RESUMO
Successful hematopoietic progenitor cell (HPC) transplant therapy is improved by mobilizing HPCs from the bone marrow niche in donors. Notch receptor-ligand interactions are known to retain HPCs in the bone marrow, and neutralizing antibodies against Notch ligands, Jagged-1 or Delta-like ligand (DLL4), or NOTCH2 receptor potentiates HPC mobilization. Notch-ligand interactions are dependent on posttranslational modification of Notch receptors with O-fucose and are modulated by Fringe-mediated extension of O-fucose moieties. We previously reported that O-fucosylglycans on Notch are required for Notch receptor-ligand engagement controlling hematopoietic stem cell quiescence and retention in the marrow niche. Here, we generated recombinant fragments of NOTCH1 or NOTCH2 extracellular domain carrying the core ligand-binding regions (EGF11-13) either as unmodified forms or as O-fucosylglycan-modified forms. We found that the addition of O-fucose monosaccharide or the Fringe-extended forms of O-fucose to EGF11-13 showed substantial increases in binding to DLL4. Furthermore, the O-fucose and Fringe-extended NOTCH1 EGF11-13 protein displayed much stronger binding to DLL4 than the NOTCH2 counterpart. When assessed in an in vitro 3D osteoblastic niche model, we showed that the Fringe-extended NOTCH1 EGF11-13 fragment effectively released lodged HPC cells with a higher potency than the NOTCH2 blocking antibody. We concluded that O-fucose and Fringe-modified NOTCH1 EGF11-13 protein can be utilized as effective decoys for stem cell niche localized ligands to potentiate HPC egress and improve HPC collection for hematopoietic cell therapy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Fucose/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Animais , Células CHO , Cricetulus , Células HEK293 , Humanos , Receptor Notch1/genética , Receptor Notch2/genéticaRESUMO
Up-regulation of human prion protein (PrP) in patients with pancreatic ductal adenocarcinoma (PDAC) is associated with a poor prognosis. However, the underlying molecular mechanism of PrP-mediated tumorigenesis is not completely understood. In this study, we found that PDAC cell lines can be divided into either PrP high expresser or PrP low expresser. In addition to filamin A (FLNA), PrP interacts with Notch1, forming a PrP/FLNA/Notch1 complex. Silencing PrP in high-expresser cells decreases Notch1 expression and Notch1 signaling. These cells exhibited decreased proliferation, xenograft growth, and tumor invasion but show increased tumor apoptosis. These phenotypes were rescued by ectopically expressed and activated Notch1. By contrast, overexpression of PrP in low expressers increases Notch1 expression and signaling, enhances proliferation, and increases tumor invasion and xenograft growth that can be blocked by a Notch inhibitor. Our data further suggest that PrP increases Notch1 stability likely through suppression of Notch proteosome degradation. Additionally, we found that targeting PrP combined with anti-Notch is much more effective than singularly targeted therapy in retarding PDAC growth. Finally, we show that coexpression of PrP and Notch1 confers an even poorer prognosis than PrP expression alone. Taken together, our results have unraveled a novel molecular pathway driven by interactions between PrP and Notch1 in the progression of PDAC, supporting a critical tumor-promoting role of Notch1 in PrP-expressing PDAC tumors.
