RESUMEN
Translocation of cytoplasmic molecules to the plasma membrane is commonplace in cell signaling. Membrane localization has been hypothesized to increase intermolecular association rates; however, it has also been argued that association should be faster in the cytosol because membrane diffusion is slow. Here, we directly compare an identical association reaction, the binding of complementary DNA strands, in solution and on supported membranes. The measured rate constants show that for a 10-µm-radius spherical cell, association is 22- to 33-fold faster at the membrane than in the cytoplasm. The kinetic advantage depends on cell size and is essentially negligible for typical ~1 µm prokaryotic cells. The rate enhancement is attributable to a combination of higher encounter rates in two dimensions and a higher reaction probability per encounter.
Asunto(s)
Transducción de Señal , Citoplasma/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Membranas , CinéticaRESUMEN
RAS genes are known to be dysregulated in cancer for several decades, and substantial effort has been dedicated to develop agents that reduce RAS expression or block RAS activation. The recent introduction of RAS inhibitors for cancer patients highlights the importance of comprehending RAS alterations in head and neck cancer (HNC). In this regard, we examine the published findings on RAS alterations and pathway activations in HNC, and summarize their role in HNC initiation, progression, and metastasis. Specifically, we focus on the intrinsic role of mutated-RAS on tumor cell signaling and its extrinsic role in determining tumor-microenvironment (TME) heterogeneity, including promoting angiogenesis and enhancing immune escape. Lastly, we summarize the intrinsic and extrinsic role of RAS alterations on therapy resistance to outline the potential of targeting RAS using a single agent or in combination with other therapeutic agents for HNC patients with RAS-activated tumors.
Asunto(s)
Antineoplásicos , Neoplasias de Cabeza y Cuello , Humanos , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/genética , Antineoplásicos/uso terapéutico , Transducción de Señal , Genes ras , Microambiente TumoralRESUMEN
The path of moving eukaryotic cells depends on the kinetics and direction of extending pseudopods. Amoeboid cells constantly change their shape with pseudopods extending in different directions. Detailed analysis has revealed that time, place and direction of pseudopod extension are not random, but highly ordered with strong prevalence for only one extending pseudopod, with defined life-times, and with reoccurring events in time and space indicative of memory. Important components are Ras activation and the formation of branched F-actin in the extending pseudopod and inhibition of pseudopod formation in the contractile cortex of parallel F-actin/myosin. In biology, order very often comes with symmetry. In this essay, I discuss cell movement and the dynamics of pseudopod extension from the perspective of symmetry and symmetry changes of Ras activation and the formation of branched F-actin in the extending pseudopod. Combining symmetry of Ras activation with kinetics and memory of pseudopod extension results in a refined model of amoeboid movement that appears to be largely conserved in the fast moving Dictyostelium and neutrophils, the slow moving mesenchymal stem cells and the fungus B.d. chytrid.
Asunto(s)
Batrachochytrium/fisiología , Movimiento Celular/fisiología , Dictyostelium/fisiología , Células Madre Mesenquimatosas/fisiología , Neutrófilos/fisiología , Seudópodos/metabolismo , Actinas/metabolismo , Animales , Quimiotaxis/fisiología , Citoesqueleto/metabolismo , Cinética , Modelos Biológicos , Miosinas/metabolismo , Transducción de Señal/fisiología , Proteínas ras/metabolismoRESUMEN
RAS GTPases are involved in a number of dynamic signaling processes and have been a major focus of research due to the prevalence of activating RAS mutations in cancer. However, despite decades of research, some fundamental aspects of RAS biology are still not well understood. Difficulty in fully defining RAS-driven signaling stems from the overall complexity of downstream pathways and a lack of tools for specifically perturbing RAS function. To better characterize RAS-driven signaling, we recently developed a chemical genetic system for activating endogenous RAS with a small molecule. In this chapter, we describe the use of chemically inducible activator of RAS (CIAR), a single-protein, chemical genetic system that allows the rapid and dose-dependent activation of endogenous RAS. Methods in this chapter also describe the validation of RAS activation with CIAR through the analysis of downstream signaling.
