Abstract Background Obsessive–compulsive disorder (OCD) is a prevalent and severe clinical condition. Robust evidence suggests a gene-environment interplay in its etiopathogenesis, yet the underlying molecular clues remain only partially understood. In order to further deepen our understanding of OCD, it is essential to ascertain how genes interact with environmental risk factors, a cross-talk that is thought to be mediated by epigenetic mechanisms. The human microbiota may be a key player, because bacterial metabolites can act as epigenetic modulators. We analyzed, in the blood and saliva of OCD subjects and healthy controls, the transcriptional regulation of the oxytocin receptor gene and, in saliva, also the different levels of major phyla. We also investigated the same molecular mechanisms in specific brain regions of socially isolated rats showing stereotyped behaviors reminiscent of OCD as well as short chain fatty acid levels in the feces of rats. Results Higher levels of oxytocin receptor gene DNA methylation, inversely correlated with gene expression, were observed in the blood as well as saliva of OCD subjects when compared to controls. Moreover, Actinobacteria also resulted higher in OCD and directly correlated with oxytocin receptor gene epigenetic alterations. The same pattern of changes was present in the prefrontal cortex of socially-isolated rats, where also altered levels of fecal butyrate were observed at the beginning of the isolation procedure. Conclusions This is the first demonstration of an interplay between microbiota modulation and epigenetic regulation of gene expression in OCD, opening new avenues for the understanding of disease trajectories and for the development of new therapeutic strategies.
Endocannabinoid (eCB)-binding receptors can be modulated by several ligands and membrane environment, yet the effect of glycosylation remains to be assessed. In this study, we used human neuroblastoma SH-SY5Y cells to interrogate whether expression, cellular localization, and activity of eCB-binding receptors may depend on N-linked glycosylation. Following treatment with tunicamycin (a specific inhibitor of N-linked glycosylation) at the non-cytotoxic dose of 1 µg/mL, mRNA, protein levels and localization of eCB-binding receptors, as well as N-acetylglucosamine (GlcNAc) residues, were evaluated in SH-SY5Y cells by means of quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR), fluorescence-activated cell sorting (FACS), and confocal microscopy, respectively. In addition, the activity of type-1 and type-2 cannabinoid receptors (CB₁ and CB₂) was assessed by means of rapid binding assays. Significant changes in gene and protein expression were found upon tunicamycin treatment for CB₁ and CB₂, as well as for GPR55 receptors, but not for transient receptor potential vanilloid 1 (TRPV1). Deglycosylation experiments with N-glycosidase-F and immunoblot of cell membranes derived from SH-SY5Y cells confirmed the presence of one glycosylated form in CB₁ (70 kDa), that was reduced by tunicamycin. Morphological studies demonstrated the co-localization of CB₁ with GlcNAc residues, and showed that tunicamycin reduced CB₁ membrane expression with a marked nuclear localization, as confirmed by immunoblotting. Cleavage of the carbohydrate side chain did not modify CB receptor binding affinity. Overall, these results support N-linked glycosylation as an unprecedented post-translational modification that may modulate eCB-binding receptors' expression and localization, in particular for CB₁.
