Short-term psychoanalytic psychotherapy with depressed adolescents: Comparing in-session interactions in good and poor outcome cases.

OBJECTIVE: To identify and describe in-session interaction patterns between psychoanalytic therapists and adolescents diagnosed with major depressive disorder, comparing good and poor outcome cases. METHOD: Audio recordings for 100 psychotherapy sessions from 10 Short-Term Psychoanalytic Psychotherapies were analysed using the Adolescent Psychotherapy Q-Set (APQ). The cases and sessions were evenly divided into two groups (poor outcome and good outcome, 5 patients and 50 sessions per group). Interaction patterns were analysed with an Exploratory Factor Analysis (EFA), while group differences were assessed through t-tests. RESULTS: The EFA revealed three factors: (1) "Open, engaged young person working collaboratively with a therapist to make sense of their experiences", (2) "Directive therapist with a young person fluctuating in emotional state and unwilling to explore", (3) "Young person expressing anger and irritation and challenging the therapist". Factor 1 was significantly more prominent in the good outcome cases, while factor 3, on the contrary, was more significantly related to the poor outcome cases. Factor 2 was equally present in both groups. CONCLUSION: Besides reinforcing to researchers and clinicians the association between a collaborative psychotherapy process with good outcomes, our findings also provide empirical data regarding the role of anger in adolescent depression and the psychotherapy process.

Prevention of Childhood Adversities and Children's Common Mental Disorders and School Grades.

Importance: Adversity during childhood can limit children's chances of achieving their optimal developmental and psychological outcomes. Well-designed observational studies might help identify adversities that are most implicated in this, thereby helping to identify potential targets for developing interventions. Objective: To compare the association between preventing childhood poverty, parental mental illness and parental separation, and the population rate of offspring common mental disorders (ages 16-21 years) or average school grades (age 16 years). Design, Setting, and Participants: A population-based, longitudinal cohort study using Swedish registries was conducted. A total of 163 529 children born in Sweden between January 1, 1996, and December 31, 1997, were followed up until their 21st birthday. They were linked to registries using Sweden's national personal identification number. Children were linked to birth parents, hospital records, and school data. Parents were linked to registries containing health, income, sociodemographic, and obstetric data. Analyses were conducted between January 10, 2021, and August 26, 2022. Exposures: Childhood adversities of relative poverty (household disposable income <50% of the median), parental inpatient admission for a mental illness, or parental separation. Adversities were categorized into developmental periods: ages 0 to 3, 4 to 7, 8 to 11, and 12 to 16 years. Main Outcomes and Measures: The main outcomes were children's hospital records with a diagnosis of anxiety or depression between ages 16 and 21 years and school grades at the end of compulsory education (age 16 years). The parametric g-formula modeled population changes in outcomes associated with the counterfactual, hypothetical preventing adversity exposures, accounting for fixed and time-varying confounders. Adjustments were made for parental demographic characteristics, obstetric variables, and socioeconomic data at birth. Results: A total of 163 529 children were included in the cohort (51.2% boys, 51.4% born in 1996). Preventing all adversities was associated with an estimated change in the prevalence of offspring common mental disorders from 10.2% to 7.6% and an improvement in school grades with an SD of 0.149 (95% CI, 0.147-0.149). Preventing parental separation provided for the greatest improvement, with an estimated 2.34% (95% CI, 2.23%-2.42%) fewer children with a common mental disorder and an improvement in school grades by 0.127 SDs (0.125-0.129). Greater improvements were shown by hypothetically targeting adolescents (age 12-16 years) and those whose parents had a mental illness when the child was born. Conclusions and Relevance: The results of this cohort modeling study suggest that preventing childhood adversity could provide notable improvements in the rates of common mental disorders and school grades. Many children might achieve better life outcomes if resources are properly allocated to the right adversities (parental separation), the right groups (children with parental mental illness), and at the right time (adolescence).

Organic Matrix Derived from Host-Microbe Interplay Contributes to Pathological Renal Biomineralization.

