Substitution Models of Protein Evolution with Selection on Enzymatic Activity.

Substitution models of evolution are necessary for diverse evolutionary analyses including phylogenetic tree and ancestral sequence reconstructions. At the protein level, empirical substitution models are traditionally used due to their simplicity, but they ignore the variability of substitution patterns among protein sites. Next, in order to improve the realism of the modeling of protein evolution, a series of structurally constrained substitution models were presented, but still they usually ignore constraints on the protein activity. Here, we present a substitution model of protein evolution with selection on both protein structure and enzymatic activity, and that can be applied to phylogenetics. In particular, the model considers the binding affinity of the enzyme-substrate complex as well as structural constraints that include the flexibility of structural flaps, hydrogen bonds, amino acids backbone radius of gyration, and solvent-accessible surface area that are quantified through molecular dynamics simulations. We applied the model to the HIV-1 protease and evaluated it by phylogenetic likelihood in comparison with the best-fitting empirical substitution model and a structurally constrained substitution model that ignores the enzymatic activity. We found that accounting for selection on the protein activity improves the fitting of the modeled functional regions with the real observations, especially in data with high molecular identity, which recommends considering constraints on the protein activity in the development of substitution models of evolution.

Selection among site-dependent structurally constrained substitution models of protein evolution by approximate Bayesian computation.

MOTIVATION: The selection among substitution models of molecular evolution is fundamental for obtaining accurate phylogenetic inferences. At the protein level, evolutionary analyses are traditionally based on empirical substitution models but these models make unrealistic assumptions and are being surpassed by structurally constrained substitution (SCS) models. The SCS models often consider site-dependent evolution, a process that provides realism but complicates their implementation into likelihood functions that are commonly used for substitution model selection. RESULTS: We present a method to perform selection among site-dependent SCS models, also among empirical and site-dependent SCS models, based on the approximate Bayesian computation (ABC) approach and its implementation into the computational framework ProteinModelerABC. The framework implements ABC with and without regression adjustments and includes diverse empirical and site-dependent SCS models of protein evolution. Using extensive simulated data, we found that it provides selection among SCS and empirical models with acceptable accuracy. As illustrative examples, we applied the framework to analyze a variety of protein families observing that SCS models fit them better than the corresponding best-fitting empirical substitution models. AVAILABILITY AND IMPLEMENTATION: ProteinModelerABC is freely available from https://github.com/DavidFerreiro/ProteinModelerABC, can run in parallel and includes a graphical user interface. The framework is distributed with detailed documentation and ready-to-use examples.

Genomic and phenotypic analysis of invasive Streptococcus suis isolated in Spain reveals genetic diversification and associated virulence traits.

Streptococcus suis is a zoonotic pathogen that causes a major health problem in the pig production industry worldwide. Spain is one of the largest pig producers in the world. This work aimed to investigate the genetic and phenotypic features of invasive S. suis isolates recovered in Spain. A panel of 156 clinical isolates recovered from 13 Autonomous Communities, representing the major pig producers, were analysed. MLST and serotyping analysis revealed that most isolates (61.6%) were assigned to ST1 (26.3%), ST123 (18.6%), ST29 (9.6%), and ST3 (7.1%). Interestingly, 34 new STs were identified, indicating the emergence of novel genetic lineages. Serotypes 9 (27.6%) and 1 (21.8%) prevailed, followed by serotypes 7 (12.8%) and 2 (12.2%). Analysis of 13 virulence-associated genes showed significant associations between ST, serotype, virulence patterns, and clinical features, evidencing particular virulence traits associated with genetic clusters. The pangenome was generated, and the core genome was distributed in 7 Bayesian groups where each group included a variable set of over- and under-represented genes of different categories. The study provides comprehensive data and knowledge to improve the design of new vaccines, antimicrobial treatments, and bacterial typing approaches.

Evolutionary dynamics of the human pseudoautosomal regions.

