Is the Association of the Rare rs35667974 IFIH1 Gene Polymorphism With Autoimmune Diseases a Case of RNA Epigenetics?

Interferon induced with helicase C domain-containing protein 1 (IFIH1) gene encodes a cytoplasmic RNA helicase otherwise known as melanoma differentiation-associated 5 (MDA5), a RIG-1-like RNA helicase that recognizes viral RNA and is involved in innate immunity through recognition of viral RNA. Upon binding to double-stranded (ds) RNA, MDA5 forms a filamentous assembly along the length of dsRNA and utilizes molecular signatures to discriminate self, versus non-self on the basis of dsRNA length and methylation. Its missense variant rs35667974 is protective for type 1 diabetes, psoriasis, and psoriatic arthritis, but is also found to be associated with an increased risk for ankylosing spondylitis, Crohn's disease, and ulcerative colitis. To gain insight into the complex role of this variant we performed a structural analysis of MDA5 in complex with dsRNA using molecular dynamics simulations. Our data suggest that while the Ile923Val mutation of the rs35667974 variant does not affect binding to native dsRNA significantly, it displays a destabilizing effect in the presence of 2'-O uridine methylation. Thus, the presence of 2'-O-methylation at the dsRNA introduces a sensing signature that leads to selective reduction of the overall MDA catalytic activity. This study represents an evaluation of the role of the shared rs35667974 variant of autoimmune locus IFIH1, reported to lead to selectively reduced catalytic activity of the modified MDA5 phenotype and, as a consequence, reduced negative feedback on cytokine and chemokine signaling and selectively protection against autoimmunity.

Structural Studies of Piperine Inclusion Complexes in Native and Derivative ß-Cyclodextrins.

Piperine (PN), the primary pungent alkaloid in black pepper shows several biological activities such as antioxidant, antimicrobial and anti-cancerogenic effects. Similar to other alkaloids, PN is characterized by poor water solubility. One way to improve its solubility and thus its biological activities is by forming inclusion complexes with suitable cyclodextrins. In this work PN inclusion complexes in native ß-cyclodextrin (ß-CD), its methylated (randomly methylated (RM-ß-CD), heptakis-(2,6-di-O-methyl)-ß-CD (DM-ß-CD) and heptakis-(2,3,6-tri-O-methyl)-ß-CD (TM-ß-CD)) and 2-hydroxypropylated (HP-ß-CD) derivatives are investigated using physicochemical methods, such as phase solubility study and X-ray crystallography complemented by theoretical (molecular dynamics simulations) studies. The determination of the crystal structure of the PN inclusion complexes in ß-CD, DM-ß-CD and TM-ß-CD, reveals the formation of 1:2 guest:host inclusion complexes in the crystalline state. The guest PN molecule threads the hydrophobic cavities of the hosts which are arranged as couples in a tail-to-tail mode in the case of PN/ß-CD and in a head-to-tail mode in the cases of PN/DM-ß-CD and PN/TM-ß-CD. MD studies based on the crystallographically determined structures and docked models show the stability of the examined complexes in an aqueous environment whereas the binding affinity of PN for the host molecules is calculated by the MM/GBSA method. Finally, phase-solubility studies of PN with ß-CD, RM-ß-CD and HP-ß-CD are presented, indicating a Bs-type for the PN/ß-CD complex and an AL-type for the PN/RM-ß-CD and PN/HP-ß-CD complexes with 1:1 guest:host stoichiometry.

Functional significance of the rare rs35667974 IFIH1 gene polymorphism, associated with multiple autoimmune diseases, using a structural biological approach.

Autoimmune diseases, which affect approximately 5% of human population, are a range of diseases in which the immune response to self-antigens results in damage or dysfunction of tissues. Recent genome wide association studies (GWAS) have successfully identified novel autoimmune disease-associated loci, with many of them shared by multiple disease-associated pathways but much of the genetics and pathophysiological mechanisms remain still obscure. Considering that most of the potential causal variants are still unknown, many studies showed that the missense variant rs35667974 at interferon-induced with helicase C domain 1 (IFIH1) gene is protective for type 1 diabetes (T1D), psoriasis (PS) and psoriatic arthritis (PsA). Recently, this variant was found to be also associated with ankylosing spondylitis (AS), Crohn's disease (CD) and ulcerative colitis (UC). The IFIH1 gene encodes a cytoplasmic RNA helicase otherwise known as melanoma differentiation-associated 5 (MDA5) that recognizes viral RNA and is involved in innate immunity through recognition of viral RNA. In the present study we sought to investigate the association of the rare rs35667974 variant of IFIH1 gene, which resides in exon 14 and changes a conserved isoleucine at position #923 to valine, in the development of various autoimmune diseases and give a reason for the selectivity affecting different autoimmune diseases. Evolutionary studies and three-dimensional (3 D) homology modelling were employed on the MDA5 protein product, through its association with dsRNA, recognition factor controlling cytokine and chemokine signalling, to investigate the protective role of the MDA5 variant for certain autoimmune diseases.

