , Homologia de Sequência de Aminoácidos , Taxa de Sobrevida
8.
PLoS One ; 7(8): e44252, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22952941

RESUMO

The ubiquitously expressed phosphatidylinositol binding clathrin assembly (PICALM) protein associates with the plasma membrane, binds clathrin, and plays a role in clathrin-mediated endocytosis. Alterations of the human PICALM gene are present in aggressive hematopoietic malignancies, and genome-wide association studies have recently linked the PICALM locus to late-onset Alzheimer's disease. Inactivating and hypomorphic Picalm mutations in mice cause different degrees of severity of anemia, abnormal iron metabolism, growth retardation and shortened lifespan. To understand PICALM's function, we studied the consequences of PICALM overexpression and characterized PICALM-deficient cells derived from mutant fit1 mice. Our results identify a role for PICALM in transferrin receptor (TfR) internalization and demonstrate that the C-terminal PICALM residues are critical for its association with clathrin and for the inhibitory effect of PICALM overexpression on TfR internalization. Murine embryonic fibroblasts (MEFs) that are deficient in PICALM display several characteristics of iron deficiency (increased surface TfR expression, decreased intracellular iron levels, and reduced cellular proliferation), all of which are rescued by retroviral PICALM expression. The proliferation defect of cells that lack PICALM results, at least in part, from insufficient iron uptake, since it can be corrected by iron supplementation. Moreover, PICALM-deficient cells are particularly sensitive to iron chelation. Taken together, these data reveal that PICALM plays a critical role in iron homeostasis, and offer new perspectives into the pathogenesis of PICALM-associated diseases.


Assuntos
Homeostase , Ferro/metabolismo , Proteínas Monoméricas de Montagem de Clatrina/metabolismo , Aminoácidos/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Clatrina/metabolismo , Embrião de Mamíferos/citologia , Endocitose/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Homeostase/efeitos dos fármacos , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Quelantes de Ferro/farmacologia , Deficiências de Ferro , Camundongos , Proteínas Monoméricas de Montagem de Clatrina/química , Proteínas Monoméricas de Montagem de Clatrina/deficiência , Fenótipo , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo
9.
Sci Signal ; 5(233): ra51, 2012 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-22810897

RESUMO

Chemokine signaling is critical for T cell function during homeostasis and inflammation and directs T cell polarity and migration through the activation of specific intracellular pathways. Here, we uncovered a previously uncharacterized role for the Abl family tyrosine kinases Abl and Arg in the regulation of T cell-dependent inflammatory responses and showed that the Abl family kinases were required for chemokine-induced T cell polarization and migration. Our data demonstrated that Abl and Arg were activated downstream of chemokine receptors and mediated the chemokine-induced tyrosine phosphorylation of human enhancer of filamentation 1 (HEF1), an adaptor protein that is required for the activity of the guanosine triphosphatase Rap1, which mediates cell adhesion and migration. Phosphorylation of HEF1 by Abl family kinases and activation of Rap1 were required for chemokine-induced T cell migration. Mouse T cells that lacked Abl and Arg exhibited defective homing to lymph nodes and impaired migration to sites of inflammation. These findings suggest that Abl family kinases are potential therapeutic targets for the treatment of T cell-dependent immune disorders that are characterized by chemokine-mediated inflammation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular/imunologia , Quimiocinas/metabolismo , Inflamação/imunologia , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Animais , Western Blotting , Adesão Celular/imunologia , Guanosina Trifosfato/metabolismo , Humanos , Camundongos , Fosforilação , Imagem com Lapso de Tempo
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