Arginines of the CGN codon family are Achilles' heels of cancer genes.

Recent studies have revealed that arginine is the most favorable target of amino acid alteration in most cancer types and it has been suggested that the high preference for arginine mutations reflects the critical roles of this amino acid in the function of proteins. High rates of mutations of arginine residues in cancer, however, might also be due to increased mutability of arginine codons of the CGN family as the CpG dinucleotides of these codons may be methylated. In the present work we have analyzed spectra of single base substitutions of cancer genes (oncogenes, tumor suppressor genes) and passenger genes in cancer tissues to assess the contributions of CpG hypermutability and selection to arginine mutations. Our studies have shown that arginines encoded by the CGN codon family display higher rates of mutation in both cancer genes and passenger genes than arginine codons AGA and AGG that are devoid of CpG dinucleotide, suggesting that the predominance of arginine mutations in cancer is primarily due to CpG hypermutability, rather than selection for arginine replacement. Nevertheless, our results also suggest that CGN codons for arginines may serve as Achilles' heels of cancer genes. CpG hypermutability of key arginines of proto-oncogenes, leading to high rates of recurrence of driver mutations, contributes significantly to carcinogenesis. Similarly, our results indicate that hypermutability of the CpG dinucleotide of CGA codons (converting them to TGA stop codons) contributes significantly to recurrent truncation and inactivation of tumor suppressor genes.

Evolution of termination codons of proteins and the TAG-TGA paradox.

In most eukaryotes and prokaryotes TGA is used at a significantly higher frequency than TAG as termination codon of protein-coding genes. Although this phenomenon has been recognized several years ago, there is no generally accepted explanation for the TAG-TGA paradox. Our analyses of human mutation data revealed that out of the eighteen sense codons that can give rise to a nonsense codon by single base substitution, the CGA codon is exceptional: it gives rise to the TGA stop codon at an order of magnitude higher rate than the other codons. Here we propose that the TAG-TGA paradox is due to methylation and hypermutabilty of CpG dinucleotides. In harmony with this explanation, we show that the coding genomes of organisms with strong CpG methylation have a significant bias for TGA whereas those from organisms that lack CpG methylation use TGA and TAG termination codons with similar probability.

Use of Publication Dynamics to Distinguish Cancer Genes and Bystander Genes.

de Magalhães has shown recently that most human genes have several papers in PubMed mentioning cancer, leading the author to suggest that every gene is associated with cancer, a conclusion that contradicts the widely held view that cancer is driven by a limited number of cancer genes, whereas the majority of genes are just bystanders in carcinogenesis. We have analyzed PubMed to decide whether publication metrics supports the distinction of bystander genes and cancer genes. The dynamics of publications on known cancer genes followed a similar pattern: seminal discoveries triggered a burst of cancer-related publications that validated and expanded the discovery, resulting in a rise both in the number and proportion of cancer-related publications on that gene. The dynamics of publications on bystander genes was markedly different. Although there is a slow but continuous time-dependent rise in the proportion of papers mentioning cancer, this phenomenon just reflects the increasing publication bias that favors cancer research. Despite this bias, the proportion of cancer papers on bystander genes remains low. Here, we show that the distinctive publication dynamics of cancer genes and bystander genes may be used for the identification of cancer genes.

Wnt Inhibitory Factor 1 Binds to and Inhibits the Activity of Sonic Hedgehog.

The hedgehog (Hh) and Wnt pathways, crucial for the embryonic development and stem cell proliferation of Metazoa, have long been known to have similarities that argue for their common evolutionary origin. A surprising additional similarity of the two pathways came with the discovery that WIF1 proteins are involved in the regulation of both the Wnt and Hh pathways. Originally, WIF1 (Wnt Inhibitory Factor 1) was identified as a Wnt antagonist of vertebrates, but subsequent studies have shown that in Drosophila, the WIF1 ortholog serves primarily to control the distribution of Hh. In the present, work we have characterized the interaction of the human WIF1 protein with human sonic hedgehog (Shh) using Surface Plasmon Resonance spectroscopy and reporter assays monitoring the signaling activity of human Shh. Our studies have shown that human WIF1 protein binds human Shh with high affinity and inhibits its signaling activity efficiently. Our observation that the human WIF1 protein is a potent antagonist of human Shh suggests that the known tumor suppressor activity of WIF1 may not be ascribed only to its role as a Wnt inhibitor.

