Modulating the Fibrillization of Parathyroid-Hormone (PTH) Peptides: Azo-Switches as Reversible and Catalytic Entities.

We here report a novel strategy to control the bioavailability of the fibrillizing parathyroid hormone (PTH)-derived peptides, where the concentration of the bioactive form is controlled by an reversible, photoswitchable peptide. PTH1-84, a human hormone secreted by the parathyroid glands, is important for the maintenance of extracellular fluid calcium and phosphorus homeostasis. Controlling fibrillization of PTH1-84 represents an important approach for in vivo applications, in view of the pharmaceutical applications for this protein. We embed the azobenzene derivate 3-{[(4-aminomethyl)phenyl]diazenyl}benzoic acid (3,4'-AMPB) into the PTH-derived peptide PTH25-37 to generate the artificial peptide AzoPTH25-37 via solid-phase synthesis. AzoPTH25-37 shows excellent photostability (more than 20 h in the dark) and can be reversibly photoswitched between its cis/trans forms. As investigated by ThT-monitored fibrillization assays, the trans-form of AzoPTH25-37 fibrillizes similar to PTH25-37, while the cis-form of AzoPTH25-37 generates only amorphous aggregates. Additionally, cis-AzoPTH25-37 catalytically inhibits the fibrillization of PTH25-37 in ratios of up to one-fifth. The approach reported here is designed to control the concentration of PTH-peptides, where the bioactive form can be catalytically controlled by an added photoswitchable peptide.

Designed Trp-Cage Proteins with Antimicrobial Activity and Enhanced Stability.

α-Helical antimicrobial peptides (αAMPs) are among the potential candidates for new anti-infectives to tackle the global crisis in antibiotic resistance, but they suffer from low bioavailability due to high susceptibility to enzymatic degradation. Here, we describe a strategy to increase the resistance of αAMPs against proteases. Fusing the 12-residue αAMP KR-12 with a Trp-cage domain induces an α-helical structure in the otherwise unfolded KR-12 moiety in solution. The resulting antimicrobial Trp-cage exhibits higher proteolytic resistance due to its stable fold as evidenced by correlating sequence-resolved digest data with structural analyses. In addition, the antimicrobial Trp-cage displays increased activity against bacteria in the presence of physiologically relevant concentrations of NaCl, while the hemolytic activity remains negligible. In contrast to previous strategies, the presented approach is not reliant on artificial amino acids and is therefore applicable to biosynthetic procedures. Our study aims to improve the pharmacokinetics of αAMPs to facilitate their use as therapeutics.

ß-Turn mimetic synthetic peptides as amyloid-ß aggregation inhibitors.

Aggregation of amyloid peptides results in severe neurodegenerative diseases. While the fibril structures of Aß40 and Aß42 have been described recently, resolution of the aggregation pathway and evaluation of potent inhibitors still remains elusive, in particular in view of the hairpin-region of Aß40. We here report the preparation of beta-turn mimetic conjugates containing synthetic turn mimetic structures in the turn region of Aß40 and Aß16-35, replacing 2 amino acids in the turn-region G25 - K28. The structure of the turn mimic induces both, acceleration of fibrillation and the complete inhibition of fibrillation, confirming the importance of the turn region on the aggregation. Replacing position G25-S26 provided the best inhibition effect for both beta-turn mimetics, the bicyclic BTD 1 and the aromatic TAA 2, while positions N27-K28 and V24-G25 showed only weaker or no inhibitory effects. When comparing different turn mimetics at the same position (G25-S26), conjugate 1a bearing the BTD turn showed the best inhibition of Aß40 aggregation, while 5-amino-valeric acid 4a showed the weakest effect. Thus there is a pronounced impact on fibrillation with the chemical nature of the embedded beta-turn-mimic: the conformationally constrained turns 1 and 2 lead to a significantly reduced fibrillation, even inhibiting fibrillation of native Aß40 when added in amounts down to 1/10, whereas the more flexible beta-turn-mimics 4-amino-benzoic acid 3a and 5-amino-valeric acid 4a lead to enhanced fibrillation. Toxicity-testing of the most successful conjugate showed only minor toxicity in cell-viability assays using the N2a cell line. Structural downsizing lead to the short fragment BTD/peptide Aß16-35 as inhibitor of the aggregation of Aß40, opening large potential for further small peptide based inhibitors.

