RESUMEN
The signal recognition particle (SRP), responsible for co-translational protein targeting and delivery to cellular membranes, depends on the native long-hairpin fold of its RNA to confer functionality. Since RNA initiates folding during its synthesis, we used high-resolution optical tweezers to follow in real time the co-transcriptional folding of SRP RNA. Surprisingly, SRP RNA folding is robust to transcription rate changes and the presence or absence of its 5'-precursor sequence. The folding pathway also reveals the obligatory attainment of a non-native hairpin intermediate (H1) that eventually rearranges into the native fold. Furthermore, H1 provides a structural platform alternative to the native fold for RNase P to bind and mature SRP RNA co-transcriptionally. Delays in attaining the final native fold are detrimental to the cell, altogether showing that a co-transcriptional folding pathway underpins the proper biogenesis of function-essential SRP RNA.
Asunto(s)
Pliegue del ARN/genética , ARN/genética , Partícula de Reconocimiento de Señal/genética , Transcripción Genética/genética , Escherichia coli/genética , Unión Proteica/genética , Ribosomas/genéticaRESUMEN
Disaggregation of amyloid fibrils is a fundamental biological process required for amyloid propagation. However, due to the lack of experimental systems, the molecular mechanism of how amyloid is disaggregated by cellular factors remains poorly understood. Here, we established a robust in vitro reconstituted system of yeast prion propagation and found that heat-shock protein 104 (Hsp104), Ssa1 and Sis1 chaperones are essential for efficient disaggregation of Sup35 amyloid. Real-time imaging of single-molecule fluorescence coupled with the reconstitution system revealed that amyloid disaggregation is achieved by ordered, timely binding of the chaperones to amyloid. Remarkably, we uncovered two distinct prion strain conformation-dependent modes of disaggregation, fragmentation and dissolution. We characterized distinct chaperone dynamics in each mode and found that transient, repeated binding of Hsp104 to the same site of amyloid results in fragmentation. These findings provide a physical foundation for otherwise puzzling in vivo observations and for therapeutic development for amyloid-associated neurodegenerative diseases.
Asunto(s)
Priones , Proteínas de Saccharomyces cerevisiae , Amiloide/química , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Factores de Terminación de Péptidos/metabolismo , Priones/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Self-propagating ß-sheet-rich fibrillar protein aggregates, amyloid fibers, are often associated with cellular dysfunction and disease. Distinct amyloid conformations dictate different physiological consequences, such as cellular toxicity. However, the origin of the diversity of amyloid conformation remains unknown. Here, we suggest that altered conformational equilibrium in natively disordered monomeric proteins leads to the adaptation of alternate amyloid conformations that have different phenotypic effects. We performed a comprehensive high-resolution structural analysis of Sup35NM, an N-terminal fragment of the Sup35 yeast prion protein, and found that monomeric Sup35NM harbored latent local compact structures despite its overall disordered conformation. When the hidden local microstructures were relaxed by genetic mutations or solvent conditions, Sup35NM adopted a strikingly different amyloid conformation, which redirected chaperone-mediated fiber fragmentation and modulated prion strain phenotypes. Thus, dynamic conformational fluctuations in natively disordered monomeric proteins represent a posttranslational mechanism for diversification of aggregate structures and cellular phenotypes.