Assuntos
Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/patologia , Proteínas Priônicas/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Animais , Apoptose , Carcinoma Ductal Pancreático/metabolismo , Sobrevivência Celular , Progressão da Doença , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica , Neoplasias Pancreáticas/metabolismo , Fenótipo , Proteínas Priônicas/genética , RNA Interferente Pequeno , Receptor Notch1/genética , Regulação para CimaRESUMO
Despite use of newer approaches, some patients being considered for autologous hematopoietic cell transplantation (HCT) may only mobilize limited numbers of hematopoietic progenitor cells (HPCs) into blood, precluding use of the procedure, or being placed at increased risk of complications due to slow hematopoietic reconstitution. Developing more efficacious HPC mobilization regimens and strategies may enhance the mobilization process and improve patient outcome. Although Notch signaling is not essential for homeostasis of adult hematopoietic stem cells (HSCs), Notch-ligand adhesive interaction maintains HSC quiescence and niche retention. Using Notch receptor blocking antibodies, we report that Notch2 blockade, but not Notch1 blockade, sensitizes hematopoietic stem cells and progenitors (HSPCs) to mobilization stimuli and leads to enhanced egress from marrow to the periphery. Notch2 blockade leads to transient myeloid progenitor expansion without affecting HSC homeostasis and self-renewal. We show that transient Notch2 blockade or Notch2-loss in mice lacking Notch2 receptor lead to decreased CXCR4 expression by HSC but increased cell cycling with CXCR4 transcription being directly regulated by the Notch transcriptional protein RBPJ. In addition, we found that Notch2-blocked or Notch2-deficient marrow HSPCs show an increased homing to the marrow, while mobilized Notch2-blocked, but not Notch2-deficient stem cells and progenitors, displayed a competitive repopulating advantage and enhanced hematopoietic reconstitution. These findings suggest that blocking Notch2 combined with the current clinical regimen may further enhance HPC mobilization and improve engraftment during HCT.
Assuntos
Antineoplásicos/farmacologia , Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Receptor Notch2/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Movimento Celular/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Autorrenovação Celular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Mobilização de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Camundongos Transgênicos , Receptor Notch2/deficiência , Receptor Notch2/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Notch is long recognized as a signaling molecule important for stem cell self-renewal and fate determination. Here, we reveal a novel adhesive role of Notch-ligand engagement in hematopoietic stem and progenitor cells (HSPCs). Using mice with conditional loss of O-fucosylglycans on Notch EGF-like repeats important for the binding of Notch ligands, we report that HSPCs with faulty ligand binding ability display enhanced cycling accompanied by increased egress from the marrow, a phenotype mainly attributed to their reduced adhesion to Notch ligand-expressing stromal cells and osteoblastic cells and their altered occupation in osteoblastic niches. Adhesion to Notch ligand-bearing osteoblastic or stromal cells inhibits wild type but not O-fucosylglycan-deficient HSPC cycling, independent of RBP-JK -mediated canonical Notch signaling. Furthermore, Notch-ligand neutralizing antibodies induce RBP-JK -independent HSPC egress and enhanced HSPC mobilization. We, therefore, conclude that Notch receptor-ligand engagement controls HSPC quiescence and retention in the marrow niche that is dependent on O-fucosylglycans on Notch.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Receptores Notch/metabolismo , Nicho de Células-Tronco/genética , Células Estromais/metabolismo , Animais , Humanos , Camundongos , Transdução de SinaisRESUMO
Endocannabinoids modulate multiple behaviors, including learning and memory. We show that the endocannabinoid anandamide (AEA) can alter neuronal cell function both through its established role in activation of the G-protein-coupled receptor CB1, and by serving as a precursor for a potent agonist of the nuclear receptor PPARß/δ, in turn up-regulating multiple cognition-associated genes. We show further that the fatty acid-binding protein FABP5 controls both of these functions in vivo. FABP5 both promotes the hydrolysis of AEA into arachidonic acid and thus reduces brain endocannabinoid levels, and directly shuttles arachidonic acid to the nucleus where it delivers it to PPARß/δ, enabling its activation. In accordance, ablation of FABP5 in mice results in excess accumulation of AEA, abolishes PPARß/δ activation in the brain, and markedly impairs hippocampus-based learning and memory. The data indicate that, by controlling anandamide disposition and activities, FABP5 plays a key role in regulating hippocampal cognitive function.
