Polymorphic Residues Research Articles

Genetic association and computational analysis of MTHFR gene polymorphisms rs1801131 and rs1801133 with breast cancer in the Bangladeshi population

Methylenetetrahydrofolate reductase (MTHFR) plays a crucial role in regulating one-carbon metabolism. Polymorphisms within the MTHFR gene have been found to increase the risk of breast cancer in different populations. In this study, we evaluated the association of polymorphisms of the MTHFR gene (rs1801133 and rs1801131) with the risk of breast cancer in the Bangladeshi population. This case‒control study included 202 breast cancer patients and 104 healthy controls. After the organic extraction of DNA, genotyping was performed via the PCR-RFLP method. Sanger sequencing was performed to validate the RFLP data. Statistical analyses were performed to evaluate the associations of the polymorphisms. Different computational tools were used to predict the structural and functional consequences of the SNPs. Our study revealed that the MTHFR gene polymorphism rs1801131 is associated with an increased risk of developing breast cancer (p < 0.001, OR = 3.85, 95% CI = 2.06–7.25 for the AC genotype and p < 0.001, OR = 7.82, 95% CI = 2.69–22.05 for the CC genotype). An association was also observed in the dominant model (AC + CC) (p < 0.001, OR = 4.19, 95% CI = 2.28–7.78). For rs1801131, premenopausal status was significantly associated with breast cancer risk (p < 0.001). For rs1801133, no significant association was found with breast cancer risk (p > 0.05, OR = 1.57, 95% CI = 0.90–2.74 for the CT genotype; p > 0.05, OR = 1.35, 95% CI = 0.36–4.92 for the TT genotype). Computational analyses predicted rs1801131 to be tolerated and rs1801133 to be deleterious. Structural analyses demonstrated no significant changes in protein structure but revealed alterations in neighboring interactions according to both bond distances and angles. In conclusion, rs1801131 but not rs1801133 is significantly associated with breast cancer risk in the Bangladeshi population. Moreover, in silico analyses demonstrated changes in the interaction pattern of polymorphic residues with adjacent amino acids.

Polymorphic residues in HLA-B that mediate HIV control distinctly modulate peptide interactions with both TCR and KIR molecules

Immunogenetic studies have shown that specific HLA-B residues (67, 70, 97, and 156) mediate the impact of HLA class I on HIV infection, but the molecular basis is not well understood. Here we evaluate the function of these residues within the protective HLA-B∗5701 allele. While mutation of Met67, Ser70, and Leu156 disrupt CD8+ Tcell recognition, substitution of Val97 had no significant impact. Thermal denaturation of HLA-B∗5701-peptide complexes revealed that Met67 and Leu156 maintain HLA-peptide stability, while Ser70 and Leu156 facilitate Tcell receptor (TCR) interactions. Analyses of existing structures and structural models suggested that Val97 mediates HLA-peptide binding to inhibitory KIR3DL1 molecules, which was confirmed by experimental assays. These data thereby demonstrate that the genetic basis by which host immunity impacts HIV outcomes occurs by modulating HLA-B-peptide stability and conformation for interaction with TCR and killer immunoglobulin receptor (KIR) molecules. Moreover, they indicate a key role for epitope specificity and HLA-KIR interactions to HIV control.

Donor KIR2DL1 Allelic Polymorphism Influences Posthematopoietic Progenitor Cell Transplantation Outcomes in the T Cell Depleted and Reduced Intensity Conditioning Setting

