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1.
Oncogene ; 42(12): 869-880, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36721000

RESUMEN

Targeting cyclin-dependent kinases (CDKs) has recently emerged as a promising therapeutic approach against cancer. However, the anticancer mechanisms of different CDK inhibitors (CDKIs) are not well understood. Our recent study revealed that selective CDK4/6 inhibitors sensitize colorectal cancer (CRC) cells to therapy-induced apoptosis by inducing Death Receptor 5 (DR5) via the p53 family member p73. In this study, we investigated if this pathway is involved in anticancer effects of different CDKIs. We found that less-selective CDKIs, including flavopiridol, roscovitine, dinaciclib, and SNS-032, induced DR5 via p73-mediated transcriptional activation. The induction of DR5 by these CDKIs was mediated by dephosphorylation of p73 at Threonine 86 and p73 nuclear translocation. Knockdown of a common target of these CDKIs, including CDK1, 2, or 9, recapitulated p73-mediated DR5 induction. CDKIs strongly synergized with 5-fluorouracil (5-FU), the most commonly used CRC chemotherapy agent, in vitro and in vivo to promote growth suppression and apoptosis, which required DR5 and p73. Together, these findings indicate p73-mediated DR5 induction as a potential tumor suppressive mechanism and a critical target engaged by different CDKIs in potentiating therapy-induced apoptosis in CRC cells. These findings help better understand the anticancer mechanisms of CDKIs and may help facilitate their clinical development and applications in CRC.


Asunto(s)
Antineoplásicos , Neoplasias del Colon , Humanos , Quinasas Ciclina-Dependientes , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis , Fluorouracilo/farmacología , Fluorouracilo/uso terapéutico
2.
Proc Natl Acad Sci U S A ; 119(51): e2211775119, 2022 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-36508676

RESUMEN

Synthetic lethality is a powerful approach for targeting oncogenic drivers in cancer. Recent studies revealed that cancer cells with microsatellite instability (MSI) require Werner (WRN) helicase for survival; however, the underlying mechanism remains unclear. In this study, we found that WRN depletion strongly induced p53 and its downstream apoptotic target PUMA in MSI colorectal cancer (CRC) cells. p53 or PUMA deletion abolished apoptosis induced by WRN depletion in MSI CRC cells. Importantly, correction of MSI abrogated the activation of p53/PUMA and cell killing, while induction of MSI led to sensitivity in isogenic CRC cells. Rare p53-mutant MSI CRC cells are resistant to WRN depletion due to lack of PUMA induction, which could be restored by wildtype (WT) p53 knock in or reconstitution. WRN depletion or treatment with the RecQ helicase inhibitor ML216 suppressed in vitro and in vivo growth of MSI CRCs in a p53/PUMA-dependent manner. ML216 treatment was efficacious in MSI CRC patient-derived xenografts. Interestingly, p53 gene remains WT in the majority of MSI CRCs. These results indicate a critical role of p53/PUMA-mediated apoptosis in the vulnerability of MSI CRCs to WRN loss, and support WRN as a promising therapeutic target in p53-WT MSI CRCs.


Asunto(s)
Neoplasias del Colon , Neoplasias Colorrectales , Humanos , Helicasa del Síndrome de Werner/genética , Proteína p53 Supresora de Tumor/genética , Inestabilidad de Microsatélites , Neoplasias Colorrectales/genética , RecQ Helicasas/genética
3.
Exp Biol Med (Maywood) ; 246(22): 2420-2441, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-33957803

