Peter K. Vlasov

Epistasis as the primary factor in molecular evolution.

The main forces directing long-term molecular evolution remain obscure. A sizable fraction of amino-acid substitutions seem to be fixed by positive selection, but it is unclear to what degree long-term protein evolution is constrained by epistasis, that is, instances when substitutions that are accepted in one genotype are deleterious in another. Here we obtain a quantitative estimate of the prevalence of epistasis in long-term protein evolution by relating data on amino-acid usage in 14 organelle proteins and 2 nuclear-encoded proteins to their rates of short-term evolution. We studied multiple alignments of at least 1,000 orthologues for each of these 16 proteins from species from a diverse phylogenetic background and found that an average site contained approximately eight different amino acids. Thus, without epistasis an average site should accept two-fifths of all possible amino acids, and the average rate of amino-acid substitutions should therefore be about three-fifths lower than the rate of neutral evolution. However, we found that the measured rate of amino-acid substitution in recent evolution is 20 times lower than the rate of neutral evolution and an order of magnitude lower than that expected in the absence of epistasis. These data indicate that epistasis is pervasive throughout protein evolution: about 90 per cent of all amino-acid substitutions have a neutral or beneficial impact only in the genetic backgrounds in which they occur, and must therefore be deleterious in a different background of other species. Our findings show that most amino-acid substitutions have different fitness effects in different species and that epistasis provides the primary conceptual framework to describe the tempo and mode of long-term protein evolution.

Local fitness landscape of the green fluorescent protein.

Fitness landscapes depict how genotypes manifest at the phenotypic level and form the basis of our understanding of many areas of biology, yet their properties remain elusive. Previous studies have analysed specific genes, often using their function as a proxy for fitness, experimentally assessing the effect on function of single mutations and their combinations in a specific sequence or in different sequences. However, systematic high-throughput studies of the local fitness landscape of an entire protein have not yet been reported. Here we visualize an extensive region of the local fitness landscape of the green fluorescent protein from Aequorea victoria (avGFP) by measuring the native function (fluorescence) of tens of thousands of derivative genotypes of avGFP. We show that the fitness landscape of avGFP is narrow, with 3/4 of the derivatives with a single mutation showing reduced fluorescence and half of the derivatives with four mutations being completely non-fluorescent. The narrowness is enhanced by epistasis, which was detected in up to 30% of genotypes with multiple mutations and mostly occurred through the cumulative effect of slightly deleterious mutations causing a threshold-like decrease in protein stability and a concomitant loss of fluorescence. A model of orthologous sequence divergence spanning hundreds of millions of years predicted the extent of epistasis in our data, indicating congruence between the fitness landscape properties at the local and global scales. The characterization of the local fitness landscape of avGFP has important implications for several fields including molecular evolution, population genetics and protein design.

Conversion and Compensatory Evolution of the γ-Crystallin Genes and Identification of a Cataractogenic Mutation That Reverses the Sequence of the Human CRYGD Gene to an Ancestral State

We identified a mutation in the CRYGD gene (P23S) of the gamma-crystallin gene cluster that is associated with a polymorphic congenital cataract that occurs with frequency of approximately 0.3% in a human population. To gain insight into the molecular mechanism of the pathogenesis of gamma-crystallin isoforms, we undertook an evolutionary analysis of the available mammalian and newly obtained primate sequences of the gamma-crystallin genes. The cataract-associated serine at site 23 corresponds to the ancestral state, since it was found in CRYGD of a lower primate and all the surveyed nonprimate mammals. Crystallin proteins include two structurally similar domains, and substitutions in mammalian CRYGD protein at site 23 of the first domain were always associated with substitutions in the structurally reciprocal sites 109 and 136 of the second domain. These data suggest that the cataractogenic effect of serine at site 23 in the N-terminal domain of CRYGD may be compensated indirectly by amino acid changes in a distal domain. We also found that gene conversion was a factor in the evolution of the gamma-crystallin gene cluster throughout different mammalian clades. The high rate of gene conversion observed between the functional CRYGD gene and two primate gamma-crystallin pseudogenes (CRYGEP1 and CRYGFP1) coupled with a surprising finding of apparent negative selection in primate pseudogenes suggest a deleterious impact of recently derived pseudogenes involved in gene conversion in the gamma-crystallin gene cluster.