Recombination between the X and Y human sex chromosomes is limited to the two pseudoautosomal regions (PARs) that present quite distinct evolutionary origins. Despite the crucial importance for male meiosis, genetic diversity patterns and evolutionary dynamics of these regions are poorly understood. In the present study, we analyzed and compared the genetic diversity of the PAR regions using publicly available genomic sequences encompassing both PAR1 and PAR2. Comparisons were performed through allele diversities, linkage disequilibrium status and recombination frequencies within and between X and Y chromosomes. In agreement with previous studies, we confirmed the role of PAR1 as a male-specific recombination hotspot, but also observed similar characteristic patterns of diversity in both regions although male recombination occurs at PAR2 to a much lower extent (at least one recombination event at PAR1 and in ≈1% in normal male meioses at PAR2). Furthermore, we demonstrate that both PARs harbor significantly different allele frequencies between X and Y chromosomes, which could support that recombination is not sufficient to homogenize the pseudoautosomal gene pool or is counterbalanced by other evolutionary forces. Nevertheless, the observed patterns of diversity are not entirely explainable by sexually antagonistic selection. A better understanding of such processes requires new data from intergenerational transmission studies of PARs, which would be decisive on the elucidation of PARs evolution and their role in male-driven heterosomal aneuploidies.

Consequences of Substitution Model Selection on Protein Ancestral Sequence Reconstruction.

The selection of the best-fitting substitution model of molecular evolution is a traditional step for phylogenetic inferences, including ancestral sequence reconstruction (ASR). However, a few recent studies suggested that applying this procedure does not affect the accuracy of phylogenetic tree reconstruction. Here, we revisited this debate topic by analyzing the influence of selection among substitution models of protein evolution, with focus on exchangeability matrices, on the accuracy of ASR using simulated and real data. We found that the selected best-fitting substitution model produces the most accurate ancestral sequences, especially if the data present large genetic diversity. Indeed, ancestral sequences reconstructed under substitution models with similar exchangeability matrices were similar, suggesting that if the selected best-fitting model cannot be used for the reconstruction, applying a model similar to the selected one is preferred. We conclude that selecting among substitution models of protein evolution is recommended for reconstructing accurate ancestral sequences.

Evolution of TOP1 and TOP1MT Topoisomerases in Chordata.

Type IB topoisomerases relax the torsional stress associated with DNA metabolism in the nucleus and mitochondria and constitute important molecular targets of anticancer drugs. Vertebrates stand out among eukaryotes by having two Type IB topoisomerases acting specifically in the nucleus (TOP1) and mitochondria (TOP1MT). Despite their major importance, the origin and evolution of these paralogues remain unknown. Here, we examine the molecular evolutionary processes acting on both TOP1 and TOP1MT in Chordata, taking advantage of the increasing number of available genome sequences. We found that both TOP1 and TOP1MT evolved under strong purifying selection, as expected considering their essential biological functions. Critical active sites, including those associated with resistance to anticancer agents, were found particularly conserved. However, TOP1MT presented a higher rate of molecular evolution than TOP1, possibly related with its specialized activity on the mitochondrial genome and a less critical role in cells. We could place the duplication event that originated the TOP1 and TOP1MT paralogues early in the radiation of vertebrates, most likely associated with the first round of vertebrate tetraploidization (1R). Moreover, our data suggest that cyclostomes present a specialized mitochondrial Type IB topoisomerase. Interestingly, we identified two missense mutations replacing amino acids in the Linker region of TOP1MT in Neanderthals, which appears as a rare event when comparing the genome of both species. In conclusion, TOP1 and TOP1MT differ in their rates of evolution, and their evolutionary histories allowed us to better understand the evolution of chordates.

Consequences of Genetic Recombination on Protein Folding Stability.

Genetic recombination is a common evolutionary mechanism that produces molecular diversity. However, its consequences on protein folding stability have not attracted the same attention as in the case of point mutations. Here, we studied the effects of homologous recombination on the computationally predicted protein folding stability for several protein families, finding less detrimental effects than we previously expected. Although recombination can affect multiple protein sites, we found that the fraction of recombined proteins that are eliminated by negative selection because of insufficient stability is not significantly larger than the corresponding fraction of proteins produced by mutation events. Indeed, although recombination disrupts epistatic interactions, the mean stability of recombinant proteins is not lower than that of their parents. On the other hand, the difference of stability between recombined proteins is amplified with respect to the parents, promoting phenotypic diversity. As a result, at least one third of recombined proteins present stability between those of their parents, and a substantial fraction have higher or lower stability than those of both parents. As expected, we found that parents with similar sequences tend to produce recombined proteins with stability close to that of the parents. Finally, the simulation of protein evolution along the ancestral recombination graph with empirical substitution models commonly used in phylogenetics, which ignore constraints on protein folding stability, showed that recombination favors the decrease of folding stability, supporting the convenience of adopting structurally constrained models when possible for inferences of protein evolutionary histories with recombination.

