Variations in MHC Class II Antigen Processing and Presentation in Health and Disease.

MHC class II (MHC-II) molecules are critical in the control of many immune responses. They are also involved in most autoimmune diseases and other pathologies. Here, we describe the biology of MHC-II and MHC-II variations that affect immune responses. We discuss the classic cell biology of MHC-II and various perturbations. Proteolysis is a major process in the biology of MHC-II, and we describe the various components forming and controlling this endosomal proteolytic machinery. This process ultimately determines the MHC-II-presented peptidome, including cryptic peptides, modified peptides, and other peptides that are relevant in autoimmune responses. MHC-II also variable in expression, glycosylation, and turnover. We illustrate that MHC-II is variable not only in amino acids (polymorphic) but also in its biology, with consequences for both health and disease.

Structural evolution of fibril polymorphs during amyloid assembly.

Cryoelectron microscopy (cryo-EM) has provided unprecedented insights into amyloid fibril structures, including those associated with disease. However, these structures represent the endpoints of long assembly processes, and their relationship to fibrils formed early in assembly is unknown. Consequently, whether different fibril architectures, with potentially different pathological properties, form during assembly remains unknown. Here, we used cryo-EM to determine structures of amyloid fibrils at different times during in vitro fibrillation of a disease-related variant of human islet amyloid polypeptide (IAPP-S20G). Strikingly, the fibrils formed in the lag, growth, and plateau phases have different structures, with new forms appearing and others disappearing as fibrillation proceeds. A time course with wild-type hIAPP also shows fibrils changing with time, suggesting that this is a general property of IAPP amyloid assembly. The observation of transiently populated fibril structures has implications for understanding amyloid assembly mechanisms with potential new insights into amyloid progression in disease.

HLAs, TCRs, and KIRs, a Triumvirate of Human Cell-Mediated Immunity.

In all human cells, human leukocyte antigen (HLA) class I glycoproteins assemble with a peptide and take it to the cell surface for surveillance by lymphocytes. These include natural killer (NK) cells and γδ T cells of innate immunity and αß T cells of adaptive immunity. In healthy cells, the presented peptides derive from human proteins, to which lymphocytes are tolerant. In pathogen-infected cells, HLA class I expression is perturbed. Reduced HLA class I expression is detected by KIR and CD94:NKG2A receptors of NK cells. Almost any change in peptide presentation can be detected by αß CD8+ T cells. In responding to extracellular pathogens, HLA class II glycoproteins, expressed by specialized antigen-presenting cells, present peptides to αß CD4+ T cells. In comparison to the families of major histocompatibility complex (MHC) class I, MHC class II and αß T cell receptors, the antigenic specificity of the γδ T cell receptors is incompletely understood.

Pharmacogenomics of GPCR Drug Targets.

Natural genetic variation in the human genome is a cause of individual differences in responses to medications and is an underappreciated burden on public health. Although 108 G-protein-coupled receptors (GPCRs) are the targets of 475 (∼34%) Food and Drug Administration (FDA)-approved drugs and account for a global sales volume of over 180 billion US dollars annually, the prevalence of genetic variation among GPCRs targeted by drugs is unknown. By analyzing data from 68,496 individuals, we find that GPCRs targeted by drugs show genetic variation within functional regions such as drug- and effector-binding sites in the human population. We experimentally show that certain variants of µ-opioid and Cholecystokinin-A receptors could lead to altered or adverse drug response. By analyzing UK National Health Service drug prescription and sales data, we suggest that characterizing GPCR variants could increase prescription precision, improving patients' quality of life, and relieve the economic and societal burden due to variable drug responsiveness. VIDEO ABSTRACT.

Immunoglobulin germline gene polymorphisms influence the function of SARS-CoV-2 neutralizing antibodies.