Exon Shuffling Played a Decisive Role in the Evolution of the Genetic Toolkit for the Multicellular Body Plan of Metazoa.

Division of labor and establishment of the spatial pattern of different cell types of multicellular organisms require cell type-specific transcription factor modules that control cellular phenotypes and proteins that mediate the interactions of cells with other cells. Recent studies indicate that, although constituent protein domains of numerous components of the genetic toolkit of the multicellular body plan of Metazoa were present in the unicellular ancestor of animals, the repertoire of multidomain proteins that are indispensable for the arrangement of distinct body parts in a reproducible manner evolved only in Metazoa. We have shown that the majority of the multidomain proteins involved in cell-cell and cell-matrix interactions of Metazoa have been assembled by exon shuffling, but there is no evidence for a similar role of exon shuffling in the evolution of proteins of metazoan transcription factor modules. A possible explanation for this difference in the intracellular and intercellular toolkits is that evolution of the transcription factor modules preceded the burst of exon shuffling that led to the creation of the proteins controlling spatial patterning in Metazoa. This explanation is in harmony with the temporal-to-spatial transition hypothesis of multicellularity that proposes that cell differentiation may have predated spatial segregation of cell types in animal ancestors.

Use of signals of positive and negative selection to distinguish cancer genes and passenger genes.

A major goal of cancer genomics is to identify all genes that play critical roles in carcinogenesis. Most approaches focused on genes positively selected for mutations that drive carcinogenesis and neglected the role of negative selection. Some studies have actually concluded that negative selection has no role in cancer evolution. We have re-examined the role of negative selection in tumor evolution through the analysis of the patterns of somatic mutations affecting the coding sequences of human genes. Our analyses have confirmed that tumor suppressor genes are positively selected for inactivating mutations, oncogenes, however, were found to display signals of both negative selection for inactivating mutations and positive selection for activating mutations. Significantly, we have identified numerous human genes that show signs of strong negative selection during tumor evolution, suggesting that their functional integrity is essential for the growth and survival of tumor cells.

The DNA in the cells of the human body is usually copied correctly when a cell divides. However, errors (mutations) are sometimes introduced during the copying process. Although the majority of mutations have no major impact on cells, many mutations are harmful: they decrease the ability of cells to survive. There are, however, mutations that can lead to cells dividing more frequently or gaining the ability to spread, which can lead to cancer. These mutations are known as 'driver mutations' because they drive the growth of tumors. Since such 'driver mutations' provide a growth advantage to tumor cells, they are subject to positive selection, this is, their frequency in the tumor increases over time. Because of their selective advantage, driver mutations accumulate at significantly higher rates than the neutral 'passenger mutations' that do not play a role in tumor growth. Genes that carry driver mutations are called driver genes, while genes that carry only passenger mutations are known as passenger genes. Certain genes, however, do not fit into either category. For example, some genes that are essential for tumor growth must get rid of harmful mutations to maintain activity. Mutations of such 'tumor essential genes' are thus subject to 'negative' or 'purifying selection'. A major goal of cancer research is to identify genes that play critical roles in tumor growth. Earlier studies have identified numerous driver genes positively selected for driver mutations, exploiting the fact that driver genes show significantly higher mutation rates than passenger genes. Identification of tumor essential genes, however, is inherently more difficult since the paucity of mutations of negatively selected genes hinders the analysis of the mutation data. The failure to provide convincing evidence for negative selection in tumors has led to suggestions that it has no role in cancer evolution. Bányai et al. used a novel approach to address the question of whether negative selection occurs in cancer. Based on characteristic differences in the patterns of mutations in cancer they distinguished clusters of passenger genes, driver genes and tumor essential genes. The group of tumor essential genes includes genes that serve to satisfy the increased demand of rapidly dividing tumor cells for nutrients' and genes that are essential for cell migration and metastasis (the spread of cancer cells to other areas of the body). The tumor essential genes that Bányai et al. identified may prove to be valuable targets for cancer therapy, illustrating the importance of genome sequencing in cancer research. Identification of additional tumor essential genes is, however, hindered by the fact that they are likely to have low levels of mutations, which can exclude them from meaningful analyses. Progress with genomic sequencing of tumors is expected to overcome this limitation and help identify additional genes that are essential for cancer growth.

The SDHB Arg230His mutation causing familial paraganglioma alters glycolysis in a new Caenorhabditis elegans model.

