Negative Selection Signals Research Articles

Small proteins, great promises: Geographic bioprospecting of Bowman–Birk protease inhibitors and domestication side‐effects in African cowpea (Vigna unguiculata L.)

Societal Impact StatementThe legume crop cowpea is grown worldwide, but 90% of the world's total share is produced in Africa. It is a promising species due to its resilience properties, balance of macro and micronutrients and presence of health‐promoting bioactive compounds. In African countries, cowpea has a crucial role in guaranteeing food security as a subsistence crop for families and commercial income for small farmers. The discovery of compounds with high nutraceutical value and bioactive properties supports socio‐economic policies to improve health and nutrition, especially in low‐ and middle‐income countries. In turn, this encourages biodiversity protection and crop enhancement programmes.Summary Bowman–Birk protease inhibitors (BBIs) are a restricted group of small proteins in plants mainly involved in defence mechanisms against pests. BBIs are demonstrated to be active components capable of reducing the viabilities of different cancer cell lines. BBI bioactivity is directly linked to the inhibition capacity, but the variability and the efficiency against the physiological targets of different BBI isoforms remain still unexplored. We analysed the natural genetic diversity of two main genes encoding BBI trypsin‐trypsin (BBI‐TT) and trypsin‐chymotrypsin (BBI‐TC) in wild and domesticated cowpea samples mainly spread in Sub‐Saharan Africa. We analysed DNA sequences and respective amino acidic isoforms/isoproteins to explore signs of natural selection and haplotype relationships. Moreover, we calculated the binding energy between BBIs and their biological targets to identify which are the most efficient inhibitors and their geographical locations. We found a high level of haplotype diversity for both genes, almost exclusively in wild accessions and detected positive and negative selection signals in the amino acid sequences. Furthermore, in the wild diversity pool, some BBI‐TT and BBI‐TC mature proteins were potentially better interactors with the physiological targets. The long interaction between plant‐pathogen has selected new and useful isoforms in wild lineages that have allowed the chances of survival of the species to improve. On the other hand, the domestication process has produced an intense bottleneck leaving only poorly efficient BBI variants. In addition to providing information on the natural diversity and evolution of BBIs, our work discusses the potential applications in agriculture and human health.

Babesia bovis RON2 binds to bovine erythrocytes through a highly conserved epitope

B. bovis invasion of bovine erythrocytes requires tight junction formation involving AMA-1/RON2 complex interaction. RON2 has been considered a vaccine candidate since antibodies targeting the protein can inhibit parasite invasion of target cells; however, the mechanism controlling B. bovis RON2 interaction with red blood cells is not yet fully understood. This study was thus aimed at identifying B. bovis RON2 protein regions associated with interaction with bovine erythrocytes. Natural selection analysis of the ron2 gene identified predominantly negative selection signals in the C-terminal region. Interestingly, protein-cell and competition assays highlighted the RON2-C region’s role in peptide 42918-mediated erythrocyte binding, probably to a sialoglycoprotein receptor. This peptide (1218SFIMVKPPALHCVLKPVETL1237) lies within an intrinsically disordered region of the RON2 secondary structure flanked by two helical residues. The study provides, for the first time, valuable insights into RON2’s role in interaction with its target cells. Future studies are required for studying the peptide’s potential as an anti-B. bovis vaccine component.

Cancer genomes tolerate deleterious coding mutations through somatic copy number amplifications of wild-type regions

Cancers evolve under the accumulation of thousands of somatic mutations and chromosomal aberrations. While most coding mutations are deleterious, almost all protein-coding genes lack detectable signals of negative selection. This raises the question of how tumors tolerate such large amounts of deleterious mutations. Using 8,690 tumor samples from The Cancer Genome Atlas, we demonstrate that copy number amplifications frequently cover haploinsufficient genes in mutation-prone regions. This could increase tolerance towards the deleterious impact of mutations by creating safe copies of wild-type regions and, hence, protecting the genes therein. Our findings demonstrate that these potential buffering events are highly influenced by gene functions, essentiality, and mutation impact and that they occur early during tumor evolution. We show how cancer type-specific mutation landscapes drive copy number alteration patterns across cancer types. Ultimately, our work paves the way for the detection of novel cancer vulnerabilities by revealing genes that fall within amplifications likely selected during evolution to mitigate the effect of mutations.

Isoform-specific functions of Ras in T-cell development and differentiation.