Matrix stones are a rare form of kidney stones. They feature a high percentage of hydrogel-like organic matter, and their formation is closely associated with urinary tract infections. Herein, comprehensive materials and biochemical approaches were taken to map the organic-inorganic interface and gather insights into the host-microbe interplay in pathological renal biomineralization. Surgically extracted soft and slimy matrix stones were examined using micro-X-ray computed tomography and various microspectroscopy techniques. Higher-mineral-density laminae were positive for calcium-bound Alizarin red. Lower-mineral-density laminae revealed periodic acid-Schiff-positive organic filamentous networks of varied thickness. These organic filamentous networks, which featured a high polysaccharide content, were enriched with zinc, carbon, and sulfur elements. Neutrophil extracellular traps (NETs) along with immune response-related proteins, including calprotectin, myeloperoxidase, CD63, and CD86, also were identified in the filamentous networks. Expressions of NETs and upregulation of polysaccharide-rich mucin secretion are proposed as a part of the host immune defense to "trap" pathogens. These host-microbe derived organic matrices can facilitate heterogeneous nucleation and precipitation of inorganic particulates, resulting in macroscale aggregates known as "matrix stones". These insights into the plausible aggregation of constituents through host-microbe interplay underscore the unique "double-edged sword" effect of the host immune response to pathogens and the resulting renal biominerals.

The Next Generation of Population-Based DFNB16 Carrier Screening and Diagnosis: STRC Copy-Number Variant Analysis from Genome Sequencing Data.

BACKGROUND: Deafness, autosomal recessive 16 (DFNB16) is caused by compound heterozygous or homozygous variants in STRC and is the second most common form of genetic hearing loss. Due to the nearly identical sequences of STRC and the pseudogene STRCP1, analysis of this region is challenging in clinical testing. METHODS: We developed a method that accurately identifies the copy number of STRC and STRCP1 using standard short-read genome sequencing. Then, we used whole genome sequencing (WGS) data to investigate the population distribution of STRC copy number in 6813 neonates and the correlation between STRC and STRCP1 copy number. RESULTS: The comparison of WGS results with multiplex ligation-dependent probe amplification demonstrated high sensitivity (100%; 95% CI, 97.5%-100%) and specificity (98.8%; 95% CI, 97.7%-99.5%) in detecting heterozygous deletion of STRC from short-read genome sequencing data. The population analysis revealed that 5.22% of the general population has STRC copy number changes, almost half of which (2.33%; 95% CI, 1.99%-2.72%) were clinically significant, including heterozygous and homozygous STRC deletions. There was a strong inverse correlation between STRC and STRCP1 copy number. CONCLUSIONS: We developed a novel and reliable method to determine STRC copy number based on standard short-read based WGS data. Incorporating this method into analytic pipelines would improve the clinical utility of WGS in the screening and diagnosis of hearing loss. Finally, we provide population-based evidence of pseudogene-mediated gene conversions between STRC and STRCP1.

The ubiquitination status of the glucagon receptor determines signal bias.

The pancreatic hormone glucagon activates the glucagon receptor (GCGR), a class B seven-transmembrane G protein-coupled receptor that couples to the stimulatory heterotrimeric G protein and provokes PKA-dependent signaling cascades vital to hepatic glucose metabolism and islet insulin secretion. Glucagon-stimulation also initiates recruitment of the endocytic adaptors, ßarrestin1 and ßarrestin2, which regulate desensitization and internalization of the GCGR. Unlike many other G protein-coupled receptors, the GCGR expressed at the plasma membrane is constitutively ubiquitinated and upon agonist-activation, internalized GCGRs are deubiquitinated at early endosomes and recycled via Rab4-containing vesicles. Herein we report a novel link between the ubiquitination status and signal transduction mechanism of the GCGR. In the deubiquitinated state, coupling of the GCGR to Gs is diminished, while binding to ßarrestin is enhanced with signaling biased to a ßarrestin1-dependent p38 mitogen activated protein kinase (MAPK) pathway. This ubiquitin-dependent signaling bias arises through the modification of lysine333 (K333) on the cytoplasmic face of transmembrane helix V. Compared with the GCGR-WT, the mutant GCGR-K333R has impaired ubiquitination, diminished G protein coupling, and PKA signaling but unimpaired potentiation of glucose-stimulated-insulin secretion in response to agonist-stimulation, which involves p38 MAPK signaling. Both WT and GCGR-K333R promote the formation of glucagon-induced ßarrestin1-dependent p38 signaling scaffold that requires canonical upstream MAPK-Kinase3, but is independent of Gs, Gi, and ßarrestin2. Thus, ubiquitination/deubiquitination at K333 in the GCGR defines the activation of distinct transducers with the potential to influence various facets of glucagon signaling in health and disease.

Corrigendum: Calcium interactions in amelogenin-derived peptide assembly.

[This corrects the article DOI: 10.3389/fphys.2022.1063970.].

Establishment of multi-stage intravenous self-administration paradigms in mice.