The majority of established KIR clinical assessment algorithms used for donor selection for hematopoietic progenitor cell transplantation (HPCT) evaluate gene content (presence/absence) of the KIR gene complex. In comparison, relatively little is known about the impact of KIR allelic polymorphism. By analyzing donors of T cell depleted (TcD) reduced intensity conditioning (RIC) HPCT, this study investigated the influence on post-transplant outcome of 2 polymorphic residues of the inhibitory KIR2DL1. The aim of this study was to expand upon existing research into the influence of KIR2DL1 allelic polymorphism upon post-transplant outcome. The effects of allele groups upon transplant outcomes were investigated within a patient cohort using a defined treatment protocol of RIC with TcD. Using phylogenetic data, KIR2DL1 allelic polymorphism was categorized into groups on the basis of variation within codons 114 and 245 (positive or negative for the following groups: KIR2DL1*002/001g, KIR2DL1*003, KIR2DL1*004g) and the identification of null alleles. The influence of these KIR2DL1 allele groups in hematopoietic progenitor cell transplantation (HPCT) donors was assessed in the post-transplant data of 86 acute myelogenous leukemia patients receiving RIC TcD HPCT at a single center. KIR2DL1 allele groups in the donor significantly impacted upon 5-year post-transplant outcomes in RIC TcD HPCT. Donor KIR2DL1*003 presented the greatest influence upon post-transplant outcomes, with KIR2DL1*003 positive donors severely reducing 5-year post-transplant overall survival (OS) compared to those receiving a transplant from a KIR2DL1*003 negative donor (KIR2DL1*003 pos versus neg: 27.0% versus 60.0%, P = .008, pc = 0.024) and disease-free survival (DFS) (KIR2DL1*003 pos versus neg: 23.5% versus 60.0%, P = .004, pc = 0.012), and increasing 5-year relapse incidence (KIR2DL1*003 pos versus neg: 63.9% versus 27.2%, P = .009, pc = 0.027). KIR2DL1*003 homozygous and KIR2DL1*003 heterozygous grafts did not present significantly different post-transplant outcomes. Donors possessing the KIR2DL1*002/001 allele group were found to significantly improve post-transplant outcomes, with donors positive for the KIR2DL1*004 allele group presenting a trend towards improvement. KIR2DL1*002/001 allele group (KIR2DL1*002/001g) positive donors improved 5-year OS (KIR2DL1*002/001g pos versus neg: 56.4% versus 27.2%, P = .009, pc = 0.024) and DFS (KIR2DL1*002/001g pos versus neg: 53.8% versus 25.5%, P = .018, pc = 0.036). KIR2DL1*004 allele group (KIR2DL1*004g) positive donors trended towards improving 5-year OS (KIR2DL1*004g pos versus neg: 53.3% versus 35.5%, P = .097, pc = 0.097) and DFS (KIR2DL1*004g pos versus neg: 50.0% versus 33.9%, P = .121, pc = 0.121), and reducing relapse incidence (KIR2DL1*004g pos versus neg: 33.1% versus 54.0%, P = .079, pc = 0.152). The presented findings suggest donor selection algorithms for TcD RIC HPCT should consider avoiding KIR2DL1*003 positive donors, where possible, and contributes to the mounting evidence that KIR assessment in donor selection algorithms should reflect the conditioning regime protocol used.

Structural principles of peptide-centric chimeric antigen receptor recognition guide therapeutic expansion.

Peptide-centric chimeric antigen receptors (PC-CARs) recognize oncoprotein epitopes displayed by cell-surface human leukocyte antigens (HLAs) and offer a promising strategy for targeted cancer therapy. We have previously developed a PC-CAR targeting a neuroblastoma-associated PHOX2B peptide, leading to robust tumor cell lysis restricted by two common HLA allotypes. Here, we determine the 2.1-angstrom crystal structure of the PC-CAR-PHOX2B-HLA-A*24:02-β2m complex, which reveals the basis for antigen-specific recognition through interactions with CAR complementarity-determining regions (CDRs). This PC-CAR adopts a diagonal docking mode, where interactions with both conserved and polymorphic HLA framework residues permit recognition of multiple HLA allotypes from the A9 serological cross-reactive group, covering a combined global population frequency of up to 46.7%. Biochemical binding assays, molecular dynamics simulations, and structural and functional analyses demonstrate that high-affinity PC-CAR recognition of cross-reactive pHLAs necessitates the presentation of a specific peptide backbone, where subtle structural adaptations of the peptide are critical for high-affinity complex formation, and CAR T cell killing. Our results provide a molecular blueprint for engineering CARs with optimal recognition of tumor-associated antigens in the context of different HLAs, while minimizing cross-reactivity with self-epitopes.

Vicia faba SV channel VfTPC1 is a hyperexcitable variant of plant vacuole Two Pore Channels.

