RESUMEN
The nonpsychoactive cannabinoid, cannabidiol (CBD), is Food and Dug Administration approved for treatment of two drug-resistant epileptic disorders and is seeing increased use among the general public, yet the mechanisms that underlie its therapeutic effects and side-effect profiles remain unclear. Here, we report a systems-level analysis of CBD action in human cell lines using temporal multiomic profiling. FRET-based biosensor screening revealed that CBD elicits a sharp rise in cytosolic calcium, and activation of AMP-activated protein kinase in human keratinocyte and neuroblastoma cell lines. CBD treatment leads to alterations in the abundance of metabolites, mRNA transcripts, and proteins associated with activation of cholesterol biosynthesis, transport, and storage. We found that CBD rapidly incorporates into cellular membranes, alters cholesterol accessibility, and disrupts cholesterol-dependent membrane properties. Sustained treatment with high concentrations of CBD induces apoptosis in a dose-dependent manner. CBD-induced apoptosis is rescued by inhibition of cholesterol synthesis and potentiated by compounds that disrupt cholesterol trafficking and storage. Our data point to a pharmacological interaction of CBD with cholesterol homeostasis pathways, with potential implications in its therapeutic use.
Asunto(s)
Cannabidiol , Cannabinoides , Humanos , Cannabidiol/farmacología , Calcio/metabolismo , Proteínas Quinasas Activadas por AMP , Línea Celular , Cannabinoides/farmacología , Homeostasis , ARN Mensajero/metabolismo , ColesterolRESUMEN
Cellular quiescence is a reversible state of cell cycle arrest whereby cells are temporarily maintained in the nondividing phase. Inducing quiescence in cancer cells by targeting growth receptors is a treatment strategy to slow cell growth in certain aggressive tumors, which in turn increases the efficacy of treatments such as surgery or systemic chemotherapy. However, ligand interactions with cell receptors induce receptor-mediated endocytosis followed by proteolytic degradation, which limits the duration of cellular quiescence. Here, we report the effects of targeted covalent affibody photoconjugation to epidermal growth factor receptors (EGFR) on EGFR-positive MDA-MB-468 breast cancer cells. First, covalently conjugating affibodies to cells increased doubling time two-fold and reduced ERK activity by 30% as compared to cells treated with an FDA-approved anti-EGFR antibody Cetuximab, which binds to EGFR noncovalently. The distribution of cells in each phase of the cell cycle was determined, and cells conjugated with the affibody demonstrated an accumulation in the G1 phase, indicative of G1 cell cycle arrest. Finally, the proliferative capacity of the cells was determined by the incorporation of 5-ethynyl-2-deoxyuridine and Ki67 Elisa assay, which showed that the percentage of proliferative cells with photoconjugated affibody was half of that found for the untreated control.
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Muerte Celular/efectos de los fármacos , Receptores ErbB , Procesos Fotoquímicos , Proteínas Recombinantes de Fusión , Neoplasias de la Mama/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Receptores ErbB/química , Receptores ErbB/metabolismo , Femenino , Humanos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
In this work, we show that a prodrug enzyme covalently photoconjugated to live cell receptors survives endosomal proteolysis and retains its catalytic activity over multiple days. Here, a fusion protein was designed with both an antiepidermal growth factor receptor (EGFR) affibody and the prodrug enzyme cytosine deaminase, which can convert prodrug 5-fluorocytosine to the anticancer drug 5-fluorouracil. A benzophenone group was added at a site-specific mutation within the affibody, and the fusion protein was selectively photoconjugated to EGFR receptors expressed on membranes of MDA-MB-468 breast cancer cells. The fusion protein was next labeled with two dyes for tracking uptake: AlexaFluor 488 and pH-sensitive pHAb. Flow cytometry showed that fusion proteins photo-cross-linked to EGFR first underwent receptor-mediated endocytosis within 12 h, followed by recycling back to the cell membrane within 24 h. These findings were also confirmed by confocal microscopy. The unique cross-linking of the affibody-enzyme fusion proteins was utilized for two anticancer treatments. First, the covalent linking of the protein to the EGFR led to inhibition of ERK signaling over a two-day period, whereas conventional antibody therapy only led to 6 h of inhibition. Second, when the affibody-CodA fusion proteins were photo-cross-linked to EGFR overexpressed on MDA-MB-468 breast cancer cells, prodrug conversion was found even 48 h postincubation without any apparent decrease in cell killing, while without photo-cross-linking no cell killing was observed 8 h postincubation. These studies show that affinity-mediated covalent conjugation of the affibody-enzymes to cell receptors allows for prolonged expression on membranes and retained enzymatic activity without genetic engineering.
