Explore the vast range of titles available.

Prolactin-releasing peptide (PrRP) is an endogenous neuropeptide involved in appetite regulation and energy homeostasis. PrRP binds with high affinity to G-protein coupled receptor 10 (GPR10) and with lesser activity towards the neuropeptide FF receptor type 2 (NPFF2R). The present study aimed to develop long-acting PrRP31 analogues with potent anti-obesity efficacy. A comprehensive series of C18 lipidated PrRP31 analogues was characterized in vitro and analogues with various GPR10 and NPFF2R activity profiles were profiled for bioavailability and metabolic effects following subcutaneous administration in diet-induced obese (DIO) mice. PrRP31 analogues acylated with a C18 lipid chain carrying a terminal acid (C18 diacid) were potent GPR10-selective agonists and weight-neutral in DIO mice. In contrast, acylation with aliphatic C18 lipid chain (C18) resulted in dual GPR10-NPFF2R co-agonists that suppressed food intake and promoted a robust weight loss in DIO mice, which was sustained for at least one week after last dosing. Rapid in vivo degradation of C18 PrRP31 analogues gave rise to circulating lipidated PrRP metabolites maintaining dual GPR10-NPFF2R agonist profile and long-acting anti-obesity efficacy in DIO mice. Combined GPR10 and NPFF2R activation may therefore be a critical mechanism for obtaining robust anti-obesity efficacy of PrRP31 analogues.

Delineating human cardiac pathologies and their basic molecular mechanisms relies on research conducted in model organisms. Yet translating findings from preclinical models to humans present a significant challenge, in part due to differences in cardiac protein expression between humans and model organisms. Proteins immediately determine cellular function, yet their large-scale investigation in hearts has lagged behind those of genes and transcripts. Here, we set out to bridge this knowledge gap: By analyzing protein profiles in humans and commonly used model organisms across cardiac chambers, we determine their commonalities and regional differences. We analyzed cardiac tissue from each chamber of human, pig, horse, rat, mouse, and zebrafish in biological replicates. Using mass spectrometry–based proteomics workflows, we measured and evaluated the abundance of approximately 7,000 proteins in each species. The resulting knowledgebase of cardiac protein signatures is accessible through an online database: atlas.cardiacproteomics.com . Our combined analysis allows for quantitative evaluation of protein abundances across cardiac chambers, as well as comparisons of cardiac protein profiles across model organisms. Up to a quarter of proteins with differential abundances between atria and ventricles showed opposite chamber-specific enrichment between species; these included numerous proteins implicated in cardiac disease. The generated proteomics resource facilitates translational prospects of cardiac studies from model organisms to humans by comparisons of disease-linked protein networks across species.

Migraine is a debilitating headache disorder. The disease has neurovascular origin and migraine attacks can be elicited by vasodilative neuropeptides such as alpha calcitonin gene-related peptide (αCGRP). Antagonizing CGRP actions in migraine patients has proven clinically efficient. Here, we present a pipeline for development of a peptide-based hCGRP receptor antagonist with increased half-life capable of antagonising the vasodilatory effect of hαCGRP. A series of hαCGRP8-37 analogues carrying a C18-or C20-diacid lipidation was screened for their antagonism against the hCGRP receptor. hαCGRP8-37 analogues with a C20-diacid were 2-6 fold more potent than analogues conjugated with a C18-diacid. Half-life of hαCGRP8-37 analogues carrying a C20-diacid was estimated in mice in a pilot study (n = 1–2). Half-lives ranged from 7.3 to 13.7 h. An hαCGRP8-37 analogue conjugated with a C20 diacid at position 25 was subjected to an amino acid substitution scan to identify mutations that could further enhance hCGRP receptor antagonism. Substituting alanine with serine at position 36 resulted in a ~ 4 fold gain of potency. Vasodilative actions of hαCGRP were successfully antagonized by hαCGRP8-37 analogues carrying a C20 diacid at position 25. Our findings demonstrate that lipidation can improve hαCGRP8-37 pharmacokinetics while maintaining hαCGRP antagonism, thus demonstrating potential for a peptide-based migraine treatment strategy.