To fire action-potential-like electrical signals, the vacuole membrane requires the two-pore channel TPC1, formerly called SV channel. The TPC1/SV channel functions as a depolarization-stimulated, non-selective cation channel that is inhibited by luminal Ca2+. In our search for species-dependent functional TPC1 channel variants with different luminal Ca2+ sensitivity, we found in total three acidic residues present in Ca2+ sensor sites 2 and 3 of the Ca2+-sensitive AtTPC1 channel from Arabidopsis thaliana that were neutral in its Vicia faba ortholog and also in those of many other Fabaceae. When expressed in the Arabidopsis AtTPC1-loss-of-function background, wild-type VfTPC1 was hypersensitive to vacuole depolarization and only weakly sensitive to blocking luminal Ca2+. When AtTPC1 was mutated for these VfTPC1-homologous polymorphic residues, two neutral substitutions in Ca2+ sensor site 3 alone were already sufficient for the Arabidopsis At-VfTPC1 channel mutant to gain VfTPC1-like voltage and luminal Ca2+ sensitivity that together rendered vacuoles hyperexcitable. Thus, natural TPC1 channel variants exist in plant families which may fine-tune vacuole excitability and adapt it to environmental settings of the particular ecological niche.

Human Leukocyte Antigen Signatures as Pathophysiological Discriminants of Microscopic Colitis Subtypes.

Microscopic colitis [MC] is currently regarded as an inflammatory bowel disease that manifests as two subtypes: collagenous colitis [CC] and lymphocytic colitis [LC]. Whether these represent a clinical continuum or distinct entities is, however, an open question. Genetic investigations may contribute important insight into their respective pathophysiologies. We conducted a genome-wide association study [GWAS] meta-analysis in 1498 CC, 373 LC patients, and 13 487 controls from Europe and the USA, combined with publicly available MC GWAS data from UK Biobank and FinnGen [2599 MC cases and 552 343 controls in total]. Human leukocyte antigen [HLA] alleles and polymorphic residues were imputed and tested for association, including conditional analyses for the identification of key causative variants and residues. Genetic correlations with other traits and diagnoses were also studied. We detected strong HLA association with CC, and conditional analyses highlighted the DRB1*03:01 allele and its residues Y26, N77, and R74 as key to this association (best p = 1.4 × 10-23, odds ratio [OR] = 1.96). Nominally significant genetic correlations were detected between CC and pneumonia [rg = 0.77; p = 0.048] and oesophageal diseases [rg = 0.45, p = 0.023]. An additional locus was identified in MC GWAS analyses near the CLEC16A and RMI2 genes on chromosome 16 [rs35099084, p = 2.0 × 10-8, OR = 1.31]. No significant association was detected for LC. Our results suggest CC and LC have distinct pathophysiological underpinnings, characterised by an HLA predisposing role only in CC. This challenges existing classifications, eventually calling for a re-evaluation of the utility of MC umbrella definitions.

Abstract 6310: Phylogenetic conservation of primate Trop-2 underscores favorable pharmacokinetics and lack of toxicity of the cancer-selective Hu2G10 anti-Trop-2