Initial Characterization of Stressed Transgenic Mice With Cardiomyocyte-Specific Overexpression of Protein Phosphatase 2C.

As part of our ongoing studies on the potential pathophysiological role of serine/threonine phosphatases (PP) in the mammalian heart, we have generated mice with cardiac-specific overexpression of PP2Cß (PP2C-TG) and compared them with littermate wild type mice (WT) serving as a control. Cardiac fibrosis was noted histologically in PP2C-TG. Collagen 1a, interleukin-6 and the natriuretic peptides ANP and BNP were augmented in PP2C-TG vs. WT (p < 0.05). Left atrial preparations from PP2C-TG were less resistant to hypoxia than atria from WT. PP2C-TG maintained cardiac function after the injection of lipopolysaccharide (LPS, a model of sepsis) and chronic isoproterenol treatment (a model of heart failure) better than WT. Crossbreeding of PP2C-TG mice with PP2A-TG mice (a genetic model of heart failure) resulted in double transgenic (DT) mice that exhibited a pronounced increase of heart weight in contrast to the mild hypertrophy noted in the mono-transgenic mice. The ejection fraction was reduced in PP2C-TG and in PP2A-TG mice compared with WT, but the reduction was the highest in DT compared with WT. PP2A enzyme activity was enhanced in PP2A-TG and DT mice compared with WT and PP2C-TG mice. In summary, cardiac overexpression of PP2Cß and co-overexpression of both the catalytic subunit of PP2A and PP2Cß were detrimental to cardiac function. PP2Cß overexpression made cardiac preparations less resistant to hypoxia than WT, leading to fibrosis, but PP2Cß overexpression led to better adaptation to some stressors, such as LPS or chronic ß-adrenergic stimulation. Hence, the effect of PP2Cß is context sensitive.

Synthesis and Aggregation of Polymer-Amyloid ß Conjugates.

Modulating the assembly of medically relevant peptides and proteins via macromolecular engineering is an important step in modifying their overall pathological effects. The synthesis of polymer-peptide conjugates composed of the amyloidogenic Alzheimer peptide, Aß1-40 , and poly(oligo(ethylene glycol)m acrylates) (m = 2,3) with different molecular weights (Mn = 1400-6600 g mol-1 ) is presented here. The challenging conjugation of a synthetic polymer to an in situ aggregating protein is established via two different coupling strategies, only successful for polymers with molecular weights not exceeding 6600 g mol-1 , relying on resin-based synthesis or solution-based coupling chemistries. The conjugates are characterized by high-performance liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The aggregation of these polymer-Aß1-40 conjugates, as monitored via thioflavine-T (ThT)-fluorescence spectroscopy, is accelerated mainly upon attaching the polymers. However, the appearance of the observed fibrils is different from those composed of native Aß1-40, specifically with respect to length and morphology of the obtained aggregates. Instead of long, unbranched fibrils characteristic for Aß1-40 , bundles of short aggregates are observed for the conjugates. Finally, the ThT kinetics and morphologies of Aß1-40 fibrils formed in the presence of the conjugates give some mechanistic insights.

Correction: Metabolites of lactic acid bacteria present in fermented foods are highly potent agonists of human hydroxycarboxylic acid receptor 3.

[This corrects the article DOI: 10.1371/journal.pgen.1008145.].

Metabolites of lactic acid bacteria present in fermented foods are highly potent agonists of human hydroxycarboxylic acid receptor 3.

The interplay of microbiota and the human host is physiologically crucial in health and diseases. The beneficial effects of lactic acid bacteria (LAB), permanently colonizing the human intestine or transiently obtained from food, have been extensively reported. However, the molecular understanding of how LAB modulate human physiology is still limited. G protein-coupled receptors for hydroxycarboxylic acids (HCAR) are regulators of immune functions and energy homeostasis under changing metabolic and dietary conditions. Most mammals have two HCAR (HCA1, HCA2) but humans and other hominids contain a third member (HCA3) in their genomes. A plausible hypothesis why HCA3 function was advantageous in hominid evolution was lacking. Here, we used a combination of evolutionary, analytical and functional methods to unravel the role of HCA3 in vitro and in vivo. The functional studies included different pharmacological assays, analyses of human monocytes and pharmacokinetic measurements in human. We report the discovery of the interaction of D-phenyllactic acid (D-PLA) and the human host through highly potent activation of HCA3. D-PLA is an anti-bacterial metabolite found in high concentrations in LAB-fermented food such as Sauerkraut. We demonstrate that D-PLA from such alimentary sources is well absorbed from the human gut leading to high plasma and urine levels and triggers pertussis toxin-sensitive migration of primary human monocytes in an HCA3-dependent manner. We provide evolutionary, analytical and functional evidence supporting the hypothesis that HCA3 was consolidated in hominids as a new signaling system for LAB-derived metabolites.

