RESUMO
GPI-anchored folate receptor α (FRα) is an attractive anticancer drug target and diagnosis marker in fundamental biology and medical research due to its significant expression on many cancer cells. Currently, analyses of FRα expression levels are usually achieved using immunological methods. Due to the continual FRα synthesis and degradation, immunological methods are not suitable for studying real-time dynamic activities of FRα in living cells. In this paper, we introduce a rapid and specific FRα protein-labeling fluorescent probe, FR1, to facilitate the study of the dynamics of expression and degradation processes of endogenous FRα in living cells. With this labeling probe, insights on FRα protein lifetime and shedding from the cell surface can be obtained using fluorescence live-cell imaging and electrophoresis techniques. We revealed that FRα undergoes soluble domain release and endocytosis degradation simultaneously. Imaging results showed that most of the membrane FRα are transported to the lysosomes after 2 h of incubation. Furthermore, we also showed that the secretion of a FRα soluble domain into the environment is most likely accomplished by phospholipase. We believe that this protein-labeling approach can be an important tool for analyzing various dynamic processes involving FRα.
Assuntos
Antineoplásicos , Receptor 1 de Folato , Receptor 1 de Folato/metabolismo , Corantes FluorescentesRESUMO
Ectodomain shedding is a form of limited proteolysis in which a protease cleaves a transmembrane protein, releasing the extracellular domain from the cell surface. Cells use this process to regulate a wide variety of biological events. Typically, immunological detection methods are employed for the analysis of ectodomains secreted into the cultured media. In this paper, we describe a new strategy using an affinity-based protein-labeling fluorescent probe to study ectodomain shedding. We analyzed the ectodomain shedding of cell surface carbonic anhydrases (CAIX and CAXII), which are important biomarkers for tumor hypoxia. Using both chemical and genetic approaches, we identified that the ADAM17 metalloprotease is responsible for the shedding of carbonic anhydrases. Compared to current immunological methods, this protein-labeling approach not only detects ectodomain released into the culture media but also allows real-time living cell tracking and quantitative analysis of remnant proteins on the cell surface, thereby providing a more detailed insight into the mechanism of ectodomain shedding as well as protein lifetime on the cell surface.
Assuntos
Anidrases Carbônicas , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Corantes Fluorescentes/metabolismo , Proteínas de Membrana/metabolismo , Membrana Celular/metabolismo , Metaloproteases/metabolismoRESUMO
To date, various affinity-based protein labeling probes have been developed and applied in biological research to modify endogenous proteins in cell lysates and on the cell surface. However, the reactive groups on the labeling probes are also the cause of probe instability and nonselective labeling in a more complex environment, e. g., intracellular and inâ vivo. Here, we show that labeling probes composed of a sterically stabilized difluorophenyl pivalate can achieve efficient and selective labeling of endogenous proteins on the cell surface, inside living cells and inâ vivo. As compared with the existing protein labeling probes, probes with the difluorophenyl pivalate exhibit several advantages, including long-term stability in stock solutions, resistance to enzymatic hydrolysis and can be customized easily with diverse fluorophores and protein ligands. With this probe design, endogenous hypoxia biomarker in living cells and nude mice were successfully labeled and validated by inâ vivo, ex vivo, and immunohistochemistry imaging.
Assuntos
Anidrase Carbônica II/análise , Corantes Fluorescentes/química , Albumina Sérica/análise , Animais , Anidrase Carbônica II/metabolismo , Linhagem Celular Tumoral , Corantes Fluorescentes/administração & dosagem , Humanos , Injeções Subcutâneas , Cinética , Ligantes , Camundongos , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/metabolismo , Imagem ÓpticaRESUMO
Currently most fluorogenic probes are developed for the analysis of enzymes, where a bond breaking or rearrangement reaction is required to transform a nonfluorescent enzymatic substrate into a fluorescent product. However, this approach cannot be used for proteins that do not possess enzymatic activities. In this article, we show that fluorogenic probes with a self-immolative difluorophenyl ester linker can mimic the bond disassembly processes of fluorogenic enzyme substrates for the rapid analysis of nonenzymatic proteins. Although numerous self-immolative reagents have shown promising applications in sensors, drug delivery systems, and material chemistry, all of them are triggered by either enzymes or small reactive molecules. In our strategy, the probe binds to the protein via a specific protein-ligand interaction, inducing a chemical reaction between the self-immolative linker and an amino acid of the protein, thereby triggering a cascade reaction that leads to the activation and release of the fluorogenic reporter. In contrast, a phenyl ester linker without the difluoro substituent cannot be triggered to release the fluorogenic reporter. With this probe design, live-cell imaging of extracellular and intracellular endogenous tumor marker proteins can be achieved with high selectivity and sensitivity.
