Dieter Palm

High molecular weight hyaluronic acid inhibits advanced glycation endproduct-induced NF-κB activation and cytokine expression

Advanced glycation endproducts (AGEs), which accumulate on long‐lived proteins and protein deposits (amyloids), induce the expression of proinflammatory cytokines through NF‐κB‐dependent pathways. Hyaluronic acid with a molecular weight above 1.2 MDa (HMW‐HA) inhibits the AGE‐induced activation of the transcription factor NF‐κB and the NF‐κB‐regulated cytokines interleukin‐1α, interleukin‐6 and tumor necrosis factor‐α. Since the molecular weight of hyaluronic acid in humans decreases with age and under conditions of oxidative stress, it is likely that the protective effect of HMW‐HA against AGE‐induced cellular activation is lost at sites of chronic inflammation and in older age.

Amino acid specificity of glycation and protein–AGE crosslinking reactivities determined with a dipeptide SPOT library

Advanced glycation end products (AGEs) contribute to changes in protein conformation, loss of function, and irreversible crosslinking. Using a library of dipeptides on cellulose membranes (SPOT library), we have developed an approach to systematically assay the relative reactivities of amino acid side chains and the N-terminal amino group to sugars and protein–AGEs. The sugars react preferentially with cysteine or tryptophan when both the α-amino group and the side chains are free. In peptides with blocked N-terminus and free side chains, cysteine, lysine, and histidine were preferred. Crosslinking of protein–AGEs to dipeptides with free side chains and blocked N termini occurred preferentially to arginine and tryptophan. Dipeptide SPOT libraries are excellent tools for comparing individual reactivities of amino acids for nonenzymatic modifications, and could be extended to other chemically reactive molecules.

Multisite contacts involved in coupling of the β‐adrenergic receptor with the stimulatory guanine‐nucleotide‐binding regulatory protein

Synthetic peptides, 12-22 amino acid residues long, comprising the presumed coupling sites of the beta-adrenergic receptor with the stimulatory guanine-nucleotide-binding regulatory protein (Gs), were examined for their ability to modulate Gs activation in turkey erythrocyte membranes. Three peptides corresponding to the second cytoplasmic loop, the N-terminal region of the third cytoplasmic loop, and the N-terminal region of the putative fourth cytoplasmic loop, compete synergistically with the hormone-stimulated receptor for Gs activation with median effector concentrations of 15-35 microM, or 3-4 microM for combinations of two peptides. One peptide, corresponding to the C-terminal region of the third cytoplasmic loop, carries the unique ability to activate the Gs-adenylate-cyclase complex independent of the signalling state of the receptor. These observations are consistent with a dynamic model of receptor-mediated G-protein activation in membranes, where domains composed of the second, third and fourth intracellular loop of the receptor bind to and are interactive with the G-protein heterotrimer, resulting in ligand-induced conformational changes of the receptor. In response to hormone binding, the extent or the number of sites involved in interaction with Gs may be readjusted using a fourth site. Modulation of coupling sites may elicit congruent conformational changes within the Gs heterotrimer, with qualitatively different effects on GTP/GDP exchange in the alpha subunit of Gs and downstream effector regulation. This model corroborates and expands a similar model suggested for activated rhodopsin-transducin interaction [König, B., Arendt, A., McDowell, J. H., Kahlert, M., Hargrave, P. A. & Hofmann, K. P. (1989) Proc. Natl Acad. Sci. USA 86, 6878-6882].

The cognition-enhancing drug tenilsetam is an inhibitor of protein crosslinking by advanced glycosylation

SummaryNon-enzymatic glycosylation of proteins, also called Maillard reaction, which occurs at an accelerated rate in diabetes, can lead to the formation of advanced glycosylation endproducts (AGEs). Tenilsetam (®CAS 997: (±)-3-(2-thienyl)-2-piperazinone), a cognition-enhancing drug successfully used for treatment of patients suffering from Alzheimers disease, when included in the Maillard reaction apparently inhibits protein crosslinking by AGEs in vitro. According to the mechanism proposed, Tenilsetam acts via covalent attachment to glycated proteins, thus blocking the reactive sites for further polymerisation reactions. A beneficial effect of Tenilsetam in Alzheimers disease could come from the interference with AGE-derived crosslinking of amyloid plaques and a decreased inflammatory response by diminished activation of phagocytosing microglia.