Involvement of the Adhesion GPCRs Latrophilins in the Regulation of Insulin Release.

Insulin secretion from pancreatic ß cells is a highly complex and tightly regulated process. Its dysregulation is one characteristic of type 2 diabetes, and thus, an in-depth understanding of the mechanisms controlling insulin secretion is essential for rational therapeutic intervention. G-protein-coupled receptors (GPCRs) have been established as major regulators of insulin exocytosis. Recent studies also suggest the involvement of adhesion GPCRs, a non-prototypical class of GPCRs. Here, we identify latrophilins, which belong to the class of adhesion GPCRs, to be highly expressed in different cell types of pancreatic islets. In vitro and ex vivo analyses show that distinct splice variants of the latrophilin LPHN3/ADGRL3 decrease insulin secretion from pancreatic ß cells by reducing intracellular cyclic AMP levels via the Gi-mediated pathway. Our data highlight the key role of LPHN3 in modulating insulin secretion and its potential as therapeutic target for type 2 diabetes.

Mechano-Dependent Phosphorylation of the PDZ-Binding Motif of CD97/ADGRE5 Modulates Cellular Detachment.

Cells respond to mechanical stimuli with altered signaling networks. Here, we show that mechanical forces rapidly induce phosphorylation of CD97/ADGRE5 (pCD97) at its intracellular C-terminal PDZ-binding motif (PBM). Biochemically, this phosphorylation disrupts CD97 binding to PDZ domains of the scaffold protein DLG1. In shear-stressed cells, pCD97 appears not only in junctions, retracting fibers, and the attachment area but also in lost membrane patches, demonstrating (intra)cellular detachment at the CD97 PBM. This motif is critical for the CD97-dependent mechanoresponse. Cells expressing CD97 without the PBM are more deformable, and under shear stress, these cells lose cell contacts faster and show changes in the actin cytoskeleton when compared with cells expressing full-length CD97. Our data indicate CD97 linkage to the cytoskeleton. Consistently, CD97 knockout phenocopies CD97 without the PBM, and membranous CD97 is organized in an F-actin-dependent manner. In summary, CD97 shapes the cellular mechanoresponse through signaling modulation via its PBM.

Mechano-dependent signaling by Latrophilin/CIRL quenches cAMP in proprioceptive neurons.

Adhesion-type G protein-coupled receptors (aGPCRs), a large molecule family with over 30 members in humans, operate in organ development, brain function and govern immunological responses. Correspondingly, this receptor family is linked to a multitude of diverse human diseases. aGPCRs have been suggested to possess mechanosensory properties, though their mechanism of action is fully unknown. Here we show that the Drosophila aGPCR Latrophilin/dCIRL acts in mechanosensory neurons by modulating ionotropic receptor currents, the initiating step of cellular mechanosensation. This process depends on the length of the extended ectodomain and the tethered agonist of the receptor, but not on its autoproteolysis, a characteristic biochemical feature of the aGPCR family. Intracellularly, dCIRL quenches cAMP levels upon mechanical activation thereby specifically increasing the mechanosensitivity of neurons. These results provide direct evidence that the aGPCR dCIRL acts as a molecular sensor and signal transducer that detects and converts mechanical stimuli into a metabotropic response.

Activation of Adhesion G Protein-coupled Receptors: AGONIST SPECIFICITY OF STACHEL SEQUENCE-DERIVED PEPTIDES.