Abstract Trop-2 is a transmembrane signal transducer that activates growth-signaling networks that converge on Akt, ERK, Cyclin D1, NFkB (1). We discovered that Trop-2 cleavage by ADAM10 is an activator switch of cancer growth and metastatic diffusion (2) and exposes novel target epitopes that are inaccessible in the unprocessed wtTrop-2. The Hu2G10 mAb selectively binds these epitopes with an affinity that is ≈10,000-fold higher than for the unprocessed molecule. To validate a reliable model for Hu2G10 binding in vivo, we explored Trop-2 phylogenetic conservation in primates. Pan troglodites (chimpanzee), Papio anubis (baboon), Macaca mulatta/fascicularis (Rhesus/Cynomolgous), Callithrix jaccus (marmoset) TROP2/TACSTD2 sequences were compared. The only difference between Pan troglodytes and human Trop-2 is one additional leucine in the Pan leader peptide, making the mature Trop-2 identical to that in man. The Macaca mulatta/fascicularis Trop-2 sequences are 97% identical, 100% similar to the human sequence. Three-dimensional modeling revealed polymorphic residues usage versus activation-driven structural rearrangement of Trop-2. COS-7 and HEK-293 cells transfected with individual primate Trop-2 showed efficient recognition by Hu2G10 in all tested species. Immunohistochemistry analysis of normal Macaca tissues showed Trop-2 expression patterns essentially corresponding to the human ones. IV injected Hu2G10 at doses of 5 to 10 mg/kg was well tolerated by Cynomolgous monkeys. No neurological, respiratory, digestive and urinary adverse effects were observed, and no body-weight loss occurred during the 28-day observation. No alterations of blood monocyte, neutrophil and basophil counts nor significant changes in biochemical parameters were detected. A pharmacokinetic (PK), study was carried out. Serum concentration of Hu2G10 was determined using anti-idiotypic antibody-based ELISA assays. At the higher dosing of 10 mg/kg Hu2G10 serum values reached a peak 2-hour after the injection (13281±4686 ng/ml). The concentration of Hu2G10 then diminished gradually, and reached baseline levels on day 21. The serum concentration peak of 5 mg/kg Hu2G10 was 5759±1729 ng/ml, and reached baseline levels on day 14. Thus, Hu2G10 is stable in plasma, and is detectable in the circulation up to three weeks after the infusion (t1/2=6.5 days). Taken together, these findings validate primate models as reliable for assessing Hu2G10 in vivo toxicity and PK. The lack of toxicity and favorable PK parameters candidate the cancer specific Hu2G10 as first-in-class anti-Trop-2 mAb. (1) Guerra E. et al., Oncogene 41:1795-1808 (2022).(2) Trerotola M. et al., Neoplasia 23: 415-428 (2021). Citation Format: Saverio Alberti, Martina Ceci, Ludovica Pantalone, Marco Trerotola, Emanuela Guerra. Phylogenetic conservation of primate Trop-2 underscores favorable pharmacokinetics and lack of toxicity of the cancer-selective Hu2G10 anti-Trop-2. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6310.

Non-random Codon Usage of Synonymous and Non-synonymous Mutations in the Human HLA-A Gene.

The structure and function of human leucocyte antigen (HLA-A) is well known and is an extremely variable protein. From the public HLA-A database, we chose 26 high frequency HLA-A alleles (45% of sequenced alleles). Using five arbitrary references from these alleles, we analyzed synonymous mutations at the third codon position (sSNP3) and non-synonymous mutations (NSM). Both mutation types showed non-random locations of 29 sSNP3 codons and 71 NSM codons in the five reference lists. Most sSNP3 codons show identical mutation types with many mutations resulting from cytosine deamination. We proposed 23 ancestral parents of sSNP3 in five reference sequences using conserved parents in five unidirectional codons and 18 majority parents in reciprocal codons. These 23 proposed ancestral parents show exclusive codon usage of G3 or C3 parents located on both DNA strands that mutate to A3 or T3 variants mostly (76%) by cytosine deamination The sSNP3 and NSM show clear separation of the two variant types with most sSNP3 located in conserved areas in exons 2, 3 and 4, compared to most NSM appearing in two Variable Areas with no sSNP3 in the latter parts of exons 2 (α1) and 3 (α2). The Variable Areas contain NSM (polymorphic) residues at the center of the groove that bind the foreign peptide. We find distinctly different mutation patterns in NSM codons from those of sSNP3. Namely, G-C to A-T mutation frequency was much smaller, suggesting that evolutional pressures of deamination and other mechanisms applied to the two areas are significantly different.

A combination of HLA-DP α and β chain polymorphisms paired with a SNP in the DPB1 3' UTR region, denoting expression levels, are associated with atopic dermatitis.

Introduction: Components of the immune response have previously been associated with the pathophysiology of atopic dermatitis (AD), specifically the Human Leukocyte Antigen (HLA) Class II region via genome-wide association studies, however the exact elements have not been identified. Methods: This study examines the genetic variation of HLA Class II genes using next generation sequencing (NGS) and evaluates the resultant amino acids, with particular attention on binding site residues, for associations with AD. The Genetics of AD cohort was used to evaluate HLA Class II allelic variation on 464 subjects with AD and 384 controls. Results: Statistically significant associations with HLA-DP α and β alleles and specific amino acids were found, some conferring susceptibility to AD and others with a protective effect. Evaluation of polymorphic residues in DP binding pockets revealed the critical role of P1 and P6 (P1: α31M + (β84G or β84V) [protection]; α31Q + β84D [susceptibility] and P6: α11A + β11G [protection]) and were replicated with a national cohort of children consisting of 424 AD subjects. Independently, AD susceptibility-associated residues were associated with the G polymorphism of SNP rs9277534 in the 3' UTR of the HLA-DPB1 gene, denoting higher expression of these HLA-DP alleles, while protection-associated residues were associated with the A polymorphism, denoting lower expression. Discussion: These findings lay the foundation for evaluating non-self-antigens suspected to be associated with AD as they potentially interact with particular HLA Class II subcomponents, forming a complex involved in the pathophysiology of AD. It is possible that a combination of structural HLA-DP components and levels of expression of these components contribute to AD pathophysiology.