Asunto(s)
Antineoplásicos/farmacología , Citosina Desaminasa/farmacología , Receptores ErbB/antagonistas & inhibidores , Flucitosina/farmacología , Fluorouracilo/farmacología , Profármacos/farmacología , Antineoplásicos/farmacocinética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Citosina Desaminasa/farmacocinética , Receptores ErbB/metabolismo , Femenino , Flucitosina/farmacocinética , Fluorouracilo/farmacocinética , Humanos , Profármacos/farmacocinética , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Sorafenib (Nexavar) is a broad-spectrum multikinase inhibitor that proves effective in treating advanced renal-cell carcinoma and liver cancer. Despite its well-characterized mechanism of action on several established cancer-related protein kinases, sorafenib causes variable responses among human tumors, although the cause for this variation is unknown. In an unbiased screening of an oncology drug library, we found that sorafenib activates recruitment of the ubiquitin E3 ligase Parkin to damaged mitochondria. We show that sorafenib inhibits the activity of both complex II/III of the electron transport chain and ATP synthase. Dual inhibition of these complexes, but not inhibition of each individual complex, stabilizes the serine-threonine protein kinase PINK1 on the mitochondrial outer membrane and activates Parkin. Unlike the protonophore carbonyl cyanide m-chlorophenylhydrazone, which activates the mitophagy response, sorafenib treatment triggers PINK1/Parkin-dependent cellular apoptosis, which is attenuated upon Bcl-2 overexpression. In summary, our results reveal a new mechanism of action for sorafenib as a mitocan and suggest that high Parkin activity levels could make tumor cells more sensitive to sorafenib's actions, providing one possible explanation why Parkin may be a tumor suppressor gene. These insights could be useful in developing new rationally designed combination therapies with sorafenib.
Asunto(s)
Complejo III de Transporte de Electrones/antagonistas & inhibidores , Complejo II de Transporte de Electrones/antagonistas & inhibidores , Mitocondrias/efectos de los fármacos , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , Niacinamida/análogos & derivados , Compuestos de Fenilurea/farmacología , Proteínas Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Células Cultivadas , Transporte de Electrón/efectos de los fármacos , Complejo II de Transporte de Electrones/metabolismo , Complejo III de Transporte de Electrones/metabolismo , Células HEK293 , Humanos , Mitocondrias/enzimología , Mitocondrias/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Niacinamida/farmacología , SorafenibRESUMEN
The Musashi-2 (Msi2) RNA-binding protein maintains stem cell self-renewal and promotes oncogenesis by enhancing cell proliferation in hematopoietic and gastrointestinal tissues. However, it is unclear how Msi2 recognizes and regulates mRNA targets in vivo and whether Msi2 primarily controls cell growth in all cell types. Here we identified Msi2 targets with HITS-CLIP and revealed that Msi2 primarily recognizes mRNA 3'UTRs at sites enriched in multiple copies of UAG motifs in epithelial progenitor cells. RNA-seq and ribosome profiling demonstrated that Msi2 promotes targeted mRNA decay without affecting translation efficiency. Unexpectedly, the most prominent Msi2 targets identified are key regulators that govern cell motility with a high enrichment in focal adhesion and extracellular matrix-receptor interaction, in addition to regulators of cell growth and survival. Loss of Msi2 stimulates epithelial cell migration, increases the number of focal adhesions and also compromises cell growth. These findings provide new insights into the molecular mechanisms of Msi2's recognition and repression of targets and uncover a key function of Msi2 in restricting epithelial cell migration.