Biophysical Studies and In Vitro Effects of Tumor Cell Lines of Cannabidiol and Its Cyclodextrin Inclusion Complexes.

Phytocannabinoids possess anticancer properties, as established in vitro and in vivo. However, they are characterized by high lipophilicity. To improve the properties of cannabidiol (CBD), such as solubility, stability, and bioavailability, CBD inclusion complexes with cyclodextrins (CDs) might be employed, offering targeted, faster, and prolonged CBD release. The aim of the present study is to investigate the in vitro effects of CBD and its inclusion complexes in randomly methylated ß-CD (RM-ß-CD) and 2-hyroxypropyl-ß-CD (HP-ß-CD). The enhanced solubility of CBD upon complexation with CDs was examined by phase solubility study, and the structure of the inclusion complexes of CBD in 2,6-di-O-methyl-ß-CD (DM-ß-CD) and 2,3,6-tri-O-methyl-ß-CD (TM-ß-CD) was determined by X-ray crystallography. The structural investigation was complemented by molecular dynamics simulations. The cytotoxicity of CBD and its complexes with RM-ß-CD and HP-ß-CD was tested on two cell lines, the A172 glioblastoma and TE671 rhabdomyosarcoma cell lines. Methylated ß-CDs exhibited the best inclusion ability for CBD. A dose-dependent effect of CBD on both cancer cell lines and improved efficacy of the CBD-CDs complexes were verified. Thus, cannabinoids may be considered in future clinical trials beyond their palliative use as possible inhibitors of cancer growth.

Association of the DNASE1L3 rs35677470 polymorphism with systemic lupus erythematosus, rheumatoid arthritis and systemic sclerosis: Structural biological insights.

Although genome­wide association studies (GWAS) have identified hundreds of autoimmune disease­associated loci, much of the genetics underlying these diseases remains unknown. In an attempt to identify potential causal variants, previous studies have determined that the rs35677470 missense variant of the Deoxyribonuclease I­like 3 (DNASE1L3) gene was associated with the development of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and systemic sclerosis (SSc). DNase1L3 is a member of the human DNase I family, representing a nuclease that cleaves double­stranded DNA during apoptosis and serving a role in the development of autoimmune diseases. The present study aimed to determine the role of the rs35677470 variant at the DNASE1L3 gene leading to the R206C mutation in SLE, RA and SSc. The underlying mechanism potentially affecting protein structure loss of function was also assessed. DNASE1L3 evolution was investigated to define conservation elements in the protein sequence. Additionally, 3D homology modeling and in silico mutagenesis was performed to localize the polymorphism under investigation. Evolutionary analysis revealed heavily conserved sequence elements among species, indicating structural/functional importance. In silico mutagenesis and 3D protein structural analysis also demonstrated the potentially varied impact of the DNASE1L3 (rs35677470) single nucleotide polymorphism (SNP), providing an explanation for its effect on the R206C variant. Structural analysis demonstrated that the rs35677470 SNP encodes a non­conservative amino acid variation, R206C, which disrupted the conserved electrostatic network holding secondary protein structure elements in position. Specifically, the R206 to E170 interaction forming part of a salt bridge network stabilizing two α­helices was interrupted, thereby affecting the molecular architecture. Previous studies on the effect of this SNP in Caucasian populations demonstrated lower DNAse1L3 activity levels, which is consistent with the current results. The present study comprehensively evaluated the shared autoimmune locus of DNASE1L3 (rs35677470), which produced an inactive form of DNaseIL3. Furthermore, structural analysis explained the potential role of the produced mutation by modifying the placement of structural elements and consequently introducing disorder in protein folding, affecting biological function.

The putative polysaccharide deacetylase Ba0331: cloning, expression, crystallization and structure determination.

Ba0331 is a putative polysaccharide deacetylase from Bacillus anthracis, the etiological agent of the disease anthrax, that contributes to adaptation of the bacterium under extreme conditions and to maintenance of the cell shape. In the present study, the crystal structure of Ba0331 was determined at 2.6 Šresolution. The structure consists of two domains: a fibronectin type 3-like (Fn3-like) domain and a NodB catalytic domain. The latter is present in all carbohydrate esterase family 4 enzymes, while a comparative analysis of the Fn3-like domain revealed structural plasticity despite the retention of the conserved Fn3-like domain characteristics.

Structural and Evolutionary Insights within the Polysaccharide Deacetylase Gene Family of Bacillus anthracis and Bacillus cereus.