Mass Spectrometry Defines Lysophospholipids as Ligands for Chicken MHCY Class I Molecules.

Chicken (Gallus gallus) MHCY class I molecules are highly polymorphic yet substantially different from polymorphic MHC class I molecules that bind peptide Ags. The binding grooves in MHCY class I molecules are hydrophobic and too narrow to accommodate peptides. An earlier structural study suggested that ligands for MHCY class I might be lipids, but the contents of the groove were not clearly identified. In this study, lysophospholipids have been identified by mass spectrometry as bound in two MHCY class I isoforms that differ substantially in sequence. The two isoforms, YF1*7.1 and YF1*RJF34, differ by 35 aa in the α1 and α2 domains that form the MHC class I ligand binding groove. Lyso-phosphatidylethanolamine (lyso-PE) 18:1 was the dominant lipid identified in YF1*7.1 and YF1*RJF34 expressed as recombinant molecules and renatured with β2-microglobulin in the presence of a total lipid extract from Escherichia coli. Less frequently detected were lyso-PE 17:1, lyso-PE 16:1, and lysophosphatidylglycerols 17:1 and 16:0. These data provide evidence that lysophospholipids are candidate ligands for MHCY class I molecules. Finding that MHCY class I isoforms differing substantially in sequence bind the same array of lysophospholipids indicates that the amino acid polymorphism that distinguishes MHCY class I molecules is not key in defining ligand specificity. The polymorphic positions lie mostly away from the binding groove and might define specificity in interactions of MHCY class I molecules with receptors that are presently unidentified. MHCY class I molecules are distinctive in bound ligand and in display of polymorphic residues.

Conformation-Dependent Influences of Hydrophobic Amino Acids in Two In-Register Parallel β-Sheet Amyloids, an α-Synuclein Amyloid and a Local Structural Model of PrPSc.

Prions are unconventional pathogens that encode the pathogenic information in conformations of the constituent abnormal isoform of prion protein (PrPSc), independently of the nucleotide genome. Therefore, conformational diversity of PrPSc underlies the existence of many prion strains and species barriers of prions, although the conformational information is extremely limited. Interestingly, differences between polymorphic or species-specific residues responsible for the species/strain barriers are often caused by conservative replacements between hydrophobic amino acids. This implies that subtle differences among hydrophobic amino acids are significant for PrPSc structures. Here we analyzed the influence of different hydrophobic residues on the structures of an in-register parallel β-sheet amyloid of α-synuclein (αSyn) using molecular dynamics (MD) simulation and applied the knowledge from the αSyn amyloid to modeling a local structure of human PrPSc encompassing residues 107–143. We found that mutations equivalent to polymorphisms that cause transmission barriers substantially affect the stabilities of the local structures; for example, the G127V mutation, which makes the host resistant to various human prion diseases, greatly destabilized the local structure of the model amyloid. Our study indicates that subtle differences among hydrophobic side chains can considerably affect the interaction network, including hydrogen bonds, and demonstrates specifically how and in what structures hydrophobic residues can exert unique effects on in-register parallel β-sheet amyloids.

Association of envelope-specific B-cell differentiation and viral selective pressure signatures in HIV-1 CRF01_AE infection.