Assuntos
Ésteres/química , Corantes Fluorescentes/química , Hidrocarbonetos Fluorados/química , Proteínas/análise , Proteínas/química , Limite de DetecçãoRESUMO
The long-term stability of affinity-based protein labeling probes is crucial to obtain reproducible protein labeling results. However, highly stable probes generally suffer from low protein labeling efficiency and pose significant challenges when labeling low abundance native proteins in living cells. In this paper, we report that protein labeling probes based on an ortho-difluorophenyl ester reactive module exhibit long-term stability in DMSO stock solution and aqueous buffer, yet they can undergo rapid and selective labeling of native proteins. This novel electrophile can be customized with a wide range of different protein ligands and is particularly well-suited for the labeling and imaging of transmembrane proteins. With this probe design, the identity and relative levels of basal and hypoxia-induced transmembrane carbonic anhydrases were revealed by live cell imaging and in-gel fluorescence analysis. We believe that the extension of this difluorophenyl ester reactive module would allow for the specific labeling of various endogenous membrane proteins, facilitating in-depth studies of their distribution and functions in biological processes.
Assuntos
Ésteres/química , Corantes Fluorescentes/química , Hidrocarbonetos Fluorados/química , Proteínas de Membrana/análise , Coloração e Rotulagem , Linhagem Celular Tumoral , Ésteres/síntese química , Corantes Fluorescentes/síntese química , Humanos , Hidrocarbonetos Fluorados/síntese química , Estrutura Molecular , Imagem ÓpticaRESUMO
Cancer metastasis is a common cause of failure in cancer therapy. However, over 60% of oral cancer patients present with advanced stage disease, and the five-year survival rates of these patients decrease from 72.6% to 20% as the stage becomes more advanced. In order to manage oral cancer, identification of metastasis biomarker and mechanism is critical. In this study, we use a pair of oral squamous cell carcinoma lines, OC3, and invasive OC3-I5 as a model system to examine invasive mechanism and to identify potential therapeutic targets. We used two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) to examine the global protein expression changes between OC3 and invasive OC3-I5. A proteomic study reveals that invasive properties alter the expression of 101 proteins in OC3-I5 cells comparing to OC3 cells. Further studies have used RNA interference technique to monitor the influence of progesterone receptor membrane component 1 (PGRMC1) protein in invasion and evaluate their potency in regulating invasion and the mechanism it involved. The results demonstrated that expression of epithelial-mesenchymal transition (EMT) markers including Twist, p-Src, Snail1, SIP1, JAM-A, vimentin and vinculin was increased in OC3-I5 compared to OC3 cells, whereas E-cadherin expression was decreased in the OC3-I5 cells. Moreover, in mouse model, PGRMC1 is shown to affect not only migration and invasion but also metastasis in vivo. Taken together, the proteomic approach allows us to identify numerous proteins, including PGRMC1, involved in invasion mechanism. Our results provide useful diagnostic markers and therapeutic candidates for the treatment of oral cancer invasion.
Assuntos
Proliferação de Células/genética , Proteínas de Membrana/genética , Neoplasias Bucais/genética , Proteínas de Neoplasias/genética , Receptores de Progesterona/genética , Animais , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Xenoenxertos , Humanos , Camundongos , Neoplasias Bucais/patologia , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Metástase Neoplásica , ProteômicaRESUMO
Although many protein labeling probes have been developed to elucidate the trafficking and turnover processes of cell surface proteins, real-time tracking of intracellular proteins remains a challenging task. Herein, we describe a new design to construct a cell-permeable, photostable, and far-red fluorescent turn-on probe to enable no-wash, organelle-specific, and long-term visualization of intracellular SNAP-tagged proteins in living cells. When the probe was used in dual-color pulse chase labeling experiments to differentiate between preLamin and mature Lamin, our results reveal that the shape of mature Lamin can be altered by the newly synthesized preLamin and that this alteration is progressive, cumulative, and due to a concentration-dependent dominant-negative effect of preLamin. We believe that this probe can also be applied to other intracellular proteins whose cellular localization and synthesis changes dynamically in response to external stimuli.