The conserved B-subunit of succinate dehydrogenase (SDH) participates in the tricarboxylic acid cycle (TCA) cycle and mitochondrial electron transport. The Arg230His mutation in SDHB causes heritable pheochromocytoma/paraganglioma (PPGL). In Caenorhabditiselegans, we generated an in vivo PPGL model (SDHB-1 Arg244His; equivalent to human Arg230His), which manifests delayed development, shortened lifespan, attenuated ATP production and reduced mitochondrial number. Although succinate is elevated in both missense and null sdhb-1(gk165) mutants, transcriptomic comparison suggests very different causal mechanisms that are supported by metabolic analysis, whereby only Arg244His (not null) worms demonstrate elevated lactate/pyruvate levels, pointing to a missense-induced, Warburg-like aberrant glycolysis. In silico predictions of the SDHA-B dimer structure demonstrate that Arg230His modifies the catalytic cleft despite the latter's remoteness from the mutation site. We hypothesize that the Arg230His SDHB mutation rewires metabolism, reminiscent of metabolic reprogramming in cancer. Our tractable model provides a novel tool to investigate the metastatic propensity of this familial cancer and our approach could illuminate wider SDH pathology.This article has an associated First Person interview with the first author of the paper.

Structure, function and disease relevance of Wnt inhibitory factor 1, a secreted protein controlling the Wnt and hedgehog pathways.

Wnts and Hedgehogs (Hh) are large, lipid-modified extracellular morphogens that play key roles in embryonic development and stem cell proliferation of Metazoa. Both morphogens signal through heptahelical Frizzled-type receptors of the G-Protein Coupled Receptor family and there are several other similarities that suggest a common evolutionary origin of the Hh and Wnt pathways. There is evidence that the secreted protein, Wnt inhibitory factor 1 (WIF1) modulates the activity of both Wnts and Hhs and may thus contribute to the intertwining of these pathways. In this article, we review the structure, evolution, molecular interactions and functions of WIF1 with major emphasis on its role in carcinogenesis.

Exon skipping-rich transcriptomes of animals reflect the significance of exon-shuffling in metazoan proteome evolution.

ᅟ: Animals are known to have higher rates of exon skipping than other eukaryotes. In a recent study, Grau-Bové et al. (Genome Biology 19:135, 2018) have used RNA-seq data across 65 eukaryotic species to investigate when and how this high prevalence of exon skipping evolved. They have found that bilaterian Metazoa have significantly increased exon skipping frequencies compared to all other eukaryotic groups and that exon skipping in nearly all animals, including non-bilaterians, is strongly enriched for frame-preserving events. The authors have hypothesized that "the increase of exon skipping rates in animals followed a two-step process. First, exon skipping in early animals became enriched for frame-preserving events. Second, bilaterian ancestors dramatically increased their exon skipping frequencies, likely driven by the interplay between a shift in their genome architectures towards more exon definition and recruitment of frame-preserving exon skipping events to functionally diversify their cell-specific proteomes." Here we offer a different explanation for the higher frequency of frame-preserving exon skipping in Metzoa than in all other eukaryotes. In our view these observations reflect the fact that the majority of multidomain proteins unique to metazoa and indispensable for metazoan type multicellularity were assembled by exon-shuffling from 'symmetrical' modules (i.e. modules flanked by introns of the same phase), whereas this type of protein evolution played a minor role in other groups of eukaryotes, including plants. The higher frequency of 'symmetrical' exons in Metazoan genomes provides an explanation for the enrichment for frame-preserving events since skipping or inclusion of 'symmetrical' modules during alternative splicing does not result in a reading-frame shift. REVIEWERS: This article was reviewed by Manuel Irimia, Ashish Lal and Erez Levanon. The reviewers were nominated by the Editorial Board.

Morphological Stasis and Proteome Innovation in Cephalochordates.