RESUMEN

Metabolic syndrome is a complex disease that involves multiple organ systems including a critical role for the liver. Non-alcoholic fatty liver disease (NAFLD) is a key component of the metabolic syndrome and fatty liver is linked to a range of metabolic dysfunctions that occur in approximately 25% of the population. A panel of experts recently agreed that the acronym, NAFLD, did not properly characterize this heterogeneous disease given the associated metabolic abnormalities such as type 2 diabetes mellitus (T2D), obesity, and hypertension. Therefore, metabolic dysfunction-associated fatty liver disease (MAFLD) has been proposed as the new term to cover the heterogeneity identified in the NAFLD patient population. Although many rodent models of NAFLD/NASH have been developed, they do not recapitulate the full disease spectrum in patients. Therefore, a platform has evolved initially focused on human biomimetic liver microphysiology systems that integrates fluorescent protein biosensors along with other key metrics, the microphysiology systems database, and quantitative systems pharmacology. Quantitative systems pharmacology is being applied to investigate the mechanisms of NAFLD/MAFLD progression to select molecular targets for fluorescent protein biosensors, to integrate computational and experimental methods to predict drugs for repurposing, and to facilitate novel drug development. Fluorescent protein biosensors are critical components of the platform since they enable monitoring of the pathophysiology of disease progression by defining and quantifying the temporal and spatial dynamics of protein functions in the biosensor cells, and serve as minimally invasive biomarkers of the physiological state of the microphysiology system experimental disease models. Here, we summarize the progress in developing human microphysiology system disease models of NAFLD/MAFLD from several laboratories, developing fluorescent protein biosensors to monitor and to measure NAFLD/MAFLD disease progression and implementation of quantitative systems pharmacology with the goal of repurposing drugs and guiding the creation of novel therapeutics.


Asunto(s)
Técnica del Anticuerpo Fluorescente/métodos , Hígado/fisiopatología , Enfermedad del Hígado Graso no Alcohólico/fisiopatología , Biomimética/métodos , Progresión de la Enfermedad , Humanos , Hígado/patología , Síndrome Metabólico/patología , Síndrome Metabólico/fisiopatología , Enfermedad del Hígado Graso no Alcohólico/patología
4.
Commun Biol ; 3(1): 312, 2020 06 16.
Artículo en Inglés | MEDLINE | ID: mdl-32546759

RESUMEN

The recent recovery of mutations in vesicular trafficking genes causing congenital heart disease (CHD) revealed an unexpected role for the endocytic pathway. We now show that mice with a C4232R missense mutation in Low density lipoprotein receptor related protein 1 (LRP1) exhibit atrioventricular septal defects with double outlet right ventricle. Lrp1m/m mice exhibit shortened outflow tracts (OFT) and dysmorphic hypocellular cushions with reduced proliferation and increased apoptosis. Lrp1m/m embryonic fibroblasts show decreased cell motility and focal adhesion turnover associated with retention of mutant LRP1 in endoplasmic reticulum and reduced LRP1 expression. Conditional deletion of Lrp1 in cardiac neural crest cells (CNC) replicates the full CHD phenotype. Cushion explants showed defective cell migration, with gene expression analysis indicating perturbation of Wnt and other signaling pathways. Thus, LRP1 function in CNCs is required for normal OFT development with other cell lineages along the CNC migratory path playing a supporting role.


Asunto(s)
Cardiopatías Congénitas/genética , Corazón/embriología , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Mutación Missense , Cresta Neural/citología , Animales , Linaje de la Célula , Movimiento Celular/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Corazón/diagnóstico por imagen , Cardiopatías Congénitas/patología , Defectos de los Tabiques Cardíacos/genética , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Miocardio/citología
5.
Proc Natl Acad Sci U S A ; 115(7): 1617-1622, 2018 02 13.
Artículo en Inglés | MEDLINE | ID: mdl-29378961

RESUMEN

Neurotransmission is mediated by synaptic exocytosis of neuropeptide-containing dense-core vesicles (DCVs) and small-molecule transmitter-containing small synaptic vesicles (SSVs). Exocytosis of both vesicle types depends on Ca2+ and shared secretory proteins. Here, we show that increasing or decreasing expression of Myopic (mop, HD-PTP, PTPN23), a Bro1 domain-containing pseudophosphatase implicated in neuronal development and neuropeptide gene expression, increases synaptic neuropeptide stores at the Drosophila neuromuscular junction (NMJ). This occurs without altering DCV content or transport, but synaptic DCV number and age are increased. The effect on synaptic neuropeptide stores is accounted for by inhibition of activity-induced Ca2+-dependent neuropeptide release. cAMP-evoked Ca2+-independent synaptic neuropeptide release also requires optimal Myopic expression, showing that Myopic affects the DCV secretory machinery shared by cAMP and Ca2+ pathways. Presynaptic Myopic is abundant at early endosomes, but interaction with the endosomal sorting complex required for transport III (ESCRT III) protein (CHMP4/Shrub) that mediates Myopic's effect on neuron pruning is not required for control of neuropeptide release. Remarkably, in contrast to the effect on DCVs, Myopic does not affect release from SSVs. Therefore, Myopic selectively regulates synaptic DCV exocytosis that mediates peptidergic transmission at the NMJ.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Exocitosis/fisiología , Neuropéptidos/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Vesículas Secretoras/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Drosophila/crecimiento & desarrollo , Unión Neuromuscular/metabolismo , Terminales Presinápticos/metabolismo , Transmisión Sináptica
6.
Dev Cell ; 37(5): 428-43, 2016 06 06.
Artículo en Inglés | MEDLINE | ID: mdl-27237791