Asunto(s)
Movimiento Celular/genética , Regulación de la Expresión Génica , Queratinocitos/fisiología , Estabilidad del ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Regiones no Traducidas 3' , Animales , Sitios de Unión , Proliferación Celular/genética , Supervivencia Celular , Estudio de Asociación del Genoma Completo , Secuenciación de Nucleótidos de Alto Rendimiento , Inmunoprecipitación , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones , Análisis de Secuencia de ARN , TranscriptomaRESUMEN
The in vitro migration of keratinocyte cell sheets displays behavioral and biochemical similarities to the in vivo wound healing response of keratinocytes in animal model systems. In both cases, ligand-dependent Epidermal Growth Factor Receptor (EGFR) activation is sufficient to elicit collective cell migration into the wound. Previous mathematical modeling studies of in vitro wound healing assays assume that physical connections between cells have a hindering effect on cell migration, but biological literature suggests a more complicated story. By combining mathematical modeling and experimental observations of collectively migrating sheets of keratinocytes, we investigate the role of cell-cell adhesion during in vitro keratinocyte wound healing assays. We develop and compare two nonlinear diffusion models of the wound healing process in which cell-cell adhesion either hinders or promotes migration. Both models can accurately fit the leading edge propagation of cell sheets during wound healing when using a time-dependent rate of cell-cell adhesion strength. The model that assumes a positive role of cell-cell adhesion on migration, however, is robust to changes in the leading edge definition and yields a qualitatively accurate density profile. Using RNAi for the critical adherens junction protein, α-catenin, we demonstrate that cell sheets with wild type cell-cell adhesion expression maintain migration into the wound longer than cell sheets with decreased cell-cell adhesion expression, which fails to exhibit collective migration. Our modeling and experimental data thus suggest that cell-cell adhesion promotes sustained migration as cells pull neighboring cells into the wound during wound healing.
Asunto(s)
Algoritmos , Movimiento Celular/fisiología , Queratinocitos/fisiología , Modelos Biológicos , Cicatrización de Heridas/fisiología , Adhesión Celular/fisiología , Línea Celular , Movimiento Celular/efectos de los fármacos , Simulación por Computador , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Cinética , Interferencia de ARN , Factores de Tiempo , alfa Catenina/genética , alfa Catenina/metabolismoRESUMEN
Mammalian cells can decode the concentration of extracellular transforming growth factor-ß (TGF-ß) and transduce this cue into appropriate cell fate decisions. How variable TGF-ß ligand doses quantitatively control intracellular signaling dynamics and how continuous ligand doses are translated into discontinuous cellular fate decisions remain poorly understood. Using a combined experimental and mathematical modeling approach, we discovered that cells respond differently to continuous and pulsating TGF-ß stimulation. The TGF-ß pathway elicits a transient signaling response to a single pulse of TGF-ß stimulation, whereas it is capable of integrating repeated pulses of ligand stimulation at short time interval, resulting in sustained phospho-Smad2 and transcriptional responses. Additionally, the TGF-ß pathway displays different sensitivities to ligand doses at different time scales. While ligand-induced short-term Smad2 phosphorylation is graded, long-term Smad2 phosphorylation is switch-like to a small change in TGF-ß levels. Correspondingly, the short-term Smad7 gene expression is graded, while long-term PAI-1 gene expression is switch-like, as is the long-term growth inhibitory response. Our results suggest that long-term switch-like signaling responses in the TGF-ß pathway might be critical for cell fate determination.
Asunto(s)
Queratinocitos/fisiología , Inhibidor 1 de Activador Plasminogénico/metabolismo , Transducción de Señal , Proteína Smad2/metabolismo , Proteína smad7/metabolismo , Biología de Sistemas/métodos , Factor de Crecimiento Transformador beta/metabolismo , Diferenciación Celular , Línea Celular , Proliferación Celular , Expresión Génica , Humanos , Queratinocitos/citología , Cómputos Matemáticos , Modelos Biológicos , Fosforilación , Inhibidor 1 de Activador Plasminogénico/genética , Proteína Smad2/genética , Proteína smad7/genética , Transfección , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Sustained cell migration is essential for wound healing and cancer metastasis. The epidermal growth factor receptor (EGFR) signaling cascade is known to drive cell migration and proliferation. While the signal transduction downstream of EGFR has been extensively investigated, our knowledge of the initiation and maintenance of EGFR signaling during cell migration remains limited. The metalloprotease TACE (tumor necrosis factor alpha converting enzyme) is responsible for producing active EGFR family ligands in the via ligand shedding. Sustained TACE activity may perpetuate EGFR signaling and reduce a cell's reliance on exogenous growth factors. Using a cultured keratinocyte model system, we show that depletion of α-catenin perturbs adherens junctions, enhances cell proliferation and motility, and decreases dependence on exogenous growth factors. We show that the underlying mechanism for these observed phenotypical changes depends on enhanced autocrine/paracrine release of the EGFR ligand transforming growth factor alpha in a TACE-dependent manner. We demonstrate that proliferating keratinocyte epithelial cell clusters display waves of oscillatory extracellular signal-regulated kinase (ERK) activity, which can be eliminated by TACE knockout, suggesting that these waves of oscillatory ERK activity depend on autocrine/paracrine signals produced by TACE. These results provide new insights into the regulatory role of adherens junctions in initiating and maintaining autocrine/paracrine signaling with relevance to wound healing and cellular transformation.