Asunto(s)
Transducción de Señal , Proteínas ras , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Aberrant activation of the RAS cascade ubiquitously occurs in human hepatocellular carcinomas (HCC), regardless of rare mutations of RAS. However, the association between the Ras cascade and hepatic steatosis during hepatocarcinogenesis remains under-investigated. Here, the variation in the constitutive activity of Ras signaling and HCC incidence was found in a nonalcoholic fatty liver disease (NAFLD)-HCC mouse model, and Ras activity was induced by hepatic steatosis. Even in hepatocyte-specific expression of KrasG12D (Alb-Cre/KrasG12D, Krashep) mice, mutagenic activation of Ras signaling was still significantly enhanced by NAFLD, with downregulation of negative regulators. Interestingly, hepatic steatosis could be alleviated by persistent activation of Ras, whereas Ras accelerated DNA damage and HCC progression through Carnitine palmitoyltransferase 1A (CPT1α). A close correlation between active Ras and CPT1α was also shown in clinical steatosis peri-tumor tissues of HCC samples and experimental models. CPT1α inhibitor etomoxir (ETO) largely ameliorated active Ras-drived HCC. These findings can provide a novel link between steatosis and Ras activity in liver cancer.
Asunto(s)
Carcinoma Hepatocelular/enzimología , Carnitina O-Palmitoiltransferasa/metabolismo , Transformación Celular Neoplásica/metabolismo , Dieta Alta en Grasa , Hígado Graso/metabolismo , Neoplasias Hepáticas Experimentales/enzimología , Hígado/enzimología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Animales , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/prevención & control , Carnitina O-Palmitoiltransferasa/antagonistas & inhibidores , Carnitina O-Palmitoiltransferasa/genética , Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/patología , Daño del ADN , Dietilnitrosamina , Progresión de la Enfermedad , Inhibidores Enzimáticos/farmacología , Compuestos Epoxi/farmacología , Hígado Graso/etiología , Hígado Graso/genética , Hígado Graso/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Metabolismo de los Lípidos , Hígado/efectos de los fármacos , Hígado/patología , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/patología , Neoplasias Hepáticas Experimentales/prevención & control , Ratones Transgénicos , Estrés Oxidativo , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de Señal , Factores de TiempoRESUMEN
RAS effector signaling instead of being simple, unidirectional and linear cascade, is actually recognized as highly complex and dynamic signaling network. RAF-MEK-ERK cascade, being at the center of complex signaling network, links to multiple scaffold proteins through feed forward and feedback mechanisms and dynamically regulate tumor initiation and progression. Three isoforms of Ras harbor mutations in a cell and tissue specific manner. Besides mutations, their epigenetic silencing also attributes them to exhibit oncogenic activities. Recent evidences support the functions of RAS oncoproteins in the acquisition of tumor cells with Epithelial-to-mesenchymal transition (EMT) features/ epithelial plasticity, enhanced metastatic potential and poor patient survival. Google Scholar electronic databases and PubMed were searched for original papers and reviews available till date to collect information on stimulation of EMT core inducers in a Ras driven cancer and their regulation in metastatic spread. Improved understanding of the mechanistic basis of regulatory interactions of microRNAs (miRs) and EMT by reprogramming the expression of targets in Ras activated cancer, may help in designing effective anticancer therapies. Apparent lack of adverse events associated with the delivery of miRs and tissue response make 'drug target miRNA' an ideal therapeutic tool to achieve progression free clinical response.
RESUMEN
Eukaryotic cells chemotax in a wide range of chemoattractant concentration gradients, and thus need inhibitory processes that terminate cell responses to reach adaptation while maintaining sensitivity to higher-concentration stimuli. However, the molecular mechanisms underlying inhibitory processes are still poorly understood. Here, we reveal a locally controlled inhibitory process in a GPCR-mediated signaling network for chemotaxis in Dictyostelium discoideum We identified a negative regulator of Ras signaling, C2GAP1, which localizes at the leading edge of chemotaxing cells and is activated by and essential for GPCR-mediated Ras signaling. We show that both C2 and GAP domains are required for the membrane targeting of C2GAP1, and that GPCR-triggered Ras activation is necessary to recruit C2GAP1 from the cytosol and retains it on the membrane to locally inhibit Ras signaling. C2GAP1-deficient c2gapA- cells have altered Ras activation that results in impaired gradient sensing, excessive polymerization of F actin, and subsequent defective chemotaxis. Remarkably, these cellular defects of c2gapA- cells are chemoattractant concentration dependent. Thus, we have uncovered an inhibitory mechanism required for adaptation and long-range chemotaxis.