Ras GTPases, well characterized for their role in oncogenesis, are the cells' molecular switches that signal to maintain immune homeostasis through cellular development, proliferation, differentiation, survival, and apoptosis. In the immune system, T cells are the central players that cause autoimmunity if dysregulated. Antigen-specific T-cell receptor (TCR) stimulation activates Ras-isoforms, which exhibit isoform-specific activator and effector requirements, functional specificities, and a selective role in T-cell development and differentiation. Recent studies show the role of Ras in T-cell-mediated autoimmune diseases; however, there is a scarcity of knowledge about the role of Ras in T-cell development and differentiation. To date, limited studies have demonstrated Ras activation in response to positive and negative selection signals and Ras isoform-specific signaling, including subcellular signaling, in immune cells. The knowledge of isoform-specific functions of Ras in T cells is essential, but still inadequate to develop the T-cell-targeted Ras isoform-specific treatment strategies for the diseases caused by altered Ras-isoform expression and activation in T cells. In this review, we discuss the role of Ras in T-cell development and differentiation, critically analyzing the isoform-specific functions.

Novel start codons introduce novel coding sequences in the human genomes

Start-gain mutations can introduce novel start codons and generate novel coding sequences that may affect the function of genes. In this study, we systematically investigated the novel start codons that were either polymorphic or fixed in the human genomes. 829 polymorphic start-gain SNVs were identified in the human populations, and the novel start codons introduced by these SNVs have significantly higher activity in translation initiation. Some of these start-gain SNVs were reported to be associated with phenotypes and diseases in previous studies. By comparative genomic analysis, we found 26 human-specific start codons that were fixed after the divergence between the human and chimpanzee, and high-level translation initiation activity was observed on them. The negative selection signal was detected in the novel coding sequences introduced by these human-specific start codons, indicating the important function of these novel coding sequences.

The complete chloroplast genome sequence of Eugenia klotzschiana O. Berg unveils the evolutionary dynamics in plastomes of Myrteae DC. Tribe (Myrtaceae)

Myrteae is the most diversified tribe in the Myrtaceae family and has great ecological and economic importance. Here, we performed the assembly and annotation of the chloroplast genome of Eugenia klotzschiana O. Berg and used this in a comparative analysis with other 13 species from the Myrteae tribe. The E. klotzschiana plastome exhibited a length of 158,977 bp and a very conserved structure and gene composition when compared with other Myrteae genomes. We identified 34 large repetitive sequences and 94 SSR repeats in E. klotzschiana plastome. The trnT-trnL, rpl32-trnL, ndhF-rpl32, psbE-petL, and ycf1 regions were identified as mutational hotspots. A negative selection signal was detected in 74 protein-coding genes while neutral evolution was detected in two genes (rps12 and psaI). Furthermore, 222 RNA editing sites were identified in the E. klotzschiana plastome. We also obtained a plastome-based Myrtales phylogenetic tree, including E. klotzschiana for the first time in a molecular phylogeny, recovering its sister relationship for all other Eugenia species. Our results illuminate how evolution shaped the chloroplast genome structure and composition in the Myrteae tribe, especially in the E. klotzschiana plastome.

RNA structure-altering mutations underlying positive selection on Spike protein reveal novel putative signatures to trace crossing host-species barriers in Betacoronavirus

ABSTRACT Similar to other RNA viruses, the emergence of Betacoronavirus relies on cross-species viral transmission, which requires careful health surveillance monitoring of protein-coding information as well as genome-wide analysis. Although the evolutionary jump from natural reservoirs to humans may be mainly traced-back by studying the effect that hotspot mutations have on viral proteins, it is largely unexplored if other impacts might emerge on the structured RNA genome of Betacoronavirus. In this survey, the protein-coding and viral genome architecture were simultaneously studied to uncover novel insights into cross-species horizontal transmission events. We analysed 1,252,952 viral genomes of SARS-CoV, MERS-CoV, and SARS-CoV-2 distributed across the world in bats, intermediate animals, and humans to build a new landscape of changes in the RNA viral genome. Phylogenetic analyses suggest that bat viruses are the most closely related to the time of most recent common ancestor of Betacoronavirus, and missense mutations in viral proteins, mainly in the S protein S1 subunit: SARS-CoV (G > T; A577S); MERS-CoV (C > T; S746R and C > T; N762A); and SARS-CoV-2 (A > G; D614G) appear to have driven viral diversification. We also found that codon sites under positive selection on S protein overlap with non-compensatory mutations that disrupt secondary RNA structures in the RNA genome complement. These findings provide pivotal factors that might be underlying the eventual jumping the species barrier from bats to intermediate hosts. Lastly, we discovered that nearly half of the Betacoronavirus genomes carry highly conserved RNA structures, and more than 90% of these RNA structures show negative selection signals, suggesting essential functions in the biology of Betacoronavirus that have not been investigated to date. Further research is needed on negatively selected RNA structures to scan for emerging functions like the potential of coding virus-derived small RNAs and to develop new candidate antiviral therapeutic strategies.

