RESUMO
The coxsackievirus and adenovirus receptor (CAR) mediates homo- and heterotopic interactions between neighboring cardiomyocytes at the intercalated disc. CAR is upregulated in the hypoxic areas surrounding myocardial infarction (MI). To elucidate whether CAR contributes to hypoxia signaling and MI pathology, we used a gain- and loss-of-function approach in transfected HEK293 cells, H9c2 cardiomyocytes and CAR knockout mice. CAR overexpression increased RhoA activity, HIF-1α expression and cell death in response to chemical and physical hypoxia. In vivo, we subjected cardiomyocyte-specific CAR knockout (KO) and wild-type mice (WT) to coronary artery ligation. Survival was drastically improved in KO mice with largely preserved cardiac function as determined by echocardiography. Histological analysis revealed a less fibrotic, more compact lesion. Thirty days after MI, there was no compensatory hypertrophy or reduced cardiac output in hearts from CAR KO mice, in contrast to control mice with increased heart weight and reduced ejection fraction as signs of the underlying pathology. Based on these findings, we suggest CAR as a therapeutic target for the improved future treatment or prevention of myocardial infarction.
Assuntos
Infarto do Miocárdio , Camundongos , Animais , Humanos , Células HEK293 , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Hipóxia/metabolismo , Camundongos KnockoutRESUMO
Rho family GTPases are central regulators of cytoskeletal dynamics controlled by guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). This protocol presents a workflow for a robust high-throughput compatible biosensor assay to analyze changes in Rho GTPase activity by these proteins in the native cellular environment. The procedure can be used for semi-quantitative comparison of GEF/GAP function and extended for analysis of additional modulators. The experimental design is applicable also to other monomolecular ratiometric FRET sensors. For complete details on the use and execution of this protocol, please refer to Müller et al. (2020).
Assuntos
Transferência Ressonante de Energia de Fluorescência , Proteínas rho de Ligação ao GTP , Proteínas rho de Ligação ao GTP/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas Ativadoras de GTPase/metabolismoRESUMO
Rho GTPases are central regulators of the cytoskeleton and, in humans, are controlled by 145 multidomain guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). How Rho signalling patterns are established in dynamic cell spaces to control cellular morphogenesis is unclear. Through a family-wide characterization of substrate specificities, interactomes and localization, we reveal at the systems level how RhoGEFs and RhoGAPs contextualize and spatiotemporally control Rho signalling. These proteins are widely autoinhibited to allow local regulation, form complexes to jointly coordinate their networks and provide positional information for signalling. RhoGAPs are more promiscuous than RhoGEFs to confine Rho activity gradients. Our resource enabled us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling CDC42-RHOA crosstalk. Moreover, we show that integrin adhesions spatially segregate GEFs and GAPs to shape RAC1 activity zones in response to mechanical cues. This mechanism controls the protrusion and contraction dynamics fundamental to cell motility. Our systems analysis of Rho regulators is key to revealing emergent organization principles of Rho signalling.
Assuntos
Citoesqueleto/genética , Proteínas Ativadoras de GTPase/genética , Integrinas/genética , Mecanotransdução Celular/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Proteínas rac1 de Ligação ao GTP/genética , Animais , Células COS , Adesão Celular , Linhagem Celular , Movimento Celular , Chlorocebus aethiops , Biologia Computacional , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Cães , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Proteínas Ativadoras de GTPase/classificação , Proteínas Ativadoras de GTPase/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Integrinas/metabolismo , Células Madin Darby de Rim Canino , Camundongos , Pan troglodytes , Domínios Proteicos , Ratos , Fatores de Troca de Nucleotídeo Guanina Rho/classificação , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Systematic analysis of human arginine methylation identifies two distinct signaling modes; either isolated modifications akin to canonical post-translational modification regulation, or clustered arrays within disordered protein sequence. Hundreds of proteins contain these methyl-arginine arrays and are more prone to accumulate mutations and more tightly expression-regulated than dispersed methylation targets. Arginines within an array in the highly methylated RNA-binding protein synaptotagmin binding cytoplasmic RNA interacting protein (SYNCRIP) were experimentally shown to function in concert, providing a tunable protein interaction interface. Quantitative immunoprecipitation assays defined two distinct cumulative binding mechanisms operating across 18 proximal arginine-glycine (RG) motifs in SYNCRIP. Functional binding to the methyltransferase PRMT1 was promoted by continual arginine stretches, whereas interaction with the methyl-binding protein SMN1 was arginine content-dependent irrespective of linear position within the unstructured region. This study highlights how highly repetitive modifiable amino acid arrays in low structural complexity regions can provide regulatory platforms, with SYNCRIP as an extreme example how arginine methylation leverages these disordered sequences to mediate cellular interactions.