Functional and folding constraints impose interdependence between interacting sites along the protein chain that are envisaged through protein sequence evolution. Studying the influence of structure in phylogenetic models requires detailed and reliable structural models. Polysaccharide deacetylases (PDAs), members of the carbohydrate esterase family 4, perform mainly metal-dependent deacetylation of O- or N-acetylated polysaccharides such as peptidoglycan, chitin and acetylxylan through a conserved catalytic core termed the NodB homology domain. Genomes of Bacillus anthracis and its relative Bacillus cereus contain multiple genes of putative or known PDAs. A comparison of the functional domains of the recently determined PDAs from B. anthracis and B. cereus and multiple amino acid and nucleotide sequence alignments and phylogenetic analysis performed on these closely related species showed that there were distinct differences in binding site formation, despite the high conservation on the protein sequence, the folding level and the active site assembly. This may indicate that, subject to biochemical verification, the binding site-forming sequence fragments are under functionally driven evolutionary pressure to accommodate and recognize distinct polysaccharide residues according to cell location, use, or environment. Finally, we discuss the suggestion of the paralogous nature of at least two genes of B. anthracis, ba0330 and ba0331, via specific differences in gene sequence, protein structure, selection pressure and available localization patterns. This study may contribute to understanding the mechanisms under which sequences evolve in their structures and how evolutionary processes enable structural variations.

Structures of the Peptidoglycan N-Acetylglucosamine Deacetylase Bc1974 and Its Complexes with Zinc Metalloenzyme Inhibitors.

The cell wall peptidoglycan is recognized as a primary target of the innate immune system, and usually its disintegration results in bacterial lysis. Bacillus cereus, a close relative of the highly virulent Bacillus anthracis, contains 10 polysaccharide deacetylases. Among these, the peptidoglycan N-acetylglucosamine deacetylase Bc1974 is the highest homologue to the Bacillus anthracis Ba1977 that is required for full virulence and is involved in resistance to the host's lysozyme. These metalloenzymes belong to the carbohydrate esterase family 4 (CE4) and are attractive targets for the development of new anti-infective agents. Herein we report the first X-ray crystal structures of the NodB domain of Bc1974, the conserved catalytic core of CE4s, in the unliganded form and in complex with four known metalloenzyme inhibitors and two amino acid hydroxamates that target the active site metal. These structures revealed the presence of two conformational states of a catalytic loop known as motif-4 (MT4), which were not observed previously for peptidoglycan deacetylases, but were recently shown in the structure of a Vibrio clolerae chitin deacetylase. By employing molecular docking of a substrate model, we describe a catalytic mechanism that probably involves initial binding of the substrate in a receptive, more open state of MT4 and optimal catalytic activity in the closed state of MT4, consistent with the previous observations. The ligand-bound structures presented here, in addition to the five Bc1974 inhibitors identified, provide a valuable basis for the design of antibacterial agents that target the peptidoglycan deacetylase Ba1977.

Τhe genetics of juvenile idiopathic arthritis: Searching for new susceptibility loci.

Juvenile idiopathic arthritis (JIA) is an autoimmune disease that is characterized by persistent chronic arthritis and affected by genetic and environmental factors. Different genetic variations have been reported as risk factors for JIA. However, given that many results could not be replicated in individuals of different ancestral origin, it was assumed that heterogeneous genetic factors are involved in this disease. In the present study, we analyzed three single nucleotide polymorphisms (SNPs), namely PTPRC (rs10919563), TYK2 (rs34536443) and PRKCQ (rs4750316), which were found to be associated with JIA in previous studies. We also investigated whether the intron­4 located 27­bp VNTR of endothelial nitric oxide synthase (eNOS), is associated with risk for JIA in Greece. In total, 125 JIA patients and 221 healthy controls from northern Greece were included in the study as a sample set. Samples were then analyzed, and genotyped for the three SNPs with TaqMan primer­probe sets, using a Real­Time PCR platform (ViiA™ 7 Real­Time PCR system), while eNOS VNTR polymorphism was genotyped by PCR. Statistical analysis was performed using a GraphPad Prism statistical program. The χ2 test was used to examine differences of genotype and allele frequencies between patients and controls. Statistical significance was defined by using the two­tailed P<0.05 test. Bioinformatics analysis was conducted by using BlastP, Pymol, Maestro and Desmond. In the case­control association study performed, eNOS only was found to be associated with JIA. Genotype a/a and allele 'a' were found in a higher frequency in JIA patients than in controls [p<0.0001, odds ratio (OR)=0.15, 95% confidence intervals (CI): 0.065­0.37; and p<0.0001, OR=0.34, 95% CI: 0.23­0.49, respectively]. No associations with JIA were detected for TYK2, PTPRC or PRKCQ. Aiming to investigate the structural consequences and the structure/function relationships accompanying the Pro1104 to Ala (rs34536443) mutation on TYK2 protein, bioinformatics analysis was performed. Combining three­dimensional (3D)­modeling and molecular dynamics simulations we identified changes in structural flexibility, affecting the functionality of the kinase domain of TYK2. To the best of our knowledge, this is the first time that eNOS VNTR polymorphism is associated with susceptibility to JIA, suggesting a differential role of allele 'a' in various complex diseases. The current data emphasize the importance of comparative studies in populations of a different ancestral background towards the clarification of the role of specific alleles in the development of JIA.