Novel receptor-derived cyclopeptides to treat heart failure caused by anti-ß1-adrenoceptor antibodies in a human-analogous rat model.

Despite recent therapeutic advances the prognosis of heart failure remains poor. Recent research suggests that heart failure is a heterogeneous syndrome and that many patients have stimulating auto-antibodies directed against the second extracellular loop of the ß1 adrenergic receptor (ß1EC2). In a human-analogous rat model such antibodies cause myocyte damage and heart failure. Here we used this model to test a novel antibody-directed strategy aiming to prevent and/or treat antibody-induced cardiomyopathy. To generate heart failure, we immunised n = 76/114 rats with a fusion protein containing the human ß1EC2 (amino-acids 195-225) every 4 weeks; n = 38/114 rats were control-injected with 0.9% NaCl. Intravenous application of a novel cyclic peptide mimicking ß1EC2 (ß1EC2-CP, 1.0 mg/kg every 4 weeks) or administration of the ß1-blocker bisoprolol (15 mg/kg/day orally) was initiated either 6 weeks (cardiac function still normal, prevention-study, n = 24 (16 treated vs. 8 untreated)) or 8.5 months after the 1st immunisation (onset of cardiomyopathy, therapy-study, n = 52 (40 treated vs. 12 untreated)); n = 8/52 rats from the therapy-study received ß1EC2-CP/bisoprolol co-treatment. We found that ß1EC2-CP prevented and (alone or as add-on drug) treated antibody-induced cardiac damage in the rat, and that its efficacy was superior to mono-treatment with bisoprolol, a standard drug in heart failure. While bisoprolol mono-therapy was able to stop disease-progression, ß1EC2-CP mono-therapy -or as an add-on to bisoprolol- almost fully reversed antibody-induced cardiac damage. The cyclo¬peptide acted both by scavenging free anti-ß1EC2-antibodies and by targeting ß1EC2-specific memory B-cells involved in antibody-production. Our model provides the basis for the clinical translation of a novel double-acting therapeutic strategy that scavenges harmful anti-ß1EC2-antibodies and also selectively depletes memory B-cells involved in the production of such antibodies. Treatment with immuno-modulating cyclopeptides alone or as an add-on to ß1-blockade represents a promising new therapeutic option in immune-mediated heart failure.

Tripeptides of RS1 (RSC1A1) inhibit a monosaccharide-dependent exocytotic pathway of Na+-D-glucose cotransporter SGLT1 with high affinity.

The human gene RSC1A1 codes for a 67-kDa protein named RS1 that mediates transcriptional and post-transcriptional regulation of Na(+)-D-glucose cotransporter SGLT1. The post-transcriptional regulation occurs at the trans-Golgi network (TGN). We identified two tripeptides in human RS1 (Gln-Cys-Pro (QCP) and Gln-Ser-Pro (QSP)) that induce posttranscriptional down-regulation of SGLT1 at the TGN leading to 40-50% reduction of SGLT1 in plasma membrane. For effective intracellular concentrations IC(50) values of 2.0 nM (QCP) and 0.16 nm (QSP) were estimated. Down-regulation of SGLT1 by tripeptides was attenuated by intracellular monosaccharides including non-metabolized methyl-alpha-D-glucopyranoside and 2-deoxyglucose. In small intestine post-transcriptional regulation of SGLT1 may contribute to glucose-dependent regulation of liver metabolism and intestinal mobility. QCP and QSP are transported by the H(+)-peptide cotransporter PepT1 that is colocated with SGLT1 in small intestinal enterocytes. Using coexpression of SGLT1 and PepT1 in Xenopus oocytes or polarized Caco-2 cells that contain both transporters we demonstrated that the tripeptides were effective when applied to the extracellular compartment. After a 1-h perfusion of intact rat small intestine with QSP, glucose absorption was reduced by 30%. The data indicate that orally applied tripeptides can be used to down-regulate small intestinal glucose absorption, e.g. in diabetes mellitus.

Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism.

Parathyroid hormone (PTH) and its related receptor (PTHR) are essential regulators of calcium homeostasis and bone physiology. PTH activates PTHR by interacting with a ligand-binding site localized within the N-terminal extracellular domain (the N-domain) and the domain comprising the seven transmembrane helices and the connecting extracellular loops (the J-domain). PTH binding triggers a conformational switch in the receptor, leading to receptor activation and subsequent cellular responses. The process of receptor activation occurs rapidly, within approximately 1 s, but the binding event preceding receptor activation is not understood. By recording FRET between tetramethyl-rhodamine in PTH(1-34) and GFP in the N-domain of the receptor, we measured the binding event in real time in living cells. We show that the association time course between PTH(1-34) and PTHR involves a two-step binding process where the agonist initially binds the receptor with a fast time constant (tau approximately 140 ms) and then with slower kinetics (tau approximately 1 s). The fast and slow phases were assigned to hormone association to the receptor N- and J domains, respectively. Our data indicate that the slow binding step to the J-domain coincides with a conformational switch in the receptor, also monitored by FRET between the enhanced cyan fluorescent protein and the enhanced yellow fluorescent protein in the PTHR sensor, PTHR enhanced cyan fluorescent protein/enhanced yellow fluorescent protein (PTHR(CFP/YFP)). These data suggest that the conformational change that switches the receptor into its active state proceeds in a sequential manner, with the first rapid binding step event preceding receptor activation by PTH(1-34).

Identification and characterization of PorH, a new cell wall channel of Corynebacterium glutamicum.

The cell wall of Corynebacterium glutamicum contains the cation-selective channel (porin) PorA(C.glut) and the anion-selective channel PorB(C.glut) for the passage of hydrophilic solutes. Lipid bilayer experiments with organic solvent extracts of whole C. glutamicum cells cultivated in minimal medium suggested that also another cation-selective channel-forming protein, named PorH(C.glut), is present in C. glutamicum. The protein was purified to homogeneity by fast-protein liquid chromatography across a HiTrap-Q column. The pure protein had an apparent molecular mass of about 12 kDa on SDS-PAGE. Western blot analysis suggested that the cell wall channel is presumably formed by protein oligomers. The purified protein forms cation-selective channels with an average single-channel conductance of about 2.5 nS in 1 M KCl in the lipid bilayer assay. The PorH(C.glut) protein was partially sequenced, and based on the resulting amino acid sequence, the corresponding gene, designated as porH(C.glut), was identified in the published genome sequence of C. glutamicum ATCC13032. PorH(C.glut) contains only the inducer methionine but no N-terminal extension, which suggests that the export and assembly of the protein follow a yet unknown pathway. PorH(C.glut) is coded in the bacterial chromosome by a gene that is localized in the vicinity of porA(C.glut), within a putative operon of 13 genes. RT-PCR revealed that both porins are cotranscribed. They coexist according to immunological detection experiments in the cell wall of C. glutamicum together with PorB(C.glut) and PorC(C.glut).

Structure-function analysis of the cysteine string protein in Drosophila: cysteine string, linker and C terminus.

Cysteine string proteins (CSPs) are conserved secretory vesicle proteins involved in regulating neurotransmitter and peptide release. While the function of the J-domain has been studied in detail, little is known about other conserved regions. We have constructed mutant genes coding for proteins with modified cysteine string, linker region or C terminus and transformed them into Csp null-mutant Drosophila: In the living animal, mutated CSP lacking all cysteines fails to associate with membranes, does not concentrate in synaptic terminals, and cannot rescue adult temperature-sensitive paralysis and short life span, both prominent null mutant phenotypes. A mutant protein with 5 instead of 11 string cysteines appears to be normally targeted but cannot rescue paralysis at 37 degrees C. We propose that the cysteine string, in addition to its role in targeting, may be essential for a function of CSP that is dependent on the number of cysteines in the string. A deletion in the linker region or the C terminus does not affect CSP targeting, and function in adults is only marginally impaired.