RESUMO
Stimulation of renal collecting duct principal cells with antidiuretic hormone (arginine-vasopressin, AVP) results in inhibition of the small GTPase RhoA and the enrichment of the water channel aquaporin-2 (AQP2) in the plasma membrane. The membrane insertion facilitates water reabsorption from primary urine and fine-tuning of body water homeostasis. Rho guanine nucleotide exchange factors (GEFs) interact with RhoA, catalyze the exchange of GDP for GTP and thereby activate the GTPase. However, GEFs involved in the control of AQP2 in renal principal cells are unknown. The A-kinase anchoring protein, AKAP-Lbc, possesses GEF activity, specifically activates RhoA, and is expressed in primary renal inner medullary collecting duct principal (IMCD) cells. Through screening of 18,431 small molecules and synthesis of a focused library around one of the hits, we identified an inhibitor of the interaction of AKAP-Lbc and RhoA. This molecule, Scaff10-8, bound to RhoA, inhibited the AKAP-Lbc-mediated RhoA activation but did not interfere with RhoA activation through other GEFs or activities of other members of the Rho family of small GTPases, Rac1 and Cdc42. Scaff10-8 promoted the redistribution of AQP2 from intracellular vesicles to the periphery of IMCD cells. Thus, our data demonstrate an involvement of AKAP-Lbc-mediated RhoA activation in the control of AQP2 trafficking.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Aquaporina 2/metabolismo , Membrana Celular/metabolismo , Túbulos Renais Coletores/citologia , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína rhoA de Ligação ao GTP/metabolismo , Membrana Celular/efeitos dos fármacos , Células HEK293 , Humanos , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Although Rho GTPases are essential molecular switches involved in many cellular processes, an unbiased experimental comparison of their interaction partners was not yet performed. Here, we develop quantitative GTPase affinity purification (qGAP) to systematically identify interaction partners of six Rho GTPases (Cdc42, Rac1, RhoA, RhoB, RhoC, and RhoD), depending on their nucleotide loading state. The method works with cell line or tissue-derived protein lysates in combination with SILAC-based or label-free quantification, respectively. We demonstrate that qGAP identifies known and novel binding partners that can be validated in an independent assay. Our interaction network for six Rho GTPases contains many novel binding partners, reveals highly promiscuous interaction of several effectors, and mirrors evolutionary relationships among Rho GTPases.
Assuntos
Encéfalo/metabolismo , Proteômica/métodos , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Células HEK293 , Células HeLa , Humanos , Espectrometria de Massas , Camundongos , Mapas de Interação de ProteínasRESUMO
Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators. Here, we report the generation of a quantitative interaction network, directly linking 14 human proteins to the AAA+ ATPase p97, an essential hexameric protein with multiple cellular functions. We show that the high-affinity interacting protein ASPL efficiently promotes p97 hexamer disassembly, resulting in the formation of stable p97:ASPL heterotetramers. High-resolution structural and biochemical studies indicate that an extended UBX domain (eUBX) in ASPL is critical for p97 hexamer disassembly and facilitates the assembly of p97:ASPL heterotetramers. This spontaneous process is accompanied by a reorientation of the D2 ATPase domain in p97 and a loss of its activity. Finally, we demonstrate that overproduction of ASPL disrupts p97 hexamer function in ERAD and that engineered eUBX polypeptides can induce cell death, providing a rationale for developing anti-cancer polypeptide inhibitors that may target p97 activity.