Asunto(s)
Amiloide , Factores de Terminación de Péptidos , Priones , Proteínas de Saccharomyces cerevisiae , Amiloide/química , Amiloide/metabolismo , Cinética , Resonancia Magnética Nuclear Biomolecular , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Priones/química , Priones/genética , Priones/metabolismo , Conformación Proteica , Pliegue de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoAsunto(s)
Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Agregado de Proteínas , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Animales , Supervivencia Celular , Humanos , Agregado de Proteínas/fisiología , Conformación Proteica , Pliegue de Proteína , Transporte de Proteínas , Proteínas/química , Proteínas/metabolismoRESUMEN
Antiangiogenesis therapies are now part of the standard repertoire of cancer therapies, but the mechanisms for the proliferation and survival of endothelial cells are not fully understood. Although endothelial cells are covered with a glycocalyx, little is known about how endothelial glycosylation regulates endothelial functions. Here, we show that alpha2,6-sialic acid is necessary for the cell-surface residency of platelet endothelial cell adhesion molecule (PECAM), a member of the immunoglobulin superfamily that plays multiple roles in cell adhesion, mechanical stress sensing, antiapoptosis, and angiogenesis. As a possible underlying mechanism, we found that the homophilic interactions of PECAM in endothelial cells were dependent on alpha2,6-sialic acid. We also found that the absence of alpha2,6-sialic acid down-regulated the tyrosine phosphorylation of PECAM and recruitment of Src homology 2 domain-containing protein-tyrosine phosphatase 2 and rendered the cells more prone to mitochondrion-dependent apoptosis, as evaluated using PECAM- deficient endothelial cells. The present findings open up a new possibility that modulation of glycosylation could be one of the promising strategies for regulating angiogenesis.
Asunto(s)
Apoptosis , Ácido N-Acetilneuramínico/fisiología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Transducción de Señal , Animales , Células Cultivadas , Células Endoteliales , Endotelio Vascular/citología , Glicosilación , Humanos , Ratones , Ratones Noqueados , Neovascularización Fisiológica , Fosforilación , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismoRESUMEN
Acyclic retinoid (ACR) is currently under clinical trial as an agent to suppress the recurrence of hepatocellular carcinoma (HCC) through its ability to induce apoptosis in premature HCC cells. ACR has an anticancer effect in vivo as well, although it shows weak apoptosis-inducing activity against mature HCC cells, suggesting the existence of an additional action mechanism. In this study, we investigated the antiangiogenic activity of ACR. ACR inhibited angiogenesis within chicken chorioallantoic membrane (CAM) in as similar a manner as all-trans retinoic acid (atRA). Although suppression of angiogenesis by atRA was partially rescued by the simultaneous addition of angiopoietin-1, suppression of angiogenesis by ACR was not rescued under the same condition at all. Conversely, although suppression of angiogenesis by ACR was partially inverted by the simultaneous addition of vascular endothelial growth factor (VEGF), suppression of angiogenesis by atRA was not affected under the same condition. These results suggested that mechanisms underlying the suppression of angiogenesis by ACR and atRA were different. ACR selectively inhibited the phosphorylation of VEGF receptor 2 (VEGFR2) and of extracellular signal-regulated kinase (ERK) without changing their protein expression levels, and inhibited endothelial cell growth, migration, and tube formation. The inhibition of the phosphorylation of ERK, endothelial growth, migration, tube formation, and angiogenesis by ACR was rescued by the overexpression of constitutively active mitogen-activated protein kinase (MAPK). Finally, ACR, but not atRA, inhibited HCC-induced angiogenesis in a xenografted CAM model. These results delineate the novel activity of ACR as an antiangiogenic through a strong inhibition of the VEGFR2 MAPK pathway.