Contribution of protein kinase A and protein kinase C signalling pathways to the regulation of HSD11B2 expression and proliferation of MCF-7 cells

Contribution of the protein kinase A (PKA) and protein kinase C (PKC) signalling pathways to the regulation of 11beta-hydroxysteroid dehydrogenase type II (HSD11B2) gene expression was investigated in human breast cancer cell line MCF-7. Treatment of the cells with an adenylyl cyclase activator, forskolin, known to stimulate the PKA pathway, resulted in an increase in HSD11B2 mRNA content. Semi-quantitative RT-PCR revealed attenuation of the effect of forskolin by phorbol ester, tetradecanoyl phorbol acetate (TPA), an activator of the PKC pathway. It was also demonstrated that specific inhibitors significantly reduced the effect of activators of the two pathways. Stimulation of the PKA pathway did not affect, whereas stimulation of the PKC pathway significantly reduced MCF-7 cell proliferation in a time-dependent manner. A cell growth inhibitor, dexamethasone, at high concentrations, caused a 40% decrease in proliferation of MCF-7 cells and this effect was abolished under conditions of increased HSD11B2 expression. It was concluded that in MCF-7 cells, stimulation of the PKA signal transduction pathway results in the induction of HSD11B2 expression and that this effect is markedly reduced by activation of the PKC pathway. Activation of the PKC pathway also resulted in inhibition of cell proliferation, while activation of the PKA pathway abolished the antiproliferative effect of dexamethasone. These effects might be due to oxidation of dexamethasone by the PKA-inducible HSD11B2.     

Global Profiling of Huntingtin-associated protein E (HYPE)-Mediated AMPylation through a Chemical Proteomic Approach

