Duplication Of Domains Research Articles

Conserved untranslated regions of multipartite viruses: Natural markers of novel viral genomic components and tags of viral evolution.

Viruses with split genomes are classified as being either segmented or multipartite based on whether their genomic segments occur within a single virion or across different virions. Despite variations in number and sequence during evolution, the genomic segments of many viruses are conserved within the untranslated regions (UTRs). In this study, we present a methodology that combines RNA sequencing with iterative BLASTn of UTRs (https://github.com/qq371260/Iterative-blast-v.1.0) to identify new viral genomic segments. Some novel multipartite-like viruses related to the phylum Kitrinoviricota were annotated using sequencing data from field plant samples and public databases. We identified potentially plant-infecting jingmen-related viruses (order Amarillovirales) and jivi-related viruses (order Martellivirales) with at least six genomic components. The number of RNA molecules associated with a genome of the novel viruses in the families Closteroviridae, Kitaviridae, and Virgaviridae within the order Martellivirales reached five. Several of these viruses seem to represent new taxa at the subgenus, genus, and family levels. The diversity of novel genomic components and the multiple duplication of proteins or protein domains within single or multiple genomic components emphasize the evolutionary roles of genetic recombination (horizontal gene transfer), reassortment, and deletion. The relatively conserved UTRs at the genome level might explain the relationships between monopartite and multipartite viruses, as well as how subviral agents such as defective RNAs and satellite viruses can coexist with their helper viruses.

Insights into the presence of multiple RecQ helicases in the ancient cyanobacterium, Nostoc sp. strain PCC7120: bioinformatics and expression analysis approach.

Owing to their crucial role in genome maintenance, RecQ helicases are ubiquitous and present across organisms. Though the multiplicity of RecQ helicases is well known in higher organisms, it is rare among bacteria. The ancient cyanobacterium Nostoc sp. strain PCC7120 was found to have three annotated RecQ helicases. This study aims at understanding its structural differences and evolution through bioinformatics approach and functionality through expression analysis studies. Nostoc RecQ helicases were found to be transcriptionally regulated by LexA and DNA damage inducing stresses. Bioinformatic analysis revealed that all three RecQ helicases of Nostoc possess helicases_C and Zn+2-binding domains. Two of the helicases (AnRecQ and AnRecQ2) lacked the complete RQC and HRDC domains, and AnRecQ2 had an additional Phosphoribosyl transferase domain (Pribosyltran), also seen in RecQ-like helicase (RqlH) protein of Mycobacterium smegmatis. AnRecQ1, which was similar to most bacterial RecQ helicases, differed in having a long C-terminal tail. STRING analysis revealed that the proteins also differed in their predicted protein interactome. Phylogenetic analysis suggested that the multiple recQ genes may have been acquired through duplication and acquisition of additional domains from the smallest of the RecQ helicases (AnRecQ) to cater multiple functions required to deal with the harsh environmental conditions. In course of evolution, however, the multiplicity was lost with the modern-day bacteria and lower eukaryotes which retained fewer RecQ helicases, while further duplication of the acquired RECQ occurred in higher animals and plants to deal with cellular complexity.

Similar protein segments shared between domains of different evolutionary lineages.

The emergence of novel proteins, beyond these that can be readily made by duplication and recombination of preexisting domains, is elusive. De novo emergence from random sequences is unlikely because the vast majority of random chains would not even fold, let alone function. An alternative explanation is that novel proteins emerge by duplication and fusion of pre‐existing polypeptide segments. In this case, traces of such ancient events may remain within contemporary proteins in the form of reused segments. Together with the late Dan Tawfik, we detected such similar segments, far shorter than intact protein domains, which are found in different environments. The detection of these, “bridging themes,” was based on a unique search strategy, where in addition to searching for similarity of shared fragments, so‐called “themes,” we also explicitly searched for cases in which the sequence segments before and after the theme are dissimilar (both in sequence and structure). Here, using a similar strategy, we further expanded the search and discovered almost 500 additional “bridging themes,” linking domains that are often from ancient folds. The themes, of 20 residues or more (average 53), do not retain their structure despite sharing 37% sequence identity on average. Indeed, conformation flexibility may confer an evolutionary advantage, in that it fits in multiple environments. We elaborate on two interesting themes, shared between Rossmann/Trefoil‐Plexin‐like domains and a β‐propeller‐like domain.For a Broad AudienceA fundamental question in molecular evolution is how protein domains emerged. Similar segments shared between domains of seemingly distinct origins, may offer clues, as these may be remnants of the evolutionary process through which these domains emerged. However, finding such cases is difficult. Here, we expand the set of such cases which we curated previously, adding segments shared between domains that are considered ancient.