Anti-leprosy drug clofazimine inhibits growth of triple-negative breast cancer cells via inhibition of canonical Wnt signaling.

Research on existing drugs often discovers novel mechanisms of their action and leads to the expansion of their therapeutic scope and subsequent remarketing. The Wnt signaling pathway is of the immediate therapeutic relevance, as it plays critical roles in cancer development and progression. However, drugs which disrupt this pathway are unavailable despite the high demand. Here we report an attempt to identify antagonists of the Wnt-FZD interaction among the library of the FDA-approved drugs. We performed an in silico screening which brought up several potential antagonists of the ligand-receptor interaction. 14 of these substances were tested using the TopFlash luciferase reporter assay and four of them identified as active and specific inhibitors of the Wnt3a-induced signaling. However, further analysis through GTP-binding and β-catenin stabilization assays showed that the compounds do not target the Wnt-FZD pair, but inhibit the signaling at downstream levels. We further describe the previously unknown inhibitory activity of an anti-leprosy drug clofazimine in the Wnt pathway and provide data demonstrating its efficiency in suppressing growth of Wnt-dependent triple-negative breast cancer cells. These data provide a basis for further investigations of the efficiency of clofazimine in treatment of Wnt-dependent cancers.

Stop codons in bacteria are not selectively equivalent

BackgroundThe evolution and genomic stop codon frequencies have not been rigorously studied with the exception of coding of non-canonical amino acids. Here we study the rate of evolution and frequency distribution of stop codons in bacterial genomes.ResultsWe show that in bacteria stop codons evolve slower than synonymous sites, suggesting the action of weak negative selection. However, the frequency of stop codons relative to genomic nucleotide content indicated that this selection regime is not straightforward. The frequency of TAA and TGA stop codons is GC-content dependent, with TAA decreasing and TGA increasing with GC-content, while TAG frequency is independent of GC-content. Applying a formal, analytical model to these data we found that the relationship between stop codon frequencies and nucleotide content cannot be explained by mutational biases or selection on nucleotide content. However, with weak nucleotide content-dependent selection on TAG, -0.5 < Nes < 1.5, the model fits all of the data and recapitulates the relationship between TAG and nucleotide content. For biologically plausible rates of mutations we show that, in bacteria, TAG stop codon is universally associated with lower fitness, with TAA being the optimal for G-content < 16% while for G-content > 16% TGA has a higher fitness than TAG.ConclusionsOur data indicate that TAG codon is universally suboptimal in the bacterial lineage, such that TAA is likely to be the preferred stop codon for low GC content while the TGA is the preferred stop codon for high GC content. The optimization of stop codon usage may therefore be useful in genome engineering or gene expression optimization applications.ReviewersThis article was reviewed by Michail Gelfand, Arcady Mushegian and Shamil Sunyaev. For the full reviews, please go to the Reviewers’ Comments section.

Translation termination in pyrrolysine-utilizing archaea

Although some data link archaeal and eukaryotic translation, the overall mechanism of protein synthesis in archaea remains largely obscure. Both archaeal (aRF1) and eukaryotic (eRF1) single release factors recognize all three stop codons. The archaeal genus Methanosarcinaceae contains two aRF1 homologs, and also uses the UAG stop to encode the 22nd amino acid, pyrrolysine. Here we provide an analysis of the last stage of archaeal translation in pyrrolysine‐utilizing species. We demonstrated that only one of two Methanosarcina barkeri aRF1 homologs possesses activity and recognizes all three stop codons. The second aRF1 homolog may have another unknown function. The mechanism of pyrrolysine incorporation in the Methanosarcinaceae is discussed.