Assuntos
Ácidos Araquidônicos/metabolismo , Encéfalo/fisiologia , Cognição/fisiologia , Endocanabinoides/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Proteínas de Neoplasias/metabolismo , PPAR delta/metabolismo , PPAR beta/metabolismo , Alcamidas Poli-Insaturadas/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Western Blotting , Encéfalo/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Redes Reguladoras de Genes , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Proteínas de Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , PPAR delta/agonistas , PPAR delta/genética , PPAR beta/agonistas , PPAR beta/genética , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiazóis/farmacologia , Transcriptoma/efeitos dos fármacosRESUMO
Retinoic acid (RA) regulates gene transcription by activating the nuclear receptors retinoic acid receptor (RAR) and peroxisome proliferator-activated receptor (PPAR) ß/δ and their respective cognate lipid-binding proteins CRABP-II and FABP5. RA induces neuronal differentiation, but the contributions of the two transcriptional pathways of the hormone to the process are unknown. Here, we show that the RA-induced commitment of P19 stem cells to neuronal progenitors is mediated by the CRABP-II/RAR path and that the FABP5/PPARß/δ path can inhibit the process through induction of the RAR repressors SIRT1 and Ajuba. In contrast with its inhibitory activity in the early steps of neurogenesis, the FABP5/PPARß/δ path promotes differentiation of neuronal progenitors to mature neurons, an activity mediated in part by the PPARß/δ target gene PDK1. Hence, RA-induced neuronal differentiation is mediated through RAR in the early stages and through PPARß/δ in the late stages of the process. The switch in RA signaling is accomplished by a transient up-regulation of RARß concomitantly with a transient increase in the CRABP-II/FABP5 ratio at early stages of differentiation. In accordance with these conclusions, hippocampi of FABP5-null mice display excess accumulation of neuronal progenitor cells and a deficit in mature neurons versus wild-type animals.
Assuntos
Antineoplásicos/farmacologia , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese/efeitos dos fármacos , Neurônios/metabolismo , PPAR beta/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Ácido Retinoico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tretinoína/farmacologia , Animais , Linhagem Celular , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Hipocampo/citologia , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Camundongos , Camundongos Mutantes , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/citologia , Neurogênese/fisiologia , Neurônios/citologia , PPAR beta/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Receptores Citoplasmáticos e Nucleares/genética , Receptores do Ácido Retinoico/genética , Transdução de Sinais/fisiologia , Sirtuína 1/genética , Sirtuína 1/metabolismoRESUMO
BACKGROUND: Resistance to standard therapy is a major reason for the poor prognosis of pancreatic ductal adenocarcinoma (PDAC). Developing novel therapy to overcome PDAC drug-resistance is urgently needed. CRABP-II was highly expressed in all PDAC but not expressed in normal pancreatic tissues and chronic pancreatitis. CRABP-II was shown to promote PDAC migration and metastasis while its potential role in promoting PDAC drug-resistance was not known. METHODS: A paired cohort of human primary and relapsing PDAC tissues was assessed for CRABP-II expression by immunohistochemistry. CRISPR/cas9 gene editing was used to establish CRABP-II knockout cell lines and MTT assays were performed to assess gemcitabine sensitivity in vitro. Cleaved caspase-3/PARP blots and Annexin V staining were conducted to detect cell apoptosis. Gene expression microarray, Q-PCR, western blots, Co-IP and RNA-IP were used to study the molecular function of CRABP-II. Sucrose gradient ultracentrifugation was applied to isolate lipid rafts and LC-MS-MS was used to assess cholesterol content. Both subcutaneous CDX models and orthotopic PDX models were established to examine the efficacy of SNIPER-11 and the synergistic effect between SNIPER-11 and gemcitabine in vivo. RESULTS: A higher expression of CRABP-II was found in relapsing PDAC tissue and was associated with poor prognosis. Gemcitabine-resistant cell lines exhibited increased level of CRABP-II, while CRABP-II knockout resensitized PDAC cells to gemcitabine. Mechanistically, aberrant expression of CRABP-II increased the stability of SREBP-1c mRNA through cooperation with HuR and upregulated the downstream genes of SREBP-1c to favor cholesterol uptake and accumulation in lipid rafts. Increased lipid raft cholesterol accumulation facilitated ATK survival signaling and PDAC drug resistance. The small compound SNIPER-11 treatment effectively induced CRABP-II protein degradation, induced apoptosis, and suppressed tumor growth. Combination of SNIPER-11 and gemcitabine significantly reduced the lipid raft cholesterol content in CDX/PDX and profoundly inhibited tumor progression. CONCLUSIONS: These findings identified CRABP-II as a novel regulator of cholesterol metabolism and suggested that CRABP-II is a selective target for overcoming PDAC drug resistance.