The Repeating, Modular Architecture of the HtrA Proteases.

A conserved, 26-residue sequence [AA(X2)[A/G][G/L](X2)GDV[I/L](X2)[V/L]NGE(X1)V(X6)] and corresponding structure repeating module were identified within the HtrA protease family using a non-redundant set (N = 20) of publicly available structures. While the repeats themselves were far from sequence perfect, they had notable conservation to a statistically significant level. Three or more repetitions were identified within each protein despite being statistically expected to randomly occur only once per 1031 residues. This sequence repeat was associated with a six stranded antiparallel β-barrel module, two of which are present in the core of the structures of the PA clan of serine proteases, while a modified version of this module could be identified in the PDZ-like domains. Automated structural alignment methods had difficulties in superimposing these β-barrels, but the use of a target human HtrA2 structure showed that these modules had an average RMSD across the set of structures of less than 2 Å (mean and median). Our findings support Dayhoff’s hypothesis that complex proteins arose through duplication of simpler peptide motifs and domains.

Two 'what' pathways for action and object recognition.

The ventral visual stream is conceived as a pathway for object recognition. However, we also recognize the actions an object can be involved in. Here, we show that action recognition critically depends on a pathway in lateral occipitotemporal cortex, partially overlapping and topographically aligned with object representations that are precursors for action recognition. By contrast, object features that are more relevant for object recognition, such as color and texture, are typically found in ventral occipitotemporal cortex. We argue that occipitotemporal cortex contains similarly organized lateral and ventral 'what' pathways for action and object recognition, respectively. This account explains a number of observed phenomena, such as the duplication of object domains and the specific representational profiles in lateral and ventral cortex.

On the Evolutionary Origins of Land Plant Auxin Biology.

Indole-3-acetic acid, that is, auxin, is a molecule found in a broad phylogenetic distribution of organisms, from bacteria to eukaryotes. In the ancestral land plant auxin was co-opted to be the paramount phytohormone mediating tropic responses and acting as a facilitator of developmental decisions throughout the life cycle. The evolutionary origins of land plant auxin biology genes can now be traced with reasonable clarity. Genes encoding the two enzymes of the land plant auxin biosynthetic pathway arose in the ancestral land plant by a combination of horizontal gene transfer from bacteria and possible neofunctionalization following gene duplication. Components of the auxin transcriptional signaling network have their origins in ancestral alga genes, with gene duplication and neofunctionalization of key domains allowing integration of a portion of the preexisting transcriptional network with auxin. Knowledge of the roles of orthologous genes in extant charophycean algae is lacking, but could illuminate the ancestral functions of both auxin and the co-opted transcriptional network.

Novel Monomeric Fungal Subtilisin Inhibitor from a Plant-Pathogenic Fungus, Choanephora cucurbitarum: Isolation and Molecular Characterization

