RESUMEN
Mammalian ACs (adenylyl cyclases) are integrating effector molecules in signal transduction regulated by a plethora of molecules in either an additive, synergistic or antagonistic manner. Out of nine different isoforms, each AC subtype uses an individual set of regulators. In the present study, we have used chimaeric constructs, point mutations and peptide competition studies with ACs to show a common mechanism of multiple contact sites for the regulatory molecules G(betagamma) and calmodulin. Despite their chemical, structural and functional variety and different target motifs on AC, G(betagamma) and calmodulin share a two-site-interaction mechanism with G(alphas) and forskolin to modulate AC activity. Forskolin and G(alphas) are known to interact with both cytosolic domains of AC, from inside the catalytic cleft as well as at the periphery. An individual interaction site located at C(1) of the specifically regulated AC subtype had been ascribed for both G(betagamma) and calmodulin. In the present study we now show for these two regulators of AC that a second isoform- and regulator-specific contact site in C(2) is necessary to render enzyme activity susceptible to G(betagamma) or calmodulin modulation. In addition to the PFAHL motif in C(1b) of ACII, G(betagamma) contacts the KF loop in C(2), whereas calmodulin requires not only the Ca2+-independent AC28 region in C(1b) but also a Ca2+-dependent domain in C(2a) of ACI containing the VLG loop to stimulate this AC isoform.
Asunto(s)
Adenilil Ciclasas/metabolismo , Calmodulina/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Adenilil Ciclasas/química , Adenilil Ciclasas/genética , Secuencia de Aminoácidos , Animales , Bovinos , Línea Celular , Eliminación de Gen , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , SpodopteraRESUMEN
In the early steps of visual signal transduction, light-activated rhodopsin (R*) catalyzes GDP/GTP exchange in the heterotrimeric G protein (Galphabetagamma) transducin. We recently reported that the catalytic interaction involves two sequential steps. An initial docking between R* and Gbetagamma leads to conformational changes which make the C-terminus of Galpha (CTalpha) available for binding to R*. Binding of CTalpha by R* then triggers GDP/GTP exchange in the Galpha subunit. To further study this two-step mechanism, we investigated different single amino acid substitutions within CTalpha and discuss the effects of high affinity mutations on nucleotide exchange catalysis.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Rodopsina/metabolismo , Segmento Externo de la Célula en Bastón/metabolismo , Transducina/metabolismo , Sustitución de Aminoácidos , Animales , Bovinos , Interacciones Hidrofóbicas e Hidrofílicas , Espectroscopía de Resonancia Magnética , Unión Proteica , Conformación Proteica , Secuencias Repetidas Terminales , Visión Ocular/fisiologíaRESUMEN
INTRODUCTION: DNA-based TLR9 agonists are potent activators of the immune system. ProMune® and dSLIM® belong to different families of TLR9 agonists and both have been established as cancer immunotherapeutics in clinical proof-of-concept studies. Unfortunately, ProMune® failed in pivotal oncological trials. dSLIM®, the active ingredient of Lefitolimod (MGN1703), successfully finished a double-blinded, placebo-controlled phase II study in patients with advanced colorectal cancer, exhibiting improved progression-free survival and durable disease control. METHODS: To explain the different systemic efficacies of dSLIM® and ProMune®, both TLR9 agonists and chimeric molecules thereof are analyzed side-by-side in a panel of in vitro assays for immune activation. RESULTS AND CONCLUSIONS: Indeed, dSLIM® exposure results in an IFN-α dependent broad activation of immune cells whereas ProMune® strongly stimulates B cells. Moreover, all functional effects of dSLIM® strictly depend on the presence of CG-motifs within its dumbbell-shaped, covalently closed structural context. Conversely, several immunological effects of ProMune® like IL-8 secretion are independent of CG-motifs and could be ascribed to the phosphorothioate-modifications of its DNA backbone, which may have caused the side effects of ProMune® in clinical trials. Finally, we showed that the implementation of ProMune® (ODN2006) base sequence into the characteristic dSLIM® dumbbell form resulted in dSLIM2006 with all beneficial effects for immunostimulation combined from both TLR9 classes without any CG-independent effects.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Oligodesoxirribonucleótidos/farmacología , Antineoplásicos/inmunología , Secuencia de Bases , Células Cultivadas , Citocinas , Células Dendríticas , Humanos , Interferón-alfa , Oligodesoxirribonucleótidos/inmunología , Receptor Toll-Like 9RESUMEN