The human immunoglobulin heavy-chain (IGH) locus is exceptionally polymorphic, with high levels of allelic and structural variation. Thus, germline IGH genotypes are personal, which may influence responses to infection and vaccination. For an improved understanding of inter-individual differences in antibody responses, we isolated SARS-CoV-2 spike-specific monoclonal antibodies from convalescent health care workers, focusing on the IGHV1-69 gene, which has the highest level of allelic variation of all IGHV genes. The IGHV1-69∗20-using CAB-I47 antibody and two similar antibodies isolated from an independent donor were critically dependent on allele usage. Neutralization was retained when reverting the V region to the germline IGHV1-69∗20 allele but lost when reverting to other IGHV1-69 alleles. Structural data confirmed that two germline-encoded polymorphisms, R50 and F55, in the IGHV1-69 gene were required for high-affinity receptor-binding domain interaction. These results demonstrate that polymorphisms in IGH genes can influence the function of SARS-CoV-2 neutralizing antibodies.

Allelic polymorphism controls autoreactivity and vaccine elicitation of human broadly neutralizing antibodies against influenza virus.

Human broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin stalk of group 1 influenza A viruses (IAVs) are biased for IGHV1-69 alleles that use phenylalanine (F54) but not leucine (L54) within their CDRH2 loops. Despite this, we demonstrated that both alleles encode for human IAV bnAbs that employ structurally convergent modes of contact to the same epitope. To resolve differences in lineage expandability, we compared F54 versus L54 as substrate within humanized mice, where antibodies develop with human-like CDRH3 diversity but are restricted to single VH genes. While both alleles encoded for bnAb precursors, only F54 IGHV1-69 supported elicitation of heterosubtypic serum bnAbs following immunization with a stalk-only nanoparticle vaccine. L54 IGHV1-69 was unproductive, co-encoding for anergic B cells and autoreactive stalk antibodies that were cleared from B cell memory. Moreover, human stalk antibodies also demonstrated L54-dependent autoreactivity. Therefore, IGHV1-69 polymorphism, which is skewed ethnically, gates tolerance and vaccine expandability of influenza bnAbs.

Circadian clock gene polymorphisms implicated in human pathologies.

Circadian rhythms, ~24 h cycles of physiological and behavioral processes, can be synchronized by external signals (e.g., light) and persist even in their absence. Consequently, dysregulation of circadian rhythms adversely affects the well-being of the organism. This timekeeping system is generated and sustained by a genetically encoded endogenous mechanism composed of interlocking transcriptional/translational feedback loops that generate rhythmic expression of core clock genes. Genome-wide association studies (GWAS) and forward genetic studies show that SNPs in clock genes influence gene regulation and correlate with the risk of developing various conditions. We discuss genetic variations in core clock genes that are associated with various phenotypes, their implications for human health, and stress the need for thorough studies in this domain of circadian regulation.

Fluorescent proteins generate a genetic color polymorphism and counteract oxidative stress in intertidal sea anemones.

Fluorescent proteins (FPs) are ubiquitous tools in research, yet their endogenous functions in nature are poorly understood. In this work, we describe a combination of functions for FPs in a clade of intertidal sea anemones whose FPs control a genetic color polymorphism together with the ability to combat oxidative stress. Focusing on the underlying genetics of a fluorescent green "Neon" color morph, we show that allelic differences in a single FP gene generate its strong and vibrant color, by increasing both molecular brightness and FP gene expression level. Natural variation in FP sequences also produces differences in antioxidant capacity. We demonstrate that these FPs are strong antioxidants that can protect live cells against oxidative stress. Finally, based on structural modeling of the responsible amino acids, we propose a model for FP antioxidant function that is driven by molecular surface charge. Together, our findings shed light on the multifaceted functions that can co-occur within a single FP and provide a framework for studying the evolution of fluorescence as it balances spectral and physiological functions in nature.

Ligand-induced incompatible curvatures control ultrathin nanoplatelet polymorphism and chirality.

