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.

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.

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.

Concluding the trilogy: The interaction of 2,2'-dihydroxy-benzophenones and their carbonyl N-analogues with human glutathione transferase M1-1 face to face with the P1-1 and A1-1 isoenzymes involved in MDR.

A series of 2,2'-dihydroxybenzophenones and their carbonyl N-analogues were studied as potential inhibitors against human glutathione transferase M1-1 (hGSTM1-1) purified from recombinant E. coli. Their screening revealed an inhibition against hGSTM1-1 within a range of 0-42% (25 µM). The IC50 values for the two stronger ones, 16 and 13, were 53.5 ± 5.6 µΜ and 28.5 ± 2.5 µΜ, respectively. The results were compared with earlier ones for isoenzymes hGSTP1-1 and hGSTA1-1 involved in MDR. All but one bind more strongly to A1-1, than M1-1 and P1-1, the latter being a poor binder. An order of potency A1-1 > > M1-1 >  P1-1 meritted 13, 14 and 16 as the most potent inhibitors with hGSTM1-1. Enzyme kinetics with hGSTM1-1 (Km(CDNB) 213 ± 10 µΜ and Km(GSH) 303 ± 11 µΜ) revealed a competitive modality for 16 (Ki(16)  = 22.3 ± 1.1 µΜ) and a mixed one for 13 versus CDNB (Ki(13)  = 33.3 ± 1.6 µM for the free enzyme and Ki(13) ' = 17.7 ± 1.7 µM for the enzyme-CDNB complex). 5- or 5'-Bromo- or phenyl-substituted (but not in combination) inhibitors, having a H-bonded oxime weakly acidic group of a small volume, are optimal candidates for binding hGSTM1-1. The outcome of the isoenzyme trilogy identified good binder leads for the investigated GSTs involved in MDR.

Isoenzyme- and allozyme-specific inhibitors: 2,2'-dihydroxybenzophenones and their carbonyl N-analogues that discriminate between human glutathione transferase A1-1 and P1-1 allozymes.

The selectivity of certain benzophenones and their carbonyl N-analogues was investigated towards the human GSTP1-1 allozymes A, B and C involved in MDR. The allozymes were purified from extracts derived from E. coli harbouring the plasmids pEXP5-CT/TOPO-TA-hGSTP1*A, pOXO4-hGSTP1*B or pOXO4-hGSTP1*C. Compound screening with each allozyme activity indicated three compounds with appreciable inhibitory potencies, 12 and 13 with P1-1A 62% and 67%, 11 and 12 with P1-1C 51% and 70%, whereas that of 15 fell behind with P1-1B (41%). These findings were confirmed by IC50 values (74-125 µm). Enzyme inhibition kinetics, aided by molecular modelling and docking, revealed that there is competition with the substrate CDNB for the same binding site on the allozyme (Ki(13/A)  = 63.6 ± 3.0 µm, Ki(15/B)  = 198.6 ± 14.3 µm, and Ki(11/C)  = 16.5 ± 2.7 µm). These data were brought into context by an in silico structural comparative analysis of the targeted proteins. Although the screened compounds showed moderate inhibitory potency against hGSTP1-1, remarkably, some of them demonstrated absolute isoenzyme and/or allozyme selectivity.

2,2'-Dihydroxybenzophenones and their carbonyl N-analogues as inhibitor scaffolds for MDR-involved human glutathione transferase isoenzyme A1-1.

The MDR-involved human GSTA1-1, an important isoenzyme overexpressed in several tumors leading to chemotherapeutic-resistant tumour cells, has been targeted by 2,2'-dihydroxybenzophenones and some of their carbonyl N-analogues, as its potential inhibitors. A structure-based library of the latter was built-up by a nucleophilic cleavage of suitably substituted xanthones to 2,2'-dihydroxy-benzophenones (5-9) and subsequent formation of their N-derivatives (oximes 11-13 and N-acyl hydrazones 14-16). Screening against hGSTA1-1 led to benzophenones 6 and 8, and hydrazones 14 and 16, having the highest inhibition potency (IC50 values in the range 0.18 ± 0.02 to 1.77 ± 0.10 µM). Enzyme inhibition kinetics, molecular modeling and docking studies showed that they interact primarily at the CDNB-binding catalytic site of the enzyme. In addition, the results from cytotoxicity studies with human colon adenocarcinoma cells showed low LC50 values for benzophenone 6 and its N-acyl hydrazone analogue 14 (31.4 ± 0.4 µM and 87 ± 1.9 µM, respectively), in addition to the strong enzyme inhibition profile (IC50(6)=1,77 ± 0.10 µM; IC50(14)=0.33 ± 0.05 µM). These structures may serve as leads for the design of new potent mono- and bi-functional inhibitors and pro-drugs against human GTSs.

Designer xanthone: an inhibitor scaffold for MDR-involved human glutathione transferase isoenzyme A1-1.

Glutathione transferases (GSTs) are cell detoxifiers involved in multiple drug resistance (MDR), hampering the effectiveness of certain anticancer drugs. To our knowledge, this is the first report on well-defined synthetic xanthones as GST inhibitors. Screening 18 xanthones revealed three derivatives bearing a bromomethyl and a methyl group (7) or two bromomethyl groups (8) or an aldehyde group (17), with high inhibition potency (>85%), manifested by low IC(50) values (7: 1.59 ± 0.25 µM, 8: 5.30 ± 0.30 µM, and 17: 8.56 ± 0.14 µM) and a competitive modality of inhibition versus CDNB (Ki(7) = 0.76 ± 0.18 and Ki(17) = 1.69 ± 0.08 µM). Of them, derivative 17 readily inhibited hGSTA1-1 in colon cancer cell lysate (IC(50) = 10.54 ± 2.41 µM). Furthermore, all three derivatives were cytotoxic to Caco-2 intact cells, with 17 being the least cytotoxic (LC(50) = 151.3 ± 16.3 µM). The xanthone scaffold may be regarded as a pharmacophore for hGSTA1-1 and the three derivatives, especially 17, as potent precursors for the synthesis of new inhibitors and conjugate prodrugs for human GSTs.

Synthesis and study of 2-(pyrrolesulfonylmethyl)-N-arylimines: a new class of inhibitors for human glutathione transferase A1-1.

Overexpression of human GSTA1-1 in tumor cells is part of MDR mechanisms. We report on the synthesis of 11 pyrrole derivatives as hGSTA1-1 inhibitors starting from 1-methyl-2-[(2-nitrobenzylsulfanyl]-1H-pyrrole. Molecular modeling revealed two locations in the enzyme H binding site: the catalytic primary one accommodating shorter and longer derivatives and the secondary one, where shorter derivatives can occupy. Derivative 9, displaying the highest inhibition and bearing a p-nitroarylimino moiety, and derivative 4, lacking this moiety, were studied kinetically. Derivative 9 binds (K(i(9)) = 71 ± 4 µM) at the primary site competitively vs CDNB. Derivative 4 binds (K(i(4)) = 135 ± 27 µM) at the primary and secondary sites, allowing the binding of a second molecule (4 or CDNB) leading to formation of unreactive and reactive complexes, respectively. The arylmethylsulfonylpyrrole core structure is a new pharmacophore for hGSTA1-1, whereas its derivative 9 may serve as a lead structure.