Structural Characterization of the Interaction of Hypoxia Inducible Factor-1 with Its Hypoxia Responsive Element at the -964G > A Variation Site of the HLA-G Promoter Region.

Human Antigen Leukocyte-G (HLA-G) gene encodes an immune checkpoint molecule that has restricted tissue expression in physiological conditions; however, the gene may be induced in hypoxic conditions by the interaction with the hypoxia inducible factor-1 (HIF1). Hypoxia regulatory elements (HRE) located at the HLA-G promoter region and at exon 2 are the major HIF1 target sites. Since the G allele of the -964G > A transversion induces higher HLA-G expression when compared to the A allele in hypoxic conditions, here we analyzed HIF1-HRE complex interaction at the pair-atom level considering both -964G > A polymorphism alleles. Mouse HIF2 dimer crystal (Protein Data Bank ID: 4ZPK) was used as template to perform homology modelling of human HIF1 quaternary structure using MODELLER v9.14. Two 3D DNA structures were built from 5'GCRTG'3 HRE sequence containing the -964G/A alleles using x3DNA. Protein-DNA docking was performed using the HADDOCK v2.4 server, and non-covalent bonds were computed by DNAproDB server. Molecular dynamic simulation was carried out per 200 ns, using Gromacs v.2019. HIF1 binding in the HRE containing -964G allele results in more hydrogen bonds and van der Waals contact formation than HRE with -964A allele. Protein-DNA complex trajectory analysis revealed that HIF1-HRE-964G complex is more stable. In conclusion, HIF1 binds in a more stable and specific manner at the HRE with G allele.

Computational and atomistic studies applied to the understanding of the structural and behavioral features of the immune checkpoint HLA-G molecule and gene.

We took advantage of the increasingly evolving approaches for in silico studies concerning protein structures, protein molecular dynamics (MD), protein-protein and protein-DNA docking to evaluate: (i) the structure and MD characteristics of the HLA-G well-recognized isoforms, (ii) the impact of missense mutations at HLA-G receptor genes (LILRB1/2), and (iii) the differential binding of the hypoxia-inducible factor 1 (HIF1) to hypoxia-responsive elements (HRE) at the HLA-G gene. Besides reviewing these topics, they were revisited including the following novel results: (i) the HLA-G6 isoforms were unstable docked or not with ß2-microglobulin or peptide, (ii) missense mutations at LILRB1/2 genes, exchanging amino acids at the intracellular domain, particularly those located within and around the ITIM motifs, may impact the HLA-G binding strength, and (iii) HREs motifs at the HLA-G promoter or exon 2 regions exhibiting a guanine at their third position present a higher affinity for HIF1 when compared to an adenine at the same position. These data shed some light into the functional aspects of HLA-G, particularly how polymorphisms may influence the role of the molecule. Computational and atomistic studies have provided alternative tools for experimental physical methodologies, which are time-consuming, expensive, demanding large quantities of purified proteins, and exhibit low output.

Human leukocyte antigen-G 3' untranslated region polymorphisms are associated with asthma severity.

Asthma is a genetically complex chronic inflammatory airway disorder, and according to disease pathogenesis, clinical manifestations may vary according to asthma severity. A gene region close to the human leukocyte antigen-G (HLA-G) gene was identified as an independent susceptibility marker for asthma. Considering that the HLA-G immune checkpoint molecule may modulate inflammation, we evaluated the diversity of the HLA-G 3' untranslated region (3'UTR) in asthmatic patients stratified according to disease severity. We evaluate the entire HLA-G 3'UTR segment in 115 Brazilian patients stratified into mild (n=29), moderate (n=21) and severe asthmatics (n=65), and in 116 healthy individuals. HLA-G 3'UTR typing was performed using Sanger sequencing. The multiple comparisons among patients stratified according to disease severity revealed several associations; however, after Bonferroni's correction, the following results remained significant: i) the +3010C and +3142G alleles were overrepresented in mild asthma patients when compared to controls; ii) the +3010G and +3142C alleles were overrepresented in severe asthma patients in comparison to patients with mild asthma. In conclusion, the +3010C/G and +3142C/G HLA-G 3'UTR variation sites were differentially associated according to asthma severity.