Lancelets, extant representatives of basal chordates, are prototypic examples of evolutionary stasis; they preserved a morphology and body-plan most similar to the fossil chordates from the early Cambrian. Such a low level of morphological evolution is in harmony with a low rate of amino acid substitution; cephalochordate proteins were shown to evolve slower than those of the slowest evolving vertebrate, the elephant shark. Surprisingly, a study comparing the predicted proteomes of Chinese amphioxus, Branchiostoma belcheri and the Florida amphioxus, Branchiostoma floridae has led to the conclusion that the rate of creation of novel domain combinations is orders of magnitude greater in lancelets than in any other Metazoa, a finding that contradicts the notion that high rates of protein innovation are usually associated with major evolutionary innovations. Our earlier studies on a representative sample of proteins have provided evidence suggesting that the differences in the domain architectures of predicted proteins of these two lancelet species reflect annotation errors, rather than true innovations. In the present work, we have extended these studies to include a larger sample of genes and two additional lancelet species, Asymmetron lucayanum and Branchiostoma lanceolatum. These analyses have confirmed that the domain architecture differences of orthologous proteins of the four lancelet species are because of errors of gene prediction, the error rate in the given species being inversely related to the quality of the transcriptome dataset that was used to aid gene prediction.

Influence of WFIKKN1 on BMP1-mediated activation of latent myostatin.

The NTR domain of WFIKKN1 protein has been shown to have significant affinity for the prodomain regions of promyostatin and latent myostatin but the biological significance of these interactions remained unclear. In view of its role as a myostatin antagonist, we tested the assumption that WFIKKN1 inhibits the release of myostatin from promyostatin and/or latent myostatin. WFIKKN1 was found to have no effect on processing of promyostatin by furin, the rate of cleavage of latent myostatin by BMP1, however, was significantly enhanced in the presence of WFIKKN1 and this enhancer activity was superstimulated by heparin. Unexpectedly, WFIKKN1 was also cleaved by BMP1 and our studies have shown that the KKN1 fragment generated by BMP1-cleavage of WFIKKN1 contributes most significantly to the observed enhancer activity. Analysis of a pro-TGF-ß -based homology model of homodimeric latent myostatin revealed that the BMP1-cleavage sites are buried and not readily accessible to BMP1. In view of this observation, the most plausible explanation for the BMP1-enhancer activity of the KKN1 fragment is that it shifts a conformational equilibrium of latent myostatin from the closed circular structure of the homodimer to a more open form, making the cleavage sites more accessible to BMP1. On the other hand, the observation that the enhancer activity of KKN1 is superstimulated in the presence of heparin is explained by the fact KKN1, latent myostatin, and BMP1 have affinity for heparin and these interactions with heparin increase the local concentrations of the reactants thereby facilitating the action of BMP1. ENZYMES: Furin: EC 3.4.21.75; BMP1, bone morphogentic protein 1 or procollagen C-endopeptidase: EC 3.4.24.19.

Putative extremely high rate of proteome innovation in lancelets might be explained by high rate of gene prediction errors.

A recent analysis of the genomes of Chinese and Florida lancelets has concluded that the rate of creation of novel protein domain combinations is orders of magnitude greater in lancelets than in other metazoa and it was suggested that continuous activity of transposable elements in lancelets is responsible for this increased rate of protein innovation. Since morphologically Chinese and Florida lancelets are highly conserved, this finding would contradict the observation that high rates of protein innovation are usually associated with major evolutionary innovations. Here we show that the conclusion that the rate of proteome innovation is exceptionally high in lancelets may be unjustified: the differences observed in domain architectures of orthologous proteins of different amphioxus species probably reflect high rates of gene prediction errors rather than true innovation.

Identification and Correction of Erroneous Protein Sequences in Public Databases.

Correct prediction of the structure of protein-coding genes of higher eukaryotes is a difficult task therefore public sequence databases incorporating predicted sequences are increasingly contaminated with erroneous sequences. The high rate of misprediction has serious consequences since it significantly affects the conclusions that may be drawn from genome-scale sequence analyses.Here we describe the MisPred and FixPred approaches that may help the identification and correction of erroneous sequences. The rationale of these approaches is that a protein sequence is likely to be erroneous if some of its features conflict with our current knowledge about proteins.

Wnts grasp the WIF domain of Wnt Inhibitory Factor 1 at two distinct binding sites.

Wnts have a structure resembling a hand with "thumb" and "index" fingers that grasp the cysteine rich domains of Frizzled receptors at two distinct binding sites. In the present work we show that the WIF domain of Wnt Inhibitory Factor 1 is also bound by Wnts at two sites. Using C-terminal domains of Wnt5a and Wnt7a and arginine-scanning mutagenesis of the WIF domain we demonstrate that, whereas the N-terminal, lipid-modified "thumb" of Wnts interacts with the alkyl-binding site of the WIF domain, the C-terminal domain of Wnts (Wnt-CTD) binds to a surface on the opposite side of the WIF domain.