Analysis of selection in protein-coding sequences accounting for common biases.

The evolution of protein-coding genes is usually driven by selective processes, which favor some evolutionary trajectories over others, optimizing the subsequent protein stability and activity. The analysis of selection in this type of genetic data is broadly performed with the metric nonsynonymous/synonymous substitution rate ratio (dN/dS). However, most of the well-established methodologies to estimate this metric make crucial assumptions, such as lack of recombination or invariable codon frequencies along genes, which can bias the estimation. Here, we review the most relevant biases in the dN/dS estimation and provide a detailed guide to estimate this metric using state-of-the-art procedures that account for such biases, along with illustrative practical examples and recommendations. We also discuss the traditional interpretation of the estimated dN/dS emphasizing the importance of considering complementary biological information such as the role of the observed substitutions on the stability and function of proteins. This review is oriented to help evolutionary biologists that aim to accurately estimate selection in protein-coding sequences.

Immunoescape of HIV-1 in Env-EL9 CD8 + T cell response restricted by HLA-B*14:02 in a Non progressor who lost twenty-seven years of HIV-1 control.

BACKGROUND: Long-Term Non-Progressors (LTNPs) are untreated Human Immunodeficiency virus type 1 (HIV-1) infected individuals able to control disease progression for prolonged periods. However, the LTNPs status is temporary, as viral load increases followed by decreases in CD4 + T-cell counts. Control of HIV-1 infection in LTNPs viremic controllers, have been associated with effective immunodominant HIV-1 Gag-CD8 + T-cell responses restricted by protective HLA-B alleles. Individuals carrying HLA-B*14:02 control HIV-1 infection is related to an immunodominant Env-CD8 + T-cell response. Limited data are available on the contribution of HLA-B*14:02 CD8 + T -cells in LTNPs. RESULTS: In this study, we performed a virological and immunological detailed analysis of an HLA-B*14:02 LNTP individual that lost viral control (LVC) 27 years after HIV-1 diagnosis. We analysed viral evolution and immune escape in HLA-B*14:02 restricted CD8 + T -cell epitopes and identified viral evolution at the Env-EL9 epitope selecting the L592R mutation. By IFN-γ ELISpot and immune phenotype, we characterized HLA- B*14:02 HIV-1 CD8 + T cell responses targeting, Gag-DA9 and Env-EL9 epitopes before and after LVC. We observed an immunodominant response against the Env-EL9 epitope and a decreased of the CD8 T + cell response over time with LVC. Loss of Env-EL9 responses was concomitant with selecting K588R + L592R mutations at Env-EL9. Finally, we evaluated the impact of Env-EL9 escape mutations on HIV-1 infectivity and Env protein structure. The K588R + L592R escape variant was directly related to HIV-1 increase replicative capacity and stability of Env at the LVC. CONCLUSIONS: These findings support the contribution of immunodominant Env-EL9 CD8 + T-cell responses and the imposition of immune escape variants with higher replicative capacity associated with LVC in this LNTP. These data highlight the importance of Env-EL9 specific-CD8 + T-cell responses restricted by the HLA-B*14:02 and brings new insights into understanding long-term HIV-1 control mediated by Env mediated CD8 + T-cell responses.

Evolutionary History of TOPIIA Topoisomerases in Animals.