Genetically tractable animal models provide needed strategies to resolve the biological basis of drug addiction. Intravenous self-administration (IVSA) is the gold standard for modeling psychostimulant and opioid addiction in animals, but technical limitations have precluded the widespread use of IVSA in mice. Here, we describe IVSA paradigms for mice that capture the multi-stage nature of the disorder and permit predictive modeling. In these paradigms, C57BL/6J mice with long-standing indwelling jugular catheters engaged in cocaine- or remifentanil-associated lever responding that was fixed ratio-dependent, dose-dependent, extinguished by withholding the drug, and reinstated by the presentation of drug-paired cues. The application of multivariate analysis suggested that drug taking in both paradigms was a function of two latent variables we termed incentive motivation and discriminative control. Machine learning revealed that vulnerability to drug seeking and relapse were predicted by a mouse's a priori response to novelty, sensitivity to drug-induced locomotion, and drug-taking behavior. The application of these behavioral and statistical-analysis approaches to genetically-engineered mice will facilitate the identification of neural circuits driving addiction susceptibility and relapse and focused therapeutic development.

Morphological Selectivity of a Protein Self-Assembly System with a Repertoire of Diverse Interaction Modes.

Multiple metal chelating sites were incorporated onto the second mitochondria-derived activator of caspase (SMAC) building blocks. The combination of different binding sites generated a repertoire of diverse binding modes, among which two different microfilament types (small and large) with distinct patterns were selected under thermodynamic control. Furthermore, the two microfilaments exhibited a pronounced secondary assembly trend due to the potential noncovalent interactions on the protein surfaces. Coupled with stereoselectivity, they presented a strong self-recognition effect and underwent two distinct reassembly patterns. That is, the large filaments self-associated in pairs to form "interlocked chain" structures, while the small ones twisted to form protein helical bundles. This work represents one of the few studies of selective self-assembly of self-assembled protein assemblies. Such an idea may provide inspiration for constructing more sophisticated protein architectures in the future.

A single phenylalanine residue in ß-arrestin2 critically regulates its binding to G protein-coupled receptors.

Arrestins and their yeast homologs, arrestin-related trafficking adaptors (ARTs), share a stretch of 29 amino acids called the ART motif. However, the functionality of that motif is unknown. We now report that deleting this motif prevents agonist-induced ubiquitination of ß-arrestin2 (ß-arr2) and blocks its association with activated G protein-coupled receptors (GPCRs). Within the ART motif, we have identified a conserved phenylalanine residue, Phe116, that is critical for the formation of ß-arr2-GPCR complexes. ß-arr2 Phe116Ala mutant has negligible effect on blunting ß2-adrenergic receptor-induced cAMP generation unlike ß-arr2, which promotes rapid desensitization. Furthermore, available structures for inactive and inositol hexakisphosphate 6-activated forms of bovine ß-arr2 revealed that Phe116 is ensconced in a hydrophobic pocket, whereas the adjacent Phe117 and Phe118 residues are not. Mutagenesis of Phe117 and Phe118, but not Phe116, preserves GPCR interaction of ß-arr2. Surprisingly, Phe116 is dispensable for the association of ß-arr2 with its non-GPCR partners. ß-arr2 Phe116Ala mutant presents a significantly reduced protein half-life compared with ß-arr2 and undergoes constitutive Lys-48-linked polyubiquitination, which tags proteins for proteasomal degradation. We also found that Phe116 is critical for agonist-dependent ß-arr2 ubiquitination with Lys-63-polyubiquitin linkages that are known mediators of protein scaffolding and signal transduction. Finally, we have shown that ß-arr2 Phe116Ala interaction with activated ß2-adrenergic receptor can be rescued with an in-frame fusion of ubiquitin. Taken together, we conclude that Phe116 preserves structural stability of ß-arr2, regulates the formation of ß-arr2-GPCR complexes that inhibit G protein signaling, and promotes subsequent ubiquitin-dependent ß-arr2 localization and trafficking.

Calcium interactions in amelogenin-derived peptide assembly.