In HIV type 1 (HIV-1) infection, virus-specific B-cell and neutralizing antibody (NAb) responses are impaired but exert selective pressure on target viral Envelope (Env) resulting in prominent sequence diversification among geographical areas. The basal induction patterns of HIV Env-specific B cells and their interaction with HIV Env awaits clarification. We investigated the relationship of Env polymorphisms and Env-specific B-cell responses in treatment-naive HIV-1 CRF01_AE-infected Vietnamese. Samples of 43 HIV-1 CRF01_AE infection-identified individuals were divided into acute-phase ( n = 12) and chronic-phase ( n = 31) by combined criteria of serological recent-infection assay and clinical parameters. We quantified subcloning-based polymorphic residue site numbers in plasma-derived Env variable region 1-5 (V1-V5)-coding regions within each individual, designating their summation within each region as variant index. Peripheral blood Env gp 140-specific B-cell responses and plasma neutralizing activity of Env pseudoviruses were examined to analyze their relationship with variant index. HIV-1 CRF01_AE Env gp140-specific total B-cell and plasma cell (CD19 + IgD - CD27 + CD38 + CD138 + ) responses were determined. In chronic-phase samples, significant correlation of variant index in all Env V1-V5 regions with Env-specific plasma cell responses was shown, and V1-V5 total variant index correlated stronger with Env-specific plasma cell as compared with total Env-specific B-cell responses. Env V5 variant index was significantly higher in chronic-phase cross-neutralizers of V5-polymorphic/VRC01-insensitive CRF01_AE Env. Results revealed the association between circulating Env-specific plasma cell responses and Env polymorphisms, implicating selective pressure on Env by plasma cell-derived antibodies and conversely suggests that Env-specific B-cell induction alone is insufficient for exerting Env selective pressure in HIV infection.

COVID-19 vaccination elicits an evolving, cross-reactive antibody response to epitopes conserved with endemic coronavirus spike proteins.

The COVID-19 pandemic has triggered the first widespread vaccination campaign against a coronavirus. Many vaccinated subjects are previously naive to SARS-CoV-2; however, almost all have previously encountered other coronaviruses (CoVs), and the role of this immunity in shaping the vaccine response remains uncharacterized. Here, we use longitudinal samples and highly multiplexed serology to identify mRNA-1273 vaccine-induced antibody responses against a range of CoV Spike epitopes, in both phylogenetically conserved and non-conserved regions. Whereas reactivity to SARS-CoV-2 epitopes shows a delayed but progressive increase following vaccination, we observe distinct kinetics for the endemic CoV homologs at conserved sites in Spike S2: these become detectable sooner and decay at later time points. Using homolog-specific antibody depletion and alanine-substitution experiments, we show that these distinct trajectories reflect an evolving cross-reactive response that can distinguish rare, polymorphic residues within these epitopes. Our results reveal mechanisms for the formation of antibodies with broad reactivity against CoVs.

Structure and Peptidomes of Swine MHC Class I with Long Peptides Reveal the Cross-Species Characteristics of the Novel N-Terminal Extension Presentation Mode.

Antigenic peptide presentation by the MHC is essential for activating T cells. The current view is that the peptide termini are tethered within the closed Ag-binding groove of MHC class I (MHC-I). Recently, the N-terminal extension mode of peptide presentation has been observed in human MHC-I (HLA-I). In this study, we found that the N terminus of the long peptide can extend beyond the groove of swine MHC-I (SLA-1*0401), confirming that this phenomenon can occur across species. Removal of the N-terminal extra (P-1) residue of the RW12 peptide significantly reduced the folding efficiency of the complex, but truncation of the second half of the peptide did not. Consistent with previous reports, the second (P1) residue of the peptide is twisted, and its side chain is inserted into the A pocket to form two hydrogen bonds with polymorphic E63 and conserved Y159. Mutations of E63 disrupt the binding of the peptide, indicating that E63 is necessary for this peptide-binding mode. Compared with W167, which exists in most MHC-Is, SLA-I-specific S167 ensures an open N-terminal groove of SLA-1*0401, enabling the P-1 residue to extend from the groove. In this MHC class II-like peptide-binding mode, the A pocket is restrictive to the P1 residue and is affected by the polymorphic residues. The peptidomes and refolding data indicated that the open N-terminal groove of SLA-1*0401 allows one to three residues to extend out of the Ag-binding groove. These cross-species comparisons can help us better understand the characteristics of this N-terminal extension presentation mode.

Characterization of Terpene synthase variation in flowers of wild aquilegia species from Northeastern Asia.