K153R polymorphism in myostatin gene increases the rate of promyostatin activation by furin.

Recent studies demonstrated an association between the K153R polymorphism in the myostatin gene with extreme longevity, lower muscle strength and obesity but the molecular basis of these associations has not been clarified. Here, we show that the K153R mutation significantly increases the rate of proteolysis of promyostatin by furin, but has no effect on the activity of the latent complex or the cleavage of the latent complex by bone morphogenetic protein 1 (BMP-1). The increased rate of activation of K153R mutant promyostatin may explain why this polymorphism is associated with obesity, lower muscle strength and extension of lifespan.

FixPred: a resource for correction of erroneous protein sequences.

Protein databases are heavily contaminated with erroneous (mispredicted, abnormal and incomplete) sequences and these erroneous data significantly distort the conclusions drawn from genome-scale protein sequence analyses. In our earlier work we described the MisPred resource that serves to identify erroneous sequences; here we present the FixPred computational pipeline that automatically corrects sequences identified by MisPred as erroneous. The current version of the associated FixPred database contains corrected UniProtKB/Swiss-Prot and NCBI/RefSeq sequences from Homo sapiens, Mus musculus, Rattus norvegicus, Monodelphis domestica, Gallus gallus, Xenopus tropicalis, Danio rerio, Fugu rubripes, Ciona intestinalis, Branchostoma floridae, Drosophila melanogaster and Caenorhabditis elegans; future releases of the FixPred database will include corrected sequences of additional Metazoan species. The FixPred computational pipeline and database (http://www.fixpred.com) are easily accessible through a simple web interface coupled to a powerful query engine and a standard web service. The content is completely or partially downloadable in a variety of formats. Database URL: http://www.fixpred.com.

Both LCCL-domains of human CRISPLD2 have high affinity for lipid A.

The LCCL-domain is a recently defined protein module present in diverse extracellular multidomain proteins. Practically nothing is known about the molecular function of these domains; based on functional features of proteins harboring LCCL-domains it has been suggested that these domains might function as lipopolysaccharide-binding domains. Here we show that the two LCCL-domains of human CRISPLD2 protein, a lipopolysaccharide-binding serum protein involved in defense against endotoxin shock, have higher affinity for the lipid A, the toxic moiety of lipopolysaccharides than for ipopolysaccharide. Our observation that the LCCL-domains of CRISPLD2 are specific for the toxic lipid A moiety of the endotoxin suggests that it may block the interaction between endotoxins and the host endotoxin receptors without interfering with the development of antibacterial immunity against the polysaccharide moiety of LPS. We suggest that the anti-inflammatory function of CRISPLD2 protein may account for its role in various pathological and developmental processes.

Latent myostatin has significant activity and this activity is controlled more efficiently by WFIKKN1 than by WFIKKN2.

Myostatin, a negative regulator of skeletal muscle growth, is produced from myostatin precursor by multiple steps of proteolytic processing. After cleavage by a furin-type protease, the propeptide and growth factor domains remain associated, forming a noncovalent complex, the latent myostatin complex. Mature myostatin is liberated from latent myostatin by bone morphogenetic protein 1/tolloid proteases. Here, we show that, in reporter assays, latent myostatin preparations have significant myostatin activity, as the noncovalent complex dissociates at an appreciable rate, and both mature and semilatent myostatin (a complex in which the dimeric growth factor domain interacts with only one molecule of myostatin propeptide) bind to myostatin receptor. The interaction of myostatin receptor with semilatent myostatin is efficiently blocked by WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 1 or growth and differentiation factor-associated serum protein 2 (WFIKKN1), a large extracellular multidomain protein that binds both mature myostatin and myostatin propeptide [Kondás et al. (2008) J Biol Chem 283, 23677-23684]. Interestingly, the paralogous protein WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 2 or growth and differentiation factor-associated serum protein 1 (WFIKKN2) was less efficient than WFIKKN1 as an antagonist of the interactions of myostatin receptor with semilatent myostatin. Our studies have shown that this difference is attributable to the fact that only WFIKKN1 has affinity for the propeptide domain, and this interaction increases its potency in suppressing the receptor-binding activity of semilatent myostatin. As the interaction of WFIKKN1 with various forms of myostatin permits tighter control of myostatin activity until myostatin is liberated from latent myostatin by bone morphogenetic protein 1/tolloid proteases, WFIKKN1 may have greater potential as an antimyostatic agent than WFIKKN2.