Inside the USCAP Journals

Inside the USCAP Journals

Inside the USCAP Journals

Inside the USCAP Journals

Extracellular vesicles extracted from young donor serum attenuate inflammaging via partially rejuvenating aged T-cell immunotolerance.

Biologic aging results in a chronic inflammatory condition, termed inflammaging, which establishes a risk for such age-related diseases as neurocardiovascular diseases; therefore, it is of great importance to develop rejuvenation strategies that are able to attenuate inflammaging as a means of intervention for age-related diseases. A promising rejuvenation factor that is present in young blood has been found that can make aged neurons younger; however, the component in the young blood and its mechanism of action are poorly elucidated. We assessed rejuvenation in naturally aged mice with extracellular vesicles (EVs) or exosomes extracted from young murine serum on the basis of different spectrums of microRNAs in these vesicles from young and old sera. We found that EVs extracted from young donor mouse serum, rather than EVs extracted from old donor mouse serum or non-EV supernatant extracted from young donor mouse serum, were able to attenuate inflammaging in old mice. Inflammaging is attributed to multiple factors, one of which is thymic aging-released self-reactive T cell-induced pathology. We found that the attenuation of inflammaging after treatment with EVs from young serum partially contributed to the rejuvenation of thymic aging, which is characterized by partially reversed thymic involution, enhancement of negative selection signals, and reduced autoreactions in the periphery. Our results provide evidence for understanding of the potential rejuvenation factor in the young donor serum, which holds great promise for the development of novel therapeutics to reduce morbidity and mortality caused by age-related inflammatory diseases.-Wang, W., Wang, L., Ruan, L., Oh, J., Dong, X., Zhuge, Q., Su, D.-M. Extracellular vesicles extracted from young donor serum attenuate inflammaging via partially rejuvenating aged T-cell immunotolerance.

Identifying Potential Plasmodium vivax Sporozoite Stage Vaccine Candidates: An Analysis of Genetic Diversity and Natural Selection.

Parasite antigen genetic diversity represents a great obstacle when designing a vaccine against malaria caused by Plasmodium vivax. Selecting vaccine candidate antigens has been focused on those fulfilling a role in invasion and which are conserved, thus avoiding specific-allele immune responses. Most antigens described to date belong to the blood stage, thereby blocking parasite development within red blood cells, whilst studying antigens from other stages has been quite restricted. Antigens from different parasite stages are required for developing a completely effective vaccine; thus, pre-erythrocyte stage antigens able to block the first line of infection becoming established should also be taken into account. However, few antigens from this stage have been studied to date. Several P. falciparum sporozoite antigens are involved in invasion. Since 77% of genes are orthologous amongst Plasmodium parasites, P. vivax sporozoite antigen orthologs to those of P. falciparum might be present in its genome. Although these genes might have high genetic diversity, conserved functionally-relevant regions (ideal for vaccine development) could be predicted by comparing genetic diversity patterns and evolutionary rates. This study was thus aimed at searching for putative P. vivax sporozoite genes so as to analyse their genetic diversity for determining their potential as vaccine candidates. Several DNA sequence polymorphism estimators were computed at each locus. The evolutionary force (drift, selection and recombination) drawing the genetic diversity pattern observed was also determined by using tests based on polymorphism frequency spectrum as well as the type of intra- and inter-species substitutions. Likewise, recombination was assessed both indirectly and directly. The results showed that sporozoite genes were more conserved than merozoite genes evaluated to date. Putative domains implied in cell traversal, gliding motility and hepatocyte interaction had a negative selection signal, being conserved amongst different species in the genus. PvP52, PvP36, PvSPATR, PvPLP1, PvMCP1, PvTLP, PvCelTOS, and PvMB2 antigens or functionally restricted regions within them would thus seem promising vaccine candidates and could be used when designing a pre-erythrocyte and/or multi-stage vaccine against P. vivax to avoid allele-specific immune responses that could reduce vaccine efficacy.

Evolutionary constraint and disease associations of post-translational modification sites in human genomes.