There are several causes for the great diversity in floral terpenes. The terpene products are determined by the catalytic fidelity, efficiency and plasticity of the active sites of terpene synthases (TPSs). However, the molecular mechanism of TPS in catalyzing terpene biosynthesis and its evolutionary fate in wild plant species remain largely unknown. In this study, the functionality of terpene synthases and their natural variants were assessed in two Northeastern Asia endemic columbine species and their natural hybrid. Synoptically, TPS7, TPS8, and TPS9 were highly expressed in these Aquilegia species from the Zuojia population. The in vitro and in vivo enzymatic assays revealed that TPS7 and TPS8 mainly produced (+)-limonene and β-sesquiphellandrene, respectively, whereas TPS9 produced pinene, similar to the major components released from Aquilegia flowers. Multiple sequence alignment of Aquilegia TPS7 and TPS8 in the Zuojia population revealed amino acid polymorphisms. Domain swapping and amino acid substitution assays demonstrated that 413A, 503I and 529D had impacts on TPS7 catalytic activity, whereas 420G, 538F and 545L affected the ratio of β-sesquiphellandrene to β-bisabolene in TPS8. Moreover, these key polymorphic amino acid residues were found in Aquilegia species from the Changbai Mountain population. Interestingly, amino acid polymorphisms in TPSs were present in individuals with low expression levels, and nonsynonymous mutations could impact the catalytic activity or product specificity of these genes. The results of this study will shed new light on the function and evolution of TPS genes in wild plant species and are beneficial to the modification of plant fragrances.

Connecting MHC-I-binding motifs with HLA alleles via deep learning

The selection of peptides presented by MHC molecules is crucial for antigen discovery. Previously, several predictors have shown impressive performance on binding affinity. However, the decisive MHC residues and their relation to the selection of binding peptides are still unrevealed. Here, we connected HLA alleles with binding motifs via our deep learning-based framework, MHCfovea. MHCfovea expanded the knowledge of MHC-I-binding motifs from 150 to 13,008 alleles. After clustering N-terminal and C-terminal sub-motifs on both observed and unobserved alleles, MHCfovea calculated the hyper-motifs and the corresponding allele signatures on the important positions to disclose the relation between binding motifs and MHC-I sequences. MHCfovea delivered 32 pairs of hyper-motifs and allele signatures (HLA-A: 13, HLA-B: 12, and HLA-C: 7). The paired hyper-motifs and allele signatures disclosed the critical polymorphic residues that determine the binding preference, which are believed to be valuable for antigen discovery and vaccine design when allele specificity is concerned.

Human leukocyte antigen class-I variation is associated with atopic dermatitis: A case-control study

Atopic dermatitis (AD) is a common immune-medicated skin disease. Previous studies have explored the relationship between Human Leukocyte Antigen (HLA) allelic variation and AD with conflicting results. The aim was to examine HLA Class I genetic variation, specifically peptide binding groove variation, and associations with AD. A case-control study was designed to evaluate HLA class I allelic variation and binding pocket polymorphisms, using next generation sequencing on 464 subjects with AD and 388 without AD. Logistic regression was used to evaluate associations with AD by estimating odds ratios (95% confidence intervals). Significant associations were noted with susceptibility to AD (B*53:01) and protection from AD (A*01:01, A*02:01, B*07:02 and C*07:02). Evaluation of polymorphic residues in Class I binding pockets revealed six amino acid residues conferring protection against AD: A9F (HLA-A, position 9, phenylalanine) [pocket B/C], A97I [pocket C/E], A152V [pocket E], A156R [pocket D/E], B163E [pocket A] and C116S [pocket F]. These findings demonstrate that specific HLA class I components are associated with susceptibility or protection from AD. Individual amino acid residues are relevant to protection from AD and set the foundation for evaluating potential HLA Class I molecules in complex with peptides/antigens that may initiate or interfere with T-cell responses.