Members of the adhesion G protein-coupled receptor (aGPCR) family carry an agonistic sequence within their large ectodomains. Peptides derived from this region, called the Stachel sequence, can activate the respective receptor. As the conserved core region of the Stachel sequence is highly similar between aGPCRs, the agonist specificity of Stachel sequence-derived peptides was tested between family members using cell culture-based second messenger assays. Stachel peptides derived from aGPCRs of subfamily VI (GPR110/ADGRF1, GPR116/ADGRF5) and subfamily VIII (GPR64/ADGRG2, GPR126/ADGRG6) are able to activate more than one member of the respective subfamily supporting their evolutionary relationship and defining them as pharmacological receptor subtypes. Extended functional analyses of the Stachel sequences and derived peptides revealed agonist promiscuity, not only within, but also between aGPCR subfamilies. For example, the Stachel-derived peptide of GPR110 (subfamily VI) can activate GPR64 and GPR126 (both subfamily VIII). Our results indicate that key residues in the Stachel sequence are very similar between aGPCRs allowing for agonist promiscuity of several Stachel-derived peptides. Therefore, aGPCRs appear to be pharmacologically more closely related than previously thought. Our findings have direct implications for many aGPCR studies, as potential functional overlap has to be considered for in vitro and in vivo studies. However, it also offers the possibility of a broader use of more potent peptides when the original Stachel sequence is less effective.

The constitutive activity of the adhesion GPCR GPR114/ADGRG5 is mediated by its tethered agonist.

Adhesion GPCRs (aGPCRs) form the second largest, yet most enigmatic class of the GPCR superfamily. Although the physiologic importance of aGPCRs was demonstrated in several studies, the majority of these receptors is still orphan with respect to their agonists and signal transduction. Recent studies reported that aGPCRs are activated through a tethered peptide agonist, coined the Stachel sequence. The Stachel sequence is the most C-terminal part of the highly conserved GPCR autoproteolysis-inducing domain. Here, we used cell culture-based assays to investigate 2 natural splice variants within the Stachel sequence of the orphan Gs coupling aGPCR GPR114/ADGRG5. There is 1 variant constitutively active in cAMP assays (∼25-fold over empty vector) and sensitive to mechano-activation. The other variant has low basal activity in cAMP assays (6-fold over empty vector) and is insensitive to mechano-activation. In-depth mutagenesis studies of these functional differences revealed that the N-terminal half of the Stachel sequence confers the agonistic activity, whereas the C-terminal part orientates the agonistic core sequence to the transmembrane domain. Sequence comparison and functional testing suggest that the proposed mechanism of Stachel-mediated activation is relevant not only to GPR114 but to aGPCRs in general.

The Activation Mechanism of Glycoprotein Hormone Receptors with Implications in the Cause and Therapy of Endocrine Diseases.

Glycoprotein hormones (GPHs) are the main regulators of the pituitary-thyroid and pituitary-gonadal axes. Selective interaction between GPHs and their cognate G protein-coupled receptors ensure specificity in GPH signaling. The mechanisms of how these hormones activate glycoprotein hormone receptors (GPHRs) or how mutations and autoantibodies can alter receptor function were unclear. Based on the hypothesis that GPHRs contain an internal agonist, we systematically screened peptide libraries derived from the ectodomain for agonistic activity on the receptors. We show that a peptide (p10) derived from a conserved sequence in the C-terminal part of the extracellular N terminus can activate all GPHRs in vitro and in GPHR-expressing tissues. Inactivating mutations in this conserved region or in p10 can inhibit activation of the thyroid-stimulating hormone receptor by autoantibodies. Our data suggest an activation mechanism where, upon extracellular ligand binding, this intramolecular agonist isomerizes and induces structural changes in the 7-transmembrane helix domain, triggering G protein activation. This mechanism can explain the pathophysiology of activating autoantibodies and several mutations causing endocrine dysfunctions such as Graves disease and hypo- and hyperthyroidism. Our findings highlight an evolutionarily conserved activation mechanism of GPHRs and will further promote the development of specific ligands useful to treat Graves disease and other dysfunctions of GPHRs.

Oriented Cell Division in the C. elegans Embryo Is Coordinated by G-Protein Signaling Dependent on the Adhesion GPCR LAT-1.