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Neovascularización Patológica/prevención & control , Neovascularización Fisiológica/efectos de los fármacos , Tretinoina/análogos & derivados , Animales , Aorta/citología , Carcinoma Hepatocelular/irrigación sanguínea , Bovinos , División Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Membrana Corioalantoides/irrigación sanguínea , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Neoplasias Hepáticas Experimentales/irrigación sanguínea , Neoplasias Hepáticas Experimentales/patología , Trasplante de Neoplasias , Fosforilación/efectos de los fármacos , Trasplante Heterólogo , Tretinoina/farmacología , Venas Umbilicales/citología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Spliceostatin A (SSA) is a methylated derivative of an antitumor natural product FR901464, which specifically binds and inhibits the SF3b spliceosome sub-complex. To investigate the selective antitumor activity of SSA, we focused on the regulation of vascular endothelial growth factor (VEGF) mRNA, since VEGF is a key regulatory component in tumor angiogenesis and known for the intricate regulation of mRNA processing, such as alternative splicing. We found that in HeLa cells SSA reduced the amount of both mRNA and protein of VEGF. Spliceostatin A not only inhibited the splicing reaction of VEGF pre-mRNA but also reduced the total amount of VEGF's transcripts, while SSA affected GAPDH mRNA to a lesser extent. Given a significant reduction in VEGF gene expression, SSA was expected to possess anti-angiogenic activity in vivo. Indeed, SSA inhibited cancer cell-derived angiogenesis in vivo in a chicken chorioallantoic membrane (CAM) assay. The inhibition of angiogenesis with SSA was abolished by addition of exogenous VEGF. We also performed global gene expression analyses of HeLa cells and found that the expression levels of 38% of total genes including VEGF decreased to <50% of the basal levels following 16 h of SSA treatment. These results suggest that the global interference of gene expression including VEGF in tumor cells is at least one of the mechanisms by which SSA (or FR901464) exhibits its strong antitumor activity.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neovascularización Patológica/genética , Piranos/farmacología , Compuestos de Espiro/farmacología , Factor A de Crecimiento Endotelial Vascular/genética , Inhibidores de la Angiogénesis/química , Animales , Western Blotting , Embrión de Pollo , Membrana Corioalantoides/irrigación sanguínea , Membrana Corioalantoides/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Estructura Molecular , Neovascularización Patológica/metabolismo , Neovascularización Patológica/prevención & control , Análisis de Secuencia por Matrices de Oligonucleótidos , Piranos/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Compuestos de Espiro/química , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
KLF6 (Krüppel-like factor 6) is a transcription factor and tumour suppressor with a growing range of biological activities and transcriptional targets. Among these, KLF6 suppresses growth through transactivation of TGF-beta1 (transforming growth factor-beta1). KLF6 can be alternatively spliced, generating lower-molecular-mass isoforms that antagonize the full-length WT (wild-type) protein and promote growth. A key target gene of full-length KLF6 is endoglin, which is induced in vascular injury. Endoglin, a homodimeric cell membrane glycoprotein and TGF-beta auxiliary receptor, has a pro-angiogenic role in endothelial cells and is also involved in malignant progression. The aim of the present work was to explore the effect of TGF-beta on KLF6 expression and splicing, and to define the contribution of TGF-beta on promoters regulated by co-operation between KLF6 and Sp1 (specificity protein 1). Using co-transfection, co-immunoprecipitation and fluorescence resonance energy transfer, our data demonstrate that KLF6 co-operates with Sp1 in transcriptionally regulating KLF6-responsive genes and that this co-operation is further enhanced by TGF-beta1 through at least two mechanisms. First, in specific cell types, TGF-beta1 may decrease KLF6 alternative splicing, resulting in a net increase in full-length, growth-suppressive KLF6 activity. Secondly, KLF6-Sp1 co-operation is further enhanced by the TGF-beta-Smad (similar to mothers against decapentaplegic) pathway via the likely formation of a tripartite KLF6-Sp1-Smad3 complex in which KLF6 interacts indirectly with Smad3 through Sp1, which may serve as a bridging molecule to co-ordinate this interaction. These findings unveil a finely tuned network of interactions between KLF6, Sp1 and TGF-beta to regulate target genes.