Assuntos
Corantes Fluorescentes/química , Laminas/análise , Corantes Fluorescentes/metabolismo , Humanos , Laminas/metabolismo , Células MCF-7 , Membrana Nuclear/química , Membrana Nuclear/metabolismo , Imagem Óptica/métodos , Processamento de Proteína Pós-TraducionalRESUMO
Enzyme-catalyzed signal amplification with an antibody-enzyme conjugate is commonly employed in many bioanalytical methods to increase assay sensitivity. However, covalent labeling of the enzyme to the antibody, laborious operating procedures, and extensive washing steps are necessary for protein recognition and signal amplification. Herein, we describe a novel label-free and washing-free enzyme-amplified protein detection method by using dual-functional synthetic molecules to impose steric effects upon protein binding. In our approach, protein recognition and signal amplification are modulated by a simple dual-functional synthetic probe which consists of a protein ligand and an inhibitor. In the absence of the target protein, the inhibitor from the dual-functional probe would inhibit the enzyme activity. In contrast, binding of the target protein to the ligand perturbs this enzyme-inhibitor affinity due to the generation of steric effects caused by the close proximity between the target protein and the enzyme, thereby activating the enzyme to initiate signal amplification. With this strategy, the fluorescence signal can be amplified to as high as 70-fold. The generality and versatility of this strategy are demonstrated by the rapid, selective, and sensitive detection of four different proteins, avidin, O6-methylguanine DNA methyltransferase (MGMT), SNAP-tag, and lactoferrin, with four different probes.
Assuntos
Avidina/análise , Anidrase Carbônica II/metabolismo , Corantes Fluorescentes/química , Lactoferrina/análise , O(6)-Metilguanina-DNA Metiltransferase/análise , Corantes Fluorescentes/síntese química , Humanos , Ligantes , Estrutura Molecular , O(6)-Metilguanina-DNA Metiltransferase/metabolismoRESUMO
Currently most of the fluorogenic probes are designed for the detection of enzymes which work by converting the non-fluorescence substrate into the fluorescence product via an enzymatic reaction. On the other hand, the design of fluorogenic probes for non-enzymatic proteins remains a great challenge. Herein, we report a general strategy to create near-IR fluorogenic probes, where a small molecule ligand is conjugated to a novel γ-phenyl-substituted Cy5 fluorophore, for the selective detection of proteins through a non-enzymatic process. Detail mechanistic studies reveal that the probes self-assemble to form fluorescence-quenched J-type aggregate. In the presence of target analyte, bright fluorescence in the near-IR region is emitted through the recognition-induced disassembly of the probe aggregate. This Cy5 fluorophore is a unique self-assembly/disassembly dye as it gives remarkable fluorescence enhancement. Based on the same design, three different fluorogenic probes were constructed and one of them was applied for the no-wash imaging of tumor cells for the detection of hypoxia-induced cancer-specific biomarker, transmembrane-type carbonic anhydrase IX.
RESUMO
The lipid modified human N-Ras protein, implicated in human cancer development, is of particular interest due to its membrane anchor that determines the activity and subcellular location of the protein. Previous solid-state NMR investigations indicated that this membrane anchor is highly dynamic, which may be indicative of backbone conformational flexibility. This article aims to address if a dynamic exchange between three structural models exist that had been determined previously. We applied a combination of solid-state nuclear magnetic resonance (NMR) methods and replica exchange molecular dynamics (MD) simulations using a Ras peptide that represents the terminal seven amino acids of the human N-Ras protein. Analysis of correlations between the conformations of individual amino acids revealed that Cys 181 and Met 182 undergo collective conformational exchange. Two major structures constituting about 60% of all conformations could be identified. The two conformations found in the simulation are in rapid exchange, which gives rise to low backbone order parameters and nuclear spin relaxation as measured by experimental NMR methods. These parameters were also determined from two 300 ns conventional MD simulations, providing very good agreement with the experimental data.