Phosphorylation of serine residues has been recognized as a pivotal event in the evolution of mineralized tissues in many biological systems. During enamel development, the extracellular matrix protein amelogenin is most abundant and appears to be critical to the extreme high aspect ratios (length:width) of apatite mineral fibers reaching several millimeters in larger mammalian teeth. A 14-residue peptide (14P2, residues Gly8 to Thr21) was previously identified as a key sequence mediating amelogenin assembly formation, the domain also contains the native single phosphoserine residue (Ser16) of the full-length amelogenin. In this research, 14P2 and its phosphorylated form (p14P2) were investigated at pH 6.0 with various calcium and phosphate ion concentrations, indicating that both peptides could self-assemble into amyloid-like conformation but with differences in structural details. With calcium, the distance between 31P within the p14P2 self-assemblies is averaged to be 4.4 ± 0.2Å, determined by solid-state NMR 31P PITHIRDS-CT experiments. Combining with other experimental results, solid-state Nuclear Magnetic Resonance (SSNMR) suggests that the p14P2 self-assemblies are in parallel in-register ß-sheet conformation and divalent calcium ions most likely connect two adjacent peptide chains by binding to the phosphate group of Ser16 and the carboxylate of Glu18 side-chain. This study on the interactions between calcium ions and amelogenin-derived peptides provides insights on how amelogenin may self-assemble in the presence of calcium ions in early enamel development.

A N-Terminus Domain Determines Amelogenin's Stability to Guide the Development of Mouse Enamel Matrix.

Amelogenins, the principal proteins in the developing enamel microenvironment, self-assemble into supramolecular structures to govern the remodeling of a proteinaceous organic matrix into longitudinally ordered hydroxyapatite nanocrystal arrays. Extensive in vitro studies using purified native or recombinant proteins have revealed the potential of N-terminal amelogenin on protein self-assembly and its ability to guide the mineral deposition. We have previously identified a 14-aa domain (P2) of N-terminal amelogenin that can self-assemble into amyloid-like fibrils in vitro. Here, we investigated how this domain affects the ability of amelogenin self-assembling and stability of enamel matrix protein scaffolding in an in vivo animal model. Mice harboring mutant amelogenin lacking P2 domain had a hypoplastic, hypomineralized, and aprismatic enamel. In vitro, the mutant recombinant amelogenin without P2 had a reduced tendency to self-assemble and was prone to accelerated hydrolysis by MMP20, the prevailing metalloproteinase in early developing enamel matrix. A reduced amount of amelogenins and a lack of elongated fibrous assemblies in the development enamel matrix of mutant mice were evident compared with that in the wild-type mouse enamel matrix. Our study is the first to demonstrate that a subdomain (P2) at the N-terminus of amelogenin controls amelogenin's assembly into a transient protein scaffold that resists rapid proteolysis during enamel development in an animal model. Understanding the building blocks of fibrous scaffold that guides the longitudinal growth of hydroxyapatites in enamel matrix sheds light on protein-mediated enamel bioengineering. © 2021 American Society for Bone and Mineral Research (ASBMR).

PCM1 is necessary for focal ciliary integrity and is a candidate for severe schizophrenia.

The neuronal primary cilium and centriolar satellites have functions in neurogenesis, but little is known about their roles in the postnatal brain. We show that ablation of pericentriolar material 1 in the mouse leads to progressive ciliary, anatomical, psychomotor, and cognitive abnormalities. RNAseq reveals changes in amine- and G-protein coupled receptor pathways. The physiological relevance of this phenotype is supported by decreased available dopamine D2 receptor (D2R) levels and the failure of antipsychotic drugs to rescue adult behavioral defects. Immunoprecipitations show an association with Pcm1 and D2Rs. Finally, we sequence PCM1 in two human cohorts with severe schizophrenia. Systematic modeling of all discovered rare alleles by zebrafish in vivo complementation reveals an enrichment for pathogenic alleles. Our data emphasize a role for the pericentriolar material in the postnatal brain, with progressive degenerative ciliary and behavioral phenotypes; and they support a contributory role for PCM1 in some individuals diagnosed with schizophrenia.

In-frame fusion of SUMO1 enhances ßarrestin2's association with activated GPCRs as well as with nuclear pore complexes.

