Christiane Kleuss

Gαs is palmitoylated at the N‐terminal glycine

Covalent lipid attachments are essential co‐ and post‐translational modifications for signalling proteins. Gαs, the α‐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 Gαs 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 (Gly2). 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 Gαi are downregulated to <10%. Thereby, Gly2‐palmitoylation of Gαs relieves cellular stimulation at the level of adenylyl cyclase whereas it renders the inhibitory modulation via Gαi more difficult.

Gβγ Activation Site in Adenylyl Cyclase Type II ADENYLYL CYCLASE TYPE III IS INHIBITED BY Gβγ

The Gβγ complex of heterotrimeric G proteins is the most outstanding example for the divergent regulation of mammalian adenylyl cyclases. The heterodimeric Gβγ 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 Gβγ, the determinant of the stimulatory effect of Gβγ has not been detected. Here, we identified the C1b domain as the stimulatory region using full-length adenylyl cyclase. The relevant Gβγ 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 Gβγ, whereas they were dispensable for Gαs or forskolin stimulation. The PFAHL motif is present in all three adenylyl cyclase isoforms that are activated by Gβγ but is absent in other adenylyl cyclase isoforms as well as other known effectors of Gβγ. The emerging concept of two contact sites on different molecule halves for effective regulation of adenylyl cyclase is discussed.

[25] Palmitoylation of G-protein α subunits

Publisher Summary This chapter describes the procedures used to detect protein palmitoylation. The protocols for metabolic radiolabeling of cells and for analysis of radioactive fatty acids incorporated into proteins are also reviewed. Covalent attachment of palmitate to protein occurs usually through a thioester linkage to cysteine. However, the lipid may also be bound to protein through oxyester linkages to serine or threonine. Fatty acylation of proteins through ester bonds is not specific for palmitate. Longer chain saturated fatty acids, such as stearate, are also detected. Modification of proteins by myristate is primarily by cotranslational addition of the fatty acid through an amide linkage to the amino terminus of the protein. Protein palmitoylation contributes to membrane association of a number of proteins including G-protein α subunits, and the neuronal growth cone protein GAP-43. Palmitoylation of proteins is usually assayed by isolating the protein from cells that have been incubated with radioactive palmitic acid. Thioester linkages are hydrolyzed by treatment with base, and fatty acids cleaved from the protein can be analyzed by reversed phase high-performance liquid chromatography (HPLC) or thin-layer chromatography (TLC).

Resolution of transducin subunits by chromatography on blue sepharose.

The retinal guanine nucleotide-binding protein, transducin (TD), was subjected to chromatography on Blue Sepharose (BLS). A simple two-step protocol was developed, allowing the resolution of the alpha-subunit and the beta gamma-complex of the protein extracted from bovine retina by the use of a poorly hydrolysable GTP analogue. If TD was applied to BLS in a divalent cation-containing buffer, the beta gamma-complex did not bind to the resin, whereas the alpha-subunit was retained; elution of the latter was achieved by removing the divalent cation from the buffer. Binding of the alpha-subunit to BLS was not affected by nucleotides or by ADP ribosylation catalysed by bacterial toxins. However, adsorption of the alpha-subunit by BLS or by a strong cation exchanger (Mono S) depended strictly on divalent cations. In contrast to previous reports, the data suggest the formation of a complex between a sulphonyl residue of Cibacron Blue, a divalent metal ion, and the alpha-subunit as the relevant binding mechanism causing adsorption of the alpha-subunit to BLS.

Two interaction sites on mammalian adenylyl cyclase type I and II: modulation by calmodulin and Gβγ

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.

Immune response induced by a linear DNA vector: Influence of dose, formulation and route of injection

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.

[27] Microinjection of antisense oligonucleotides to assess G-protein subunit function

Publisher Summary Heterotrimeric G proteins represent a family of closely related regulatory proteins, composed of one α , β , and γ subunit each. About 20 different cDNA sequences of α subunits are known, and at least 4 different cDNAs coding for G-protein β ubunits and 6 cDNAs of γ subunits have been identified. The G proteins are grouped into subfamilies classified according their α subunits. The functions of the various G proteins are highly diverse. For some subfamilies, the physiological roles, namely, receptors stimulating the G protein and the G-protein-regulated effectors, are at least partially known; for others the functions remain largely unknown. Assignment of a function to individual members of the G-protein family, to specific subunits, and to αβγ -subunit heterotrimers is an important task in future studies on G-protein-mediated signal transduction. This chapter presents a new approach to determine the functional identity of G proteins involved in hormonal regulations that can be studied on a single cell level. Antisense oligonucleotides selectively hybridizing with the mRNA of a G-protein subunit were microinjected into the nuclei of single cells. Following incubation to wait for reduction in the concentration of the target subunit, the resulting biological effects were measured electrophysiologically by the whole-cell modification of the patch-clamp method.

Cationic Lipid-Formulated DNA Vaccine against Hepatitis B Virus: Immunogenicity of MIDGE-Th1 Vectors Encoding Small and Large Surface Antigen in Comparison to a Licensed Protein Vaccine

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.

Genuine Immunomodulation With dSLIM

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.

Combination of MIDGE-Th1 DNA vaccines with the cationic lipid SAINT-18: Studies on formulation, biodistribution and vector clearance

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.