Asunto(s)
Proteína ADAM17/metabolismo , Uniones Adherentes/metabolismo , alfa Catenina/metabolismo , Proteína ADAM17/fisiología , Uniones Adherentes/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular , Factor de Crecimiento Epidérmico/metabolismo , Células Epiteliales/metabolismo , Receptores ErbB/metabolismo , Células HaCaT , Humanos , Metaloproteasas/metabolismo , Comunicación Paracrina/fisiología , Fosforilación , Transducción de Señal , Factor de Crecimiento Transformador alfa/metabolismo , alfa Catenina/fisiologíaRESUMEN
Haemophilus influenzae beta-carbonic anhydrase (HICA) is hypothesized to be an allosteric protein that is regulated by the binding of bicarbonate ion to a non-catalytic (inhibitory) site that controls the ligation of Asp44 to the catalytically essential zinc ion. We report here the X-ray crystallographic structures of two variants (W39F and Y181F) involved in the binding of bicarbonate ion in the non-catalytic site and an active-site variant (D44N) that is incapable of forming a strong zinc ligand. The alteration of Trp39 to Phe increases the apparent K(i) for bicarbonate inhibition by 4.8-fold. While the structures of W39F and Y181F are very similar to the wild-type enzyme, the X-ray crystal structure of the D44N variant reveals that it has adopted an active-site conformation nearly identical to that of non-allosteric beta-carbonic anhydrases. We propose that the structure of the D44N variant is likely to be representative of the active conformation of the enzyme. These results lend additional support to the hypothesis that HICA is an allosteric enzyme that can adopt active and inactive conformations, the latter of which is stabilized by bicarbonate ion binding to a non-catalytic site.
Asunto(s)
Sitio Alostérico/genética , Sustitución de Aminoácidos , Anhidrasas Carbónicas/química , Haemophilus influenzae/enzimología , Bicarbonatos/química , Biocatálisis , Anhidrasas Carbónicas/genética , Dominio Catalítico , Cristalografía por Rayos X , Haemophilus influenzae/genética , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Isótopos de Oxígeno/química , Conformación Proteica , Proteínas Recombinantes/química , Zinc/químicaRESUMEN
Living cells employ complex and highly dynamic signaling networks and transcriptional circuits to maintain homeostasis and respond appropriately to constantly changing environments. These networks enable cells to maintain tight control on intracellular concentrations of ions, metabolites, proteins, and other biomolecules and ensure a careful balance between a cell's energetic needs and catabolic processes required for growth. Establishing molecular mechanisms of genetic and pharmacological perturbations remains challenging, due to the interconnected nature of these networks and the extreme sensitivity of cellular systems to their external environment. Live cell imaging with genetically encoded fluorescent biosensors provides a powerful new modality for nondestructive spatiotemporal tracking of ions, small molecules, enzymatic activities, and molecular interactions in living systems, from cells, tissues, and even living organisms. By deploying large panels of cell lines, each with distinct biosensors, many critical biochemical pathways can be monitored in a highly parallel and high-throughput fashion to identify pharmacological vulnerabilities and combination therapies unique to a given cell type or genetic background. Here we describe the experimental and analytical methods required to conduct multiplexed parallel fluorescence microscopy experiments on live cells expressing stable transgenic synthetic protein biosensors.
Asunto(s)
Técnicas Biosensibles/métodos , Enzimas/química , Microscopía Fluorescente/métodos , Proteínas/química , Enzimas/genética , Transferencia Resonante de Energía de Fluorescencia , Iones/química , Proteínas/genética , Transducción de Señal/genéticaRESUMEN
Biomolecule-functionalized hydrogels have emerged as valuable cell culture platforms to recapitulate the mechanical and biochemical properties of the extracellular niche. The typical strategy to functionalize hydrogels with biomolecules involves directly tethering them to the hydrogel backbone resulting in a static material. Thus, this approach fails to capture the dynamic changes in biomolecule composition that occur during biological processes or that may be required for regenerative medicine applications. Moreover, it also limits the scope of biomolecules to simple peptides, as signaling proteins generally have poor stability under cell culture conditions and lose their bioactivity over time. To that end, we sought to develop a bioconjugation reaction that would enable reversible and repeatable tethering of signaling proteins to hydrogels, so that spent protein could be released on-demand and replaced with fresh protein as needed. Specifically, we designed an allyl sulfide chain-transfer agent that enables a reversible, photomediated, thiol-ene bioconjugation of signaling proteins to hydrogels. Upon addition of a thiolated protein to the allyl sulfide moiety, the previously tethered protein is released, and the "ene" functionality is regenerated. Using this approach, we demonstrate that protein patterning can be achieved in hydrogels through a thiol-ene reaction, and the patterned protein can then be released through a subsequent thiol-ene reaction of a PEG thiol. Importantly, this process is repeatable through multiple iterations and proceeds at physiologically relevant signaling protein concentrations. Finally, we demonstrate that whole signaling proteins can be patterned and released in the presence of cells, and that cells respond to their presentation with spatial fidelity. Combined, these data represent the first example of a methodology that enables fully reversible and repeatable patterning and release of signaling proteins from hydrogels.