The bacterial protease inhibitor domains known as Streptomyces subtilisin inhibitors (SSI) are rarely found in fungi. Genome analysis of a fungal pathogen, Choanephora cucurbitarum KUS-F28377, revealed 11 SSI-like domains that are horizontally transferred and sequentially diverged during evolution. We investigated the molecular function of fungal SSI-like domains of C. cucurbitarum, designated "choanepins." Among the proteins tested, only choanepin9 showed inhibitory activity against subtilisin as the target protease, accounting for 47% of the inhibitory activity of bacterial SSI. However, the binding affinity (expressed as the dissociation constant [Kd ]) of choanepin9 measured via microscale thermophoresis was 21 nM, whereas that for bacterial SSI is 34 nM. The trend of binding and inhibitory activity suggests that the two inhibitors exhibit different inhibitory mechanisms for subtilisin protease. Interestingly, choanepin9 was identified as a monomer in studies in vitro, whereas bacterial SSI is a homodimer. Based on these observations, we constructed a monomeric bacterial SSI protein with decreased binding affinity to abrogate its inhibitory activity. By altering the reactive sites of choanepin9 deduced from the P1 and P4 sites of bacterial SSI, we reestablished that these residues in choanepins are also crucial for modulating inhibitory activity. These findings suggest that the fungal SSI evolved to target specific cognate proteases by altering the residues involved in inhibitory reactivity (reactive sites) and binding affinity (structural integrity). The function of fungal SSI proteins identified in this study provides not only a clue to fungal pathogenesis via protease inhibition but also a template for the design of novel serine protease inhibitors.IMPORTANCE Until recently, Streptomyces subtilisin inhibitors (SSI) were reported and characterized only in bacteria. We found SSI-like domains in a plant-pathogenic fungus, Choanephora cucurbitarum KUS-F28377, which contains 11 sequentially diverged SSI-like domains. None of these fungal SSI-like domains were functionally characterized before. The active form of fungal SSI-like protein is a monomer, in contrast to the homodimeric bacterial SSI. We constructed a synthetic monomer of bacterial SSI to demonstrate the modulation of its activity based on structural integrity and not reactive sites. Our results suggest the duplication and divergence of SSI-like domains of C. cucurbitarum within the genome to inhibit various cognate proteases during evolution by modulating both binding and reactivity. The molecular functional characterization of fungal SSI-like domains will be useful in understanding their biological role and future biotechnological applications.

Duplication of clostridial binding domains for enhanced macromolecular delivery into neurons.

Neurological diseases constitute a quarter of global disease burden and are expected to rise worldwide with the ageing of human populations. There is an increasing need to develop new molecular systems which can deliver drugs specifically into neurons, non-dividing cells meant to last a human lifetime. Neuronal drug delivery must rely on agents which can recognise neurons with high specificity and affinity. Here we used a recently introduced ‘stapling’ system to prepare macromolecules carrying duplicated binding domains from the clostridial family of neurotoxins. We engineered individual parts of clostridial neurotoxins separately and combined them using a strong alpha-helical bundle. We show that combining two identical binding domains of tetanus and botulinum type D neurotoxins, in a sterically defined way by protein stapling, allows enhanced intracellular delivery of molecules into neurons. We also engineered a botulinum neurotoxin type C variant with a duplicated binding domain which increased enzymatic delivery compared to the native type C toxin. We conclude that duplication of the binding parts of tetanus or botulinum neurotoxins will allow production of high avidity agents which could deliver imaging reagents and large therapeutic enzymes into neurons with superior efficiency.

Duplication of neuronal binding domains derived from clostridial neurotoxins greatly enhances intraneuronal delivery of therapeutics

Duplication of neuronal binding domains derived from clostridial neurotoxins greatly enhances intraneuronal delivery of therapeutics

Evolutionarily conserved partial gene duplication in the Triticeae tribe of grasses confers pathogen resistance

