Evolution of the plasma and tissue kallikreins, and their alternative splicing isoforms.

Kallikreins are secreted serine proteases with important roles in human physiology. Human plasma kallikrein, encoded by the KLKB1 gene on locus 4q34-35, functions in the blood coagulation pathway, and in regulating blood pressure. The human tissue kallikrein and kallikrein-related peptidases (KLKs) have diverse expression patterns and physiological roles, including cancer-related processes such as cell growth regulation, angiogenesis, invasion, and metastasis. Prostate-specific antigen (PSA), the product of the KLK3 gene, is the most widely used biomarker in clinical practice today. A total of 15 KLKs are encoded by the largest contiguous cluster of protease genes in the human genome (19q13.3-13.4), which makes them ideal for evolutionary analysis of gene duplication events. Previous studies on the evolution of KLKs have traced mammalian homologs as well as a probable early origin of the family in aves, amphibia and reptilia. The aim of this study was to address the evolutionary and functional relationships between tissue KLKs and plasma kallikrein, and to examine the evolution of alternative splicing isoforms. Sequences of plasma and tissue kallikreins and their alternative transcripts were collected from the NCBI and Ensembl databases, and comprehensive phylogenetic analysis was performed by Bayesian as well as maximum likelihood methods. Plasma and tissue kallikreins exhibit high sequence similarity in the trypsin domain (>50%). Phylogenetic analysis indicates an early divergence of KLKB1, which groups closely with plasminogen, chymotrypsin, and complement factor D (CFD), in a monophyletic group distinct from trypsin and the tissue KLKs. Reconstruction of the earliest events leading to the diversification of the tissue KLKs is not well resolved, indicating rapid expansion in mammals. Alternative transcripts of each KLK gene show species-specific divergence, while examination of sequence conservation indicates that many annotated human KLK isoforms are missing the catalytic triad that is crucial for protease activity

A holistic evolutionary and structural study of flaviviridae provides insights into the function and inhibition of HCV helicase

Viral RNA helicases are involved in duplex unwinding during the RNA replication of the virus. It is suggested that these helicases represent very promising antiviral targets. Viruses of the flaviviridae family are the causative agents of many common and devastating diseases, including hepatitis, yellow fever and dengue fever. As there is currently no available anti-Flaviviridae therapy, there is urgent need for the development of efficient anti-viral pharmaceutical strategies. Herein, we report the complete phylogenetic analysis across flaviviridae alongside a more in-depth evolutionary study that revealed a series of conserved and invariant amino acids that are predicted to be key to the function of the helicase. Structural molecular modelling analysis revealed the strategic significance of these residues based on their relative positioning on the 3D structures of the helicase enzymes, which may be used as pharmacological targets. We previously reported a novel series of highly potent HCV helicase inhibitors, and we now re-assess their antiviral potential using the 3D structural model of the invariant helicase residues. It was found that the most active compound of the series, compound C4, exhibited an IC50 in the submicromolar range, whereas its stereoisomer (compound C12) was completely inactive. Useful insights were obtained from molecular modelling and conformational search studies via molecular dynamics simulations. C12 tends to bend and lock in an almost “U” shape conformation, failing to establish vital interactions with the active site of HCV. On the contrary, C4 spends most of its conformational time in a straight, more rigid formation that allows it to successfully block the passage of the oligonucleotide in the ssRNA channel of the HCV helicase. This study paves the way and provides the necessary framework for the in-depth analysis required to enable the future design of new and potent anti-viral agents

Evolution of the F₀F₁ ATP Synthase Complex in Light of the Patchy Distribution of Different Bioenergetic Pathways across Prokaryotes