RESUMEN

Clathrin-coated vesicles form by rapid assembly of discrete coat constituents into a cargo-sorting lattice. How the sequential phases of coat construction are choreographed is unclear, but transient protein-protein interactions mediated by short interaction motifs are pivotal. We show that arrayed Asp-Pro-Phe (DPF) motifs within the early-arriving endocytic pioneers Eps15/R are differentially decoded by other endocytic pioneers Fcho1/2 and AP-2. The structure of an Eps15/R⋅Fcho1 µ-homology domain complex reveals a spacing-dependent DPF triad, bound in a mechanistically distinct way from the mode of single DPF binding to AP-2. Using cells lacking FCHO1/2 and with Eps15 sequestered from the plasma membrane, we establish that without these two endocytic pioneers, AP-2 assemblies are fleeting and endocytosis stalls. Thus, distinct DPF-based codes within the unstructured Eps15/R C terminus direct the assembly of temporary Fcho1/2⋅Eps15/R⋅AP-2 ternary complexes to facilitate conformational activation of AP-2 by the Fcho1/2 interdomain linker to promote AP-2 cargo engagement.


Asunto(s)
Complejo 2 de Proteína Adaptadora/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de la Membrana/metabolismo , Complejo 2 de Proteína Adaptadora/química , Proteínas Adaptadoras Transductoras de Señales/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Clatrina/metabolismo , Vesículas Cubiertas por Clatrina/metabolismo , Endocitosis , Proteínas de Unión a Ácidos Grasos , Células HeLa , Humanos , Modelos Biológicos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Mapas de Interacción de Proteínas , Ratas , Transfección
7.
Elife ; 32014 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-25303365

RESUMEN

Clathrin-mediated endocytosis is an evolutionarily ancient membrane transport system regulating cellular receptivity and responsiveness. Plasmalemma clathrin-coated structures range from unitary domed assemblies to expansive planar constructions with internal or flanking invaginated buds. Precisely how these morphologically-distinct coats are formed, and whether all are functionally equivalent for selective cargo internalization is still disputed. We have disrupted the genes encoding a set of early arriving clathrin-coat constituents, FCHO1 and FCHO2, in HeLa cells. Endocytic coats do not disappear in this genetic background; rather clustered planar lattices predominate and endocytosis slows, but does not cease. The central linker of FCHO proteins acts as an allosteric regulator of the prime endocytic adaptor, AP-2. By loading AP-2 onto the plasma membrane, FCHO proteins provide a parallel pathway for AP-2 activation and clathrin-coat fabrication. Further, the steady-state morphology of clathrin-coated structures appears to be a manifestation of the availability of the muniscin linker during lattice polymerization.


Asunto(s)
Clatrina/metabolismo , Endonucleasas/metabolismo , Proteínas de la Membrana/metabolismo , Edición de ARN , Transactivadores/metabolismo , Complejo 2 de Proteína Adaptadora/metabolismo , Regulación Alostérica , Animales , Secuencia de Bases , Membrana Celular/metabolismo , Clatrina/ultraestructura , Secuencia Conservada , Endocitosis , Proteínas de Unión a Ácidos Grasos , Sitios Genéticos , Células HeLa , Humanos , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Péptidos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Filogenia , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/metabolismo
8.
Methods Mol Biol ; 1174: 349-60, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24947394

RESUMEN

In oviparous animals, clathrin-dependent endocytosis is often critical to stockpile a necessary supply of yolk within the maturing oocyte, which enables subsequent embryonic development. In the physically linked chains of maturing egg chambers within the Drosophila melanogaster ovary, a distinct, morphologically discernable subset undergoes a massive burst clathrin-mediated endocytosis to accumulate yolk in a process termed vitellogenesis. Here, we describe how to prepare isolated ovaries to follow endocytosis, and detail approaches to follow live uptake of soluble reporters into vitellogenic Drosophila egg chambers.