Interpreting the impact of human genome variation on phenotype is challenging. The functional effect of protein-coding variants is often predicted using sequence conservation and population frequency data, however other factors are likely relevant. We hypothesized that variants in protein post-translational modification (PTM) sites contribute to phenotype variation and disease. We analyzed fraction of rare variants and non-synonymous to synonymous variant ratio (Ka/Ks) in 7,500 human genomes and found a significant negative selection signal in PTM regions independent of six factors, including conservation, codon usage, and GC-content, that is widely distributed across tissue-specific genes and function classes. PTM regions are also enriched in known disease mutations, suggesting that PTM variation is more likely deleterious. PTM constraint also affects flanking sequence around modified residues and increases around clustered sites, indicating presence of functionally important short linear motifs. Using target site motifs of 124 kinases, we predict that at least ∼180,000 motif-breaker amino acid residues that disrupt PTM sites when substituted, and highlight kinase motifs that show specific negative selection and enrichment of disease mutations. We provide this dataset with corresponding hypothesized mechanisms as a community resource. As an example of our integrative approach, we propose that PTPN11 variants in Noonan syndrome aberrantly activate the protein by disrupting an uncharacterized cluster of phosphorylation sites. Further, as PTMs are molecular switches that are modulated by drugs, we study mutated binding sites of PTM enzymes in disease genes and define a drug-disease network containing 413 novel predicted disease-gene links.

Experimental evidence indicating that mastreviruses probably did not co-diverge with their hosts.

BackgroundDespite the demonstration that geminiviruses, like many other single stranded DNA viruses, are evolving at rates similar to those of RNA viruses, a recent study has suggested that grass-infecting species in the genus Mastrevirus may have co-diverged with their hosts over millions of years. This "co-divergence hypothesis" requires that long-term mastrevirus substitution rates be at least 100,000-fold lower than their basal mutation rates and 10,000-fold lower than their observable short-term substitution rates. The credibility of this hypothesis, therefore, hinges on the testable claim that negative selection during mastrevirus evolution is so potent that it effectively purges 99.999% of all mutations that occur.ResultsWe have conducted long-term evolution experiments lasting between 6 and 32 years, where we have determined substitution rates of between 2 and 3 × 10-4 substitutions/site/year for the mastreviruses Maize streak virus (MSV) and Sugarcane streak Réunion virus (SSRV). We further show that mutation biases are similar for different geminivirus genera, suggesting that mutational processes that drive high basal mutation rates are conserved across the family. Rather than displaying signs of extremely severe negative selection as implied by the co-divergence hypothesis, our evolution experiments indicate that MSV and SSRV are predominantly evolving under neutral genetic drift.ConclusionThe absence of strong negative selection signals within our evolution experiments and the uniformly high geminivirus substitution rates that we and others have reported suggest that mastreviruses cannot have co-diverged with their hosts.

Characterizing Thymocyte Migration in the Medulla Using Two-Photon Microscopy (138.20)

Abstract To establish tolerance, a developing thymocyte must be screened for self-reactivity before exiting the thymus. However, the mechanism by which a thymocyte searches the medulla and antigen-presenting cells for self-antigens is not clear. We have developed a cut thymic lobe preparation that allows us to document thymocyte migration within the medulla by two-photon laser scanning microscopy. Medullary thymocytes migrate rapidly through confined random walk making short and dynamic contacts with dendritic cells. In response to negative selection signals, thymocyte migration is significantly slower and constrained to smaller areas. In addition, self-reactive thymocytes engage in long lasting contacts with dendritic cells. Polyclonal medullary thymocytes consist of a small but significant population of relatively slow thymocytes with more constrained migration that may correspond to auto-reactive thymocytes undergoing negative selection. Taken together, our data defines scanning strategies used by medullary thymocytes during negative selection and suggests that a significant proportion of medullary thymocytes are actively receiving negative selecting signals.

Thymocyte Negative Selection Is Mediated by Protein Kinase C- and Ca2+-Dependent Transcriptional Induction of Bim

The processes of positive and negative selection in the thymus both determine the population of T cells that will enter the peripheral immune system and eliminate self-reactive T cells by apoptosis. Substantial evidence indicates that TCR signal intensity mediates this cell fate choice: low-intensity signals lead to survival and differentiation, whereas high-intensity signals generated by self-Ag lead to cell death. The molecular mechanism by which these graded signals are converted to discrete outcomes is not understood. Positive selection requires the Ca(2+)-dependent phosphatase calcineurin, whereas negative selection requires the proapoptotic Bcl-2 family member Bcl-2-interacting mediator of cell death (Bim). In this study, we investigated the regulation of Bim expression and the role of Ca(2+) in mediating negative selection. Our results show that transcription is necessary for both negative selection and Bim induction. Surprisingly, we also found that Ca(2+) is necessary for Bim induction. Induction of bim transcription appears to involve protein kinase C, but not calcineurin, JNK, p38 MAPK, or MEK. These results localize the decision point in positive vs negative selection to a step downstream of Ca(2+) signaling and suggest that negative selection signals induce Ca(2+)-dependent bim transcription through PKC.