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Receptores do Ácido Retinoico , Apoptose , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Colesterol , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Microdomínios da Membrana , Recidiva Local de Neoplasia/genética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores do Ácido Retinoico/metabolismoRESUMO
The endoplasmic reticulum unfolded protein response (UPR) is a conserved adaptive signaling in ER homeostasis and has emerged as critical in highly proliferating cells and potential treatment target for acute T-cell lymphoblastic leukemia (T-ALL). Methods: in this study, we assessed the transcriptomic and phenotypic alterations in UPR response of the bone marrow endothelial cells (ECs) in mice engrafted with T-ALL and in bone marrow specimens from patients who have T-ALL. We used PERK inhibitor and generated endothelial specific PERK knockout mice to study the impact of PERK on leukemia progression and hematopoiesis. We performed chromatin immunoprecipitation (ChIP) to study the mechanistic regulation of JAG1 by ATF4. We characterized small extracellular vesicles (SEV) from leukemia-developing mice and studied the effect of SEVs on EC function. Results: we found that T-ALL development induced a robust activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK)-dominant UPR in the bone marrow endothelial vascular niche. The activation of PERK-eIF2a-ATF4 axis remodels the vascular niche, upregulates angiogenic factors including VEGFα and ATF4-regulated JAG1, and suppresses the expression of SCF and CXCL12, which are important to HSC maintenance and regeneration. Further, targeting endothelial PERK significantly improved T-ALL outcome. EC-specific deletion of PERK abolished the aberrant JAG1 up-regulation, improved HSC maintenance, promoted leukemia apoptosis, and improved overall survival. Finally, we showed that small extracellular vesicles are critical mediators of endothelial PERK-eIF2a-ATF4 activation and JAG1 up-regulation in leukemia. Corroborating animal model studies, activation of PERK-ATF4-JAG1 is prominent in human T-ALL bone marrow and T-ALL xenografts. Conclusion: our studies thus revealed for the first time that the leukemia-initiated PERK-ATF4-JAG1 axis plays a critical role in the remodeling of the bone marrow vascular niche and that targeting vascular niche UPR is a potential therapeutic opportunity in T-ALL.
Assuntos
Células Endoteliais , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Resposta a Proteínas não Dobradas , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Medula Óssea/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Células Endoteliais/metabolismo , Humanos , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Proteína Jagged-1/farmacologia , Camundongos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , eIF-2 Quinase/metabolismoRESUMO
Filamin A (FLNA) is an integrator of cell mechanics and signaling. The spreading and migration observed in FLNA sufficient A7 melanoma cells but not in the parental FLNA deficient M2 cells have been attributed to FLNA. In A7 and M2 cells, the normal prion (PrP) exists as pro-PrP, retaining its glycosylphosphatidyl-inositol (GPI) anchor peptide signal sequence (GPI-PSS). The GPI-PSS of PrP has a FLNA binding motif and binds FLNA. Reducing PrP expression in A7 cells alters the spatial distribution of FLNA and organization of actin and diminishes cell spreading and migration. Integrin ß1 also binds FLNA. In A7 cells, FLNA, PrP, and integrin ß1 exist as two independent, yet functionally linked, complexes; they are FLNA with PrP or FLNA with integrin ß1. Reducing PrP expression in A7 cells decreases the amount of integrin ß1 bound to FLNA. A PrP GPI-PSS synthetic peptide that crosses the cell membrane inhibits A7 cell spreading and migration. Thus, in A7 cells FLNA does not act alone; the binding of pro-PrP enhances association between FLNA and integrin ß1, which then promotes cell spreading and migration. Pro-PrP is detected in melanoma in situ but not in melanocyte. Invasive melanoma has more pro-PrP. The binding of pro-PrP to FLNA, therefore, contributes to melanomagenesis.