<div><p>Bacteria and archaea are characterized by an amazing metabolic diversity, which allows them to persist in diverse and often extreme habitats. Apart from oxygenic photosynthesis and oxidative phosphorylation, well-studied processes from chloroplasts and mitochondria of plants and animals, prokaryotes utilize various chemo- or lithotrophic modes, such as anoxygenic photosynthesis, iron oxidation and reduction, sulfate reduction, and methanogenesis. Most bioenergetic pathways have a similar general structure, with an electron transport chain composed of protein complexes acting as electron donors and acceptors, as well as a central cytochrome complex, mobile electron carriers, and an ATP synthase. While each pathway has been studied in considerable detail in isolation, not much is known about their relative evolutionary relationships. Wanting to address how this metabolic diversity evolved, we mapped the distribution of nine bioenergetic modes on a phylogenetic tree based on 16S rRNA sequences from 272 species representing the full diversity of prokaryotic lineages. This highlights the patchy distribution of many pathways across different lineages, and suggests either up to 26 independent origins or 17 horizontal gene transfer events. Next, we used comparative genomics and phylogenetic analysis of all subunits of the F₀F₁ ATP synthase, common to most bacterial lineages regardless of their bioenergetic mode. Our results indicate an ancient origin of this protein complex, and no clustering based on bioenergetic mode, which suggests that no special modifications are needed for the ATP synthase to work with different electron transport chains. Moreover, examination of the ATP synthase genetic locus indicates various gene rearrangements in the different bacterial lineages, ancient duplications of <i>atpI</i> and of the beta subunit of the F₀ subcomplex, as well as more recent stochastic lineage-specific and species-specific duplications of all subunits. We discuss the implications of the overall pattern of conservation and flexibility of the F₀F₁ ATP synthase genetic locus.</p></div

KLK gene clusters in selected species, as determined by examining the NCBI Gene database.

<p>The genetic locus neighborhood for <i>KLK</i>s in cattle, opossum, chicken, lizard, and frog are shown. The <i>KLK</i> gene cluster in cattle (<i>Bos taurus</i> chromosome 18) has a similar structure to the one in human, mouse and dog, except that it is missing the KLK2 and KLK3 genes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068074#pone.0068074.s002" target="_blank">Figure S2</a>). The <i>KLK</i> gene cluster in the opossum (<i>Monodelphis domestica</i> chromosome 4) contains a smaller number of genes, but again has a similar structure, except that KLK13 and KLK14 are not adjacent to the other KLKs and that they are transcribed in the opposite direction, indicating a chromosomal rearrangement. In the chicken (<i>Gallus gallus</i> chromosome 1), three trypsin-like proteins adjacent to the annotated KLK7 were identified as putative novel KLKs (GgKLK, GgKLKL, GgPRSS3). Similarly, in the lizard (<i>Anolis carolinensis</i>) two putative novel KLKs (AcKLK14L, AcKLKL) were identified adjacent to the annotated KLK14. Finally, in the frog (<i>Xenopus tropicalis</i>) ten putative novel KLKs (XtKLKun1-10) were identified adjacent to annotated KLKs. Note that the lizard and frog sequences are on parts of the genome that are not fully assembled yet.</p

Features of the tissue KLK isoforms in human, mouse, dog, cattle, opossum and platypus.

<p>Panel A: The general structure of the reference KLK isoform includes 5 coding exons, and may include one 5′ and/or one 3′ UTR exon shown with a +/− sign <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068074#pone.0068074-Lawrence1" target="_blank">[20]</a>. Coding regions are shown in black, UTR in grey, dotted lines indicate that the length of introns may vary. Exon1 usually includes at least part of the 5′UTR, exon5 usually includes at least part of the 3′UTR. Panel B: Alternative splicing patterns for all annotated KLK isoforms in the NCBI Gene and Ensembl66 databases are shown. The different types of alternative splicing used to generate structural diversity (alternative promoter, exon skipping, alternative 3′ SS selection, alternative 5′ SS selection, alternative poly(A)) are as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068074#pone.0068074-Keren1" target="_blank">[27]</a>. Purple boxes indicate that a feature is used in a protein-coding isoform, blue boxes are for features only found in NMD or RNA-only isoforms. “p” indicates that only part of an exon has become non-coding. For each KLK, we chose as reference the isoform containing 5 coding exons, with or without one 5′ UTR and one 3′ UTR, to infer the most parsimonious diversity pattern (see panel A). Some of the Ensembl gene summaries, which form the basis of the data summarized in this panel, are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068074#pone.0068074.s004" target="_blank">Figure S4</a>, for human, mouse and dog KLKs, which display significant structural variability.</p

Phylogenetic reconstruction based on 16S rRNA sequences to map the taxonomic distribution of bioenergetic pathways.