AMPylation of mammalian small GTPases by bacterial virulence factors can be a key step in bacterial infection of host cells, and constitutes a potential drug target. This posttranslational modification also exists in eukaryotes, and AMP transferase activity was recently assigned to HYPE Filamentation induced by cyclic AMP domain containing protein (FICD) protein, which is conserved from Caenorhabditis elegans to humans. In contrast to bacterial AMP transferases, only a small number of HYPE substrates have been identified by immunoprecipitation and mass spectrometry approaches, and the full range of targets is yet to be determined in mammalian cells. We describe here the first example of global chemoproteomic screening and substrate validation for HYPE-mediated AMPylation in mammalian cell lysate. Through quantitative mass-spectrometry-based proteomics coupled with novel chemoproteomic tools providing MS/MS evidence of AMP modification, we identified a total of 25 AMPylated proteins, including the previously validated substrate endoplasmic reticulum (ER) chaperone BiP (HSPA5), and also novel substrates involved in pathways of gene expression, ATP biosynthesis, and maintenance of the cytoskeleton. This dataset represents the largest library of AMPylated human proteins reported to date and a foundation for substrate-specific investigations that can ultimately decipher the complex biological networks involved in eukaryotic AMPylation.Covalent posttranslational modification (PTM) of hydroxyl-containing amino acids in proteins by adenosine monophosphate (AMP), called AMPylation or adenylylation, was first discovered almost a half century ago as a mechanism controlling the activity of bacterial glutamine synthetase (1). This unusual PTM was unknown in eukaryotes until it was identified in 2009 in the context of bacterial infection, when Yarbrough et al. reported AMPylation of host small GTPases by bacterial virulence factor Vibrio outer protein S (VopS) from Vibrio parahemeolyticus. In this context, AMPylation precludes interactions with downstream binding partners and causes actin cytoskeleton collapse leading to cell death (2). Since then, the field of AMPylation has grown substantially, with reports describing AMPylation activity of other bacterial effectors, like Immunoglobulin binding protein A (IbpA) in Histophilus somni (3) and Defects in Rab1 recruitment protein A (DrrA) in Legionella pneumophila (4). These new bacterial AMPylators share a common substrate class (small GTPases); however, they differed in the identity of their catalytic residues and architecture of their active sites. Accordingly, bacterial AMP transferases have been classified as either filamentation induced by cyclic AMP (FIC) or adenylyl transferase (AT)¹ domain containing enzymes, with catalytic His or Asp residues, respectively.Although adenylylation has been most extensively described in the context of bacterial infection, there is a growing interest in elucidating the scope of this PTM in a native eukaryotic context. Among the ca. 3000 FIC proteins identified so far by sequence alignment, only a single enzyme has been identified in eukaryotes: Huntingtin-associated protein E (HYPE), also known as FICD. HYPE is conserved from C. elegans to humans, and mRNA expression data suggest that it is present at low levels in all human tissues (3). Apart from the catalytic FIC domain, the protein consists of one transmembrane helix and two tetratricopeptide repeat motifs that point to localization at a membrane and amenability toward protein–protein interactions, respectively. We recently added to this picture by solving the first crystal structure of Homo sapiens HYPE (5), illustrating that the only human FIC is substantially different from its bacterial cousins (6, 7). HYPE was shown to form stable asymmetric dimers supported by the extended network of contacts exclusive to the FIC domains, while the tetratricopeptide repeat motifs have a more flexible arrangement and appear to be exposed for protein–protein interactions in the vicinity of the membrane. In addition, we confirmed the similarity of the active site architecture to other FIC proteins for which a crystal structure is available, with the catalytic loop comprising the invariant catalytic His³⁶³ (8), and further substantiated the role of a critical residue Glu²³⁴ in an inhibitory helix (9) that may be responsible for regulating HYPE enzymatic activity.Various catalytic activities have been demonstrated for FIC proteins, including nucleotide (AMP, GMP, and UMP) transfer as well as phosphorylation and phosphocholination (10–13). We and others (3, 5, 14, 15) have demonstrated that HYPE can function in protein AMPylation, although the activity of the wild-type (WT) enzyme is very weak, consistent with active site obstruction by Glu²³⁴. It is hypothesized that this intramolecular inhibition can be relieved by specific but as yet unknown protein–protein interactions or by the removal of the conserved Glu. Indeed, the E234G mutation substantially boosts HYPE''s activity as demonstrated by the elevated auto-AMPylation of HYPE itself (5, 9) and a few of its recently reported substrates, including the ER chaperone BiP in vivo (14, 15) and several histone proteins in vitro (16, 17). HYPE activity was initially implicated in visual neurotransmission in flies (18) and later in regulation of the unfolded protein response (UPR) in transfected cells, although there is limited consensus over the mechanism (14, 15). Most recently, it has been proposed that HYPE activity might have a role in regulation of gene expression; however, the mechanistic details remain to be elucidated (17).AMPylation profiling is not a trivial task (19), and several strategies have emerged over the past few years ranging from labeling with radioactive ATP (2, 3) and immunoprecipitation with AMPylation-specific antibodies (20, 21) to mass spectrometry (MS) approaches focused on AMP fragmentation (22, 23). Although these methods contributed significantly to developments in the field, they also suffer from certain drawbacks, including low sensitivity, high background, limited quantitative power, and limited amenability to high-throughput (HT) substrate identification. In contrast, chemoproteomic strategies involving application of substrate analogues (substrate probes) equipped with small and inert chemical handles in combination with sensitive detection by MS can facilitate rapid visualization and/or robust enrichment of modified proteins and can provide superior performance in HT profiling of numerous challenging PTMs (24). AMPylation-specific substrate probes have been developed, and their robust performance was evaluated in vitro, albeit to date only in the context of bacterial effector-mediated AMPylation (25–27). We previously showed that a bioorthogonal substrate probe (26) is well tolerated in the active site of human HYPE and, moreover, that it has potential for chemoproteomic profiling of HYPE substrates in vitro when combined with ligation through copper-catalyzed azide alkyne cycloaddition (CuAAC) to a dedicated capture reagent decorated with a biotin affinity handle and carboxytetramethylrhodamine (TAMRA) fluorophore (5).Herein, we present the first global AMPylation profile in a native eukaryotic context utilizing a bioorthogonal ATP analogue and chemoproteomic methodology. We first demonstrate efficient enrichment and fast visualization of potential HYPE substrates in cell lysates by in-gel fluorescence, followed by robust identification via shotgun proteomics on a QExactive mass spectrometer. Furthermore, we extensively validate candidate substrates via HYPE titration and ATP competition experiments with a quantitative MS-based readout, as well as Western blotting and direct MS/MS evidence for AMP modification. Finally, we analyze HYPE interaction partners in vivo, providing a link between our discoveries in lysates and a physiologically relevant context, delivering the first experimentally validated library of HYPE substrate proteins.     

Validation,reproducibility and safety of trans dermal electrical stimulation in chronic pain patients and healthy volunteers

Background  

Surrogate pain models have been extensively tested in Normal Human Volunteers (NHV). There are few studies that examined pain models in chronic pain patients. Patients are likely to have altered pain mechanisms. It is of interest to test patient pain responses to selective pain stimuli under controlled laboratory conditions.     

Lipase-mediated kinetic resolution of racemic and desymmetrization of prochiral organophosphorus P-boranes

Enantiomerically enriched alkoxy(hydroxymethyl)phenylphosphine boranes were obtained via a lipase-catalyzed acetylation performed under kinetic resolution conditions. The reaction was slow and proceeded with a rather low enantioselectivity. A lipase-catalyzed acetylation of prochiral bis(2-hydroxyethyl)phenylphosphine borane resulted in its desymmetrization and gave the enantiomerically enriched monoacetyl derivative with ee up to 90%.     