TOPIIA topoisomerases are required for the regulation of DNA topology by DNA cleavage and re-ligation and are important targets of antibiotic and anticancer agents. Humans possess two TOPIIA paralogue genes (TOP2A and TOP2B) with high sequence and structural similarity but distinct cellular functions. Despite their functional and clinical relevance, the evolutionary history of TOPIIA is still poorly understood. Here we show that TOPIIA is highly conserved in Metazoa. We also found that TOPIIA paralogues from jawed and jawless vertebrates had different origins related with tetraploidization events. After duplication, TOP2B evolved under a stronger purifying selection than TOP2A, perhaps promoted by the more specialized role of TOP2B in postmitotic cells. We also detected genetic signatures of positive selection in the highly variable C-terminal domain (CTD), possibly associated with adaptation to cellular interactions. By comparing TOPIIA from modern and archaic humans, we found two amino acid substitutions in the TOP2A CTD, suggesting that TOP2A may have contributed to the evolution of present-day humans, as proposed for other cell cycle-related genes. Finally, we identified six residues conferring resistance to chemotherapy differing between TOP2A and TOP2B. These six residues could be targets for the development of TOP2A-specific inhibitors that would avoid the side effects caused by inhibiting TOP2B. Altogether, our findings clarify the origin, diversification and selection pressures governing the evolution of animal TOPIIA.

ProteinEvolverABC: coestimation of recombination and substitution rates in protein sequences by approximate Bayesian computation.

MOTIVATION: The evolutionary processes of mutation and recombination, upon which selection operates, are fundamental to understand the observed molecular diversity. Unlike nucleotide sequences, the estimation of the recombination rate in protein sequences has been little explored, neither implemented in evolutionary frameworks, despite protein sequencing methods are largely used. RESULTS: In order to accommodate this need, here I present a computational framework, called ProteinEvolverABC, to jointly estimate recombination and substitution rates from alignments of protein sequences. The framework implements the approximate Bayesian computation approach, with and without regression adjustments and includes a variety of substitution models of protein evolution, demographics and longitudinal sampling. It also implements several nuisance parameters such as heterogeneous amino acid frequencies and rate of change among sites and, proportion of invariable sites. The framework produces accurate coestimation of recombination and substitution rates under diverse evolutionary scenarios. As illustrative examples of usage, I applied it to several viral protein families, including coronaviruses, showing heterogeneous substitution and recombination rates. AVAILABILITY AND IMPLEMENTATION: ProteinEvolverABC is freely available from https://github.com/miguelarenas/proteinevolverabc, includes a graphical user interface for helping the specification of the input settings, extensive documentation and ready-to-use examples. Conveniently, the simulations can run in parallel on multicore machines. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Molecular Evolution of DNA Topoisomerase III Beta (TOP3B) in Metazoa.

DNA topoisomerase III beta (TOP3B) is unique by operating on both DNA and RNA substrates to regulate gene expression and genomic stability. Mutations in human TOP3B are linked to neurodevelopmental and cognitive disorders, highlighting its relevance for human health. Despite the emerging importance of TOP3B, its precise cellular functions and evolutionary history remain poorly understood. Here, we show that TOP3B is conserved across main metazoan groups and evolved under strong purifying selection. Subdomain IV was identified as the most conserved TOP3B region, in agreement with its role in providing the structural foundation of the protein. On the contrary, subdomain II is the less conserved, possibly because it is the most structurally flexible region of all TOP3B regions. Interestingly, TOP3B residue at position 472, previously associated with schizophrenia, is highly variable across animals, suggesting a more specific role in humans and related species. Finally, we show that all TOP3B CXXC zinc finger motifs previously identified at the protein C-terminal region are retained across metazoans. We also found that the two major methylation sites known to regulate TOP3B activity are located in the most conserved region of the C-terminal arginine-glycine-glycine (RGG) box, suggesting that a similar regulatory mechanism may operate throughout animals. Overall, our results provide a better understanding of the evolution and functional roles of TOP3B.

The influence of heterogeneous codon frequencies along sequences on the estimation of molecular adaptation.