Toll-like receptors are sensing modulators of the innate immune system. One member of this protein family, Toll-like receptor (TLR)-9, is increasingly being investigated as therapeutic target for infectious diseases and cancer. Double-Stem Loop ImmunoModulator (dSLIM) is a new TLR-9 agonist in clinical development for patients with metastatic colorectal carcinoma. Compared with other TLR-9 ligands developed as immunomodulators, dSLIM comprises single- and double-stranded DNA, is covalently closed, and consists of natural nucleotide components only. All investigated biologic effects of dSLIM are strongly dependent on CG motifs, and the relevant cellular activation profile of dSLIM is distinct to that of other TLR-9 agonists. Here we describe the structure and biologic profile of dSLIM: in isolated human peripheral blood mononuclear cells (PBMCs), dSLIM induced a unique pattern of cytokine secretion, activated within the PBMC pool particular cell subpopulations, and exhibited specific cytotoxicity on target cells. Using cellular isolation and depletion setups, the mechanism of immunoactivation by dSLIM was deduced to be dependent on, but not restricted to, TLR-9-bearing plasmacytoid dendritic cells. The dSLIM-promoted cellular stimulation directs systemic activation of the immune response as revealed in cancer patients. The observed cellular activation cascades are discussed in the context of cancer therapy.
RESUMEN
We have previously shown that the combination of MIDGE-Th1 DNA vectors with the cationic lipid SAINT-18 increases the immune response to the encoded antigen in mice. Here, we report on experiments to further optimize and characterize this approach. We evaluated different formulations of MIDGE-Th1 vectors with SAINT-18 by assessing their influence on the transfection efficiency in cell culture and on the immune response in mice. We found that high amounts of SAINT-18 in formulations with a w/w ratio MIDGE Th1/SAINT-18 of 1:4.8 are beneficial for cell transfection in vitro. In contrast, the formulation of HBsAg-encoding MIDGE-Th1 DNA vectors with the lowest amount of SAINT-18 (w/w ratio MIDGE Th1/SAINT-18 of 1:0.5) resulted in the highest serum IgG1 and IgG2a levels after intradermal immunization of mice. Consequently, latter formulation was selected for a comparative biodistribution study in rats. Following intradermal administration of both naked and formulated MIDGE-Th1 DNA, the vectors localized primarily at the site of injection. Vector DNA levels decreased substantially over the two months duration of the study. When administered in combination with SAINT-18, the vectors were found in significantly higher amounts in draining lymph nodes in comparison to administration of naked MIDGE-Th1 DNA. We propose that the high immune responses induced by MIDGE-Th1/SAINT-18 lipoplexes are mediated by enhanced transfection of cells in vivo, resulting in stronger antigen expression and presentation. Importantly, the combination of MIDGE-Th1 vectors with SAINT-18 was well tolerated in mice and rats and is expected to be safe in human clinical applications.
Asunto(s)
Vectores Genéticos/química , Compuestos de Piridinio/química , Vacunas de ADN/inmunología , Animales , Anticuerpos Antivirales/sangre , Cationes , Femenino , Vectores Genéticos/farmacocinética , Antígenos de Superficie de la Hepatitis B/inmunología , Inmunoglobulina G/sangre , Ganglios Linfáticos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Compuestos de Piridinio/farmacocinética , Ratas , Ratas Wistar , Células TH1/inmunología , Distribución Tisular , Transfección , Vacunas de ADN/farmacocinéticaRESUMEN
Currently marketed vaccines against hepatitis B virus (HBV) based on the small (S) hepatitis B surface antigen (HBsAg) fail to induce a protective immune response in about 10% of vaccinees. DNA vaccination and the inclusion of PreS1 and PreS2 domains of HBsAg have been reported to represent feasible strategies to improve the efficacy of HBV vaccines. Here, we evaluated the immunogenicity of SAINT-18-formulated MIDGE-Th1 vectors encoding the S or the large (L) protein of HBsAg in mice and pigs. In both animal models, vectors encoding the secretion-competent S protein induced stronger humoral responses than vectors encoding the L protein, which was shown to be retained mainly intracellularly despite the presence of a heterologous secretion signal. In pigs, SAINT-18-formulated MIDGE-Th1 vectors encoding the S protein elicited an immune response of the same magnitude as the licensed protein vaccine Engerix-B, with S protein-specific antibody levels significantly higher than those considered protective in humans, and lasting for at least six months after the third immunization. Thus, our results provide not only the proof of concept for the SAINT-18-formulated MIDGE-Th1 vector approach but also confirm that with a cationic-lipid formulation, a DNA vaccine at a relatively low dose can elicit an immune response similar to a human dose of an aluminum hydroxide-adjuvanted protein vaccine in large animals.