BackgroundThe large and highly repetitive genomes of the cultivated species Hordeum vulgare (barley), Triticum aestivum (wheat), and Secale cereale (rye) belonging to the Triticeae tribe of grasses appear to be particularly rich in gene-like sequences including partial duplicates. Most of them have been classified as putative pseudogenes. In this study we employ transient and stable gene silencing- and over-expression systems in barley to study the function of HvARM1 (for H. vulgare Armadillo 1), a partial gene duplicate of the U-box/armadillo-repeat E3 ligase HvPUB15 (for H. vulgare Plant U-Box 15).ResultsThe partial ARM1 gene is derived from a gene-duplication event in a common ancestor of the Triticeae and contributes to quantitative host as well as nonhost resistance to the biotrophic powdery mildew fungus Blumeria graminis. In barley, allelic variants of HvARM1 but not of HvPUB15 are significantly associated with levels of powdery mildew infection. Both HvPUB15 and HvARM1 proteins interact in yeast and plant cells with the susceptibility-related, plastid-localized barley homologs of THF1 (for Thylakoid formation 1) and of ClpS1 (for Clp-protease adaptor S1) of Arabidopsis thaliana. A genome-wide scan for partial gene duplicates reveals further events in barley resulting in stress-regulated, potentially neo-functionalized, genes.ConclusionThe results suggest neo-functionalization of the partial gene copy HvARM1 increases resistance against powdery mildew infection. It further links plastid function with susceptibility to biotrophic pathogen attack. These findings shed new light on a novel mechanism to employ partial duplication of protein-protein interaction domains to facilitate the expansion of immune signaling networks.

MOCASSIN-prot: a multi-objective clustering approach for protein similarity networks.

Proteins often include multiple conserved domains. Various evolutionary events including duplication and loss of domains, domain shuffling, as well as sequence divergence contribute to generating complexities in protein structures, and consequently, in their functions. The evolutionary history of proteins is hence best modeled through networks that incorporate information both from the sequence divergence and the domain content. Here, a game-theoretic approach proposed for protein network construction is adapted into the framework of multi-objective optimization, and extended to incorporate clustering refinement procedure. The new method, MOCASSIN-prot, was applied to cluster multi-domain proteins from ten genomes. The performance of MOCASSIN-prot was compared against two protein clustering methods, Markov clustering (TRIBE-MCL) and spectral clustering (SCPS). We showed that compared to these two methods, MOCASSIN-prot, which uses both domain composition and quantitative sequence similarity information, generates fewer false positives. It achieves more functionally coherent protein clusters and better differentiates protein families. MOCASSIN-prot, implemented in Perl and Matlab, is freely available at http://bioinfolab.unl.edu/emlab/MOCASSINprot. emoriyama2@unl.edu. Supplementary data are available at Bioinformatics online.

Aquerium: A web application for comparative exploration of domain-based protein occurrences on the taxonomically clustered genome tree.

Gene duplication and loss are major driving forces in evolution. While many important genomic resources provide information on gene presence, there is a lack of tools giving equal importance to presence and absence information as well as web platforms enabling easy visual comparison of multiple domain-based protein occurrences at once. Here, we present Aquerium, a platform for visualizing genomic presence and absence of biomolecules with a focus on protein domain architectures. The web server offers advanced domain organization querying against the database of pre-computed domains for ∼26,000 organisms and it can be utilized for identification of evolutionary events, such as fusion, disassociation, duplication, and shuffling of protein domains. The tool also allows alternative inputs of custom entries or BLASTP results for visualization. Aquerium will be a useful tool for biologists who perform comparative genomic and evolutionary analyses. The web server is freely accessible at http://aquerium.utk.edu. Proteins 2016; 85:72-77. © 2016 Wiley Periodicals, Inc.

Dual Constant Domain-Fab: A novel strategy to improve half-life and potency of a Met therapeutic antibody

The kinase receptor encoded by the Met oncogene is a sensible target for cancer therapy. The chimeric monovalent Fab fragment of the DN30 monoclonal antibody (MvDN30) has an odd mechanism of action, based on cell surface removal of Met via activation of specific plasma membrane proteases. However, the short half-life of the Fab, due to its low molecular weight, is a severe limitation for the deployment in therapy. This issue was addressed by increasing the Fab molecular weight above the glomerular filtration threshold through the duplication of the constant domains, in tandem (DCD-1) or reciprocally swapped (DCD-2). The two newly engineered molecules showed biochemical properties comparable to the original MvDN30 in vitro, acting as full Met antagonists, impairing Met phosphorylation and activation of downstream signaling pathways. As a consequence, Met-mediated biological responses were inhibited, including anchorage-dependent and -independent cell growth. In vivo DCD-1 and DCD-2 showed a pharmacokinetic profile significantly improved over the original MvDN30, doubling the circulating half-life and reducing the clearance. In pre-clinical models of cancer, generated by injection of tumor cells or implant of patient-derived samples, systemic administration of the engineered molecules inhibited the growth of Met-addicted tumors.

