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Annette G Beck-Sickinger

Publications and source records attributed to Annette G Beck-Sickinger.

At least 55 records · Page 3Linked to original sources
PubMed2004

Cellular uptake but low permeation of human calcitonin-derived cell penetrating peptides and Tat(47-57) through well-differentiated epithelial models.

PURPOSE: To investigate whether cell penetrating peptides (CPP) derived from human calcitonin (hCT) possess, in addition to cellular uptake, the capacity to deliver their cargo through epithelial barriers. METHODS: Cellular uptake of hCT(9-32) and permeation of six hCT-derived peptides, namely, hCT(9-32), hCT(12-32), hCT(15-32), hCT(18-32), hCT(21-32), and a random sequence of hCT(9-32) were evaluated in fully organized confluent Madin-Darby canine kidney (MDCK), Calu-3, and TR146 cell culture models. For comparison, Tat(47-57) and penetratin(43-58) were investigated. The peptides were N-terminally labeled with carboxyfluorescein (CF). Uptake in the well-differentiated epithelial models was observed by confocal laser scanning microscopy (CLSM), whereas permeation through the models was analyzed by reversed-phase (RP)-HPLC. RESULTS: In MDCK epithelium hCT(9-32), Tat(47-57) and penetratin(43-58) demonstrated punctuated cytoplasmic distribution. In Calu-3, Tat(47-57) and penetratin(43-58) were simultaneously localized in a punctuated cytoplasmic and paracellular distribution, whereas hCT(9-32) showed strict paracellular distribution. By contrast, in TR146 cells, Tat(47-57) was located strictly paracellularily, whereas penetratin(43-58) showed a punctuated cytoplasmic pattern and hCT(9-32) both. The transepithelial permeability of all tested peptides and their cargo was lower than that of paracellular markers. CONCLUSIONS: The CPP uptake pattern depends on both the type of peptide and the cell culture model. In general, the investigated CPP have no apparent potential for systemic drug delivery across epithelia. Nevertheless, distinct patterns of cellular distribution may offer a potential for localized epithelial delivery.

Animals↗
PubMed2004

From transcription profile to expression: the signaling repertoire of the SK-N-MC neuroepithelioma cell-line.

SK-N-MC neuroepithelioma cells are routinely cultured and widely used as a model system in biochemical and pharmacological experiments. To clarify the gene expression patterns of SK-N-MC cells with respect to G protein-coupled receptors and signaling network components, we describe in this report the transcription profile of the cell line. Following the traditional pathway from genome to proteome, selected examples are further examined at the level of protein expression and by functional assays. cRNA targets derived from total RNA extracts were hybridized to Affymetrix Human Genome U133A GeneChip arrays, and the data were analyzed and grouped according to functional aspects. Results obtained for neuropeptide Y (NPY) Y1, Y5, and orexin Ox1 receptors were confirmed by RT-PCR. It is surprising that we found the presence of both NPY receptor subtypes and the absence of the orexin receptor at the mRNA level. Receptor-binding experiments confirmed NPY binding of the Y1 receptor in the nanomolar range but gave no evidence for high expression levels of Y5 receptor subtypes on the cell surface. Protein expression was assayed with immunoblots by using antibodies directed against selected Galpha protein subunits. The presence of at least Galphas, Galphai3, and Galphai2 subunits was indicated.

Binding Sites↗
PubMed2004

Expressed protein ligation. Method and applications.

The introduction of noncanonical amino acids and biophysical probes into peptides and proteins, and total or segmental isotopic labelling has the potential to greatly aid the determination of protein structure, function and protein-protein interactions. To obtain a peptide as large as possible by solid-phase peptide synthesis, native chemical ligation was introduced to enable synthesis of proteins of up to 120 amino acids in length. After the discovery of inteins, with their self-splicing properties and their application in protein synthesis, the semisynthetic methodology, expressed protein ligation, was developed to circumvent size limitation problems. Today, diverse expression vectors are available that allow the production of N- and C-terminal fragments that are needed for ligation to produce large amounts and high purity protein(s) (protein alpha-thioesters and peptides or proteins with N-terminal Cys). Unfortunately, expressed protein ligation is still limited mainly by the requirement of a Cys residue. Of course, additional Cys residues can be introduced into the sequence by site directed mutagenesis or synthesis, however, those mutations may disturb protein structure and function. Recently, alternative ligation approaches have been developed that do not require Cys residues. Accordingly, it is theoretically possible to obtain each modified protein using ligation strategies.