Novel way of capping mRNA trimer and studies of its interaction with human nuclear cap-binding complex

Binding of mRNA 5' cap by the nuclear cap-binding complex (CBC) is crucial for a wide variety of mRNA metabolic events. The interaction involving the CBP20 subunit of CBC is mediated by numerous hydrogen bonds and by stacking of the tyrosine sidechains with two first bases of the capped mRNA. To examine a possible role of a longer mRNA chain in the CBC-cap recognition, we have synthesized an mRNA tetramer using a novel way of capping an RNA trimer and determined its affinity for CBC by fluorescence titration.     

Argireline: Needle-Free Botox as Analytical Challenge

Argireline-containing cosmetics attract public interest due to their confirmed reduction of facial wrinkles. Argireline is a peptide that works by inhibiting the release of neurotransmitters in the neuromuscular junction, producing a botox-like effect. Therefore, it is used as a safe needle-free alternative to botox treatment. In this work we investigated the presence of Argireline in cosmetic creams and sera by application of reversed phase liquid chromatography and tandem mass spectrometry (RP-HPLC/MS and MS/MS). The analysis revealed the presence of argireline and its oxidized form in several different cosmetics. The methionine residue in Argireline sequence was indicated as oxidation point according to neutral loss MS studies. The developed sample preparation strategy minimizes and monitors methionine oxidation, bringing to our attention the question of impact of ingredients on the stability of cosmetic product.     

The helical hairpin structure of the influenza fusion peptide can be seen on a hydrophobic moment map

An assignment of the helical hairpin of the influenza fusion peptide has been made based on the hydrophobic moments, represented in a form of two-dimensional map. Such assignment holds for all serotypes, even for the cases of mutations altering the amino acid character. Similar results are obtained for the experimentally developed hydrophobicity scales, whose values reflect the transfer energies between aqueous and membrane environments. A distinct, however still structure-related hydrophobic map corresponds to a helical and contiguous HIV gp41 fp. The method may be used as a simple tool for sequence-based prediction of structures adopted by viral fusion peptides.     

Single cell analysis of ligand binding and complex formation of interleukin-4 receptor subunits

Interleukin-4 (IL-4) is an important class I cytokine involved in adaptive immunity. IL-4 binds with high affinity to the single-pass transmembrane receptor IL-4Rα. Subsequently, IL-4Rα/IL-4 is believed to engage a second receptor chain, either IL-2Rγ or IL-13Rα1, to form type I or II receptor complexes, respectively. This ternary complex formation then triggers downstream signaling via intracellular Janus kinases bound to the cytoplasmic receptor tails. Here, we study the successive steps of complex formation at the single cell level with confocal fluorescence imaging and correlation spectroscopy. We characterize binding and signaling of fluorescently labeled IL-4 by flow cytometry of IL-4-dependent BaF3 cells. The affinity to ectopically expressed IL-4Rα was then measured by single-color fluorescence correlation spectroscopy in adherent HEK293T cells that express the components of the type II IL-4R but not type I. Finally, IL-4-induced complex formation was tested by dual-color fluorescence cross-correlation spectroscopy. The data provide evidence for codiffusion of IL-4-A647 bound IL-4Rα and the type II subunit IL-13Rα1 fused to enhanced green fluorescent protein, whereas type I complexes containing IL-2Rγ and JAK3 were not detected at the cell surface. This behavior may reflect hitherto undefined differences in the mode of receptor activation between type I (lymphoid) and type II (epithelial) receptor expressing cells.     

Effect of temperature on the formation of liquid phase-separating giant unilamellar vesicles (GUV)

Giant unilamellar vesicles (GUVs) are widely used as model systems to study both, lipid and membrane protein behavior. During their preparation by the commonly applied electroformation method, a number of issues must be considered to avoid the production of artifacts due to a poor lipid hydration and protein degradation. Here we focus on the effect of preparation temperature on GUVs composed of the most commonly used domain-forming mixture dioleoylelphospatidylcholine/shingomyelin/cholesterol (DOPC/SM/chol) (2/2/1). Lower production temperatures are generally preferable when aiming at a functional reconstitution of proteins into the membrane. On the other hand, lower growth temperature is suspected to alter the lipid composition and the yield of phase-separating vesicles. By confocal imaging, we find that vesicles prepared significantly above and below the melting temperature T(m) have the same overall morphology, similar size distributions of vesicles and a similar area coverage by liquid-ordered (L(o)) domains. However, a large population analysis indeed reveals a different overall yield of phase-separating vesicles. Two-focus scanning fluorescence correlation spectroscopy measurements did not show any divergence of lipid analog mobility in (L(o)) and (L(d)) phases in vesicles prepared at different temperatures, indicating that the lowered growth temperature did not influence the lipid organization within the two phases. Moreover, the expected advantages of lower preparation temperature for proteo-GUVs could be exemplified by the reconstitution of voltage dependent anion channel (VDAC) into DOPC/SM/chol GUVs, which aggregates at high, but not at low preparation temperatures.