A Model of Substitution Trajectories in Sequence Space and Long-Term Protein Evolution

The nature of factors governing the tempo and mode of protein evolution is a fundamental issue in evolutionary biology. Specifically, whether or not interactions between different sites, or epistasis, are important in directing the course of evolution became one of the central questions. Several recent reports have scrutinized patterns of long-term protein evolution claiming them to be compatible only with an epistatic fitness landscape. However, these claims have not yet been substantiated with a formal model of protein evolution. Here, we formulate a simple covarion-like model of protein evolution focusing on the rate at which the fitness impact of amino acids at a site changes with time. We then apply the model to the data on convergent and divergent protein evolution to test whether or not the incorporation of epistatic interactions is necessary to explain the data. We find that convergent evolution cannot be explained without the incorporation of epistasis and the rate at which an amino acid state switches from being acceptable at a site to being deleterious is faster than the rate of amino acid substitution. Specifically, for proteins that have persisted in modern prokaryotic organisms since the last universal common ancestor for one amino acid substitution approximately ten amino acid states switch from being accessible to being deleterious, or vice versa. Thus, molecular evolution can only be perceived in the context of rapid turnover of which amino acids are available for evolution.

A tetrapeptide-based method for polyproline II-type secondary structure prediction

We describe a new method for polyproline II‐type (PPII) secondary structure prediction based on tetrapeptide conformation properties using data obtained from all globular proteins in the Protein Data Bank (PDB). This is the first method for PPII prediction with a relatively high level of accuracy (∼60%). Our method uses only frequencies of different conformations among oligopeptides without any additional parameters. We also attempted to predict α‐helices and β‐strands using the same approach. We find that the application of our method reveals interrelation between sequence and structure even for very short oligopeptides (tetrapeptides). Proteins 2005.

The intramolecular impact to the sequence specificity of B-->A transition: low energy conformational variations in AA/TT and GG/CC steps.

Abstract It is well known, that local B→A transformation in DNA is involved in several biological processes. In vitro B↔A transition is sequence-specific. The physical basis of this specificity is not known yet. Here we analyze the effect of intramolecular interactions on the structural behavior of the GG/CC and AA/TT steps. These steps exemplify sequence specific bias to the B- or A-form structure. Optimization of potential energy of the molecular systems composed of an octanucle-otide, neutralized by Na+ and solvated with TIP3P water molecules in rectangular box with periodic boundary conditions gives the statistically representative sets of low energy structures for GG/CC and AA/TT steps in the middle of the diverse flanking sequences. Permissible 3D variations of GG/CC and AA/TT, and correlation of the relative motion of base pairs in these steps were analyzed. AA/TT step permits high variability for low energy conformers in the B-form DNA and small variability for low energy conformers in the A-form DNA. In contrast GG/CC step permits high variability for low energy conformers in the A-form DNA and small variability for low energy conformers in the B-form DNA. The relative motion of base pairs in GG/CC step is high correlated, while in AA/TT step this correlation is notably less. Atom-atom interactions inside-the-step always favors the B-form and their component - stacking interactions (atomatom interactions between nucleic bases) is crucial for the duplex stabilization. Formation of the A-form for both steps is a result of interactions with the flanking sequences and water-cation environment in the box. The average energy difference between conformations presenting B-form and A-form for the GG/CC step is high, while for the AA/TT step it is rather low. Thus, intramolecular interactions in GG/CC and AA/TT steps affect the possible conformational diversity (“conformational entropy”) of the A- and B- type structures of DNA step. This determines the known bias of the A-form DNA depending on the enrichment of sequences with GG/CC. If structural tuning during the process of protein-DNA complex formation lead to the local B→A transformation of DNA, it is largely directed by high conformational diversity of GG/CC step in the A-form. In such a case the presence in the target site of both kinds of examined steps ensures the reversible character of ligand binding.

Breen et al. reply

replying to D. M. McCandlish, E. Rajon, P. Shah, Y. Ding & J. B. Plotkin 497, 10.1038/nature12219 (2013)Understanding fitness landscapes, a conceptual depiction of the genotype-to-phenotype relationship, is crucial to many areas of biology. Two aspects of fitness landscapes are the focus of contemporary studies of molecular evolution. First, the local shape of the fitness landscape defined by the contribution of individual alleles to fitness that is independent of all genetic interactions. Second, the global, multidimensional fitness landscape shape determined by how interactions between alleles at different loci change each other’s fitness impact, or epistasis. In explaining the high amino-acid usage (u), we focused on the global shape of the fitness landscape, ignoring the perturbations at individual sites.