Intra-Serotype Polyprotein Variation and its Effect on Antigenicity of Dengue Virus

Dengue virus is a mosquito-borne human pathogen, causing disease that ranges from mild febrile illness to life-threatening hemorrhage fever/ shock syndrome. The altered antigenicity and virulence in the dengue virus, resulting from the accumulation and fixation of the favorable mutations in the genome, is the cause of concern nowadays. The present study focuses on the comparative study of polyproteins of viral strains within each dengue serotype to understand the trend of intra-serotype polyprotein variation and its effect on the antigenicity. Polyprotein sequences of viral strains in each serotype were investigated using multivariate statistical analysis, phylogenetic analysis and multiple sequence alignment methods. Epitope prediction was done by Bepipred-1.0 server and experimental epitope data were extracted from Immune Epitope Database with BLAST search. The study reveals that the polyproteins of viral strains of a serotype have variable amino acid composition that corresponds to the geographical regions of origin. This compositional variation has occurred due to the presence of polymorphic residues at different positions along the polyprotein sequence. The polymorphic residues have also been identified at epitope regions of structural proteins as well as NS1 of viral strains, possessing dissimilar physicochemical properties and occupy surface accessible positions. These positions on epitopes with polymorphic, dissimilar and surface accessible residues might act as putative sites for generation of antigenic variation among viral strains of a serotype of different geographical origin. Thus, these polymorphic residue positions on epitopes might be considered as putative target for development of drug or vaccine, in future.

CD4 receptor diversity represents an ancient protection mechanism against primate lentiviruses

Infection with human and simian immunodeficiency viruses (HIV/SIV) requires binding of the viral envelope glycoprotein (Env) to the host protein CD4 on the surface of immune cells. Although invariant in humans, the Env binding domain of the chimpanzee CD4 is highly polymorphic, with nine coding variants circulating in wild populations. Here, we show that within-species CD4 diversity is not unique to chimpanzees but found in many African primate species. Characterizing the outermost (D1) domain of the CD4 protein in over 500 monkeys and apes, we found polymorphic residues in 24 of 29 primate species, with as many as 11 different coding variants identified within a single species. D1 domain amino acid replacements affected SIV Env-mediated cell entry in a single-round infection assay, restricting infection in a strain- and allele-specific fashion. Several identical CD4 polymorphisms, including the addition of N-linked glycosylation sites, were found in primate species from different genera, providing striking examples of parallel evolution. Moreover, seven different guenons (Cercopithecus spp.) shared multiple distinct D1 domain variants, pointing to long-term trans-specific polymorphism. These data indicate that the HIV/SIV Env binding region of the primate CD4 protein is highly variable, both within and between species, and suggest that this diversity has been maintained by balancing selection for millions of years, at least in part to confer protection against primate lentiviruses. Although long-term SIV-infected species have evolved specific mechanisms to avoid disease progression, primate lentiviruses are intrinsically pathogenic and have left their mark on the host genome.

In-silico structural analysis of Pseudomonas syringae effector HopZ3 reveals ligand binding activity and virulence function.

Bacterial acetyltransferase effectors belonging to the Yersinia outer protein J (YopJ) group inhibit multiple immune signaling pathways in human and plants. The present study determines in-silico acetyl-coenzyme A (AcCoA) binding and Arabidopsis immune regulator RPM1-interacting protein4 (RIN4) peptide interactions to YopJ effector hypersensitivity and pathogenesis-dependent outer proteinZ3 (HopZ3) from Pseudomonas syringae. Phylogenetic analysis revealed that HopZ3 was clustered by acetyltransferase effectors from plant bacterial pathogens. Structural juxtaposition shows HopZ3 comprises topology matched closer with HopZ1a than PopP2 effectors, respectively. AcCoA binds HopZ3 at two sites i.e., substrate binding pocket and catalytic site. AcCoA interactions to substrate binding pocket was transient and dissipated upon in-silico mutation of Ser 279 residue whereas, attachment to catalytic site was found to be stable in the presence of inositol hexaphosphate (IP6) as a co-factor. Interface atoms used for measuring hydrogen bond distances, bound or accessible surface area, and root-mean-square fluctuation (RMSF) values, suggests that the HopZ3 complex stabilizes after binding to AcCoA ligand and RIN4 peptide. The few non-conserved polymorphic residues that have been displayed on HopZ3 surface presumably confer intracellular recognitions within hosts. Collectively, homology modeling and interactive docking experiments were used to substantiate Arabidopsis immune 'guardee' interactions to HopZ3.