Asunto(s)
Clatrina/metabolismo , Drosophila melanogaster/fisiología , Endocitosis/fisiología , Óvulo/citología , Óvulo/fisiología , Animales , Drosophila melanogaster/anatomía & histología , Femenino , Microscopía/métodos , Microscopía Fluorescente/métodos
9.
Mol Biol Cell ; 23(9): 1742-64, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22398720

RESUMEN

Clathrin-mediated endocytosis and phagocytosis are both selective surface internalization processes but have little known mechanistic similarity or interdependence. Here we show that the phosphotyrosine-binding (PTB) domain protein Ced-6, a well-established phagocytosis component that operates as a transducer of so-called "eat-me" signals during engulfment of apoptotic cells and microorganisms, is expressed in the female Drosophila germline and that Ced-6 expression correlates with ovarian follicle development. Ced-6 exhibits all the known biochemical properties of a clathrin-associated sorting protein, yet ced-6-null flies are semifertile despite massive accumulation of soluble yolk precursors in the hemolymph. This is because redundant sorting signals within the cytosolic domain of the Drosophila vitellogenin receptor Yolkless, a low density lipoprotein receptor superfamily member, occur; a functional atypical dileucine signal binds to the endocytic AP-2 clathrin adaptor directly. Nonetheless, the Ced-6 PTB domain specifically recognizes the noncanonical Yolkless FXNPXA sorting sequence and in HeLa cells promotes the rapid, clathrin-dependent uptake of a Yolkless chimera lacking the distal dileucine signal. Ced-6 thus operates in vivo as a clathrin adaptor. Because the human Ced-6 orthologue GULP similarly binds to clathrin machinery, localizes to cell surface clathrin-coated structures, and is enriched in placental clathrin-coated vesicles, new possibilities for Ced-6/Gulp operation during phagocytosis must be considered.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Clatrina/metabolismo , Drosophila/metabolismo , Proteínas del Huevo/metabolismo , Receptores de Superficie Celular/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Línea Celular , Vesículas Cubiertas por Clatrina/metabolismo , Embrión no Mamífero , Endocitosis , Femenino , Células HeLa , Humanos , Oocitos/química , Oocitos/metabolismo , Fagocitosis , Unión Proteica , Vitelogénesis
10.
Carcinogenesis ; 29(10): 1878-84, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18579560

RESUMEN

p53-upregulated modulator of apoptosis (PUMA) plays an essential role in p53-dependent apoptosis following DNA damage. PUMA also mediates apoptosis independent of p53. In this study, we investigated the role and mechanism of PUMA induction in response to serum starvation in p53-deficient cancer cells. Following serum starvation, the binding of Sp1 to the PUMA promoter significantly increased, whereas inhibition of Sp1 completely abrogated PUMA induction. p73 was found to be upregulated by serum starvation and mediate PUMA induction through the p53-binding sites in the PUMA promoter. Sp1 and p73beta appeared to cooperatively activate PUMA transcription, which is inhibited by the phosphoinsitide 3-kinase (PI3K)-protein kinase B (AKT) pathway. Furthermore, knockdown of PUMA suppressed serum starvation-induced apoptosis in leukemia cells. Our results suggest that transcription factors Sp1 and p73 mediate p53-independent induction of PUMA following serum starvation to trigger apoptosis in human cancer cells.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Proteínas de Unión al ADN/fisiología , Proteínas Nucleares/fisiología , Proteínas Proto-Oncogénicas/genética , Factor de Transcripción Sp1/fisiología , Activación Transcripcional , Proteínas Supresoras de Tumor/fisiología , Apoptosis , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Línea Celular Tumoral , Medio de Cultivo Libre de Suero , Humanos , Fosfatidilinositol 3-Quinasas/fisiología , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/fisiología , ARN Interferente Pequeño/farmacología , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/fisiología
11.
J Cell Sci ; 121(Pt 8): 1264-74, 2008 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-18388314