Asunto(s)
Vectores Genéticos/química , Antígenos de Superficie de la Hepatitis B/inmunología , Compuestos de Piridinio/química , Células TH1/inmunología , Vacunas de ADN/inmunología , Animales , Células CHO , Cationes , Cricetulus , Femenino , Vacunas contra Hepatitis B/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , PorcinosRESUMEN
Previously, minimalistic, immunogenetically defined gene expression (MIDGE) vectors were developed as effective and sophisticated carriers for DNA vaccination. Here we evaluate the influence of dose, formulation and delivery route on the immune response after vaccination with MIDGE-Th1 vectors encoding hepatitis B virus surface antigen (HBsAg). An HBsAg-specific IgG1 and IgG2a antibody response was induced in a dose-dependent manner, whereas the IgG2a/IgG1 ratio was independent of the injected DNA dose. Formulation of MIDGE-HBsAg-Th1 with the cationic pyridinium amphiphile SAINT-18 significantly increased antibody levels of IgG1 and IgG2a compared to the unformulated vector. In contrast, SAINT-18 had neither a significant effect on the IgG2a/IgG1 ratio nor on the type and strength of cellular immunity. Overall, the strongest immune response was generated after intradermal injection, followed by intramuscular and subcutaneous (s.c.) injection. The results show that the formulation of MIDGE-Th1 with SAINT-18 increased the efficacy of the MIDGE-Th1 DNA vaccine and is therefore a suitable approach to improve the efficacy of DNA vaccines also in large animals and humans.
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Formación de Anticuerpos/inmunología , Inmunidad Celular/inmunología , Vacunación/métodos , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Animales , Relación Dosis-Respuesta Inmunológica , Femenino , Anticuerpos Antihepatitis/sangre , Antígenos de Superficie de la Hepatitis B/inmunología , Inmunoglobulina G/sangre , Inyecciones Intradérmicas , Inyecciones Intramusculares , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos BALB C , Células TH1/inmunologíaRESUMEN
The Gbetagamma complex of heterotrimeric G proteins is the most outstanding example for the divergent regulation of mammalian adenylyl cyclases. The heterodimeric Gbetagamma complex inhibits some isoforms, e.g. ACI, and stimulates the isoforms ACII, -IV, and -VII. Although former studies identified the QEHA region located in the C2 domain of ACII as an important interaction site for Gbetagamma, the determinant of the stimulatory effect of Gbetagamma has not been detected. Here, we identified the C1b domain as the stimulatory region using full-length adenylyl cyclase. The relevant Gbetagamma signal transfer motif in IIC1b was determined as MTRYLESWGAAKPFAHL (amino acids 493-509). Amino acids of this PFAHL motif were absolutely necessary for ACII to be stimulated by Gbetagamma, whereas they were dispensable for Galpha(s) or forskolin stimulation. The PFAHL motif is present in all three adenylyl cyclase isoforms that are activated by Gbetagamma but is absent in other adenylyl cyclase isoforms as well as other known effectors of Gbetagamma. The emerging concept of two contact sites on different molecule halves for effective regulation of adenylyl cyclase is discussed.