Asunto(s)
Empalme Alternativo/genética , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteína smad3/metabolismo , Factor de Transcripción Sp1/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Colágeno/metabolismo , Citometría de Flujo , Transferencia Resonante de Energía de Fluorescencia , Células HeLa , Humanos , Inmunoprecipitación , Factor 6 Similar a Kruppel , Reacción en Cadena de la Polimerasa , Unión Proteica/genética , Unión Proteica/fisiología , Proteína smad3/genética , Factor de Transcripción Sp1/genéticaRESUMEN
Shikonin and beta-hydroxyisovalerylshikonin (beta-HIVS) from Lithospermum erythrorhizon inhibit angiogenesis via inhibition of vascular endothelial growth factor receptors (VEGFR) in an adenosine triphosphate-non-competitive manner, although the underlying molecular mechanism has not been fully understood. In the present study, we found that beta-HIVS inhibited angiogenesis within chicken chorioallantoic membrane approximately threefold more efficiently than shikonin. beta-HIVS also significantly inhibited angiogenesis in two other assays, induced either by Lewis lung carcinoma cells implanted in mouse dorsal skin or by VEGF in s.c. implanted Matrigel plugs and metastasis of Lewis lung carcinoma cells to lung. Therefore, using beta-HIVS as a bioprobe, we investigated the molecular mechanism of shikonin's anti-angiogenic actions. beta-HIVS inhibited the phosphorylation and expression of VEGFR2 and Tie2 without affecting VEGFR1 and fibroblast growth factor receptor 1 levels. beta-HIVS suppressed the phosphorylation but not the expression of extracellular signal-regulated kinase, and an Sp1-dependent transactivation of the VEGFR2 and Tie2 promoters, thereby suppressing the proliferation of vascular endothelial and progenitor cells. This was mimicked by an Sp1 inhibitor mithramycin A and partially rescued by Sp1 overexpression. These results implicate potential use of shikonin and beta-HIVS as leading compounds for clinical application in the future by virtue of their unique properties including: (i) inhibition of VEGFR2 and Tie2 phosphorylation in an adenosine triphosphate-non-competitive manner; (ii) simultaneous inhibition of the phosphorylation and expression of VEGFR2 and Tie2; and (iii) bifunctional inhibition of the growth in endothelial cells and vascular remodeling.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Medicamentos Herbarios Chinos/farmacología , Naftoquinonas/farmacología , Neovascularización Patológica/prevención & control , Animales , Apoptosis , Western Blotting , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Adhesión Celular , Movimiento Celular , Proliferación Celular , Células Cultivadas , Pollos , Membrana Corioalantoides/metabolismo , Membrana Corioalantoides/patología , Ensayo de Cambio de Movilidad Electroforética , Femenino , Técnicas para Inmunoenzimas , Inmunoprecipitación , Luciferasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosforilación , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Transfección , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Angiogenesis is an inevitable event in tumor progression and metastasis, and thus has been a compelling target for cancer therapy in recent years. Effective inhibition of tumor progression and metastasis could become a promising way to treat tumor-induced angiogenesis. We discovered that a fungus, Neosartorya sp., isolated from a soil sample, produced a new angiogenesis inhibitor, which we designated azaspirene. Azaspirene was previously shown to inhibit human umbilical vein endothelial cell (HUVEC) migration induced by vascular endothelial growth factor (VEGF) at an effective dose, 100% of 27 micromol/L without significant cell toxicity. In the present study, we investigated the antiangiogenic activity of azaspirene in vivo. Azaspirene treatment reduced the number of tumor-induced blood vessels. Administration of azaspirene at 30 microg/egg resulted in inhibition of angiogenesis (23.6-45.3% maximum inhibition relative to the controls) in a chicken chorioallantoic membrane assay. Next, we elucidated the molecular mechanism of antiangiogenesis of azaspirene. We investigated the effects of azaspirene on VEGF-induced activation of the mitogen-activated protein kinase signaling pathway in HUVEC. In vitro experiments indicated that azaspirene suppressed Raf-1 activation induced by VEGF without affecting the activation of kinase insert domain-containing receptor/fetal liver kinase 1 (VEGF receptor 2). Additionally, azaspirene preferentially inhibited the growth of HUVEC but not that of the non-vascular endothelial cells NIH3T3, HeLa, MSS31, and MCF-7. Taken together, these results demonstrate that azaspirene is a novel inhibitor of angiogenesis and Raf-1 activation that contains a unique carbon skeleton in its molecular structure.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Células Endoteliales/efectos de los fármacos , Neovascularización Patológica/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-raf/metabolismo , Pirrolidinonas/farmacología , Compuestos de Espiro/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Embrión de Pollo , HumanosRESUMEN