Small ubiquitin like modifier (SUMO) conjugation or SUMOylation of ßarrestin2 promotes its association with the clathrin adaptor protein AP2 and facilitates rapid ß2 adrenergic receptor (ß2AR) internalization. However, disruption of the consensus SUMOylation site in ßarrestin2, did not prevent ßarrestin2's association with activated ß2ARs, dopamine D2 receptors (D2Rs), angiotensin type 1a receptors (AT1aRs) and V2 vasopressin receptors (V2Rs). To address the role of SUMOylation in the trafficking of ßarrestin and GPCR complexes, we generated and characterized a yellow fluorescent protein (YFP) tagged ßarrestin2-SUMO1 chimeric protein, which is resistant to de-SUMOylation. In HEK-293 cells, YFP-SUMO1 predominantly localized in the nucleus, whereas YFP-ßarrestin2 is cytoplasmic. YFP-ßarrestin2-SUMO1 in addition to being cytoplasmic, is localized at the nuclear membrane. Nonetheless, ßarrestin2-SUMO1 associated robustly with agonist-activated ß2ARs as evaluated by co-immunoprecipitation, confocal microscopy and bioluminescence resonance energy transfer (BRET). ßarrestin2-SUMO1 associated strongly with the D2R, which forms transient complexes with ßarrestin2. But, ßarrestin2-SUMO1 and ßarrestin2 showed equivalent binding with the V2R, which forms stable complexes with ßarrestin2. ßarrestin2 expression level directly correlated with the steady state levels of the unmodified form of RanGAP1, which upon SUMOylation associates with nuclear membrane. On the other hand, ßarrestin2-SUMO1 not only localized at the nuclear membrane, but also formed a macromolecular complex with RanGAP1. Taken together, our data suggest that SUMOylation of ßarrestin2 promotes its protein interactions at both cell and nuclear membranes. Furthermore, ßarrestin2-SUMO1 presents as a useful tool to characterize ßarrestin2 recruitment to GPCRs, which form transient and unstable complex with ßarrestin2.

Protein nanoribbons template enamel mineralization.

As the hardest tissue formed by vertebrates, enamel represents nature's engineering masterpiece with complex organizations of fibrous apatite crystals at the nanometer scale. Supramolecular assemblies of enamel matrix proteins (EMPs) play a key role as the structural scaffolds for regulating mineral morphology during enamel development. However, to achieve maximum tissue hardness, most organic content in enamel is digested and removed at the maturation stage, and thus knowledge of a structural protein template that could guide enamel mineralization is limited at this date. Herein, by examining a gene-modified mouse that lacked enzymatic degradation of EMPs, we demonstrate the presence of protein nanoribbons as the structural scaffolds in developing enamel matrix. Using in vitro mineralization assays we showed that both recombinant and enamel-tissue-based amelogenin nanoribbons are capable of guiding fibrous apatite nanocrystal formation. In accordance with our understanding of the natural process of enamel formation, templated crystal growth was achieved by interaction of amelogenin scaffolds with acidic macromolecules that facilitate the formation of an amorphous calcium phosphate precursor which gradually transforms into oriented apatite fibers along the protein nanoribbons. Furthermore, this study elucidated that matrix metalloproteinase-20 is a critical regulator of the enamel mineralization as only a recombinant analog of a MMP20-cleavage product of amelogenin was capable of guiding apatite mineralization. This study highlights that supramolecular assembly of the scaffold protein, its enzymatic processing, and its ability to interact with acidic carrier proteins are critical steps for proper enamel development.

ß-Arrestin-Biased Allosteric Modulator of NTSR1 Selectively Attenuates Addictive Behaviors.

Small molecule neurotensin receptor 1 (NTSR1) agonists have been pursued for more than 40 years as potential therapeutics for psychiatric disorders, including drug addiction. Clinical development of NTSR1 agonists has, however, been precluded by their severe side effects. NTSR1, a G protein-coupled receptor (GPCR), signals through the canonical activation of G proteins and engages ß-arrestins to mediate distinct cellular signaling events. Here, we characterize the allosteric NTSR1 modulator SBI-553. This small molecule not only acts as a ß-arrestin-biased agonist but also extends profound ß-arrestin bias to the endogenous ligand by selectively antagonizing G protein signaling. SBI-553 shows efficacy in animal models of psychostimulant abuse, including cocaine self-administration, without the side effects characteristic of balanced NTSR1 agonism. These findings indicate that NTSR1 G protein and ß-arrestin activation produce discrete and separable physiological effects, thus providing a strategy to develop safer GPCR-targeting therapeutics with more directed pharmacological action.

Discovery of ß-Arrestin Biased, Orally Bioavailable, and CNS Penetrant Neurotensin Receptor 1 (NTR1) Allosteric Modulators.

Neurotensin receptor 1 (NTR1) is a G protein coupled receptor that is widely expressed throughout the central nervous system where it acts as a neuromodulator. Neurotensin receptors have been implicated in a wide variety of CNS disorders, but despite extensive efforts to develop small molecule ligands there are few reports of such compounds. Herein we describe the optimization of a quinazoline based lead to give 18 (SBI-553), a potent and brain penetrant NTR1 allosteric modulator.