Animals↗
PubMed2004

Fluorescence resonance energy transfer to study receptor dimerization in living cells.

The versatility, sensitivity, and feasibility of fluorescence methods are very attractive to study protein-protein interaction at low levels of protein expression. However, one of the most severe limits in protein chemistry has been the difficulty of introducing site-specific fluorescent labels. The development of genetically encoded fluorescent probes, that is, green fluorescent protein (GFP) and its variants therefore opened up a broad field of novel applications. To characterize protein-protein interactions and determine detailed spatio-temporal dynamics of partners that are molecularly well characterized, fluorescence energy transfer methods are excellent nondestructive tools in living cells. Cellular responses to external factors are extensively based on direct molecular interaction and especially G-protein-coupled receptors (GPCRs) have been shown to interact with an unexpected level of complexity. Classical models of signal transduction describe GPCRs as monomeric proteins, while recent studies using fluorescence resonance energy transfer (FRET) and other methods show that GPCRs can also function as homo- or heterodimers. Theoretical background information on FRET technology and its diverse applications are summarized here. A detailed description of a spectroscopic method for FRET studies in the field of GPCR interaction is presented to facilitate and propagate studies to increase our understanding of protein-protein interactions involving GPCRs.

Animals↗
PubMed2003

Facile and selective nanoscale labeling of peptides in solution by using photolabile protecting groups.

For the selective labeling of peptides, a novel strategy was developed that combines the advantages of solid-phase peptide synthesis with the flexibility of labeling reactions in solution. To direct a label at a distinct position within the peptide sequence, other reactive positions are blocked with photolabile protecting groups that could be easily removed after the labeling reaction. Therefore selective labeling may become feasible for the first time even in nanomol amounts.

Animals↗
PubMed2003

Semisynthesis and characterization of the first analogues of pro-neuropeptide y.

Enzymatic cleavage of prohormone neuropeptide Y (proNPY) leads to mature neuropeptide Y (NPY), a widely distributed neuropeptide with multiple functions both peripherally and centrally. A single dibasic pair of amino acids, Lys38-Arg39, represents the recognition motif for a class of hormone-processing enzymes known as prohormone convertases (PCs). Two members of this PC family, PC1/3 and PC2, are involved in proNPY cleavage. The aim of this work was to establish an effective method for the generation of full-length 69-amino acid proNPY analogues for further studies of prohormone convertase interaction. We have chosen two ligation sites in order to perform the semisynthesis of proNPY analogues by expressed protein ligation (EPL). By using the intein-mediated purification system (IMPACT) with improved conditions for intein splicing, we were able to isolate proNPY 1-40 and proNPY 1-54 fragments as Cterminal thioesters. Peptides bearing Nterminal cysteine instead of the naturally occurring Ser41 and Thr55 residues, respectively, were generated by solid-phase peptide synthesis. Moreover, labels (carboxyfluorescein and biotin) were inserted into the peptide sequences. The synthesis of the [C41]proNPY 41-69 fragment, which proved to be a difficult peptide sequence, could be achieved by the incorporation of two pseudo-proline derivatives. Western blot analysis revealed that all five proNPY analogues are recognized by monoclonal antibodies directed against NPY as well as against the Cflanking peptide of NPY (CPON).

Amino Acid Sequence↗
PubMed2003

Homodimerization of neuropeptide y receptors investigated by fluorescence resonance energy transfer in living cells.

Up to now neuropeptide Y (NPY) receptors, which belong to the large family of G-protein-coupled receptors and are involved in a broad range of physiological processes, are believed to act as monomers. Studies with the Y(1)-receptor antagonist and Y(4)-receptor agonist GR231118, which binds with a 250-fold higher affinity than its monomer, led to the first speculation that NPY receptors can form homodimers. In the present work we used the fluorescence resonance energy transfer (FRET) to study homodimerization of the hY(1)-, hY(2)-, and hY(5)-receptors in living cells. For this purpose, we generated fusion proteins of NPY receptors and green fluorescent protein or spectral variants of green fluorescent protein (cyan, yellow, and red fluorescent protein), which can be used as FRET pairs. Two different FRET techniques, fluorescence microscopy and fluorescence spectroscopy, were applied. Both techniques clearly showed that the hY(1)-, hY(2)-, and hY(5)-NPY receptor subtypes are able to form homodimers. By using transiently transfected cells, as well as a stable cell line expressing the hY(2)-GFP fusion protein, we could demonstrate that the Y-GFP fusion proteins are still functional and that dimerization varies from 26 to 44% dependent on the receptor. However, homodimerization is influenced neither by NPY nor by Galpha protein binding.