Orientation of spindles and cell division planes during development of many species ensures that correct cell-cell contacts are established, which is vital for proper tissue formation. This is a tightly regulated process involving a complex interplay of various signals. The molecular mechanisms underlying several of these pathways are still incompletely understood. Here, we identify the signaling cascade of the C. elegans latrophilin homolog LAT-1, an essential player in the coordination of anterior-posterior spindle orientation during the fourth round of embryonic cell division. We show that the receptor mediates a G protein-signaling pathway revealing that G-protein signaling in oriented cell division is not solely GPCR-independent. Genetic analyses showed that through the interaction with a Gs protein LAT-1 elevates intracellular cyclic AMP (cAMP) levels in the C. elegans embryo. Stimulation of this G-protein cascade in lat-1 null mutant nematodes is sufficient to orient spindles and cell division planes in the embryo in the correct direction. Finally, we demonstrate that LAT-1 is activated by an intramolecular agonist to trigger this cascade. Our data support a model in which a novel, GPCR-dependent G protein-signaling cascade mediated by LAT-1 controls alignment of cell division planes in an anterior-posterior direction via a metabotropic Gs-protein/adenylyl cyclase pathway by regulating intracellular cAMP levels.

Identification of the tethered peptide agonist of the adhesion G protein-coupled receptor GPR64/ADGRG2.

The epididymis-specific adhesion G protein-coupled receptor (aGPCR) GPR64/ADGRG2 has been shown to be a key-player in the male reproductive system. As its disruption leads to infertility, GPR64 has drawn attention as potential target for male fertility control or improvement. Like the majority of aGPCRs GPR64 is an orphan receptor regarding its endogenous agonist and signal transduction. In this study we examined the G protein-coupling abilities of GPR64 and showed that it is activated through a tethered agonist sequence, which we have previously identified as the Stachel sequence. Synthetic peptides derived from the Stachel region can activate the receptor, opening for the first time the possibility to externally manipulate the receptor activity.

A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133.

Adhesion G protein-coupled receptors (aGPCRs) comprise the second largest yet least studied class of the GPCR superfamily. aGPCRs are involved in many developmental processes and immune and synaptic functions, but the mode of their signal transduction is unclear. Here, we show that a short peptide sequence (termed the Stachel sequence) within the ectodomain of two aGPCRs (GPR126 and GPR133) functions as a tethered agonist. Upon structural changes within the receptor ectodomain, this intramolecular agonist is exposed to the seven-transmembrane helix domain, which triggers G protein activation. Our studies show high specificity of a given Stachel sequence for its receptor. Finally, the function of Gpr126 is abrogated in zebrafish with a mutated Stachel sequence, and signaling is restored in hypomorphic gpr126 zebrafish mutants upon exogenous Stachel peptide application. These findings illuminate a mode of aGPCR activation and may prompt the development of specific ligands for this currently untargeted GPCR family.

Therapeutic potential of the peptide leucine arginine as a new nonplant bowman-birk-like serine protease inhibitor.

The peptide leucine arginine (pLR) belongs to a new class of cyclic peptides isolated from frog skin. Its primary sequence is similar to the reactive loop of plant Bowman-Birk inhibitors (BBI), and the recently discovered circular sunflower trypsin inhibitor-1 (SFTI-1). The conformational properties of pLR in solution were determined by NMR spectroscopy and revealed excellent structural similarity to BBI and SFTI-1. Moreover, pLR is a highly potent trypsin inhibitor, with Ki values in the nanomolar range, and, due to its small size, a potential inhibitor of the serine protease tryptase. Since tryptase plays a crucial role in the development of allergic airway inflammation, the therapeutic potential of pLR in a murine asthma model was investigated. Treatment of ovalbumin-sensitized mice with pLR during allergen challenge reduced the acute asthma phenotype. Most importantly, application even at the end of a long-lasting chronic asthma model decreased the development of chronic airway inflammation and tissue remodeling.

Dynamics of amyloid ß fibrils revealed by solid-state NMR.

We have investigated the site-specific backbone dynamics of mature amyloid ß (Aß) fibrils using solid-state NMR spectroscopy. Overall, the known ß-sheet segments and the turn linking these two ß-strands exhibit high order parameters between 0.8 and 0.95, suggesting low conformational flexibility. The first approximately eight N-terminal and the last C-terminal residues exhibit lower order parameters between ∼0.4 and 0.8. Interestingly, the order parameters increase again for the first two residues, Asp(1) and Ala(2), suggesting that the N terminus could carry some structural importance.