In the field of molecular biology and biochemistry in which structural genomics comes as a complement to genome sequencing, Small-Angle X-ray Scattering (SAXS) is an experimental technique that, though not widely known and applied, represents a very powerful tool in the framework of post-genome structural studies. The aim of this review is to present a synthetic description of the basic principles of the theory of SAXS and to discuss in more detail its applications to the study of different biological molecules, focusing the attention to the recent advances in the structural analysis of proteins. These studies are presently undergoing a spectacular expansion associated with the development of powerful data analysis software, with the improvement of the quality of data recorded with synchrotron radiation, and finally with the increasing availability of high resolution three-dimensional structures which can constitute a starting point for the analysis of protein conformations in solution. The advantage of SAXS with respect to other techniques in the structural studies of proteins resides in the possibility of performing the measurements in any desired solvent and in the ability to follow changes of the protein structure which may occur as a response to a variety of stimuli: pH or temperature changes, interaction with small ligands, influence of substrate analogues, chemical or genetic modifications, etc. In this review we describe the principles of SAXS and the most recent methods for data analysis in the field of structural biology and, finally, we report some examples of the use of this technique as a powerful tool to the structural study of proteins in solution. Keywords: electromagnetic wave, zero-angle scattering intensity, Debye Method, ab initio shape, Allosteric Transitions, Multidomain protein, DAM Model
Lipoxygenases (LOXs) are a family of enzymes that includes different fatty acid oxygenases with a common tridimensional structure. The main functions of LOXs are the production of signaling compounds and the structural modifications of biological membranes. These features of LOXs, their widespread presence in all living organisms, and their involvement in human diseases have attracted the attention of the scientific community over the last decades, leading to several studies mainly focused on understanding their catalytic mechanism and designing effective inhibitors. The aim of this review is to discuss the state-of-the-art of a different, much less explored aspect of LOXs, that is, their interaction with lipid bilayers. To this end, the general architecture of six relevant LOXs (namely human 5-, 12-, and 15-LOX, rabbit 12/15-LOX, coral 8-LOX, and soybean 15-LOX), with different specificity towards the fatty acid substrates, is analyzed through the available crystallographic models. Then, their putative interface with a model membrane is examined in the frame of the conformational flexibility of LOXs, that is due to their peculiar tertiary structure. Finally, the possible future developments that emerge from the available data are discussed.
Obsessive Compulsive Disorder (OCD) is a mental health condition still classified and diagnosed with subjective interview-based assessments and which molecular clues have not completely been elucidated. We have recently identified a new regulator of anxiety and OCD-like behavior called Immuno-moodulin (IMOOD) and, here, we report that IMOOD gene promoter is differentially methylated in OCD subjects when compared to genomic material collected from healthy controls and this alteration is significantly correlated with the increased expression of the gene in OCD. We also demonstrated that IMOOD promoter can form G-quadruplexes and we suggest that, in homeostatic conditions, these structures could evoke DNA-methylation silencing the gene, whereas in pathological conditions, like OCD, could induce gene expression making the promoter more accessible to transcriptional factors. We here thus further suggest IMOOD as a new biomarker for OCD and also hypothesize new mechanisms of gene regulation.
Abstract Background Binge-eating spectrum disorders, including bulimia nervosa (BN) and binge-eating disorder (BED), have psychological, behavioral, and physical effects, which present significant challenges for accurate diagnosis and treatment. Identifying biomarkers is thus of relevance to improve diagnostic and treatment strategies. Methods Saliva collected from female individuals with BED (n = 20), BN (n = 17), and normal weight healthy controls (NW-HC) (n = 20) was analyzed to assess salivary microbiome, exosomal miRNA expression, and DNA methylation of dopaminergic system gene components. Results Microbial diversity was significantly reduced in BED and BN groups compared to NW-HC. Differential abundance analysis revealed that Bacilli (class-level) were enriched in BN and BED, while Lachnospirales (order-level) were significantly depleted in BN compared to NW-HC. In total, 79 miRNAs resulted differentially expressed in patients compared with controls. Alteration in four of these miRNAs (let-7b-5p, mir-15b-5p, mir-429, and mir-221-3p) identified via network analysis as potentially relevant to psychiatric disorders, were confirmed to be significantly upregulated in both BED and BN compared with controls. Significant hypomethylation at specific CpG sites of the DAT1 gene was also observed in BED and BN groups relative to controls. Correlation analysis highlighted significant associations between specific microbiota genera, miRNA expression, and DNA methylation of DAT1 in both the BED and BN groups. Conclusions Our findings provide new evidence on the role of epigenetic modifications linked to alterations in salivary microbial composition and diversity in BED and BN, opening new avenues for future research and therapeutic interventions in eating disorders targeting miRNAs and microbiota.