Animals↗
PubMed2003

Nano-high-performance liquid chromatography in combination with nano-electrospray ionization Fourier transform ion-cyclotron resonance mass spectrometry for proteome analysis.

Fourier transform ion-cyclotron resonance (FTICR) mass spectrometry offers several advantages for the analysis of biological samples, including excellent mass resolution, ultra-high mass measurement accuracy, high sensitivity, and wide mass range. We report the application of a nano-HPLC system coupled to an FTICR mass spectrometer equipped with nanoelectrospray source (nano-HPLC/nano-ESI-FTICRMS) for proteome analysis. Protein identification in proteomics is usually conducted by accurately determining peptide masses resulting from enzymatic protein digests and comparing them with theoretically digested protein sequences from databases. A tryptic in-solution digest of bovine serum albumin was used to optimize experimental conditions and data processing. Spots from Coomassie Blue and silver-stained two-dimensional (2D) gels of human thyroid tissue were excised, in-gel digested with trypsin, and subsequently analyzed by nano-HPLC/nano-ESI-FTICRMS. Additionally, we analyzed 1D-gel bands of membrane preparations of COS-6 cells from African green monkey kidney as an example of more complex protein mixtures. Nano-HPLC was performed using 1-mm reverse-phase C-18 columns for pre-concentration of the samples and reverse-phase C-18 capillary columns for separation, applying water/acetonitrile gradient elution conditions at flow rates of 200 nL/min. Mass measurement accuracies smaller than 3 ppm were routinely obtained. Different methods for processing the raw data were compared in order to identify a maximum number of peptides with the highest possible degree of automation. Parallel identification of proteins from complex mixtures down to low-femtomole levels makes nano-HPLC/nano-ESI-FTICRMS an attractive approach for proteome analysis.

Animals↗
PubMed2003

Agonists for neuropeptide Y receptors Y1 and Y5 stimulate different phases of feeding in guinea pigs.

1. The stimulatory effect of neuropeptide Y (NPY) on food intake is well established but the roles of the receptor subtypes Y(1) and Y(5) have been difficult to define. We have studied the effects of two novel Y(1)-preferring and two Y(5)-preferring agonists on feeding in guinea pigs. 2. The Y(1)-preferring receptor agonists [Arg(6),Pro(34)]pNPY and [Phe(7),Pro(34)]pNPY had high affinity for the Y(1) receptor (K(i) values 0.07 and 0.04 nM, respectively) and nanomolar affinity for the Y(5) receptor. Administration of either compound into the third brain ventricle increased food intake equally to NPY. 3. The Y(5) agonist [Ala(31),Aib(32)]pNPY displayed a moderate affinity for the Y(5) receptor (K(i) 7.42 nM) and a low affinity for Y(1) (K(i) 1.7 micro M). This compound had only a modest effect on feeding. 4. The other Y(5)-preferring peptide [cPP(1-7),NPY(19-23),Ala(31),Aib(32),Gln(34)]hPP had a higher affinity at the Y(5) receptor (K(i) 1.32 nM) and also at the Y(1) receptor (K(i) 85 nM). It potently stimulated feeding: the food consumption after administration of this peptide was two-fold compared to NPY. 5. Our results support the view that both the receptor subtypes Y(1) and Y(5) are involved in the stimulation of feeding. As the action profiles of the Y(1) and Y(5) agonists on feeding parameters were different, it seems that they influence different phases of eating.

Animals↗
PubMed2003

Presence of neuropeptide Y and the Y1 receptor in the plasma membrane and nuclear envelope of human endocardial endothelial cells: modulation of intracellular calcium.

The aims of the present study were to investigate the presence and distribution of NPY and the Y1 receptor in endocardial endothelial cells (EECs), to verify if EECs can release NPY, and to determine if the effect of NPY on intracellular calcium is mediated via the Y1 receptor. Immunofluorescence, 3-D confocal microscopy and radioimmunoassay techniques were used on 20-week-old human fetal EECs. Our results showed that NPY and the Y1 receptor are present in human EECs (hEECs) and that their distributions are similar, the fluorescence labelling being higher in the nucleus and more particularly at the level of the nuclear envelope when compared with the cytosol. Using radioimmunoassay, we demonstrated that EECs are a source of NPY and can secrete this peptide upon a sustained increase of intracellular calcium ([Ca]i). Using fluo-3 and 3-D confocal microscopy technique, superfusion of hEECs as well as EECs isolated from rat adult hearts with increasing concentrations of NPY induced a dose-dependent, sustained increase in free cytosolic and nuclear Ca2+ levels. This effect of NPY on EEC [Ca]i was completely reversible upon washout of NPY and was partially blocked by BIBP3226, a selective Y1 receptor antagonist. The results suggest that NPY and Y1 receptors are present in the EECs of 20-week-old human fetal heart and they share the same distribution and localization inside the cell. In addition, EECs are able to secrete NPY in response to an increase in [Ca]i, and the Y1 receptor as well as other NPY receptors seem to participate in mediating the effects of NPY on [Ca]i in these cells. Thus, NPY released by EECs may modulate excitation-secretion coupling of these cells.