Posttranslational modifications (PTMs) of the histone H3 tail such as methylation, acetylation and phosphorylation play important roles in epigenetic signaling. Here we study the effect of some of these PTMs on the demethylation rates of methylated lysine 9 in vitro using peptide substrates mimicking histone H3. Various combinations with other PTMs were employed to study possible cross-talk effects by comparing enzyme kinetic characteristics. We compared the kinetics of histone tail substrates for truncated histone lysine demethylases KDM4A and KDM4C containing only the catalytic core (cc) and some combinations were characterized on full length (FL) KDM4A and KDM4C. We found that the substrates combining trimethylated K4 and K9 resulted in a significant increase in the catalytic activity for FL-KDM4A. For the truncated versions of KDM4A and KDM4C a two-fold increase in the catalytic activity toward bis-trimethylated substrates could be observed. Furthermore, a significant difference in the catalytic activity between dimethylated and trimethylated substrates was found for full length demethylases in line with what has been reported previously for truncated demethylases. Histone peptide substrates phosphorylated at T11 could not be demethylated by neither truncated nor full length KDM4A and KDM4C, suggesting that phosphorylation of threonine 11 prevents demethylation of the H3K9me3 mark on the same peptide. Acetylation of K14 was also found to influence demethylation rates significantly. Thus, for truncated KDM4A, acetylation on K14 of the substrate leads to an increase in enzymatic catalytic efficiency (k cat/K m), while for truncated KDM4C it induces a decrease, primarily caused by changes in K m. This study demonstrates that demethylation activities towards trimethylated H3K9 are significantly influenced by other PTMs on the same peptide, and emphasizes the importance of studying these interactions at the peptide level to get a more detailed understanding of the dynamics of epigenetic marks.

Ca2+/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) is a key neuronal signaling protein and an emerging drug target. The central hub domain regulates the activity of CaMKIIα by organizing the holoenzyme complex into functional oligomers, yet pharmacological modulation of the hub domain has never been demonstrated. Here, using a combination of photoaffinity labeling and chemical proteomics, we show that compounds related to the natural substance γ-hydroxybutyrate (GHB) bind selectively to CaMKIIα. By means of a 2.2-Å x-ray crystal structure of ligand-bound CaMKIIα hub, we reveal the molecular details of the binding site deep within the hub. Furthermore, we show that binding of GHB and related analogs to this site promotes concentration-dependent increases in hub thermal stability believed to alter holoenzyme functionality. Selectively under states of pathological CaMKIIα activation, hub ligands provide a significant and sustained neuroprotection, which is both time and dose dependent. This is demonstrated in neurons exposed to excitotoxicity and in a mouse model of cerebral ischemia with the selective GHB analog, HOCPCA (3-hydroxycyclopent-1-ene-carboxylic acid). Together, our results indicate a hitherto unknown mechanism for neuroprotection by a highly specific and unforeseen interaction between the CaMKIIα hub domain and small molecule brain-penetrant GHB analogs. This establishes GHB analogs as powerful tools for investigating CaMKII neuropharmacology in general and as potential therapeutic compounds for cerebral ischemia in particular.

The spreadsheet file reported herein provides centroid data, descriptive of deuterium uptake, for the FabFragment of NISTmAb (PDB: 5K8A) reference material, as measured by the bottom-up hydrogen-deuterium exchange mass spectrometry (HDX-MS) method. The protein sample was incubated in deuterium-rich solutions under uniform pH and salt concentrations between 3.6 oC and 25.4 oC for seven intervals ranging over (0 to 14,400) s plus a ∞pseudo s control. The deuterium content of peptic peptide fragments were measured by mass spectrometry. These data were reported by fifteen laboratories, which conducted the measurements using orbitrap and Q-TOF mass spectrometers. The cohort reported ≈ 78,900 centroids for 430 proteolytic peptide sequences of the heavy and light chains of NISTmAb, providing nearly 100 % coverage. In addition, some groups reported ≈ 10,900 centroid measurements for 77 peptide sequences of the Fc fragment. The instrumentation and physical and chemical conditions under which these data were acquired are documented.

A subset of the GABAA receptors expressed in recombinant systems and neurons is known to exhibit both constitutive- and agonist-induced gating. Two such receptors are the δ-subunit containing GABAA receptors α4β1δ and α4β3δ, which are expressed in adult rodent hippocampal dentate gyrus granule cells (DGGCs). Here we show that the GABAA receptor mediated tonic current recorded in the presence of tetrodotoxin in adult rodent DGGCs is almost exclusively mediated by constitutively active δ-subunit containing GABAA receptors and that the constitutive current is absent in recordings at 24 °C or in recordings at 34 °C including an intracellular inhibitor of protein kinase C. These factors simultaneously govern the efficacy of an orthosteric agonist at α4β1/3δ receptors, Thio-THIP, in a reciprocal manner. In the absence of constitutive receptor activity, the efficacy of Thio-THIP was increased approximately four-fold relative to recording conditions that favors constitutive activity. Further, only under conditions of an absent constitutive current, the classified neutral antagonist gabazine (GBZ) alone, induced a tonic current in DGGCs (EC50 2.1 μM). This effect of GBZ was not seen in recording conditions of high constitutive activity, was inhibited by picrotoxin (PTX), potentiated by DS2, completely absent in δ-/- mice and reduced in β1-/- mice, but could not be replicated in human α4β1/3δ receptors expressed recombinantly in HEK cells. We hypothesize that specific intracellular components in neurons interact with receptors to determine constitutive gating and receptor responsiveness to orthosteric ligands. The presented data highlight how recording conditions for whole cell patch clamp analysis of α4β1/3δ GABAA receptors can mask important pharmacological effects. Specifically, orthosteric agonists appear with reduced efficacy, and other ligands, here exemplified with the well-known antagonist GBZ, are misinterpreted as being inactive/neutral, although they could have effect in constitutively silent receptors. Unmasking of potential hidden effects are easily done using recording conditions of reduced kinase activity in a relevant neuronal context. It follows that in pathologies with changes in phosphorylation level of δ-subunit containing GABAA receptors, the efficacy of an agonist of these receptors, measured by whole-cell recordings in vitro, will not match the efficacy of the same agonist in an unperturbed neuron in vivo.