BACKGROUND: Neurodegenerative diseases involving protein aggregation often accompany psychiatric symptoms. Frontotemporal lobar degeneration (FTLD) associated with TAR DNA-binding protein 43 (TDP-43) aggregation is characterized by progressive neuronal atrophy in frontal and temporal lobes of cerebral cortex. Furthermore, patients with FTLD display mental dysfunction in multiple behavioral dimensions. Nevertheless, their molecular origin for psychiatric symptoms remains unclear. METHODS: In FTLD neurons and mouse models with TDP-43 aggregates, we examined coaggregation between TDP-43 and disrupted in schizophrenia 1 (DISC1), a key player in the pathology of mental conditions and its effects on local translation in dendrites and psychiatric behaviors. The protein coaggregation and the expression level of synaptic proteins were also investigated with postmortem brains from patients with FTLD (n = 6). RESULTS: We found cytosolic TDP-43/DISC1 coaggregates in brains of both FTLD mouse model and patients with FTLD. At the mechanistic levels, the TDP-43/DISC1 coaggregates disrupted the activity-dependent dendritic local translation through impairment of translation initiation and, in turn, reduced synaptic protein expression. Behavioral deficits detected in FTLD model mice were ameliorated by exogenous DISC1 expression. CONCLUSIONS: Our findings reveal a novel role of the aggregate-prone TDP-43/DISC1 protein complex in regulating local translation, which affects aberrant behaviors relevant to multiple psychiatric dimensions.
Asunto(s)
Conducta Animal , Encéfalo/metabolismo , Proteínas de Unión al ADN/metabolismo , Degeneración Lobar Frontotemporal/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Agregado de Proteínas , Agregación Patológica de Proteínas/metabolismo , Biosíntesis de Proteínas , Animales , Encéfalo/fisiopatología , Modelos Animales de Enfermedad , Degeneración Lobar Frontotemporal/fisiopatología , Humanos , RatonesRESUMEN
Most of the angiogenesis inhibitors clinically used in cancer treatment target the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) pathway. However, the current strategies for treating angiogenesis have limited efficacy. The issue of how to treat angiogenesis and endothelial dysfunction in cancer remains a matter of substantial debate. Here we demonstrate a glycosylation-dependent regulatory mechanism for tumor angiogenesis. St6gal1-/- mice, lacking the α2,6-sialylation enzyme, were shown to exhibit impaired tumor angiogenesis through enhanced endothelial apoptosis. In a previous study, St6gal1-/- endothelial cells exhibited a reduction in the cell surface residency of platelet endothelial cell adhesion molecule (PECAM). In this study, we found that cooperative functionality of PECAM-VEGFR2-integrin ß3 was disturbed in St6gal1-/- mice. First, cell surface PECAM-VEGFR2 complexes were lost, and both VEGFR2 internalization and the VEGFR-dependent signaling pathway were enhanced. Second, enhanced anoikis was observed, suggesting that the absence of α2,6-sialic acid leads to dysregulated integrin signaling. Notably, ectopic expression of PECAM increased cell surface integrin-ß3, indicating that the reduction of cell surface integrin-ß3 involves loss-of-endothelial PECAM. The results suggest that the cell surface stability of these glycoproteins is significantly reduced by the lack of α2,6-sialic acid, leading to abnormal signal transduction. The present findings highlight that α2,6-sialylation is critically involved in endothelial survival by controlling the cell surface stability and signal transduction of angiogenic molecules, and could be a novel target for anti-angiogenesis therapy.