Aniline Compounds↗
PubMed2003

Semisynthesis and application of carboxyfluorescein-labelled biologically active human interleukin-8.

Human interleukin 8 (hIL-8), a neutrophil-activating and chemotactic cytokine, is known to play an important role in the pathogenesis of a large number of neutrophil-driven inflammatory diseases. This cytokine belongs to the family of CXC chemokines, mediating the response through binding to the seven-transmembrane helical G protein-coupled receptors CXCR1 and CXCR2. For the first time, we employed the expressed protein ligation (EPL) strategy to chemokine synthesis and subsequent modification. The ligation site was chosen with respect to the position of four cysteine residues within the hIL-8 sequence. Ligation with synthetic peptides that carry cysteine at their N-termini resulted in full-length hIL-8 and the specifically carboxyfluorescein-labelled analogue [K69(CF)]hIL-8(1-77). [K69(CF)]hIL-8(1-77) was fully active as shown by inhibition of cAMP production. Furthermore, this analogue was used to study receptor internalisation in human promyelotic HL60 cells that express CXCR1 and CXCR2 receptors. Binding and quenching studies were performed on HL60 membranes and suggest that the C-terminus of IL-8 is accessible to solvent in the receptor-bound state. Thus, we introduce here a powerful approach that allows the site-specific incorporation of chemical modifications into the sequence of chemokines, which opens new avenues for studying IL-8 function.

Amino Acid Sequence↗
PubMed2002

Cellular internalization of enhanced green fluorescent protein ligated to a human calcitonin-based carrier peptide.

Carrier peptides offer new opportunities to overcome problems in cellular drug delivery. Their objectives are improved cellular uptake or permeation of biological membranes, which are important pharmacokinetic features for the cellular distribution of therapeutics. Previously, human calcitonin (hCT) and selected C-terminal hCT fragments have been shown to be internalized and to permeate the epithelium of the nasal mucosa. To assess the potential of hCT-derived carrier peptides for cellular internalization of a model protein we fused enhanced green fluorescent protein (EGFP) and the [C(8)]hCT8-32 fragment by using expressed protein ligation (EPL). EGFP thioester was obtained by intein-mediated purification with an affinity chitin-binding tag (the IMPACT system, based on protein splicing). Internalization of EGFP-[C(8)]hCT8-32 by excised bovine nasal mucosa was monitored by confocal laser scanning microscopy. This novel conjugate displayed internalization into some sectors of the mucosa, whereas EGFP itself was not capable of translocation. Thus, we demonstrate successful internalization of a model protein through ligation to an hCT-derived carrier peptide, which has potential for the delivery of therapeutics. At this point the respective mechanism of translocation is unknown.

Aequorin↗
PubMed2002

Key motif to gain selectivity at the neuropeptide Y5-receptor: structure and dynamics of micelle-bound [Ala31, Pro32]-NPY.