Asunto(s)
Quimiotaxis/genética , Dictyostelium/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Protozoarias/genética , Proteínas ras/genética , Actinas/genética , Actinas/metabolismo , Adaptación Fisiológica , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Quimiotaxis/efectos de los fármacos , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Citosol/efectos de los fármacos , Citosol/metabolismo , Dictyostelium/efectos de los fármacos , Dictyostelium/genética , Dictyostelium/ultraestructura , Proteínas Activadoras de GTPasa/deficiencia , Regulación de la Expresión Génica , Transporte de Proteínas , Proteínas Protozoarias/metabolismo , Transducción de Señal , Proteínas ras/metabolismoRESUMEN
Receptor activation upon ligand binding induces activation of multiple signaling pathways. To fully understand how these signaling pathways coordinate, it is essential to determine the dynamic nature of the spatiotemporal activation profile of signaling components at the level of single living cells. Here, we outline a detailed methodology for visualizing and quantitatively measuring the spatiotemporal activation of Ras and PKD1 by applying advanced fluorescence imaging techniques, including multichannel, simultaneous imaging and Förster resonance energy transfer (FRET).
Asunto(s)
Microscopía Confocal , Imagen Molecular , Canales Catiónicos TRPP/metabolismo , Proteínas ras/metabolismo , Línea Celular Tumoral , Factores Quimiotácticos/metabolismo , Factores Quimiotácticos/farmacología , Activación Enzimática , Transferencia Resonante de Energía de Fluorescencia , Expresión Génica , Genes Reporteros , Células HL-60 , Humanos , Microscopía Confocal/métodos , Fosforilación , Canales Catiónicos TRPP/genética , Proteínas ras/genéticaRESUMEN
The aim of this study was to assess the renoprotective effect of renal human liver-type fatty acid binding protein (hL-FABP) and angiotensin II (ANG II) type 1A receptor (AT1a) loss in renal injury caused by renin-angiotensin system (RAS) activation. We established hL-FABP chromosomal transgenic mice (L-FABP(+/-)AT1a(+/+)), crossed the L-FABP(+/-)AT1a(+/+) with AT1a knockdown homo mice (L-FABP(-/-)AT1a(-/-)), and generated L-FABP(+/-)AT1a hetero mice (L-FABP(+/-)AT1a(+/-)). After the back-cross of these cubs, L-FABP(+/-)AT1a(-/-) were obtained. To activate the renal RAS, wild-type mice (L-FABP(-/-)AT1a(+/+)), L-FABP(+/-)AT1a(+/+), L-FABP(-/-)AT1a(+/-), L-FABP(+/-)AT1a(+/-), L-FABP(-/-)AT1a(-/-), and L-FABP(+/-)AT1a(-/-) were administered high-dose systemic ANG II infusion plus a high-salt diet for 28 days. In the L-FABP(-/-)AT1a(+/+), RAS activation (L-FABP(-/-)AT1a(+/+)RAS) caused hypertension and tubulointerstitial damage. In the L-FABP(+/-)AT1a(+/+)RAS, tubulointerstitial damage was significantly attenuated compared with L-FABP(-/-)AT1a(+/+)RAS. In the AT1a partial knockout (AT1a(+/-)) or complete knockout (AT1a(-/-)) mice, reduction of AT1a expression led to a significantly lower degree of renal injury compared with L-FABP(-/-)AT1a(+/+)RAS or L-FABP(+/-)AT1a(+/+)RAS mice. Renal injury in L-FABP(+/-)AT1a(+/-)RAS mice was significantly attenuated compared with L-FABP(-/-)AT1a(+/-)RAS mice. In both L-FABP(-/-)AT1a(-/-)RAS and L-FABP(+/-)AT1a(-/-)RAS mice, renal damage was rarely found. The degrees of renal hL-FABP expression and urinary hL-FABP levels increased by RAS activation and gradually decreased along with reduction of AT1a expression levels. In conclusion, in this mouse model, renal hL-FABP expression and a decrease in AT1a expression attenuated tubulointerstitial damage due to RAS activation.