A subset of the GABAA receptors expressed in recombinant systems and neurons is known to exhibit both constitutive- and agonist-induced gating. Two such receptors are the -subunit containing GABAA receptors 4 1 and 4 3 , which are expressed in adult rodent hippocampal dentate gyrus granule cells (DGGCs). Here we show that the GABAA receptor mediated tonic current recorded in the presence of tetrodotoxin in adult rodent DGGCs is almost exclusively mediated by constitutively active -subunit containing GABAA receptors and that the constitutive current is absent in recordings at 24 C or in recordings at 34 C including an intracellular inhibitor of protein kinase C. These factors simultaneously govern the efficacy of an orthosteric agonist at 4 1/3 receptors, Thio-THIP, in a reciprocal manner. In the absence of constitutive receptor activity, the efficacy of Thio-THIP was increased approximately four-fold relative to recording conditions that favors constitutive activity. Further, only under conditions of an absent constitutive current, the classified neutral antagonist gabazine (GBZ) alone, induced a tonic current in DGGCs (EC50 2.1 M). This effect of GBZ was not seen in recording conditions of high constitutive activity, was inhibited by picrotoxin (PTX), potentiated by DS2, completely absent in -/- mice and reduced in 1-/- mice, but could not be replicated in human 4 1/3 receptors expressed recombinantly in HEK cells. We hypothesize that specific intracellular components in neurons interact with receptors to determine constitutive gating and receptor responsiveness to orthosteric ligands.

The study of human cardiac pathologies often relies on research conducted in model organisms to gain molecular insight into disease and to develop novel treatment strategies; however, translating findings from model organisms back to human can present a significant challenge, in part due to a lack of knowledge about the differences across species in cardiac protein abundances and their interactions. Here we set out to bridge this knowledge gap by presenting a global analysis of cardiac protein expression profiles in humans and commonly used model organisms. Using quantitative mass spectrometry-based proteomics, we measured the abundance of ~7,000 proteins in samples from the separate chambers of human, pig, horse, rat, mouse and zebrafish hearts. This knowledgebase of cardiac protein signatures is accessible through an online database at: atlas.cardiacproteomics.com. Quantitative comparison of the protein profiles support the pig as model organism of choice for arrhythmogenic right ventricular cardiomyopathy whereas comparison of profiles from the two-chambered zebrafish heart suggests a better resemblance to the right side of mammalian hearts. This proteomics resource facilitates translational prospect of cardiac studies from model organisms to humans by enabling direct comparison of disease-linked protein networks across species. Footnotes * https://atlas.cardiacproteomics.com/

ABSTRACT Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is an abundant neuronal signaling protein involved in synaptic plasticity and memory formation1,2. The central hub domain regulates the activity of CaMKIIα by organizing the holoenzyme complex into functional oligomers3-6. Recent findings have suggested that the hub is also an allosteric determinant of kinase activity7, and is thus an emerging target for therapies to correct CaMKIIα dysregulation8,9. However, pharmacological modulation of the hub domain has never been demonstrated. Here we show that stabilization of the CaMKIIα hub domain confers neuroprotection. By combining photoaffinity labeling and chemical proteomics using small molecule analogs of the natural metabolite γ-hydroxybutyrate (GHB)10 we reveal that CaMKIIα is the selective target for GHB. We further find that these GHB analogs bind to the hub interior by solving a 2.2 Å crystal structure of CaMKIIα with bound ligand. Using differential scanning fluorimetry, we show that binding of ligands to the hub interior increases the thermal stability of hub oligomers in a concentration-dependent manner. Moreover, we demonstrate the functional significance of this hub stabilization by showing substantial neuroprotective effects in cellular excitotoxicity assays and in a mouse model of cerebral ischemia. Together, our results reveal that CaMKIIα hub stabilization is the mechanism by which GHB provides endogenous neuroprotection and that small-molecule CaMKIIα-selective ligands have therapeutic potential. Competing Interest Statement The University of Copenhagen and Otago Innovation Ltd. have licensed the patent rights for GHB derivatives and their uses (WO/2019/149329) to Ceremedy Ltd. of which B.F., B.R.K. and P.W. are co-founders.