<p>272 prokaryotic species are shown, whose full genome sequence is available, and which represent the full diversity of bacteria and archaea, colour-coded based on their bioenergetic mode. Bootstrap values for highly supported nodes have been replaced by symbols, as indicated. The full species names, as well as details and accession numbers for all sequences used are given in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003821#pcbi.1003821.s008" target="_blank">Table S1</a>. The tree shown was produced by RaxML, and its topology broadly agrees with the one produced by PhyML (the analysis based on MrBayes did not converge after 5 million generations when all sequences were included; however, when the bacteria and the archaea were examined separately, the MrBayes analysis also agreed with the RaxML and PhyML results).</p

Phylogenetic reconstruction of ATPF0A.

<p>The tree shown is the best Bayesian topology, based on 215 sequences and 232 amino acid positions (length after trimming; median sequence length before trimming: 254). Numerical values at the nodes of the tree (x/y/z) indicate statistical support by MrBayes, PhyML and RAxML (posterior probability, bootstrap and bootstrap, respectively). Values for highly supported nodes have been replaced by symbols, as indicated. Species names are colour-coded based on their bioenergetic mode as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003821#pcbi-1003821-g001" target="_blank">Figure 1</a>. Full details and accession numbers for all protein sequences used are given in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003821#pcbi.1003821.s008" target="_blank">Table S1</a>. The tree is rooted at the N-ATPase clade, previously reported to be the result of horizontal gene transfer in a variety of species, all of which also contain a canonical ATPF₀F₁ (apart from the two <i>Methanosarcina</i> species shown, which also have a canonical ATPV). The tree confidently separates the major bacterial taxonomic lineages, but with limited support for their branching order: strong support is provided for a subgroup containing the verrucomicrobia and chloroflexi, while another subgroup containing the alpha-proteobacteria, actinobacteria, chlorobi, bacteroidetes and planctomycetes also has reasonable support. This group also includes the spirochaete <i>Leptospira interrogans</i> and the gemmatimonadete <i>Gemmatimonas aurantiaca</i>, as well as <i>Candidatus Nitrospira defluvii</i> which groups with the alpha-proteobacteria. Reasonable support is also provided for the grouping of dictyoglomi and cyanobacteria, and for a subgroup containing the fusobacteria, firmicutes, tenericutes, thermotogae, and beta-gamma-proteobacteria. Two species-specific duplications (in <i>Saccharopolyspora erythraea</i> and <i>Pelobacter carbinolicus</i>) are highlighted with a red “>”. Two further duplications are highlighted with a red “-” after the species name; in <i>Photobacterium profundum</i> the duplication either occurred before the split from other closely-related species or represents HGT from other gamma-proteobacteria; the duplication in <i>Desulfococcus oleovorans</i> possibly represents HGT from thermotogae (also see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003821#pcbi.1003821.s001" target="_blank">Figures S1</a> and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003821#pcbi.1003821.s002" target="_blank">S2</a>).</p

The distribution of bioenergetic modes across taxonomic lineages suggests rampant horizontal gene transfer, or multiple independent origins.