Assuntos
Movimento Celular , Proteínas Contráteis/metabolismo , Regulação Neoplásica da Expressão Gênica , Cadeias beta de Integrinas/metabolismo , Melanoma/metabolismo , Proteínas dos Microfilamentos/metabolismo , Príons/metabolismo , Motivos de Aminoácidos , Linhagem Celular Tumoral , Proteínas Contráteis/genética , Filaminas , Glicosilfosfatidilinositóis/genética , Glicosilfosfatidilinositóis/metabolismo , Humanos , Cadeias beta de Integrinas/genética , Melanoma/genética , Proteínas dos Microfilamentos/genética , Príons/genética , Ligação ProteicaRESUMO
Aberrant protein aggregation causes numerous neurological diseases including Creutzfeldt-Jakob disease (CJD), but the aggregation mechanisms remain poorly understood. Here, we report AFM results on the formation pathways of ß-oligomers and nonfibrillar aggregates from wild-type full-length recombinant human prion protein (WT) and an insertion mutant (10OR) with five additional octapeptide repeats linked to familial CJD. Upon partial denaturing, seeds consisting of 3-4 monomers quickly appeared. Oligomers of ~11-22 monomers then formed through direct interaction of seeds, rather than by subsequent monomer attachment. All larger aggregates formed through association of these ß-oligomers. Although both WT and 10OR exhibited identical aggregation mechanisms, the latter oligomerized faster due to lower solubility and, hence, thermodynamic stability. This novel aggregation pathway has implications for prion diseases as well as others caused by protein aggregation.
Assuntos
Modelos Biológicos , Oligopeptídeos/química , Príons/química , Humanos , Microscopia de Força Atômica , Mutação , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Tamanho da Partícula , Príons/genética , Príons/metabolismoRESUMO
Conventional dendritic cells (cDC) play a central role in T-cell antitumor responses. We studied the significance of Notch-regulated DC immune responses in a mouse model of colitis-associated colorectal cancer in which there is epithelial downregulation of Notch/Hes1 signaling. This defect phenocopies that caused by GMDS (GDP-mannose 4,6-dehydratase) mutation in human colorectal cancers. We found that, although wild-type immune cells restrained dysplasia progression and decreased the incidence of adenocarcinoma in chimeric mice, the immune system with Notch2 deleted in all blood lineages or in only DCs promoted inflammation-associated transformation. Notch2 signaling deficiency not only impaired cDC terminal differentiation, but also downregulated CCR7 expression, reduced DC migration, and suppressed antigen cross-presentation to CD8+ T cells. Transfer of Notch-primed DCs restrained inflammation-associated dysplasia progression. Consistent with the mouse data, we observed a correlation between infiltrating cDC1 and Notch2 signaling in human colorectal cancers and found that GMDS-mutant colorectal cancers showed decreased CCR7 expression and suppressed cDC1 signature gene expression. Suppressed cDC1 gene signature expression in human colorectal cancer was associated with a poor prognosis. In summary, our study supports an important role for Notch2 signaling in cDC1-mediated antitumor immunity and indicates that Notch2-controlled DCs restrain inflammation-associated colon cancer development in mice.