The ability of thin materials to shape-shift is a common occurrence that leads to dynamic pattern formation and function in natural and man-made structures. However, harnessing this concept to rationally design inorganic structures at the nanoscale has remained far from reach due to a lack of fundamental understanding of the essential physical components. Here, we show that the interaction between organic ligands and the nanocrystal surface is responsible for the full range of chiral shapes seen in colloidal nanoplatelets. The adsorption of ligands results in incompatible curvatures on the top and bottom surfaces of the NPL, causing them to deform into helicoïds, helical ribbons, or tubes depending on the lateral dimensions and crystallographic orientation of the NPL. We demonstrate that nanoplatelets belong to the broad class of geometrically frustrated assemblies and exhibit one of their hallmark features: a transition between helicoïds and helical ribbons at a critical width. The effective curvature [Formula: see text] is the single aggregate parameter that encodes the details of the ligand/surface interaction, determining the nanoplatelets' geometry for a given width and crystallographic orientation. The conceptual framework described here will aid the rational design of dynamic, chiral nanostructures with high fundamental and practical relevance.

Crystal size, shape, and conformational changes drive both the disappearance and reappearance of ritonavir polymorphs in the mill.

Organic compounds can crystallize in different forms known as polymorphs. Discovery and control of polymorphism is crucial to the pharmaceutical industry since different polymorphs can have significantly different physical properties which impacts their utilization in drug delivery. Certain polymorphs have been reported to 'disappear' from the physical world, irreversibly converting to new ones. These unwanted polymorph conversions, initially prevented by slow nucleation kinetics, are eventually observed driven by significant gains in thermodynamic stabilities. The most infamous of these cases is that of the HIV drug ritonavir (RVR): Once its reluctant form was unwillingly nucleated for the first time, its desired form could no longer be produced with the same manufacturing process. Here we show that RVR's extraordinary disappearing polymorph as well as its reluctant form can be consistently produced by ball-milling under different environmental conditions. We demonstrate that the significant difference in stability between its polymorphs can be changed and reversed in the mill-a process we show is driven by crystal size as well as crystal shape and conformational effects. We also show that those effects can be controlled through careful design of milling conditions since they dictate the kinetics of crystal breakage, dissolution, and growth processes that eventually lead to steady-state crystal sizes and shapes in the mill. This work highlights the huge potential of mechanochemistry in polymorph discovery of forms initially difficult to nucleate, recovery of disappearing polymorphs, and polymorph control of complex flexible drug compounds such as RVR.

Sexual conflict drive in the rapid evolution of new gametogenesis genes.

The evolutionary forces underlying the rapid evolution in sequences and functions of new genes remain a mystery. Adaptation by natural selection explains the evolution of some new genes. However, many new genes perform sex-biased functions that have rapidly evolved over short evolutionary time scales, suggesting that new gene evolution may often be driven by conflicting selective pressures on males and females. It is well established that such sexual conflict (SC) plays a central role in maintaining phenotypic and genetic variation within populations, but the role of SC in driving new gene evolution remains essentially unknown. This review explores the connections between SC and new gene evolution through discussions of the concept of SC, the phenotypic and genetic signatures of SC in evolving populations, and the molecular mechanisms by which SC could drive the evolution of new genes. We synthesize recent work in this area with a discussion of the case of Apollo and Artemis, two extremely young genes (<200,000 years) in Drosophila melanogaster, which offered the first empirical insights into the evolutionary process by which SC could drive the evolution of new genes. These new duplicate genes exhibit the hallmarks of sexually antagonistic selection: rapid DNA and protein sequence evolution, essential sex-specific functions in gametogenesis, and complementary sex-biased expression patterns. Importantly, Apollo is essential for male fitness but detrimental to female fitness, while Artemis is essential for female fitness but detrimental to male fitness. These sexually antagonistic fitness effects and complementary changes to expression, sequence, and function suggest that these duplicates were selected for mitigating SC, but that SC has not been fully resolved. Finally, we propose Sexual Conflict Drive as a self-driven model to interpret the rapid evolution of new genes, explain the potential for SC and sexually antagonistic selection to contribute to long-term evolution, and suggest its utility for understanding the rapid evolution of new genes in gametogenesis.

Diverse mating consequences of the evolutionary breakdown of the sexual polymorphism heterostyly.