RESUMEN

In the anautogenous disease vector mosquitoes Anopheles gambiae and Aedes aegypti, egg development is nutritionally controlled. A blood meal permits further maturation of developmentally repressed previtellogenic egg chambers. This entails massive storage of extraovarian yolk precursors by the oocyte, which occurs through a burst of clathrin-mediated endocytosis. Yolk precursors are concentrated at clathrin-coated structures on the oolemma by two endocytic receptors, the vitellogenin and lipophorin receptors. Both these mosquito receptors are members of the low-density-lipoprotein-receptor superfamily that contain FxNPxY-type internalization signals. In mammals, this tyrosine-based signal is not decoded by the endocytic AP-2 adaptor complex directly. Instead, two functionally redundant phosphotyrosine-binding domain adaptors, Disabled 2 and the autosomal recessive hypercholesterolemia protein (ARH) manage the internalization of the FxNPxY sorting signal. Here, we report that a mosquito ARH-like protein, which we designate trephin, possess similar functional properties to the orthologous vertebrate proteins despite engaging AP-2 in an atypical manner, and that mRNA expression in the egg chamber is strongly upregulated shortly following a blood meal. Temporally regulated trephin transcription and translation suggests a mechanism for controlling yolk uptake when vitellogenin and lipophorin receptors are expressed and clathrin coats operate in previtellogenic ovaries.


Asunto(s)
Culicidae/fisiología , Endocitosis , Perfilación de la Expresión Génica , Oogénesis , Receptores de LDL/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Animales , Hibridación in Situ , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos Híbridos
12.
J Biol Chem ; 279(44): 46191-203, 2004 Oct 29.
Artículo en Inglés | MEDLINE | ID: mdl-15292237

RESUMEN

Clathrin-mediated endocytosis depends upon the coordinated assembly of a large number of discrete clathrin coat components to couple cargo selection with rapid internalization from the cell surface. Accordingly, the heterotetrameric AP-2 adaptor complex binds not only to clathrin and select cargo molecules, but also to an extensive family of endocytic accessory factors and alternate sorting adaptors. Physical associations between accessory proteins and AP-2 occur primarily through DP(F/W) or FXDXF motifs, which engage an interaction surface positioned on the C-terminal platform subdomain of the independently folded alpha subunit appendage. Here, we find that the WXX(F/W)X(D/E) interaction motif found in several endocytic proteins, including synaptojanin 1, stonin 2, AAK1, GAK, and NECAP1, binds a second interaction site on the bilobal alpha appendage, located on the N-terminal beta sandwich subdomain. Both alpha appendage binding sites can be engaged synchronously, and our data reveal that varied assemblies of interaction motifs with different affinities for two sites upon the alpha appendage can provide a mechanism for temporal ordering of endocytic accessory proteins during clathrin-mediated endocytosis.


Asunto(s)
Complejo 2 de Proteína Adaptadora/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Clatrina/fisiología , Endocitosis , Datos de Secuencia Molecular , Filogenia , Subunidades de Proteína , Ratas
13.
J Biol Chem ; 279(3): 2281-90, 2004 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-14565955

RESUMEN

Phosphoinositides play a fundamental role in clathrin-coat assembly at the cell surface. Several endocytic components and accessory factors contain independently folded phosphoinositide-binding modules that facilitate, in part, membrane placement at the bud site. As the clathrin-coat assembly process progresses toward deeply invaginated buds, focally synthesized phosphoinositides are dephosphorylated, principally through the action of the phosphoinositide polyphosphatase synaptojanin 1. Failure to catabolize polyphosphoinositides retards the fission process and endocytic activity. The long-splice isoform of synaptojanin 1, termed SJ170, contains a carboxyl-terminal extension that harbors interaction motifs for engaging several components of the endocytic machinery. Here, we demonstrate that in addition to DPF and FXDXF sequences, the SJ170 carboxyl terminus contains a novel AP-2 binding sequence, the WXXF motif. The WXXF sequence engages the independently folded alpha-subunit appendage that projects off the heterotetrameric AP-2 adaptor core. The endocytic protein kinases AAK1 and GAK also contain functional WXX(FW) motifs in addition to two DPF repeats, whereas stonin 2 harbors three tandem WXXF repeats. Each of the discrete SJ170 adaptor-interaction motifs bind to appendages relatively weakly but, as tandemly arrayed within the SJ170 extension, can cooperate to bind bivalent AP-2 with good apparent affinity. These interactions likely contribute to the appropriate targeting of certain endocytic components to clathrin bud sites assembling at the cell surface.


Asunto(s)
Complejo 2 de Proteína Adaptadora/química , Proteínas del Tejido Nervioso/química , Monoéster Fosfórico Hidrolasas/química , Complejo 2 de Proteína Adaptadora/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Endocitosis , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Conejos , Ratas , Dominios Homologos src
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