Assuntos
Adenocarcinoma/imunologia , Neoplasias Associadas a Colite/imunologia , Células Dendríticas/imunologia , Receptor Notch2/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Animais , Transplante de Medula Óssea , Linfócitos T CD8-Positivos/imunologia , Carboidratos Epimerases/genética , Carcinogênese/genética , Carcinogênese/imunologia , Linhagem Celular Tumoral , Neoplasias Associadas a Colite/genética , Neoplasias Associadas a Colite/mortalidade , Neoplasias Associadas a Colite/patologia , Apresentação Cruzada , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Hidroliases/genética , Cetona Oxirredutases/genética , Camundongos , Camundongos Knockout , Mutação , Prognóstico , Receptor Notch2/genética , Receptores CCR7/genética , Análise de Sobrevida , Quimeras de TransplanteRESUMO
Susceptibility to scrapie disease in sheep, the archetypal prion disease, correlates with polymorphisms within the ovine PrP (prion-related protein) gene. The VRQ (Val136Arg154Gln171) and AL141RQ (Ala136Leu141Arg154Gln171) allelic variants are associated with classical scrapie, whereas the ARR (Ala136Arg154Arg171), AF141RQ (Ala136Phe141Arg154Gln171) and AHQ (Ala136His154Gln171) allelic variants are associated with atypical scrapie. Recent studies have suggested that there are differences in the stability of PrPSc (abnormal disease-specific conformation of PrP) associated with these different forms of scrapie. To address which structural features of ovine PrP may contribute to this difference, in the present study we have investigated the conformational stability and susceptibility to aggregation of allelic variants of ovine PrP associated with classical or atypical scrapie. We find that the melting temperature of ovine recombinant VRQ and AL141RQ PrP is higher than that of AF141RQ, AHQ and ARR. In addition, monoclonal-antibody studies show that the region around helix-1 of VRQ and AL141RQ is less accessible compared with other ovine PrP allelic variants. Furthermore, the extent of both the structural change to copper-ion-treatment and denaturant-induced aggregation was reduced in PrP associated with atypical scrapie compared with PrP associated with classical scrapie. Through the use of molecular dynamics simulations we have found that these biochemical and biophysical properties of ovine PrP correlate with the ease of unwinding of helix-2 and a concurrent conformational change of the helix-2-helix-3 loop. These results reveal significant differences in the overall stability and potential for aggregation of different allelic variants of ovine PrP and consequently have implications for the differences in stability of PrPSc associated with classical and atypical scrapie.
Assuntos
Proteínas PrPSc/química , Proteínas PrPSc/genética , Ovinos/genética , Alelos , Animais , Anticorpos Monoclonais/imunologia , Cobre/metabolismo , Variação Genética , Genótipo , Modelos Moleculares , Proteínas PrPSc/metabolismo , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ovinos/metabolismoRESUMO
The human prion gene, PRNP, has two allelic forms that encode either a methionine or valine at codon 129. This polymorphism strongly influences the pathogenesis of prion disease. However, the underlying mechanism remains unclear. We compared the conformation between wild-type human prion protein (rPrP(C)) with either a valine or methionine at position 129, using a panel of monoclonal antibodies that are specific for epitopes along the entire protein. We found that rPrP(C(129M)) has a more exposed helix 1 region compared to rPrP(C(129V)). Helix 1 is important in the aggregation process. Accordingly, rPrP(C(129M)) aggregates at a faster rate and forms more aggregate than rPrP(C(129V)). In addition, by using a rPrP with a pathogenic mutation of five additional octapeptide repeat insertions, rPrP((129M)/10OR), as "seeds", we showed that rPrP((129M)/10OR) promotes the aggregation of rPrP(C(129M)) more efficiently than rPrP(C(129V)). These findings provide a possible mechanism underlying the influence of residue 129 on human prion disease.
Assuntos
Príons/química , Príons/genética , Estrutura Quaternária de Proteína , Sequência de Aminoácidos , Epitopos , Humanos , Metionina , Proteínas Priônicas , Conformação ProteicaRESUMO
Mutation in the prion gene, PRNP, accounts for approx. 10-15% of human prion diseases. However, little is known about the mechanisms by which a mutant prion protein (PrP) causes disease. We compared the biochemical properties of a wild-type human prion protein, rPrP(C) (recombinant wild-type PrP), which has five octapeptide-repeats, with two recombinant human prion proteins with insertion mutations, one with three more octapeptide repeats, rPrP(8OR), and the other with five more octapeptide repeats, rPrP(10OR). We found that the insertion mutant proteins are more prone to aggregate, and the degree and kinetics of aggregation are proportional to the number of inserts. The octapeptide-repeat and alpha-helix 1 regions are important in aggregate formation, because aggregation is inhibited with monoclonal antibodies that are specific for epitopes in these regions. We also showed that a small amount of mutant protein could enhance the formation of mixed aggregates that are composed of mutant protein and wild-type rPrP(C). Accordingly, rPrP(10OR) is also more efficient in promoting the aggregation of rPrP(C) than rPrP(8OR). These findings provide a biochemical explanation for the clinical observations that the severity of the disease in patients with insertion mutations is proportional to the number of inserts, and thus have implications for the pathogenesis of inherited human prion disease.