The structure of [Ala(31), Pro(32)]-NPY, a neuropeptide Y mutant with selectivity for the NPY Y(5)-receptor (Cabrele, C., Wieland, H. A., Stidsen, C., Beck-Sickinger, A. G., (2002) Biochemistry XX, XXXX-XXXX (companion paper)), has been characterized in the presence of the membrane mimetic dodecylphosphocholine (DPC) micelles using high-resolution NMR techniques. The overall topology closely resembles the fold of the previously described Y(5)-receptor-selective agonist [Ala(31), Aib(32)]-NPY (Cabrele, C., Langer, M., Bader, R., Wieland, H. A., Doods, H. N., Zerbe, O., and Beck-Sickinger, A. G. (2000) J. Biol. Chem 275, 36043-36048). Similar to wild-type neuropeptide Y (NPY) and [Ala(31), Aib(32)]-NPY, the N-terminal residues Tyr(1)-Asp(16) are disordered in solution. Starting from residue Leu(17), an alpha helix extends toward the C-terminus. The decreased density of medium-range NOEs for the C-terminal residues resulting in larger RMSD values for the backbone atoms of Ala(31)-Tyr(36) indicates that the alpha helix has become interrupted through the [Ala(31), Pro(32)] mutation. This finding is further supported by (15)N-relaxation data through which we can demonstrate that the well-defined alpha helix is restricted to residues 17-31, with the C-terminal tetrapeptide displaying increased flexibility as compared to NPY. Surprisingly, increased generalized order parameter as well as decreased (3)J(HN)(alpha) scalar coupling constants reveal that the central helix is stabilized in comparison to wild-type NPY. Micelle-integrating spin labels were used to probe the mode of association of the helix with the membrane mimetic. The Y(5)-receptor-selective mutant and NPY share a similar orientation, which is parallel to the lipid surface. However, signal reductions due to efficient electron, nuclear spin relaxation were much less pronounced for the surface-averted residues in [Ala(31), Pro(32)]-NPY when compared to wild-type DPC-bound NPY. Only the signals of residues Asn(29) and Leu(30) were significantly more reduced in the mutant. The postulation of a different membrane binding mode of [Ala(31), Pro(32)]-NPY is further supported by the faster H/D exchange at the C-terminal amide protons. We conclude that arginine residues 33 and 35, which are believed to be directly involved in forming contacts to acidic receptor residues at the membrane-water interface, are no longer fixed in a well-defined conformation close to the membrane surface in [Ala(31), Pro(32)]-NPY.

Alanine↗
PubMed2002

Ala31-Aib32: identification of the key motif for high affinity and selectivity of neuropeptide Y at the Y5-receptor.

The turn-inducing sequence Ala-Aib introduced into positions 31 and 32 of neuropeptide Y (NPY) and its analogues has been identified as the key structure for Y(5)-receptor selectivity. Analogues of NPY and PP/NPY chimera containing the motif Ala-Aib were prepared; these peptides turned out to be selective for the Y(5)-receptor. The affinity of the NPY-based peptides was in the range of 6-150 nM, while the affinity of three (Ala-Aib)-containing PP/NPY chimera was in the range of 0.2-0.9 nM. The circular dichroism spectra of the Aib analogues in aqueous solution were all characteristic of an alpha helix; however, they had different intensities of the two negative bands at 220 and 208 nm. Affinity and selectivity for the Y(5)-receptor were correlated with the ratio of the ellipticity at 220 nm versus the one at 208 nm (R), which indicates the presence of a pronounced helix (R > 1) versus a less stabile one (R < 1). When R was in the range 0.74-0.96, the affinity at the Y(5)-receptor was in the range >5 nM, while there was complete loss of affinity at the Y(4)-receptor. R > 1.15 was associated with very high affinity at the Y(5)-receptor and weak affinity at the Y(4)-receptor. These results suggest that the selectivity of the Ala(31)-Aib(32) motif for the Y(5)-receptor derives from a specific conformation that must be correlated with the bioactive conformation of NPY at this subtype.

Alanine↗
PubMed2002

Biochemical characterisation and genetic analysis of aureocin A53, a new, atypical bacteriocin from Staphylococcus aureus.

Aureocin A53 is produced by Staphylococcus aureus A53. It is encoded on a 10.4 kb plasmid, pRJ9, and is active against Listeria monocytogenes. Aureocin A53 is a highly cationic 51-residue peptide containing ten lysine and five tryptophan residues. Aureocin A53 was purified to homogeneity by hydrophobic-interaction, cation-exchange, and reverse-phase chromatography. MALDI-TOF mass spectrometry yielded a molecular mass of 6012.5 Da, which was 28 Da higher than predicted from the structural gene sequence of the bacteriocin. The mass increment resulted from an N-formylmethionine residue, indicating that the aureocin A53 is synthesised and secreted without a typical bacteriocin leader sequence or sec-dependent signal peptide. The structural identity of aureocin A53 was verified by Edman sequencing after de-blocking with cyanogen bromide and extensive mass spectrometry analysis of enzymatically and laser-generated fragments. The complete sequence of pRJ9 was determined and none of the open reading frames identified in the vicinity of the structural gene aucA showed similarity to genes that are typically found in bacteriocin gene clusters. Thus, neither a dedicated protease or transporter, nor modifying enzymes and regulatory elements seemed to be involved in the production of aureocin A53. Further unique features that distinguish aureocin A53 from other peptide bacteriocins include remarkable protease stability and a defined, rigid structure in aqueous solution.

Amino Acid Sequence↗