<p>He: heterotrophs/respiration.</p><p>OP: oxygenic photosynthesis.</p><p>AP: anoxygenic photosynthesis.</p><p>Me: methanogenesis.</p><p>SR/AR: sulfate/arsenate reduction.</p><p>Sfr: sulfur reduction.</p><p>SO: sulfur oxidation.</p><p>FR: iron reduction.</p><p>FO: iron oxidation.</p>a<p> = lineages represented by only one species are shown in bold and the species name abbreviation is given.</p>b<p> = only found in bacteria.</p>c<p> = only found in the archaea.</p>d<p> = ancient origin assumed (patchy distribution likely due to secondary loss, but see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003821#pcbi.1003821-NelsonSathi1" target="_blank">[37]</a>).</p>e<p> = assuming a common origin for the subgroups of the proteobacteria.</p>f<p> = ancient origin before common ancestor of the crenarchaea.</p>g<p> = assuming a common origin for the archaeglobi, the thermoplasmata and Ac_booFO.</p>h<p> = assuming one origin.</p><p>The distribution of bioenergetic modes across taxonomic lineages suggests rampant horizontal gene transfer, or multiple independent origins.</p

Phylogenetic reconstruction of plasma and tissue kallikreins in the context of other serine proteases, including trypsin, chymotrypsin, plasminogen, and complement factor D.

<p>The tree shown is the best Bayesian topology. Numerical values at the nodes of the tree (x/y/z) indicate statistical support by MrBayes, PhyML and RAxML (posterior probability, bootstrap and bootstrap, respectively). Values for highly supported nodes have been replaced by symbols, as indicated. Species names are abbreviated and colored as follows: Hs - <i>Homo sapiens</i> (human, black); Mm - <i>Mus musculus</i> (mouse, grey); Bt - <i>Bos taurus</i> (cattle, blue); Cf - <i>Canis familiaris</i> (dog, light blue); Md - <i>Monodelphis domestica</i> (opossum, green); Me - <i>Macropus eugenii</i> (wallaby, light green); Pc - <i>Procavia capensis</i> (hyrax, purple); Xt - <i>Xenopus tropicalis</i> (frog, pink); Oa - <i>Ornithorhynchus anatinus</i> (platypus, light purple); Ac - <i>Anolis carolinensis</i> (lizard, red); <i>Meleagris gallopavo</i> (turkey, brown); Gg - <i>Gallus gallus</i> (chicken, orange); Tg - <i>Taeniopygia guttata</i> (Zebra Finch, khaki); Dr - <i>Danio rerio</i> (zebrafish, black). Full details and accession numbers for all protein sequences used are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068074#pone.0068074.s005" target="_blank">Table S1</a>; also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068074#pone.0068074.s008" target="_blank">Alignments S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068074#pone.0068074.s009" target="_blank">S4</a>, for the masked and trimmed alignments used to construct the tree.</p

ATPF₀F₁ gene locus organization per lineage.

<p>The ATPF₀F₁ gene locus organization was checked for all species in the IMG database <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003821#pcbi.1003821-Markowitz1" target="_blank">[47]</a>, and is summarized per lineage. The gene order shown follows the order in which the genes are transcribed in each genome (upstream to downstream). Semicolons indicate that the separated gene groups are on non-adjacent genetic locations (and can be very far upstream or downstream; e.g. separated by only 4 intervening ORFs in <i>Geobacter</i> sp. FRC-32, and by up to 5026 intervening ORFs, or 6 Mb, in <i>Nostoc</i> sp. PCC 7120; see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003821#pcbi.1003821.s008" target="_blank">Table S1</a>). When the locus is split, the genes are shown in the order they are usually found in when the locus is intact. ATPF0B (K02109) is often duplicated, so one copy is called 0B, and the other 0B′, based on the gene order. ATPI (K02116) is also often duplicated, and is designated “I” “sI” and “R” based on the presence of distinct pfam domains, as discussed in the text. Question marks indicate that the ATPI subunit is sometimes not clearly assigned to the orthology group. “X” denotes hypothetical intervening ORFs. Notable variations within some lineages are shown. *Especially for lineages represented by relatively few species, please see TableS1 for variations between the species examined within each lineage.</p