Assuntos
Mutagênese Insercional/genética , Mutação/genética , Príons/genética , Príons/metabolismo , Anticorpos Monoclonais/imunologia , Humanos , Príons/química , Príons/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
Our previous study shows that cellular retinoic acid binding protein II (CRABP-II) is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and pre-cancerous lesions, but not detected in normal pancreatic tissues. In this study, we show that deletion of CRABP-II in PDAC cells by CRISPR/Cas9 does not affect cancer cell proliferation, but decreases cell migration and invasion. Gene expression microarray analysis reveals that IL-8 is one of the top genes whose expression is down-regulated upon CRABP-II deletion, while expression of MMP-2 and MMP-14, two targets of IL-8 are also significantly down-regulated. Moreover, we found that CRABP-II is able to form a complex with HuR, which binds to the 3'UTR of IL-8 messenger RNA (mRNA) and enhances IL-8 mRNA stability. Ectopic expression of flag-CRABP-II in CRABP-II knockout cells is able to rescue the expression of IL-8, MMP-2/MMP-14 and recovers cell migration. Using the orthotopic xenograft model, we further demonstrate that CRABP-II deletion impairs tumor metastasis to nearby lymph nodes. Taken together, our results reveal a novel pathway linking CRABP-II expression to enhanced PDAC metastasis, and hence we propose CRABP-II may serve as a new PDAC therapeutic target.
RESUMO
More than half of T-cell acute lymphoblastic leukemia (T-ALL) patients harbor gain-of-function mutations in the intracellular domain of Notch1. Diffuse infiltration of the bone marrow commonly occurs in T-ALL and relapsed B-cell acute lymphoblastic leukemia patients, and is associated with worse prognosis. However, the mechanism of leukemia outgrowth in the marrow and the resulting biologic impact on hematopoiesis are poorly understood. Here, we investigated targetable cellular and molecular abnormalities in leukemia marrow stroma responsible for the suppression of normal hematopoiesis using a T-ALL mouse model and human T-ALL xenografts. We found that actively proliferating leukemia cells inhibited normal hematopoietic stem and progenitor cell (HSPC) proliferation and homing to the perivascular region. In addition, leukemia development was accompanied by the suppression of the endosteum-lining osteoblast population. We further demonstrated that aberrant Notch activation in the stroma plays an important role in negatively regulating the expression of CXLC12 on osteoblasts and their differentiation. Notch blockade reversed attenuated HSPC cycling, leukemia-associated abnormal blood lineage distribution, and thrombocytopenia as well as recovered osteoblast and HSPC abundance and improved the hematopoietic-supportive functions of osteoblasts. Finally, we confirmed that reduced osteoblast frequency and enhanced Notch signaling were also features of the marrow stroma of human ALL tissues. Collectively, our findings suggest that therapeutically targeting the leukemia-infiltrated hematopoietic niche may restore HSPC homeostasis and improve the outcome of ALL patients.