Assuntos
Degradação Associada com o Retículo Endoplasmático/fisiologia , Proteínas de Fusão Oncogênica/metabolismo , Domínios Proteicos/fisiologia , Proteína com Valosina/metabolismo , Encéfalo/patologia , Proliferação de Células , Cristalografia por Raios X , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mutação , Proteínas de Fusão Oncogênica/química , Proteínas de Fusão Oncogênica/isolamento & purificação , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Engenharia de Proteínas , Mapas de Interação de Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteína com Valosina/química , Proteína com Valosina/isolamento & purificaçãoRESUMO
Protein kinase A is a key mediator of cAMP signalling downstream of G-protein-coupled receptors, a signalling pathway conserved in all eukaryotes. cAMP binding to the regulatory subunits (PKAR) relieves their inhibition of the catalytic subunits (PKAC). Here we report that ARHGAP36 combines two distinct inhibitory mechanisms to antagonise PKA signalling. First, it blocks PKAC activity via a pseudosubstrate motif, akin to the mechanism employed by the protein kinase inhibitor proteins. Second, it targets PKAC for rapid ubiquitin-mediated lysosomal degradation, a pathway usually reserved for transmembrane receptors. ARHGAP36 thus dampens the sensitivity of cells to cAMP. We show that PKA inhibition by ARHGAP36 promotes derepression of the Hedgehog signalling pathway, thereby providing a simple rationale for the upregulation of ARHGAP36 in medulloblastoma. Our work reveals a new layer of PKA regulation that may play an important role in development and disease.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Hedgehog/metabolismo , Meduloblastoma/patologia , Células 3T3 , Animais , Carcinogênese/patologia , Domínio Catalítico/fisiologia , Linhagem Celular Tumoral , Neoplasias Cerebelares/patologia , Cães , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Camundongos , Ligação Proteica/fisiologia , Inibidores de Proteínas Quinases/metabolismo , Proteólise , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/fisiologia , Ubiquitinação/fisiologiaRESUMO
Chemotherapy is one of the pillars of anti-cancer therapy. Although chemotherapeutics cause regression of the primary tumor, many chemotherapeutics are often shown to induce or accelerate metastasis formation. Moreover, metastatic tumors are largely resistant against chemotherapy. As more than 90% of cancer patients die due to metastases and not due to primary tumor formation, novel drugs are needed to overcome these shortcomings. In this study, we identified the anticancer phytochemical Rocaglamide (Roc-A) to be an inhibitor of cancer cell migration, a crucial event in metastasis formation. We show that Roc-A inhibits cellular migration and invasion independently of its anti-proliferative and cytotoxic effects in different types of human cancer cells. Mechanistically, Roc-A treatment induces F-actin-based morphological changes in membrane protrusions. Further investigation of the molecular mechanisms revealed that Roc-A inhibits the activities of the small GTPases RhoA, Rac1 and Cdc42, the master regulators of cellular migration. Taken together, our results provide evidence that Roc-A may be a lead candidate for a new class of anticancer drugs that inhibit metastasis formation.
Assuntos
Antineoplásicos/farmacologia , Benzofuranos/farmacologia , Movimento Celular/efeitos dos fármacos , Proteínas rho de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Humanos , Proteínas rho de Ligação ao GTP/efeitos dos fármacosRESUMO
ß2-chimaerin is a Rac1-specific negative regulator and a candidate tumor suppressor in breast cancer but its precise function in mammary tumorigenesis in vivo is unknown. Here, we study for the first time the role of ß2-chimaerin in breast cancer using a mouse model and describe an unforeseen role for this protein in epithelial cell-cell adhesion. We demonstrate that expression of ß2-chimaerin in breast cancer epithelial cells reduces E-cadherin protein levels, thus loosening cell-cell contacts. In vivo, genetic ablation of ß2-chimaerin in the MMTV-Neu/ErbB2 mice accelerates tumor onset, but delays tumor progression. Finally, analysis of clinical databases revealed an inverse correlation between ß2-chimaerin and E-cadherin gene expressions in Her2+ breast tumors. Furthermore, breast cancer patients with low ß2-chimaerin expression have reduced relapse free survival but develop metastasis at similar times. Overall, our data redefine the role of ß2-chimaerin as tumor suppressor and provide the first in vivo evidence of a dual function in breast cancer, suppressing tumor initiation but favoring tumor progression.