Assuntos
Lipídeos/química , Modelos Moleculares , Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas p21(ras)/química , Simulação por Computador , Humanos , Peptídeos/metabolismo , Estrutura Secundária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Relação Estrutura-AtividadeRESUMO
Rab geranylgeranyl transferase (RabGGTase) catalyzes the attachment of geranylgeranyl isoprenoids to Rab guanine triphosphatases, which are key regulators in vesicular transport. Because geranylgeranylation is required for proper function and overexpression of Rabs has been observed in various cancers, RabGGTase may be a target for novel therapeutics. The development of selective inhibitors is, however, difficult because two related enzymes involved in other cellular processes exist in eukaryotes and because RabGGTase recognizes protein substrates indirectly, resulting in relaxed specificity. We report the synthesis of a peptidic library based on the farnesyl transferase inhibitor pepticinnamin E. Of 469 compounds investigated, several were identified as selective for RabGGTase with low micromolar IC(50) values. The compounds were not generally cytotoxic and inhibited Rab isoprenylation in COS-7 cells. Crystal structure analysis revealed that selective inhibitors interact with a tunnel unique to RabGGTase, implying that this structural motif is an attractive target for improved RabGGTase inhibitors.
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Alquil e Aril Transferases/química , Animais , Células COS , Domínio Catalítico , Chlorocebus aethiops , Inibidores Enzimáticos/síntese química , Cinética , Modelos Moleculares , Oligopeptídeos/síntese química , Biblioteca de Peptídeos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Human posttranslationally modified N-ras oncogenes are known to be implicated in numerous human cancers. Here, we applied a combination of experimental and computational techniques to determine structural and dynamical details of the lipid chain modifications of an N-ras heptapeptide in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes. Experimentally, 2H NMR spectroscopy was used to study oriented membranes that incorporated ras heptapeptides with two covalently attached perdeuterated hexadecyl chains. Atomistic molecular dynamics simulations of the same system were carried out over 100 ns including 60 DMPC and 4 ras molecules. Several structural and dynamical experimental parameters could be directly compared to the simulation. Experimental and simulated 2H NMR order parameters for the methylene groups of the ras lipid chains exhibited a systematic difference attributable to the absence of collective motions in the simulation and to geometrical effects. In contrast, experimental 2H NMR spin-lattice relaxation rates for Zeeman order were well reproduced in the simulation. The lack of slower collective motions in the simulation did not appreciably influence the relaxation rates at a Larmor frequency of 115.1 MHz. The experimental angular dependence of the 2H NMR relaxation rates with respect to the external magnetic field was also relatively well simulated. These relaxation rates showed a weak angular dependence, suggesting that the lipid modifications of ras are very flexible and highly mobile in agreement with the low order parameters. To quantify these results, the angular dependence of the 2H relaxation rates was calculated by an analytical model considering both molecular and collective motions. Peptide dynamics in the membrane could be modeled by an anisotropic diffusion tensor with principal values of Dparallel=2.1x10(9) s(-1) and Dperpendicular=4.5x10(5) s(-1). A viscoelastic fitting parameter describing the membrane elasticity, viscosity, and temperature was found to be relatively similar for the ras peptide and the DMPC host matrix. Large motional amplitudes and relatively short correlation times facilitate mixing and dispersal with the lipid bilayer matrix, with implications for the role of the full-length ras protein in signal transduction and oncogenesis.
Assuntos
Dimiristoilfosfatidilcolina/química , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética/métodos , Fluidez de Membrana , Modelos Químicos , Modelos Moleculares , Proteínas ras/química , Proteínas ras/ultraestrutura , Simulação por Computador , Hidrogênio , Movimento (Física) , Conformação ProteicaRESUMO
Many proteins involved in signal transduction are equipped with covalently attached lipid chains providing a hydrophobic anchor targeting these molecules to membranes. Despite the considerable biological significance of this membrane binding mechanism for 5-10% of all cellular proteins, to date very little is known about structural and dynamical features of lipidated membrane binding domains. Here we report the first comprehensive study of the molecular dynamics of the C-terminus of membrane-associated full-length lipidated Ras protein determined by solid-state NMR. Fully functional lipid-modified N-Ras protein was obtained by chemical-biological synthesis ligating the expressed water soluble N-terminus with a chemically synthesized (2)H or (13)C labeled lipidated heptapeptide. Dynamical parameters for the lipid chain modification at Cys 181 were determined from static (2)H NMR order parameter and relaxation measurements. Order parameters describing the amplitude of motion in the protein backbone and the side chain were determined from site-specific measurements of (1)H-(13)C dipolar couplings for all seven amino acids in the membrane anchor of Ras. Finally, the correlation times of motion were determined from temperature dependent relaxation time measurements and analyzed using a modified Lipari Szabo approach. Overall, the C-terminus of Ras shows a versatile dynamics with segmental fluctuations and axially symmetric overall motions on the membrane surface. In particular, the lipid chain modifications are highly flexible in the membrane.