Regulation of Bim by TCR Signals in CD4/CD8 Double-Positive Thymocytes

Bim, a BH3-only Bcl-2 family member, is required for apoptosis of thymocytes in response to negative selection signals. Regulation of the apoptotic activity of Bim during negative selection is not understood. In this study we demonstrate that in murine thymocytes undergoing apoptosis in response to anti-CD3epsilon injection, levels of Bim protein expression do not change. In immature thymocytes, Bim is associated with mitochondria before stimulation and is not regulated by a change in subcellular localization during apoptosis. We also show that Bim(EL) is rapidly phosphorylated in thymocytes in response to CD3epsilon cross-linking both in vivo and in vitro, and that phosphorylation is sustained for at least 24 h. Analysis of MHC-deficient mice shows that phosphorylation of Bim occurs in CD4/CD8 double-positive thymocytes and does not depend on activation of mature T cells. We also find that TCR cross-linking on thymocytes induces an increase in the proportion of Bcl-x(L) bound to Bim at late time points. Our results favor a model in which strong TCR signals regulate the apoptotic activity of Bim by phosphorylation and subsequent changes in binding to Bcl-x(L) in immature thymocytes.

Receptor editing in positive and negative selection of B lymphopoiesis.

In B lymphopoiesis, Ag receptor expression and signaling are critical to determine developmental progression, survival, and activation. Several positive and negative selection checkpoints to test this receptor have been described in B lymphopoiesis, aiming to ensure the generation of functionally competent, nonautoimmune repertoire. Secondary Ag receptor gene recombination allows B lymphocytes to replace an inappropriate receptor with a new receptor, a mechanism called receptor editing. This salvage mechanism uncouples the Ag receptor fate from that of the cell itself, suggesting that B cell repertoire is regulated by a process of receptor selection. Secondary rearrangements are stimulated in different stages of B cell development, where editing of the receptor is necessary to fulfill stage-specific requirements. In this study, we discuss the contribution of receptor editing in B lymphopoiesis and its regulation by positive and negative selection signals.

BCL-XL-regulated apoptosis in T cell development.

Thymocyte differentiation progresses through well-defined stages in which apoptosis is central to the selection of a functional TCR repertoire. In the present study, we explored the developmental effects of BCL-XL, a repressor of apoptosis. We found that endogenous BCL-XL is down-regulated by both positive and negative selection signals at the CD4+ CD8+ stage of thymocyte development. We examined the role of BCL-XL regulatable apoptosis in T cell development in the context of an alpha beta TCR transgene, Rag-1 deficiency and the scid model. We found that BCL-XL expression promoted accumulation of CD8 single-positive thymocytes even in MHC class II-restricted TCR transgenic mice. However, the apoptotic resistance conferred by BCL-XL could not fully substitute for TCR-mediated positive selection signals nor did it prevent negative selection. Overexpression of BCL-XL promoted partial maturation of CD4-CD8- thymocytes to CD4+CD8+ cells in a Rag-deficient, but not a scid background. Thus, TCR-mediated signals mediate the regulation of endogenous BCL-XL during thymocyte development, indicating that the principal protection of double-positive thymocytes by BCL-XL occurs prior to selection. The impact of BCL-XL on the CD8 but not CD4 lineage supports the asymmetric model of lineage commitment. Moreover, several critical control points in T cell development could be distinguished as BCL-XL responsive or unresponsive.

Defective depletion of CD45-null thymocytes by the Staphylococcus aureus Enterotoxin B superantigen

The development of a normal T-cell repertoire is critically dependent on the negative and positive selection events which occur at the CD4 +CD8 + (double positive, DP) stage of thymic development. Depending on the avidity of the T-cell antigen receptor (TCR) for peptides presented within the thymus, DP thymocytes are either positively selected for maturation to CD4 +CD8 + single positive cells or are depleted by apoptosis. The addition of superantigen to thymocytes within foetal thymic organ culture (FTOC) mimics the negative selection signal of potentially autoreactive thymocytes and induces the responding population of thymocytes to apoptose. Here we present evidence that the transmembrane phosphotyrosine phosphatase CD45 critically regulates TCR-induced signals in thymic differentiation and present data to show defective depletion of CD45-null transgenic TCR-Vβ8 DP thymocytes in FTOC by the Staphylococcus aureus Enterotoxin B (SEB) superantigen.