Assuntos
Neoplasias da Mama/patologia , Adesão Celular/fisiologia , Transformação Celular Neoplásica/patologia , Proteínas de Neoplasias/metabolismo , Animais , Neoplasias da Mama/metabolismo , Transformação Celular Neoplásica/metabolismo , Progressão da Doença , Feminino , Humanos , Células MCF-7 , Camundongos , Camundongos KnockoutRESUMO
SH2D5 is a mammalian-specific, uncharacterized adaptor-like protein that contains an N-terminal phosphotyrosine-binding domain and a C-terminal Src homology 2 (SH2) domain. We show that SH2D5 is highly enriched in adult mouse brain, particularly in Purkinjie cells in the cerebellum and the cornu ammonis of the hippocampus. Despite harboring two potential phosphotyrosine (Tyr(P)) recognition domains, SH2D5 binds minimally to Tyr(P) ligands, consistent with the absence of a conserved Tyr(P)-binding arginine residue in the SH2 domain. Immunoprecipitation coupled to mass spectrometry (IP-MS) from cultured cells revealed a prominent association of SH2D5 with breakpoint cluster region protein, a RacGAP that is also highly expressed in brain. This interaction occurred between the phosphotyrosine-binding domain of SH2D5 and an NxxF motif located within the N-terminal region of the breakpoint cluster region. siRNA-mediated depletion of SH2D5 in a neuroblastoma cell line, B35, induced a cell rounding phenotype correlated with low levels of activated Rac1-GTP, suggesting that SH2D5 affects Rac1-GTP levels. Taken together, our data provide the first characterization of the SH2D5 signaling protein.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Proteínas Proto-Oncogênicas c-bcr/metabolismo , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Linhagem Celular Tumoral , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Humanos , Immunoblotting , Imuno-Histoquímica , Células K562 , Masculino , Camundongos Endogâmicos C57BL , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Neurônios/metabolismo , Fosfotirosina/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcr/genética , Interferência de RNA , Ratos , Proteínas Adaptadoras da Sinalização Shc/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Dynamin 1-like protein (DNM1L) mediates fission of mitochondria and peroxisomes, and dysfunction of DNM1L has been implicated in several neurological disorders. To study the molecular basis of mitochondrial remodelling, we determined the crystal structure of DNM1L that is comprised of a G domain, a bundle signalling element and a stalk. DNM1L assembled via a central stalk interface, and mutations in this interface disrupted dimerization and interfered with membrane binding and mitochondrial targeting. Two sequence stretches at the tip of the stalk were shown to be required for ordered assembly of DNM1L on membranes and its function in mitochondrial fission. In the crystals, DNM1L dimers further assembled via a second, previously undescribed, stalk interface to form a linear filament. Mutations in this interface interfered with liposome tubulation and mitochondrial remodelling. Based on these results and electron microscopy reconstructions, we propose an oligomerization mode for DNM1L which differs from that of dynamin and might be adapted to the remodelling of mitochondria.
Assuntos
GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Multimerização Proteica/fisiologia , Animais , Células COS , Chlorocebus aethiops , Cristalografia por Raios X , Dinaminas , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/genética , Humanos , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Tamanho Mitocondrial/efeitos dos fármacos , Tamanho Mitocondrial/genética , Modelos Biológicos , Modelos Moleculares , Mutação de Sentido Incorreto/fisiologia , Dobramento de Proteína , Estrutura Quaternária de Proteína/fisiologia , Estrutura Secundária de Proteína , RNA Interferente Pequeno/farmacologiaRESUMO
GTPases of immunity-associated proteins (GIMAPs) are regulators of lymphocyte survival and homeostasis. We previously determined the structural basis of GTP-dependent GIMAP2 scaffold formation on lipid droplets. To understand how its GTP hydrolysis is activated, we screened for other GIMAPs on lipid droplets and identified GIMAP7. In contrast to GIMAP2, GIMAP7 displayed dimerization-stimulated GTP hydrolysis. The crystal structure of GTP-bound GIMAP7 showed a homodimer that assembled via the G domains, with the helical extensions protruding in opposite directions. We identified a catalytic arginine that is supplied to the opposing monomer to stimulate GTP hydrolysis. GIMAP7 also stimulated GTP hydrolysis by GIMAP2 via an analogous mechanism. Finally, we found GIMAP2 and GIMAP7 expression differentially regulated in several human T cell lymphoma lines. Our findings suggest that GTPase activity in the GIMAP family is controlled by homo- and heterodimerization. This may have implications for the differential roles of some GIMAPs in lymphocyte survival.