Characterization of antibody affinities using an AGE-modified dipeptide spot library.

Recent immunological approaches have greatly helped understanding the significance of advanced glycation end products (AGEs) in age-related diseases. However, immunohistochemical localization of AGEs in tissues or their quantitative determination in biological fluids sometimes yields inconsistent results among different investigators. Since these differences might be caused by the heterogeneity of the AGE antibodies, a systematic mapping of their epitope recognition pattern would help in the evaluation of these different results. For this purpose, an N-terminally acetylated combinatorial dipeptide library with 400 positionally defined dipeptides was modified by glucose to yield AGEs, which are thought to be present on the protein side chains. Using this library, we have characterized six different AGE antibodies in respect to their immunoreactivity towards AGE-modified dipeptides. All antibodies predominantly recognized only one AGE-modified amino acid side chain (and not a particular dipeptide). In most cases, arginine- and lysine- derived AGEs, but also some epitopes on asparagine and on heterocyclic amino acids were recognized. Very interestingly, the immunization of different animals with the same antigen produced AGE antibodies with a totally different epitope recognition pattern. Defining the antigen recognition pattern with this method might help in the quality control of polyclonal AGE antibodies for immunodetection. In addition, defined AGE antibodies (as neutralizing antibodies) could also help to define "signal-active" AGEs in terms of specific AGE-mediated cellular responses.

Secondary structure of the third extracellular loop responsible for ligand selectivity of a mammalian gonadotropin-releasing hormone receptor.

The extracellular loop 3 (ECL3) of the mammalian gonadotropin-releasing hormone receptor (GnRH-R) contains an acidic amino acid (Glu(301) in the mouse GnRH-R) that confers agonist selectivity for Arg(8) in mammalian GnRH. It is proposed that a specific conformation of ECL3 is necessary to orientate the carboxyl side chain of the acidic residue for interaction with Arg(8) of GnRH, which is supported by decreased affinity for Arg(8) GnRH but not Gln(8) GnRH when an adjacent Pro is mutated to Ala. To probe the structural contribution of the loop domain to the proposed presentation of the carboxyl side chain, we synthesized a model peptide (CGPEMLNRVSEPGC) representing residues 293-302 of mouse ECL3, where Cys and Gly residues are added symmetrically at the N and C termini, respectively, allowing the introduction of a disulfide bridge to simulate the distances at which the ECL3 is tethered to the transmembrane domains 6 and 7 of the receptor. The ability of the ECL3 peptide to bind GnRH with low affinity was demonstrated by its inhibition of GnRH stimulation of inositol phosphate production in cells expressing the GnRH-R. The CD bands of the ECL3 peptides exhibited a superposition of predominantly unordered structure and partial contributions from beta-sheet structure. Likewise, the analysis of the amide I and amide III bands from micro-Raman and FT Raman experiments revealed mainly unordered conformations of the cyclic and of the linear peptide. NMR data demonstrated the presence of a beta-hairpin among an ensemble of largely disordered structures in the cyclic peptide. The location of the turn linking the two strands of the hairpin was assigned to the three central residues L(296), N(297), and R(298). A small population of structured species among an ensemble of predominantly random coil conformation suggests that the unliganded receptor represents a variety of structural conformers, some of which have the potential to make contacts with the ligand. We propose a mechanism of receptor activation whereby binding of the agonist to the inactive receptor state induces and stabilizes a particular structural state of the loop domain, leading to further conformational rearrangements across the transmembrane domain and signal propagating interaction with G proteins. Interaction of the Glu(301) of the receptor with Arg(8) of GnRH induces a folded configuration of the ligand. Our proposal thus suggests that conformational changes of both ligand and receptor result from this interaction.