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Integrina beta3/metabolismo , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Apoptosis/fisiología , Células CHO , Células Cultivadas , Cricetulus , Glicosilación , Humanos , Ratones , Sialiltransferasas/metabolismo , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: Sangivamycin, an antibiotic with anti-tumor and anti-herpes virus activities by inhibiting both DNA/RNA synthesis and protein kinase C activity, was reported to suppress selectively DNA synthesis and growth of human umbilical vein endothelial cells and their tube formation in vitro. Here, to address the potential clinical use of sangivamycin in future, we investigated its anti-angiogenic effect in in vivo chicken chorioallantoic membrane (CAM) and mouse dorsal air sac (DAS) assays, and investigated underlying mechanism. METHODS: The effect of sangivamycin on blood vessel formation in CAM was observed under the microscope after treating for two days. For DAS assays, chambers fulfilled with tumor cells were implanted beneath mouse dorsal skin. After the mice were administered with sangivamycin, tumor-induced angiogenesis was observed under the microscope. The effect of sangivamycin on ATP synthesis on the endothelial cell surface was assayed by measuring ATP production with bioluminescence assay. RESULTS: Sangivamycin suppressed angiogenesis within CAM down to 94-71%, which was partially blocked by simultaneous addition of a 40-fold excess of adenosine. Sangivamycin also inhibited tumor-angiogenesis in the DAS assay by 61%, and suppressed ATP production on the endothelial cell surface by 75%. CONCLUSION: Sangivamycin inhibits the in vivo angiogenesis within CAM and tumor-induced angiogenesis within mouse dorsal skin, at least in part via inhibiting endothelial cell surface ATP metabolism in addition to inhibition of DNA/RNA synthesis and/or protein kinase C activity, suggesting a potential clinical use of sangivamycin as a novel anti-cancer reagent capable of targeting not only cancer cells but also endothelial cells.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antibióticos Antineoplásicos/farmacología , Membrana Corioalantoides/irrigación sanguínea , Membrana Corioalantoides/efectos de los fármacos , Endotelio/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Nucleósidos de Pirimidina/farmacología , Sacos Aéreos , Animales , Línea Celular , Pollos , Femenino , Ratones , Ratones Endogámicos ICR , Neovascularización Patológica/tratamiento farmacológico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Huntington's disease (HD) is a polyglutamine (polyQ) disease caused by aberrant expansion of the polyQ tract in Huntingtin (HTT). While motor impairment mediated by polyQ-expanded HTT has been intensively studied, molecular mechanisms for nonmotor symptoms in HD, such as psychiatric manifestations, remain elusive. Here we have demonstrated that HTT forms a ternary protein complex with the scaffolding protein DISC1 and cAMP-degrading phosphodiesterase 4 (PDE4) to regulate PDE4 activity. We observed pathological cross-seeding between DISC1 and mutant HTT aggregates in the brains of HD patients as well as in a murine model that recapitulates the polyQ pathology of HD (R6/2 mice). In R6/2 mice, consequent reductions in soluble DISC1 led to dysregulation of DISC1-PDE4 complexes, aberrantly increasing the activity of PDE4. Importantly, exogenous expression of a modified DISC1, which binds to PDE4 but not mutant HTT, normalized PDE4 activity and ameliorated anhedonia in the R6/2 mice. We propose that cross-seeding of mutant HTT and DISC1 and the resultant changes in PDE4 activity may underlie the pathology of a specific subset of mental manifestations of HD, which may provide an insight into molecular signaling in mental illness in general.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Enfermedad de Huntington/enzimología , Proteínas del Tejido Nervioso/metabolismo , Agregación Patológica de Proteínas/enzimología , Animales , Femenino , Células HEK293 , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Ratones Transgénicos , MutaciónRESUMEN
Inhibition by all-trans retinoic acid (atRA) of the microvasculature formation in chicken chorioallantoic membrane (CAM) accompanied remarkably reduced numbers of endothelial cells (ECs) and increased numbers of mural cells (MCs) under the chorionic epithelial layer. Ro41-5253 (retinoid antagonist) exerted the opposite effect. Although atRA did not affect the differentiation of murine embryonic stem cell-derived vascular progenitor cells (VPCs) into ECs or MCs, atRA suppressed EC-MC interaction, leading to impaired branching. In both atRA-treated VPC cultures and CAM tissues underneath the chorionic epithelial layer, the expression of angiopoietin-2 (Ang-2; competitor for Ang-1) was enhanced, whereas that of Tie2 (a receptor for Angs) was reduced. Simultaneous treatment with Ang-1 partially blocked RA induction of EC-MC malinteraction and reduction in blood vessel formation. These results suggest that retinoid(s) may reduce EC-MC interaction by down-regulating Tie2 signaling as well as decreased EC numbers, which lead to impaired vascular remodeling.