Could the IgA isotype provide additional information in systemic sclerosis patients? A retrospective study entailing IgA isotyping in a Mediterranean systemic sclerosis cohort.

OBJECTIVES: Anti-CENP-B (ACA), anti-topoisomerase I (ATA) and anti-RNA polymerase III (RP3) autoantibodies are included in the 2013 SSc-ACR/EULAR classification criteria. The detection of additional autoantibodies is of interest when those are negative. Additionally, we wonder if the IgA isotype might play a role in SSc. The aims of the study were to assess the prevalence of ACA, ATA, RP3, and Ro52 autoantibodies of IgG and IgA isotype and to describe their association with clinical manifestations in a cohort of patients with SSc. METHODS: Samples from 97 patients with SSc fulfilling the 2013 ACR/EULAR classification criteria, and 50 blood donors were included and tested for IgA and IgG isotypes of ACA, ATA, RP3, and Ro52 by FEIA. RESULTS: The prevalence of IgG+IgA isotypes for the same specificity was 62.5%, 82.6%, 80.0%, 36.8%, for ACA, ATA, RP3 and Ro52, respectively. Isolated IgG was present in 35.4%, 13.0%, 20.0% and 42.1% of patients for ACA, ATA, RP3 and Ro52, respectively. Only six patients were isolated IgA for a unique specificity. Clinically, ILD tended to be associated with ATA-IgG and ATA-IgG+IgA, telangiectasias with ACA-IgG+IgA and arthritis with ACA-IgA. Indeed, digital ulcers were more frequent in ATA-IgG patients. CONCLUSIONS: Most of the patients presented ACA, ATA, or RP3 autoantibodies of IgA isotype in addition to IgG. Regarding clinical relevance, Ro52-IgG+IgA and ACA-IgG had a tendency towards sineSSc phenotype, while ACA-IgG+IgA to lcSSc phenotype. Thus, if confirmed, the determination of ACA-IgA could provide a tool to stratify patients according to the cutaneous phenotype.

Flavonoid allosteric modulation of mutated visual rhodopsin associated with retinitis pigmentosa.

Dietary flavonoids exhibit many biologically-relevant functions and can potentially have beneficial effects in the treatment of pathological conditions. In spite of its well known antioxidant properties, scarce structural information is available on the interaction of flavonoids with membrane receptors. Advances in the structural biology of a specific class of membrane receptors, the G protein-coupled receptors, have significantly increased our understanding of drug action and paved the way for developing improved therapeutic approaches. We have analyzed the effect of the flavonoid quercetin on the conformation, stability and function of the G protein-coupled receptor rhodopsin, and the G90V mutant associated with the retinal degenerative disease retinitis pigmentosa. By using a combination of experimental and computational methods, we suggest that quercetin can act as an allosteric modulator of opsin regenerated with 9-cis-retinal and more importantly, that this binding has a positive effect on the stability and conformational properties of the G90V mutant associated with retinitis pigmentosa. These results open new possibilities to use quercetin and other flavonoids, in combination with specific retinoids like 9-cis-retinal, for the treatment of retinal degeneration associated with retinitis pigmentosa. Moreover, the use of flavonoids as allosteric modulators may also be applicable to other members of the G protein-coupled receptors superfamily.

Zinc Is Involved in Depression by Modulating G Protein-Coupled Receptor Heterodimerization.

5-Hydroxytryptamine 1A receptor and galanin receptor 1 belong to the G protein-coupled receptors superfamily, and they have been described to heterodimerize triggering an anomalous physiological state that would underlie depression. Zinc supplementation has been widely reported to improve treatment against major depressive disorder. Our work has focused on the study and characterization of these receptors and its relationships with zinc both under purified conditions and in cell culture. To this aim, we have designed a strategy to purify the receptors in a conformationally active state. We have used receptors tagged with the monoclonal Rho-1D4 antibody and employed ligand-assisted purification in order to successfully purify both receptors in a properly folded and active state. The interaction between both purified receptors has been analyzed by surface plasmon resonance in order to determine the kinetics of dimerization. Zinc effect on heteromer has also been tested using the same methodology but exposing the 5-hydroxytryptamine 1A receptor to zinc before the binding experiment. These results, combined with Förster resonance energy transfer (FRET) measurements, in the absence and presence of zinc, suggest that this ion is capable of disrupting this interaction. Moreover, molecular modeling suggests that there is a coincidence between zinc-binding sites and heterodimerization interfaces for the serotonin receptor. Our results establish a rational explanation for the role of zinc in the molecular processes associated with receptor-receptor interactions and its relationship with depression, in agreement with previously reported evidence for the positive effects of zinc in depression treatment, and the involvement of our target dimer in the same disease.

The zinc binding receptor GPR39 interacts with 5-HT1A and GalR1 to form dynamic heteroreceptor complexes with signaling diversity.

GPR39 is a class A G protein-coupled receptor involved in zinc binding and glucose homeostasis regulation, among other physiological processes. GPR39 was originally thought to be the receptor for obestatin peptide but this view has been challenged. However, activation of this receptor by zinc has been clearly established. Recent studies suggest that low GPR39 expression, due to deficient zinc levels, is involved in major depressive disorder. We have previously reported that zinc can alter receptor-receptor interactions and favor specific receptor interactions. In order to unravel the effect of zinc on specific G protein-coupled receptor association processes, we have performed FRET and co-immunopurification studies with GPR39 and 5-HT1A and GalR1 which have been shown to dimerize. Our results suggest that zinc can modulate the formation of specific 5-HT1A-GPR39 and GalR1-5-HT1A-GPR39 heteroreceptor complexes under our experimental conditions. We have analyzed the differences in signaling between the mono-homomeric receptors 5-HT1A, GalR1 and GPR39 and the heteroreceptor complexes between them Our results show that the GPR39-5-HT1A heterocomplex has additive functionalities when compared to the monomeric-homomeric receptors upon receptor activation. In addition, the heterocomplex including also GalR1 shows a different behavior, upon exposure to the same agonists. Furthermore, these processes appear to be regulated by zinc. These findings provide a rationale for the antidepressive effect widely described for zinc because pro-depressive heterocomplexes are predominant at low zinc concentration levels.

G-protein-coupled receptors oligomerization: emerging signaling units and new opportunities for drug design.

G-protein-coupled receptors (GPCRs) are a widespread family of transmembrane receptors with different physiologically relevant functions. Alterations in the structure and function of these receptors at different levels (ligand binding, signaling and trafficking) may result in a number of pathological conditions which represent a major health problem. Mutations in these receptors are also linked to different inherited diseases for which there is no cure to date. Rationale design, based on receptor structural knowledge, is needed for the discovery of novel drugs with higher selectivity and less side effects. In fact, about 50% of the drugs currently under development target this kind of receptors. Oligomerization among GPCRs has been clearly established from experimental, particularly in vitro, studies. Moreover, homo and heterodimerization provide new unexpected clues for explaining the molecular mechanisms underlying some diseases in which GPCRs signaling might be affected. In this review we will analyze GPCRs structure and function for a better understanding of the dimerization process and the experimental approaches currently used to detect such interactions. Furthermore, how drugs targeting heteromers can represent new opportunities to tackle novel and safer treatments of some pathologies will be described. Recent results, in this regard, will be reported as encouraging examples in the field. Finally, the newest technologies available for developing drugs targeting heteromers will also be reviewed highlighting the importance of bivalent ligands that emerge as very powerful molecules interacting with heteromers.