Reproductive systems of flowering plants are evolutionarily fluid, with mating patterns changing in response to shifts in abiotic conditions, pollination systems, and population characteristics. Changes in mating should be particularly evident in species with sexual polymorphisms that become ecologically destabilized, promoting transitions to alternative reproductive systems. Here, we decompose female mating portfolios (incidence of selfing, outcross mate number, and intermorph mating) in eight populations of Primula oreodoxa, a self-compatible insect-pollinated herb. This species is ancestrally distylous, with populations subdivided into two floral morphs that usually mate with each other (disassortative mating). Stages in the breakdown of polymorphism also occur, including "mixed" populations of distylous and homostylous (self-pollinating) morphs and purely homostylous populations. Population morph ratios vary with elevation in association with differences in pollinator availability, providing an unusual opportunity to investigate changes in mating patterns accompanying transitions in reproductive systems. Unexpectedly, individuals mostly outcrossed randomly, with substantial disassortative mating in at most two distylous populations. As predicted, mixed populations had higher selfing rates than distylous populations, within mixed populations, homostyles selfed almost twice as much as the distylous morphs, and homostylous populations exhibited the highest selfing rates. Populations with homostyles outcrossed with fewer mates and mate number varied negatively with population selfing rates. These differences indicate maintenance of distyly at low elevation, transition to monomorphic selfing at high elevation, and uncertain, possibly variable fates at intermediate elevation. By quantifying the earliest changes in mating that initiate reproductive transitions, our study highlights the key role of mating in promoting evolutionary divergence.

Predicting the effect of mutations to investigate recent events of selection across 60,472 Escherichia coli strains.

Microbial genomics studies focusing on the dynamics of selection have often used a small number of distant genomes. As a result, they could only analyze mutations that had become fixed during the divergence between species. However, thousands of genomes of some species are now available in public databases, thanks to high-throughput sequencing. These data provide a more complete picture of the polymorphisms segregating within a species, offering a unique insight into the processes that shape the recent evolution of a species. In this study, we present GLASS (Gene-Level Amino-acid Score Shift), a selection test that is based on the predicted effects of amino acid changes. By comparing the distribution of effects of mutations observed in a gene to the expectation in the absence of selection, GLASS can quantify the intensity of selection. We applied GLASS to a dataset of 60,472 Escherichia coli strains and used this to reexamine the longstanding debate about the role of essentiality versus expression level in the rate of protein evolution. We found that selection has contrasting short-term and long-term dynamics, with essential genes being subject to strong purifying selection in the short term, while expression level determines the rate of gene evolution in the long term. GLASS also found an overrepresentation of inactivating mutations in specific transcription factors, such as efflux pump repressors, which is consistent with selection for antibiotic resistance. These gene-inactivating polymorphisms do not reach fixation, suggesting another contrast between short-term fitness gains and long-term counterselection.

Reducing overprediction of molecular crystal structures via threshold clustering.

Crystal structure prediction is becoming an increasingly valuable tool for assessing polymorphism of crystalline molecular compounds, yet invariably, it overpredicts the number of polymorphs. One of the causes for this overprediction is in neglecting the coalescence of potential energy minima, separated by relatively small energy barriers, into a single basin at finite temperature. Considering this, we demonstrate a method underpinned by the threshold algorithm for clustering potential energy minima into basins, thereby identifying kinetically stable polymorphs and reducing overprediction.

Natural selection has driven the recurrent loss of an immunity gene that protects Drosophila against a major natural parasite.

Polymorphisms in immunity genes can have large effects on susceptibility to infection. To understand the origins of this variation, we have investigated the genetic basis of resistance to the parasitoid wasp Leptopilina boulardi in Drosophila melanogaster. We found that increased expression of the gene lectin-24A after infection by parasitic wasps was associated with a faster cellular immune response and greatly increased rates of killing the parasite. lectin-24A encodes a protein that is strongly up-regulated in the fat body after infection and localizes to the surface of the parasite egg. In certain susceptible lines, a deletion upstream of the lectin-24A has largely abolished expression. Other mutations predicted to abolish the function of this gene have arisen recurrently in this gene, with multiple loss-of-expression alleles and premature stop codons segregating in natural populations. The frequency of these alleles varies greatly geographically, and in some southern African populations, natural selection has driven them near to fixation. We conclude that natural selection has favored the repeated loss of an important component of the immune system, suggesting that in some populations, a pleiotropic cost to lectin-24A expression outweighs the benefits of resistance.