MOTIVATION: The nonsynonymous/synonymous substitution rate ratio (dN/dS) is a commonly used parameter to quantify molecular adaptation in protein-coding data. It is known that the estimation of dN/dS can be biased if some evolutionary processes are ignored. In this concern, common ML methods to estimate dN/dS assume invariable codon frequencies among sites, despite this characteristic is rare in nature, and it could bias the estimation of this parameter. RESULTS: Here we studied the influence of variable codon frequencies among genetic regions on the estimation of dN/dS. We explored scenarios varying the number of genetic regions that differ in codon frequencies, the amount of variability of codon frequencies among regions and the nucleotide frequencies at each codon position among regions. We found that ignoring heterogeneous codon frequencies among regions overall leads to underestimation of dN/dS and the bias increases with the level of heterogeneity of codon frequencies. Interestingly, we also found that varying nucleotide frequencies among regions at the first or second codon position leads to underestimation of dN/dS while variation at the third codon position leads to overestimation of dN/dS. Next, we present a methodology to reduce this bias based on the analysis of partitions presenting similar codon frequencies and we applied it to analyze four real datasets. We conclude that accounting for heterogeneous codon frequencies along sequences is required to obtain realistic estimates of molecular adaptation through this relevant evolutionary parameter. AVAILABILITY AND IMPLEMENTATION: The applied frameworks for the computer simulations of protein-coding data and estimation of molecular adaptation are SGWE and PAML, respectively. Both are publicly available and referenced in the study. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Protein Evolution in the Flaviviruses.

Proteins are commonly used as molecular targets against pathogens such as viruses and bacteria. However, pathogens can evolve rapidly permitting their populations to increase in protein diversity over time and thus escape to the activity of a molecular therapy. Subsequently, in order to design more durable and robust therapies as well as to understand viral evolution in a host and subsequent transmission, it is central to understand the evolution of pathogen proteins. This understanding can enable the detection of protein regions that can be potential targets for therapies and predict the emergence of molecular resistance against therapies. In this direction, two articles published recently in the Journal of Molecular Evolution investigated the evolution of proteomes of diverse flaviviruses, including Zika virus, Dengue virus and West Nile virus. Here I discuss the importance of considering the evolution of viral proteins, with the use of as realistic as possible models and methods that mimic protein evolution, to improve the design of antiviral therapies.

SPLATCHE3: simulation of serial genetic data under spatially explicit evolutionary scenarios including long-distance dispersal.

SUMMARY: SPLATCHE3 simulates genetic data under a variety of spatially explicit evolutionary scenarios, extending previous versions of the framework. The new capabilities include long-distance migration, spatially and temporally heterogeneous short-scale migrations, alternative hybridization models, simulation of serial samples of genetic data and a large variety of DNA mutation models. These implementations have been applied independently to various studies, but grouped together in the current version. AVAILABILITY AND IMPLEMENTATION: SPLATCHE3 is written in C++ and is freely available for non-commercial use from the website http://www.splatche.com/splatche3. It includes console versions for Linux, MacOs and Windows and a user-friendly GUI for Windows, as well as detailed documentation and ready-to-use examples.

Influence of Paleolithic range contraction, admixture and long-distance dispersal on genetic gradients of modern humans in Asia.

Cavalli-Sforza and coauthors originally explored the genetic variation of modern humans throughout the world and observed an overall east-west genetic gradient in Asia. However, the specific environmental and population genetics processes causing this gradient were not formally investigated and promoted discussion in recent studies. Here we studied the influence of diverse environmental and population genetics processes on Asian genetic gradients and identified which could have produced the observed gradient. To do so, we performed extensive spatially-explicit computer simulations of genetic data under the following scenarios: (a) variable levels of admixture between Paleolithic and Neolithic populations, (b) migration through long-distance dispersal (LDD), (c) Paleolithic range contraction induced by the last glacial maximum (LGM), and (d) Neolithic range expansions from one or two geographic origins (the Fertile Crescent and the Yangzi and Yellow River Basins). Next, we estimated genetic gradients from the simulated data and we found that they were sensible to the analysed processes, especially to the range contraction induced by LGM and to the number of Neolithic expansions. Some scenarios were compatible with the observed east-west genetic gradient, such as the Paleolithic expansion with a range contraction induced by the LGM or two Neolithic range expansions from both the east and the west. In general, LDD increased the variance of genetic gradients among simulations. We interpreted the obtained gradients as a consequence of both allele surfing caused by range expansions and isolation by distance along the vast east-west geographic axis of this continent.

The Molecular Clock in the Evolution of Protein Structures.