Mapping of β-adrenoceptor coupling domains to Gs-protein by site-specific synthetic peptides

Peptides corresponding to the known sequence of turkey erythrocyte β1‐adrenergic receptor were synthesized and the effects on receptor‐mediated cyclase activation were measured. Peptides corresponding to the first and second intracellular loops (T61‐71 and T138‐159) inhibited at micromolar concentrations the hormone‐dependent cyclase activation in turkey erythrocyte membranes. In contrast, the peptide corresponding to the C‐terminal part of the third intracellular loop (T284‐295) increased the cyclase activity in a hormone‐independent manner. Peptides T338‐353 and T2‐10 and a number of synthetic peptides unrelated to the β‐adrenoceptor had no effect.

Two sites in the third inner loop of the dopamine D2 receptor are involved in functional G protein-mediated coupling to adenylate cyclase

Synthetic peptides, corresponding to the amino acid sequences of the N‐ and C‐terminal parts of the 3rd intracellular loop of the dopamine D2 receptor, attenuate dopaminergic adenylate cyclase inhibition in membranes. Both peptides also activate directly GTPase activity in membranes. We suggest a functional model for Gi‐coupled receptors where two sites in the 3rd inner loop compose the links for the receptor‐G protein interaction thus providing the tools for a selective and adjustable response. Functional coupling was not affected by a peptide representing the insert in the long form of the dopamine D; receptor (D2(long)). The selectivity pattern of conventional G protein‐linked receptors also sheds some light on the recently observed interaction of β‐amyloid protein precursor (APP) complexes with G proteins.

Oligosaccharide substrate binding in Escherichia coli maltodextrin phosphorylase.

The crystal structure of E. coli maltodextrin phosphorylase co-crystallized with an oligosaccharide has been solved at 3.0 Å resolution, providing the first structure of an oligosaccharide bound at the catalytic site of an α-glucan phosphorylase. An induced fit mechanism brings together two domains across the catalytic site tunnel. A stacking interaction between the glucosyl residue and the aromatic group of a tyrosine residue at a sub-site remote (8 Å) from the catalytic site provides a key element in substrate recognition; mutation of this residue to Ala decreases the kcat/Km by 104. Extrapolation of the results to substrate binding across the site of attack by phosphorolysis indicates a likely alteration in the glycosidic torsion angles from their preferred values, an alteration that appears to be important for the catalytic mechanism.

Identification of a Gs-protein coupling domain to the β-aderenoceptor using site-specific synthetic peptides: Carboxyl terminus of Gsα is involved in coupling to β-adrenoceptors

Competition between Gs‐protein and the synthetic peptide, GSA 379‐394, derived from the carboxyl‐terminal region of the αs‐subunit, led to complete inhibition of receptor‐mediated adenylate cyclase activation in turkey erythrocyte membranes. Related peptides corresponding to the homologous carboxyl‐terminal region of αt‐,αil‐ or αo‐subunits did not interfere with β‐receptor‐Gs coupling. The direct coupling between Gs and adenylate cyclase was not influenced by any of these peptides. These results emphasize the important role of the carboxyl‐terminus of G‐protein α‐subunits for the specific recognition of their corresponding receptors and for signal transduction.

NMR and circular dichroism studies of synthetic peptides derived from the third intracellular loop of the β-adrenoceptor

The C‐terminal part of the third intracellular loop of the β‐adrenoceptor is capable of stimulating adenylate cyclase in the presence of phospholipid vesicles via the stimulatory guanine nucleotide binding protein (Gs) [Palm et al. (1989) FEBS Lett. 254, 89–93]. We have investigated the structure of synthetic peptides corresponding to residues 284–295 of the turkey erythrocyte adrenoceptor in micelles, trifluoroethanol and aqueous solution, by using 2D 1H NMR and CD. In the presence of phospholipid micelles the peptides display a C‐terminal α‐helical region, whereas the N‐terminal part was found to be highly flexible.

Chemical and functional analysis of components of adenylyl cyclase from human platelets treated with phorbolesters

Human platelets, prelabeled with [32P]phospate were treated with tetradecanoylphorbol acetate (TPA) for 5 min at 37°C. Phosphorylation of the components of adenylyl cyclase was determined in membranes using specific antibodies against G‐proteins and the catalytic moiety. Less than 0.01 mol of [32P]phosphate/mol could be detected in immunoprecipitates using antibodies against sequences within the α‐subunit of the GTP binding protein Gi. TPA, however, caused the incorporation of 0.67–1.1 mol of [32P]phosphate per mol of catalyst while 0.13‐0.2 mol were found in the absence or TPA. Lack of modification of the α‐subunit of Gi was also indicated by the results of reconstitution experiments with purified Giα from bovine brain: adenylyl cyclase in membranes from untreated platelets was significantly more inhibited by added Giα, than that from TPA treated cells. While β,γ‐subunits were like‐wise inhibitory no difference dependent on platelet‐pretreatment could be observed.