Heterozygosity for cervid S138N polymorphism results in subclinical CWD in gene-targeted mice and progressive inhibition of prion conversion.

Prions are proteinaceous infectious particles that replicate by structural conversion of the host-encoded cellular prion protein (PrPC), causing fatal neurodegenerative diseases in mammals. Species-specific amino acid substitutions (AAS) arising from single nucleotide polymorphisms within the prion protein gene (Prnp) modulate prion disease pathogenesis, and, in several instances, reduce susceptibility of homo- or heterozygous AAS carriers to prion infection. However, a mechanistic understanding of their protective effects against clinical disease is missing. We generated gene-targeted mouse infection models of chronic wasting disease (CWD), a highly contagious prion disease of cervids. These mice express wild-type deer or PrPC harboring the S138N substitution homo- or heterozygously, a polymorphism found exclusively in reindeer (Rangifer tarandus spp.) and fallow deer (Dama dama). The wild-type deer PrP-expressing model recapitulated CWD pathogenesis including fecal shedding. Encoding at least one 138N allele prevented clinical CWD, accumulation of protease-resistant PrP (PrPres) and abnormal PrP deposits in the brain tissue. However, prion seeding activity was detected in spleens, brains, and feces of these mice, suggesting subclinical infection accompanied by prion shedding. 138N-PrPC was less efficiently converted to PrPres in vitro than wild-type deer (138SS) PrPC. Heterozygous coexpression of wild-type deer and 138N-PrPC resulted in dominant-negative inhibition and progressively diminished prion conversion over serial rounds of protein misfolding cyclic amplification. Our study indicates that heterozygosity at a polymorphic Prnp codon can confer the highest protection against clinical CWD and highlights the potential role of subclinical carriers in CWD transmission.

Optimizing CRISPR/Cas9 approaches in the polymorphic tunicate Ciona intestinalis.

CRISPR/Cas9 became a powerful tool for genetic engineering and in vivo knockout also in the invertebrate chordate Ciona intestinalis. Ciona (ascidians, tunicates) is an important model organism because it shares developmental features with the vertebrates, considered the sister group of tunicates, and offers outstanding experimental advantages: a compact genome and an invariant developmental cell lineage that, combined with electroporation mediated transgenesis allows for precise and cell type specific targeting in vivo. A high polymorphism and the mosaic expression of electroporated constructs, however, often hamper the efficient CRISPR knockout, and an optimization in Ciona is desirable. Furthermore, seasonality and artificial maintenance settings can profit from in vitro approaches that would save on animals. Here we present improvements for the CRISPR/Cas9 protocol in silico, in vitro and in vivo. Firstly, in designing sgRNAs, prior sequencing of target genomic regions from experimental animals and alignment with reference genomes of C. robusta and C. intestinalis render a correction possible of subspecies polymorphisms. Ideally, the screening for efficient and non-polymorphic sgRNAs will generate a database compatible for worldwide Ciona populations. Secondly, we challenged in vitro assays for sgRNA validation towards reduced in vivo experimentation and report their suitability but also overefficiency concerning mismatch tolerance. Thirdly, when comparing Cas9 with Cas9:Geminin, thought to synchronize editing and homology-direct repair, we could indeed increase the in vivo efficiency and notably the access to an early expressed gene. Finally, for in vivo CRISPR, genotyping by next generation sequencing (NGS) ex vivo streamlined the definition of efficient single guides. Double CRISPR then generates large deletions and reliable phenotypic excision effects. Overall, while these improvements render CRISPR more efficient in Ciona, they are useful when newly establishing the technique and very transferable to CRISPR in other organisms.