RESUMEN
Diverse environmental conditions stimulate protein "shedding" from the cell surface through proteolytic cleavage. The protease TACE [tumor necrosis factor-α (TNFα)--converting enzyme, encoded by ADAM17] mediates protein shedding, thereby regulating the maturation and release of various extracellular substrates, such as growth factors and cytokines, that induce diverse cellular responses. We developed a FRET (fluorescence resonance energy transfer)-based biosensor called TSen that quantitatively reports the kinetics of TACE activity in live cells. In combination with chemical biology approaches, we used TSen to probe the dependence of TACE activation on the induction of the kinases p38 and ERK (extracellular signal-regulated kinase) in various epithelial cell lines. Using TSen, we found that disruption of the actin cytoskeleton in keratinocytes induced rapid and robust TSen cleavage and the accumulation of TACE at the plasma membrane. Cytoskeletal disruption also increased the cleavage of endogenous TACE substrates, including transforming growth factor-α. Thus, TSen is a useful tool for unraveling the mechanisms underlying the spatiotemporal activation of TACE in live cells.
Asunto(s)
Proteínas ADAM/metabolismo , Técnicas Biosensibles/métodos , Células Epiteliales/enzimología , Transferencia Resonante de Energía de Fluorescencia/métodos , Queratinocitos/enzimología , Proteínas ADAM/genética , Proteína ADAM17 , Animales , Citoesqueleto/genética , Citoesqueleto/metabolismo , Activación Enzimática/genética , Células Epiteliales/citología , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Queratinocitos/citología , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Noqueados , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
During wound healing and cancer metastasis, cells are frequently observed to migrate in collective groups. This mode of migration relies on the cooperative guidance of leader and follower cells throughout the collective group. The upstream determinants and molecular mechanisms behind such cellular guidance remain poorly understood. We use live-cell imaging to track the behavior of epithelial sheets of keratinocytes in response to transforming growth factor ß (TGFß), which stimulates collective migration primarily through extracellular regulated kinase 1/2 (Erk1/2) activation. TGFß-treated sheets display a spatial pattern of Erk1/2 activation in which the highest levels of Erk1/2 activity are concentrated toward the leading edge of a sheet. We show that Erk1/2 activity is modulated by cellular density and that this functional relationship drives the formation of patterns of Erk1/2 activity throughout sheets. In addition, we determine that a spatially constrained pattern of Erk1/2 activity results in collective migration that is primarily wound directed. Conversely, global elevation of Erk1/2 throughout sheets leads to stochastically directed collective migration throughout sheets. Our study highlights how the spatial patterning of leader cells (cells with elevated Erk1/2 activity) can influence the guidance of a collective group of cells during wound healing.
Asunto(s)
Movimiento Celular/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Queratinocitos/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Cicatrización de Heridas/fisiología , Línea Celular , Células Epiteliales , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/biosíntesis , Humanos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Interferencia de ARN , ARN Interferente Pequeño , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Proteína Smad2/genética , Proteína smad3/genética , Proteínas de Dominio T Box/antagonistas & inhibidoresRESUMEN
Understanding how cells migrate individually and collectively during development and cancer metastasis can be significantly aided by a computation tool to accurately measure not only cellular migration speed, but also migration direction and changes in migration direction in a temporal and spatial manner. We have developed such a tool for cell migration researchers, named Pathfinder, which is capable of simultaneously measuring the migration speed, migration direction, and changes in migration directions of thousands of cells both instantaneously and over long periods of time from fluorescence microscopy data. Additionally, we demonstrate how the Pathfinder software can be used to quantify collective cell migration. The novel capability of the Pathfinder software to measure the changes in migration direction of large populations of cells in a spatiotemporal manner will aid cellular migration research by providing a robust method for determining the mechanisms of cellular guidance during individual and collective cell migration.