Assuntos
Ativação Enzimática/fisiologia , GTP Fosfo-Hidrolases/química , Proteínas de Ligação ao GTP/química , Proteínas de Membrana/química , Modelos Moleculares , Conformação Proteica , Linfócitos T/metabolismo , Calorimetria , Linhagem Celular , Cristalização , Dimerização , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Hidrólise , Metabolismo dos Lipídeos/fisiologia , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , UltracentrifugaçãoRESUMO
Growth factor stimulation generates transient H-Ras activity at the plasma membrane but sustained activity at the Golgi. Two overlapping regulatory networks control compartmentalized H-Ras activity: the guanosine diphosphate-guanosine triphosphate cycle and the acylation cycle, which constitutively traffics Ras isoforms that can be palmitoylated between intracellular membrane compartments. Quantitative imaging of H-Ras activity after decoupling of these networks revealed regulation of H-Ras activity at the plasma membrane but not at the Golgi. Nevertheless, upon stimulation with epidermal growth factor, Ras activity at the Golgi displayed a pulse-like profile similar to that at the plasma membrane but also remained high after the initial stimulus. A compartmental model that included the acylation cycle and H-Ras regulation at the plasma membrane accounted for the pulse-like profile of H-Ras activity at the Golgi but implied that sustained H-Ras activity at the Golgi required H-Ras activation at an additional compartment, which we experimentally determined to be the endoplasmic reticulum. Thus, in addition to maintaining the localization of Ras, the acylation cycle underlies a previously unknown form of signal propagation similar to radio transmission in its generation of a constitutive Ras "carrier wave" that transmits Ras activity between subcellular compartments.
Assuntos
Membrana Celular/metabolismo , Complexo de Golgi/metabolismo , Modelos Biológicos , Transdução de Sinais/fisiologia , Proteínas ras/metabolismo , Acilação , Animais , Compartimento Celular/fisiologia , Linhagem Celular , Cães , Fator de Crescimento Epidérmico/metabolismo , Microscopia de Fluorescência/métodosRESUMO
Reversible S-palmitoylation of cysteine residues critically controls transient membrane tethering of peripheral membrane proteins. Little is known about how the palmitoylation machinery governs their defined localization and function. We monitored the spatially resolved reaction dynamics and substrate specificity of the core mammalian palmitoylation machinery using semisynthetic substrates. Palmitoylation is detectable only on the Golgi, whereas depalmitoylation occurs everywhere in the cell. The reactions are not stereoselective and lack any primary consensus sequence, demonstrating that substrate specificity is not essential for de-/repalmitoylation. Both palmitate attachment and removal require seconds to accomplish. This reaction topography and rapid kinetics allows the continuous redirection of mislocalized proteins via the post-Golgi sorting apparatus. Unidirectional secretion ensures the maintenance of a proper steady-state protein distribution between the Golgi and the plasma membrane, which are continuous with endosomes. This generic spatially organizing system differs from conventional receptor-mediated targeting mechanisms and efficiently counteracts entropy-driven redistribution of palmitoylated peripheral membrane proteins over all membranes.
Assuntos
Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Lipoilação , Dados de Sequência Molecular , Filogenia , Alinhamento de SequênciaRESUMO
Cycles of depalmitoylation and repalmitoylation critically control the steady-state localization and function of various peripheral membrane proteins, such as Ras proto-oncogene products. Interference with acylation using small molecules is a strategy to modulate cellular localization--and thereby unregulated signaling--caused by palmitoylated Ras proteins. We present the knowledge-based development and characterization of a potent inhibitor of acyl protein thioesterase 1 (APT1), a bona fide depalmitoylating enzyme that is, so far, poorly characterized in cells. The inhibitor, palmostatin B, perturbs the cellular acylation cycle at the level of depalmitoylation and thereby causes a loss of the precise steady-state localization of palmitoylated Ras. As a consequence, palmostatin B induces partial phenotypic reversion in oncogenic HRasG12V-transformed fibroblasts. We identify APT1 as one of the thioesterases in the acylation cycle and show that this protein is a cellular target of the inhibitor.