A small-molecule allele-selective transcriptional inhibitor of the MIF immune susceptibility locus.

Functional variants of the gene for the cytokine macrophage migration inhibitory factor (MIF) are defined by a 4-nucleotide promoter microsatellite (-794 CATT5-8, rs5844572) and confer risk for autoimmune, infectious, and oncologic diseases. We describe herein the discovery of a prototypic, small molecule inhibitor of MIF transcription with selectivity for high microsatellite repeat number and correspondingly high gene expression. Utilizing a high-throughput luminescent proximity screen, we identify 1-carbomethoxy-5-formyl-4,6,8-trihydroxyphenazine (CMFT) to inhibit the functional interaction between the transcription factor ICBP90 (namely, UHRF1) and the MIF -794 CATT5-8 promoter microsatellite. CMFT inhibits MIF mRNA expression in a -794 CATT5-8 length-dependent manner with an IC50 of 470 nM, and preferentially reduces ICBP90-dependent MIF mRNA and protein expression in high-genotypic versus low-genotypic MIF-expressing macrophages. RNA expression analysis also showed CMFT to downregulate MIF-dependent, inflammatory gene expression with little evidence of off-target metabolic toxicity. These findings provide proof-of-concept for advancing the pharmacogenomic development of precision-based MIF inhibitors for diverse autoimmune and inflammatory conditions.

Alternative splicing of lncRNA LAIR fine-tunes the regulation of neighboring yield-related gene LRK1 expression in rice.

The diversity in alternative splicing of long noncoding RNAs (lncRNAs) poses a challenge for functional annotation of lncRNAs. Moreover, little is known on the effects of alternatively spliced lncRNAs on crop yield. In this study, we cloned nine isoforms resulting from the alternative splicing of the lncRNA LAIR in rice. The LAIR isoforms are generated via alternative 5'/3' splice sites and different combinations of specific introns. All LAIR isoforms activate the expression of the neighboring LRK1 gene and enhance yield-related rice traits. In addition, there are slight differences in the binding ability of LAIR isoforms to the epigenetic modification-related proteins OsMOF and OsWDR5, which affect the enrichment of H4K16ac and H3K4me3 at the LRK1 locus, and consequently fine-tune the regulation of LRK1 expression and yield-related traits. These differences in binding may be caused by polymorphic changes to the RNA secondary structure resulting from alternative splicing. It was also observed that the composition of LAIR isoforms was sensitive to abiotic stress. These findings suggest that the alternative splicing of LAIR leads to the formation of a functional transcript population that precisely regulates yield-related gene expression, which may be relevant for phenotypic polymorphism-based crop breeding under changing environmental conditions.

Evolution of Chromosomal Inversions across an Avian Radiation.

Chromosomal inversions are structural mutations that can play a prominent role in adaptation and speciation. Inversions segregating across species boundaries (trans-species inversions) are often taken as evidence for ancient balancing selection or adaptive introgression, but can also be due to incomplete lineage sorting. Using whole-genome resequencing data from 18 populations of 11 recognized munia species in the genus Lonchura (N = 176 individuals), we identify four large para- and pericentric inversions ranging in size from 4 to 20 Mb. All four inversions cosegregate across multiple species and predate the numerous speciation events associated with the rapid radiation of this clade across the prehistoric Sahul (Australia, New Guinea) and Bismarck Archipelago. Using coalescent theory, we infer that trans-specificity is improbable for neutrally segregating variation despite substantial incomplete lineage sorting characterizing this young radiation. Instead, the maintenance of all three autosomal inversions (chr1, chr5, and chr6) is best explained by selection acting along ecogeographic clines not observed for the collinear parts of the genome. In addition, the sex chromosome inversion largely aligns with species boundaries and shows signatures of repeated positive selection for both alleles. This study provides evidence for trans-species inversion polymorphisms involved in both adaptation and speciation. It further highlights the importance of informing selection inference using a null model of neutral evolution derived from the collinear part of the genome.