Asunto(s)
Movimiento Celular , Procesamiento de Imagen Asistido por Computador/métodos , Programas Informáticos , Línea Celular , Humanos , Microscopía Fluorescente , Factores de TiempoRESUMEN
The physiological responses to TGF-ß stimulation are diverse and vary amongst different cell types and environmental conditions. Even though the principal molecular components of the canonical and the non-canonical TGF-ß signaling pathways have been largely identified, the mechanism that underlies the well-established context dependent physiological responses remains a mystery. Understanding how the components of TGF-ß signaling function as a system and how this system functions in the context of the global cellular regulatory network requires a more quantitative and systematic approach. Here, we review the recent progress in understanding TGF-ß biology using integration of mathematical modeling and quantitative experimental analysis. These studies reveal many interesting dynamics of TGF-ß signaling and how cells quantitatively decode variable doses of TGF-ß stimulation.
Asunto(s)
Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Secuencias de Aminoácidos , Animales , Comunicación Celular , Relación Dosis-Respuesta a Droga , Humanos , Cinética , Ratones , Modelos Biológicos , Modelos Teóricos , Oscilometría , Transducción de SeñalRESUMEN
Protein degradation via the ubiquitin-proteasome pathway is important for a diverse number of cellular processes ranging from cell signaling to development. Disruption of the ubiquitin pathway occurs in a variety of human diseases, including several cancers and neurological disorders. Excessive proteolysis of tumor suppressor proteins, such as p27, occurs in numerous aggressive human tumors. To discover small-molecule inhibitors that potentially prevent p27 degradation, we developed a series of screening assays, including a cell-based screen of a small-molecule compound library and two novel nucleotide exchange assays. Several small-molecule inhibitors, including NSC624206, were identified and subsequently verified to prevent p27 ubiquitination in vitro. The mechanism of NSC624206 inhibition of p27 ubiquitination was further unraveled using the nucleotide exchange assays and shown to be due to antagonizing ubiquitin activating enzyme (E1). We determined that NSC624206 and PYR-41, a recently reported inhibitor of ubiquitin E1, specifically block ubiquitin-thioester formation but have no effect on ubiquitin adenylation. These studies reveal a novel E1 inhibitor that targets a specific step of the E1 activation reaction. NSC624206 could, therefore, be potentially useful for the control of excessive ubiquitin-mediated proteolysis in vivo.
Asunto(s)
Disulfuros/farmacología , Inhibidores Enzimáticos/farmacología , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Disulfuros/química , Ensayos de Selección de Medicamentos Antitumorales , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Humanos , Ratones , Proteolisis/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas , Ubiquitinación/efectos de los fármacosRESUMEN
The TGFß and Ras-MAPK pathways play critical roles in cell development and cell cycle regulation, as well as in tumor formation and metastasis. In the absence of cellular transformation, these pathways operate in opposition to one another, where TGFß maintains an undifferentiated cell state and suppresses proliferation, while Ras-MAPK pathways promote proliferation, survival and differentiation. However, in colorectal and pancreatic cancers, the opposing pathways' mechanisms are simultaneously activated in order to promote cancer progression and metastasis. Here, we highlight the roles of the TGFß and Ras-MAPK pathways in normal and malignant states, and provide an explanation for how the concomitant activation of these pathways drives tumor biology. Finally, we survey potential therapeutic targets in these pathways.
RESUMEN
The growth inhibition of dividing cells and most of the transcriptional responses upon TGF-beta treatment depend on the Smad2, Smad3, and Smad4 transcription factors. These proteins shuttle continuously between the cytoplasm and the nucleus, transmitting the ligand status of the TGF-beta receptors to the nuclear transcription machinery. In the absence of TGF-beta ligand, Smads 2/3/4 reside predominantly in the cytoplasm. Following ligand binding to the TGF-beta receptors, the dynamic equilibrium of shuttling Smads 2/3/4 shifts toward a predominantly nuclear state, where a high concentration of these transcription factors drives transcriptional activation and repression of genes required for proper cellular response. Here, we describe live cell imaging and immunofluorescence microscopy methods for tracking Smads subcellular localization in response to TGF-beta and leptomycin B treatment. In addition, a method of fractionating nuclear and cytoplasmic proteins used to confirm the imaging results was presented. Our results support the notion that the R-Smad shuttling mechanism is distinct from Co-Smad.