Artificial domain duplication replicates evolutionary history of ketol-acid reductoisomerases.

The duplication of protein structural domains has been proposed as a common mechanism for the generation of new protein folds. A particularly interesting case is the class II ketol-acid reductoisomerase (KARI), which putatively arose from an ancestral class I KARI by duplication of the C-terminal domain and corresponding loss of obligate dimerization. As a result, the class II enzymes acquired a deeply embedded figure-of-eight knot. To test this evolutionary hypothesis we constructed a novel class II KARI by duplicating the C-terminal domain of a hyperthermostable class I KARI. The new protein is monomeric, as confirmed by gel filtration and X-ray crystallography, and has the deeply knotted class II KARI fold. Surprisingly, its catalytic activity is nearly unchanged from the parent KARI. This provides strong evidence in support of domain duplication as the mechanism for the evolution of the class II KARI fold and demonstrates the ability of domain duplication to generate topological novelty in a function-neutral manner.

Circular Permutation Directs Orthogonal Assembly in Complex Collagen Peptide Mixtures

Multiple types of natural collagens specifically assemble and co-exist in the extracellular matrix. Although noncollagenous trimerization domains facilitate the folding of triple-helical regions, it is intriguing to ask whether collagen sequences are also capable of controlling heterospecific association. In this study, we designed a model system mimicking simultaneous specific assembly of two collagen heterotrimers using a genetically inspired operation, circular permutation. Previously, surface charge-pair interactions were optimized on three collagen peptides to promote the formation of an abc-type heterotrimer. Circular permutation of these sequences retained networks of stabilizing interactions, preserving both triple-helical structure and heterospecificity of assembly. Combining original peptides A, B, and C and permuted peptides D, E, and F resulted primarily in formation of A:B:C and D:E:F, a heterospecificity of 2 of 56 possible stoichiometries. This degree of specificity in collagen molecular recognition is unprecedented in natural or synthetic collagens. Analysis of natural collagen sequences indicates low similarity between the neighboring exons. Combining the synthetic collagen model and bioinformatic analysis provides insight on how fibrillar collagens might have arisen from the duplication of smaller domains.

Fgf and Hh signalling act on a symmetrical pre-pattern to specify anterior and posterior identity in the zebrafish otic placode and vesicle.

Specification of the otic anteroposterior axis is one of the earliest patterning events during inner ear development. In zebrafish, Hedgehog signalling is necessary and sufficient to specify posterior otic identity between the 10 somite (otic placode) and 20 somite (early otic vesicle) stages. We now show that Fgf signalling is both necessary and sufficient for anterior otic specification during a similar period, a function that is completely separable from its earlier role in otic placode induction. In lia(-/-) (fgf3(-/-)) mutants, anterior otic character is reduced, but not lost altogether. Blocking all Fgf signalling at 10-20 somites, however, using the pan-Fgf inhibitor SU5402, results in the loss of anterior otic structures and a mirror image duplication of posterior regions. Conversely, overexpression of fgf3 during a similar period, using a heat-shock inducible transgenic line, results in the loss of posterior otic structures and a duplication of anterior domains. These phenotypes are opposite to those observed when Hedgehog signalling is altered. Loss of both Fgf and Hedgehog function between 10 and 20 somites results in symmetrical otic vesicles with neither anterior nor posterior identity, which, nevertheless, retain defined poles at the anterior and posterior ends of the ear. These data suggest that Fgf and Hedgehog act on a symmetrical otic pre-pattern to specify anterior and posterior otic identity, respectively. Each signalling pathway has instructive activity: neither acts simply to repress activity of the other, and, together, they appear to be key players in the specification of anteroposterior asymmetries in the zebrafish ear.