The molecular clock hypothesis, which states that substitutions accumulate in protein sequences at a constant rate, plays a fundamental role in molecular evolution but it is violated when selective or mutational processes vary with time. Such violations of the molecular clock have been widely investigated for protein sequences, but not yet for protein structures. Here, we introduce a novel statistical test (Significant Clock Violations) and perform a large scale assessment of the molecular clock in the evolution of both protein sequences and structures in three large superfamilies. After validating our method with computer simulations, we find that clock violations are generally consistent in sequence and structure evolution, but they tend to be larger and more significant in structure evolution. Moreover, changes of function assessed through Gene Ontology and InterPro terms are associated with large and significant clock violations in structure evolution. We found that almost one third of significant clock violations are significant in structure evolution but not in sequence evolution, highlighting the advantage to use structure information for assessing accelerated evolution and gathering hints of positive selection. Clock violations between closely related pairs are frequently significant in sequence evolution, consistent with the observed time dependence of the substitution rate attributed to segregation of neutral and slightly deleterious polymorphisms, but not in structure evolution, suggesting that these substitutions do not affect protein structure although they may affect stability. These results are consistent with the view that natural selection, both negative and positive, constrains more strongly protein structures than protein sequences. Our code for computing clock violations is freely available at https://github.com/ugobas/Molecular_clock.

Forensic genetics and genomics: Much more than just a human affair.

While traditional forensic genetics has been oriented towards using human DNA in criminal investigation and civil court cases, it currently presents a much wider application range, including not only legal situations sensu stricto but also and, increasingly often, to preemptively avoid judicial processes. Despite some difficulties, current forensic genetics is progressively incorporating the analysis of nonhuman genetic material to a greater extent. The analysis of this material-including other animal species, plants, or microorganisms-is now broadly used, providing ancillary evidence in criminalistics in cases such as animal attacks, trafficking of species, bioterrorism and biocrimes, and identification of fraudulent food composition, among many others. Here, we explore how nonhuman forensic genetics is being revolutionized by the increasing variety of genetic markers, the establishment of faster, less error-burdened and cheaper sequencing technologies, and the emergence and improvement of models, methods, and bioinformatics facilities.

Survey of Crimean-Congo Hemorrhagic Fever Enzootic Focus, Spain, 2011-2015.

During 2011-2015, we conducted a Crimean-Congo hemorrhagic fever virus (CCHFV) survey in captured ticks that were feeding mainly on wild and domestic ungulates in Spain, where presence of this virus had been reported previously. We detected CCHFV RNA in Hyalomma lusitanicum and H. marginatum ticks for 3 of the 5 years. The rate of infected ticks was 2.78% (44/1,579), which was similar to those for other countries in Europe with endemic foci for CCHFV (Kosovo, Bulgaria, and Albania). These data confirm the established spread of CCHFV into western Europe. Phylogenetic study of the small RNA segment showed Africa-3 clade as the only genotype identified, although we observed cocirculation of genetic variants during 2011 and 2015. We could not rule out genetic reassortments because of lack of sequence data for the medium and large RNA segments of the virus genome.

Substitution Rates Predicted by Stability-Constrained Models of Protein Evolution Are Not Consistent with Empirical Data.

Protein structures strongly influence molecular evolution. In particular, the evolutionary rate of a protein site depends on the number of its native contacts. Stability-constrained models of protein evolution consider this influence of protein structure on evolution by predicting the effect of mutations on the stability of the native state, but they currently neglect how mutations affect the protein structure. These models predict that buried protein sites with more native contacts are more constrained by natural selection and less variable, as observed. Nevertheless, previous work did not consider the stability against compact misfolded conformations, although it is known that the negative design that destabilizes these misfolded conformations influences protein evolution significantly. Here, we show that stability-constrained models that consider misfolding predict that site-specific sequence entropy and substitution rate peak at amphiphilic sites with an intermediate number of contacts, as these sites are less constrained than exposed sites with few contacts whose hydrophobicity must be limited. This result holds both for a mean-field model with independent sites and for a pairwise model that takes as a reference the wild-type sequence, but it contrasts with the observations that indicate that the entropy and the substitution rate decrease monotonically with the number of contacts. Our work suggests that stability-constrained models overestimate the tolerance of amphiphilic sites against mutations, either because of the limits of the free energy function or, more importantly in our opinion, because they do not consider how mutations perturb the native protein structure.