Asunto(s)
Adenilil Ciclasas/química , Adenilil Ciclasas/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Adenilil Ciclasas/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Estructura Terciaria de ProteínaRESUMEN
Covalent lipid attachments are essential co- and post-translational modifications for signalling proteins. Galpha(s), the alpha-subunit of the heterotrimeric G protein that activates adenylyl cyclase, is known to be palmitoylated at the third N-terminal amino acid, a cysteine. Palmitoylation is involved in anchoring Galpha(s) to the membrane by increasing its intrinsic hydrophobicity. We identified by mass spectrometry a second, functionally even more important, covalent modification. It consists of another palmitoyl residue attached to the preceding glycine (Gly(2)). Palmitoylation at this position has profound consequences for levels of signal transduction. It sensitizes the cell up to 200-fold for adenylyl cyclase-stimulating agents. The inhibitory inputs mediated by Galpha(i) are downregulated to <10%. Thereby, Gly(2)-palmitoylation of Galpha(s) relieves cellular stimulation at the level of adenylyl cyclase whereas it renders the inhibitory modulation via Galpha(i) more difficult.
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Proteínas de Caenorhabditis elegans , Subunidades alfa de la Proteína de Unión al GTP Gi-Go , Glicina/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Palmitoil Coenzima A/metabolismo , Inhibidores de Adenilato Ciclasa , Adenilil Ciclasas/metabolismo , Cisteína/metabolismo , Activación Enzimática/fisiología , Proteínas de Unión al GTP Heterotriméricas/química , Humanos , Espectrometría de MasasRESUMEN
Guanine nucleotide exchange in heterotrimeric G proteins catalyzed by G protein-coupled receptors (GPCRs) is a key event in many physiological processes. The crystal structures of the GPCR rhodopsin and two G proteins as well as binding sites on both catalytically interacting proteins are known, but the temporal sequence of events leading to nucleotide exchange remains to be elucidated. We employed time-resolved near infrared light scattering to study the order in which the Galpha and Ggamma C-terminal binding sites on the holo-G protein interact with the active state of the GPCR rhodopsin (R*) in native membranes. We investigated these key binding sites within mass-tagged peptides and G proteins and found that their binding to R* is mutually exclusive. The interaction of the holo-G protein with R* requires at least one of the lipid modifications of the G protein (i.e. myristoylation of the Galpha N terminus and/or farnesylation of the Ggamma C terminus). A holo-G protein with a high affinity Galpha C terminus shows a specific change of the reaction rate in the GDP release and GTP uptake steps of catalysis. We interpret the data by a sequential fit model where (i) the initial encounter between R* and the G protein occurs with the Gbetagamma subunit, and (ii) the Galpha C-terminal tail then interacts with R* to release bound GDP, thereby decreasing the affinity of R* for the Gbetagamma subunit. The mechanism limits the time in which both C-terminal binding sites of the G protein interact simultaneously with R* to a short lived transitory state.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Catálisis , Bovinos , Clonación Molecular , Cristalografía por Rayos X , ADN Complementario/metabolismo , Dimerización , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Insectos , Cinética , Luz , Lípidos/química , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Unión Proteica , Conformación Proteica , Prenilación de Proteína , Estructura Terciaria de Proteína , Células Fotorreceptoras Retinianas Bastones , Rodopsina/química , Dispersión de Radiación , Factores de Tiempo , Transducina/químicaRESUMEN
S100A1, a Ca2+-sensing protein of the EF-hand family, is most highly expressed in myocardial tissue, and cardiac S100A1 overexpression in vitro has been shown to enhance myocyte contractile properties. To study the physiological consequences of S100A1 in vivo, transgenic mice were developed with cardiac-restricted overexpression of S100A1. Characterization of two independent transgenic mouse lines with approximately 4-fold overexpression of S100A1 in the myocardium revealed a marked augmentation of in vivo basal cardiac function that remained elevated after beta-adrenergic receptor stimulation. Contractile function and Ca2+ handling properties were increased in ventricular cardiomyocytes isolated from S100A1 transgenic mice. Enhanced cellular Ca2+ cycling by S100A1 was associated both with increased sarcoplasmic reticulum Ca2+ content and enhanced sarcoplasmic reticulum Ca2+-induced Ca2+ release, and S100A1 was shown to associate with the cardiac ryanodine receptor. No alterations in beta-adrenergic signal transduction or major cardiac Ca2+-cycling proteins occurred, and there were no signs of hypertrophy with chronic cardiac S100A1 overexpression. Our findings suggest that S100A1 plays an important in vivo role in the regulation of cardiac function perhaps through interacting with the ryanodine receptor. Because S100A1 protein expression is down-regulated in heart failure, increasing S100A1 expression in the heart may represent a novel means to augment contractility.