Assuntos
Inibidores Enzimáticos/farmacologia , Propiolactona/análogos & derivados , Tioléster Hidrolases/antagonistas & inibidores , Tioléster Hidrolases/química , Proteínas ras/fisiologia , Animais , Linhagem Celular , Cães , Regulação para Baixo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Rim/efeitos dos fármacos , Rim/fisiologia , Ligantes , Lipase/química , Lipase/metabolismo , Lipoilação/efeitos dos fármacos , Modelos Moleculares , Propiolactona/síntese química , Propiolactona/química , Propiolactona/farmacologia , Conformação Proteica , Proto-Oncogene Mas , Transdução de Sinais , Estômago/enzimologia , Tioléster Hidrolases/genética , Proteínas ras/efeitos dos fármacos , Proteínas ras/metabolismoRESUMO
Dynamic assembly of spatially separated signaling platforms enables a cell to tune cellular outputs in response to different input stimuli. Understanding how a vast diversity in signaling responses can be generated from a limited protein repertoire requires knowledge of how cells maintain the segregation of proteins and thereby orchestrate their local activities. Ras proteins are subject to this type of precise regulation of localization, and thus activity, in space and time. A model emerges where different lipid anchors dynamically shuttle Ras between specific membrane compartments, where differences in the accessibility of signaling environments and in the residence time of Ras therein account for isoform-specific signaling responses.
Assuntos
Proteínas ras/metabolismo , Animais , Membrana Celular/fisiologia , Complexo de Golgi/fisiologia , Humanos , Modelos Biológicos , Transdução de Sinais/fisiologiaRESUMO
This protocol describes a method for measuring fluorescence resonance energy transfer (FRET) by the detection of acceptor-sensitized emission. This approach is useful in situations where donor intensities are low and/or there is contamination with high background (auto) fluorescence in the donor channel. However, absorption spectra characteristically exhibit long tails in the higher-energy, shorter-wavelength (blue) region, which may result in the direct excitation of the acceptor molecule in addition to that of the donor, thus resulting in mixing of direct and sensitized emission. Conversely, fluorescence emission tends to tail into the red part of the spectrum, causing donor fluorescence bleed-through into the acceptor detection channel. Corrections for these effects involve the acquisition of fluorescence images of samples containing the donor, the acceptor, and both of these for three different filter settings. The result is an estimation of the sensitized emission, i.e., the emission induced by FRET from the donor to the acceptor alone. Excitation of a donor fluorophore in a FRET pair leads to quenching of the donor fluorescence and increased emission from the acceptor (sensitized emission). This can be normalized using the acceptor emission, measured after specific excitation of the acceptor, to define apparent energy transfer efficiency in each pixel of the image. It is also proportional to the fraction of acceptor molecules that is bound to a donor-tagged molecule. Alternatively, an apparent energy transfer efficiency can also be defined that is proportional to the bound fraction of donor-tagged molecules.
RESUMO
This protocol describes the detection of fluorescence resonance energy transfer (FRET) by measuring the quenching of donor emission alone. As opposed to sensitized emission measurements, photobleaching can be performed with high selectivity of the acceptor because absorption spectra are steep at their red edge, allowing the acceptor to be bleached without excitation of the donor. When using acceptor photobleaching FRET measurements, care should be taken that the photochemical product of the bleached acceptor does not have residual absorption at the donor emission and, more importantly, that it does not fluoresce in the donor spectral region. Because of mass movement of protein during the extended time required for photobleaching (typically 1-20 min), it is preferable to perform this type of FRET determination on fixed cell samples. Live-cell FRET measurements based only on donor fluorescence are more feasible using fluorescence lifetime imaging (FLIM), because lifetimes are independent of probe concentration and light path length. The former is not easy to determine in cells, and the latter means that cell shape is not a factor.
RESUMO
This image acquisition protocol is a basic plan for taking a fluorescence lifetime imaging (FLIM) series. FLIM makes live-cell FRET measurements based only on donor fluorescence more feasible, because lifetimes are independent of probe concentration and light path length. The former is not easy to determine in cells, and the latter means that cell shape is not a factor.