Insights into plant biomass conversion from the genome of the anaerobic thermophilic bacterium Caldicellulosiruptor bescii DSM 6725

Caldicellulosiruptor bescii DSM 6725 utilizes various polysaccharides and grows efficiently on untreated high-lignin grasses and hardwood at an optimum temperature of ∼80°C. It is a promising anaerobic bacterium for studying high-temperature biomass conversion. Its genome contains 2666 protein-coding sequences organized into 1209 operons. Expression of 2196 genes (83%) was confirmed experimentally. At least 322 genes appear to have been obtained by lateral gene transfer (LGT). Putative functions were assigned to 364 conserved/hypothetical protein (C/HP) genes. The genome contains 171 and 88 genes related to carbohydrate transport and utilization, respectively. Growth on cellulose led to the up-regulation of 32 carbohydrate-active (CAZy), 61 sugar transport, 25 transcription factor and 234 C/HP genes. Some C/HPs were overproduced on cellulose or xylan, suggesting their involvement in polysaccharide conversion. A unique feature of the genome is enrichment with genes encoding multi-modular, multi-functional CAZy proteins organized into one large cluster, the products of which are proposed to act synergistically on different components of plant cell walls and to aid the ability of C. bescii to convert plant biomass. The high duplication of CAZy domains coupled with the ability to acquire foreign genes by LGT may have allowed the bacterium to rapidly adapt to changing plant biomass-rich environments.

Specificity in Computational Protein Design

A long-standing goal of computational protein design is to create proteins similar to those found in Nature. One motivation is to harness the exquisite functional capabilities of proteins for our own purposes. The extent of similarity between designed and natural proteins also reports on how faithfully our models represent the selective pressures that determine protein sequences. As the field of protein design shifts emphasis from reproducing native-like protein structure to function, it has become important that these models treat the notion of specificity in molecular interactions. Although specificity may, in some cases, be achieved by optimization of a desired protein in isolation, methods have been developed to address directly the desire for proteins that exhibit specific functions and interactions.

A genomics method to identify pathogenicity-related proteins. Application to aminoacyl-tRNA synthetase-like proteins

During their extended evolution genes coding for aminoacyl-tRNA synthetases (ARS) have experienced numerous instances of duplication, insertion and deletion of domains. The ARS-related proteins that have resulted from these genetic events are generally known as aminoacyl-tRNA synthetase-like proteins (ARS-like). This heterogeneous group of polypeptides carries out an equally varied number of functions that need not be related to gene translation. Several of these proteins remain uncharacterized. At least 16 different ARS-like proteins have been identified to date, but their functions remain incompletely understood. Here we review the individual phylogenetic distribution of these proteins in bacteria, and apply a new genomics method to determine their potential implication in pathogenicity.

Comparative Genomics of Phylogenetically Diverse Unicellular Eukaryotes Provide New Insights into the Genetic Basis for the Evolution of the Programmed Cell Death Machinery

Programmed cell death (PCD) represents a significant component of normal growth and development in multicellular organisms. Recently, PCD-like processes have been reported in single-celled eukaryotes, implying that some components of the PCD machinery existed early in eukaryotic evolution. This study provides a comparative analysis of PCD-related sequences across more than 50 unicellular genera from four eukaryotic supergroups: Unikonts, Excavata, Chromalveolata, and Plantae. A complex set of PCD-related sequences that correspond to domains or proteins associated with all main functional classes--from ligands and receptors to executors of PCD--was found in many unicellular lineages. Several PCD domains and proteins previously thought to be restricted to animals or land plants are also present in unicellular species. Noteworthy, the yeast, Saccharomyces cerevisiae--used as an experimental model system for PCD research, has a rather reduced set of PCD-related sequences relative to other unicellular species. The phylogenetic distribution of the PCD-related sequences identified in unicellular lineages suggests that the genetic basis for the evolution of the complex PCD machinery present in extant multicellular lineages has been established early in the evolution of eukaryotes. The shaping of the PCD machinery in multicellular lineages involved the duplication, co-option, recruitment, and shuffling of domains already present in their unicellular ancestors.