Achieving biopolymer synergy in systems chemistry.

Synthetic and materials chemistry initiatives have enabled the translation of the macromolecular functions of biology into synthetic frameworks. These explorations into alternative chemistries of life attempt to capture the versatile functionality and adaptability of biopolymers in new orthogonal scaffolds. Information storage and transfer, however, so beautifully represented in the central dogma of biology, require multiple components functioning synergistically. Over a single decade, the emerging field of systems chemistry has begun to catalyze the construction of mutualistic biopolymer networks, and this review begins with the foundational small-molecule-based dynamic chemical networks and peptide amyloid-based dynamic physical networks on which this effort builds. The approach both contextualizes the versatile approaches that have been developed to enrich chemical information in synthetic networks and highlights the properties of amyloids as potential alternative genetic elements. The successful integration of both chemical and physical networks through ß-sheet assisted replication processes further informs the synergistic potential of these networks. Inspired by the cooperative synergies of nucleic acids and proteins in biology, synthetic nucleic-acid-peptide chimeras are now being explored to extend their informational content. With our growing range of synthetic capabilities, structural analyses, and simulation technologies, this foundation is radically extending the structural space that might cross the Darwinian threshold for the origins of life as well as creating an array of alternative systems capable of achieving the progressive growth of novel informational materials.

Correction: Achieving biopolymer synergy in systems chemistry.

Correction for 'Achieving biopolymer synergy in systems chemistry' by Yushi Bai et al., Chem. Soc. Rev., 2018, DOI: 10.1039/c8cs00174j.

A highly controllable protein self-assembly system with morphological versatility induced by reengineered host-guest interactions.

Manipulating proteins to self-assemble into highly ordered nanostructures not only provides insights into the natural protein assembly process but also allows access to advanced biomaterials. Host-guest interactions have been widely used in the construction of artificial protein assemblies in recent years. CB[8] can selectively associate with two tripeptide Phe-Gly-Gly (FGG) tags with an extraordinarily high binding affinity (Kter = 1.5 × 1011 M-2). However, the FGG tags utilized before are all fixed to the N-termini via genetic fusion; this spatial limitation greatly confined the availability of the CB[8]/FGG pair in the construction of more sophisticated protein nanostructures. Here we first designed and synthesized a maleimide-functionalized Phe-Gly-Gly tag as a versatile site-specific protein modification tool; this designed tag can site-selectively introduce desired guest moieties onto protein surfaces for host-guest driven protein assembly. When regulating the self-assembly process of proteins and CB[8], the constructed protein nanosystem can exhibit distinctive morphological diversities ranging from nanorings, nanospirals, nanowires to superwires. This work developed a new strategy for site-specific protein modification of the CB[8] binding tag and provides a possible direction for the construction of 'smart', dynamic self-assembly systems.

Protamine is an antagonist of apelin receptor, and its activity is reversed by heparin.

Apelin signaling plays an important role during embryo development and regulates angiogenesis, cardiovascular activity, and energy metabolism in adulthood. Overexpression and hyperactivity of this signaling pathway is observed in various pathologic states, such as cardiovascular diseases and cancer, which highlights the importance of inhibiting apelin receptor (APJ); therefore, we developed a cell-based screening assay that uses fluorescence microscopy to identify APJ antagonists. This approach led us to identify the U.S. Food and Drug Administration-approved compound protamine-already used clinically after cardiac surgery-as an agent to bind to heparin and thereby reverse its anticlotting activity. Protamine displays a 390-nM affinity for APJ and behaves as a full antagonist with regard to G protein and ß-arrestin-dependent intracellular signaling. Ex vivo and in vivo, protamine abolishes well-known apelin effects, such as angiogenesis, glucose tolerance, and vasodilatation. Remarkably, protamine antagonist activity is fully reversed by heparin treatment both in vitro and in vivo Thus, our results demonstrate a new pharmacologic property of protamine-blockade of APJ-that could explain some adverse effects observed in protamine-treated patients. Moreover, our data reveal that the established antiangiogenic activity of protamine would rely on APJ antagonism.-Le Gonidec, S., Chaves-Almagro, C., Bai, Y., Kang, H. J., Smith, A., Wanecq, E., Huang, X.-P., Prats, H., Knibiehler, B., Roth, B. L., Barak, L. S., Caron, M. G., Valet, P., Audigier, Y., Masri, B. Protamine is an antagonist of apelin receptor, and its activity is reversed by heparin.