Assuntos
Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Osteoblastos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Receptores Notch/metabolismo , Microambiente Tumoral/fisiologia , Adolescente , Adulto , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Medula Óssea/patologia , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Criança , Pré-Escolar , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/patologia , Humanos , Lactente , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Transdução de Sinais/fisiologia , Adulto JovemRESUMO
The two common polymorphisms (385A > G: M129V and 655G > A: E219 K) in the human prion gene (PRNP) play important roles in the pathogenesis of Creutzfeldt-Jakob diseases. We screened a total of 626 individuals, who represent three ethnic populations of China, Han, Hui, and Uyghur, for the two polymorphisms. The frequencies of M/M homozygote at residue 129 in these three groups differ significantly. The Han has a much higher frequency (98%) than Hui (85%) and Uyghur (60%). On the other hand, the frequencies of the E/E at residue 219 are higher in Uyghur (98%) and Hui (96%) than in Han (90%). We also investigated two other less common variants of PRNP, a silent substitution at residue 117 (351A > G: A117A), and one octapeptide-repeat deletion (1-OPRD) in the octapeptide-coding region. We found three Uyghur individuals with silent substitution at residue 117. Four Hui (2.0%) and one Han (0.5%) donors were found to be heterozygous for 1-OPRD. A novel three extra-repeat (72 bp) insertion within the octapeptide-coding region was identified in one healthy 11-years-old Hui. Identical mutation was also found in her mother but not her father.
Assuntos
Amiloide/genética , Povo Asiático/genética , Mutagênese Insercional/genética , Polimorfismo Genético , Precursores de Proteínas/genética , China/etnologia , Síndrome de Creutzfeldt-Jakob/etnologia , Síndrome de Creutzfeldt-Jakob/genética , Feminino , Frequência do Gene , Genótipo , Haplótipos , Humanos , Masculino , Mutação/genética , População/genética , Proteínas Priônicas , PríonsRESUMO
The RNA-binding protein HuR binds at 3' untranslated regions (UTRs) of target transcripts, thereby protecting them against degradation. We show that HuR directly interacts with cellular retinoic acid-binding protein 2 (CRABP2), a protein known to transport RA from the cytosol to the nuclear retinoic acid receptor (RAR). Association with CRABP2 dramatically increases the affinity of HuR toward target mRNAs and enhances the stability of such transcripts, including that of Apaf-1, the major protein in the apoptosome. We show further that its cooperation with HuR contributes to the ability of CRABP2 to suppress carcinoma cell proliferation. The data show that CRABP2 displays antioncogenic activities both by cooperating with RAR and by stabilizing antiproliferative HuR target transcripts. The observation that CRABP2 controls mRNA stabilization by HuR reveals that in parallel to participating in transcriptional regulation, the protein is closely involved in posttranscriptional regulation of gene expression.
Assuntos
Proteínas ELAV/metabolismo , Estabilidade de RNA , Receptores do Ácido Retinoico/metabolismo , Animais , Apoproteínas/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Fator Apoptótico 1 Ativador de Proteases/genética , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Proliferação de Células/efeitos dos fármacos , Células HEK293 , Humanos , Camundongos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Estabilidade de RNA/efeitos dos fármacos , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tretinoína/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
CRABP-II, a retinoic acid binding protein, shuffles retinoic acid from cytoplasm into nucleus and forms a complex with nuclear retinoic acid receptor to facilitate transcriptional activities of retinoic acid. In this study, we studied the expression patterns of CRABP-II in pancreatic ductal adenocarcinoma (PDAC) compared with those in normal pancreas, chronic pancreatitis, and precancerous lesions. We showed no detectable expressions of CRABP-II in normal pancreatic parenchyma, normal ductal epithelium, and chronic pancreatitis. In contrast, the expression of CRABP-II was readily detected in all PDACs including metastatic PDACs. CRABP-II staining was also observed and progressively increased from pancreatic intraepithelial neoplasia 1 to 3. In addition, when fine needle aspiration specimens were evaluated from patients with PDAC, CRABP-II was positive in 55.6% cases if cytology diagnosis was "atypia," and in 87.5% cases, if "malignancy." Our study suggests that CRABP-II is highly and specifically expressed in PDAC and is more commonly expressed in high-grade precursor cancerous lesions than in low-grade lesions. Therefore, overexpression of CRABP-II is a late event of pancreatic carcinogenesis, and it could be used as a diagnostic marker to distinguish PDAC from other benign pancreatic conditions in both resection and cytology specimens.