Primary Sequence Analysis Research Articles

Identification of phlorizin binding domains in sodium-glucose cotransporter family: SGLT1 as a unique model system.

The sodium glucose cotransporter SGLT1 expressed mainly in the intestine and kidney has been explored extensively for understanding the mechanism of sugar cotransport and its inhibition by a classical competitive inhibitor, phlorizin (Pz). It has been shown that inhibition of SGLT1 by Pz involves its interaction followed by major conformational changes in the Pz binding domain (PBD) in C-terminal loop 13. However, the mechanism of Pz inhibition and its interaction with other members of SGLT is not known. In this hypothesis, we performed molecular modeling of SGLT1-loop 13 with Pz and carried out primary sequence analyses and secondary structure predictions to determine qualitatively similar PBDs in C-termini of human SGLT2-4, except for vSGLT, which contains an unstructured short C-terminus. The ranking of predictions of Pz interaction strongly agrees with the following ranking of previously reported Pz inhibition: SGLT2>SGLT1>SGLT4>SGLT3>>vSGLT. In addition, the sugar binding residues were found to be quite conserved among all SGLT members investigated here. Based on these preliminary analyses, we propose that other Pz-sensitive SGLTs are also inhibited via mechanism similar to SGLT1 where an aglucone of Pz, phloretin, interacts with PBD and glucoside moiety with sugar binding residues. Our hypothesis sets the stage for future analyses on investigation of Pz interaction with SGLT family and further suggests that Pz modeling may be explored to design novel inhibitors targeting several SGLT members.

Comparative analysis of protein primary sequences with graph energy

We propose in this paper, the graph energy and Laplacian energy of 20 amino acids based on the codons coding the amino acids and apply them to put forward a novel 2-D graphical representation of proteins. The novel graphical representation has no circuit or degeneracy, uniquely represents proteins and allows one to easily and quickly visually observe and inspect similarity/dissimilarity between them. It also leads to two novel protein descriptors, the graph energy of a protein sequence, and the increment of graph energy between two protein sequences. We develop similarities/dissimilarities model and successfully analyze the similarities/dissimilarities of ND5, 36PDs, 24TFs and 27AFPs with good results consistent with ClustalW even better ones.

Mutational Analysis of a Putative Drug Resistance Transporter in the Human Parasite T. gondii

Toxoplasma gondii is an obligate intracellular parasite that causes toxoplasmosis in animals and humans. At least 30% of the human population is infected. The infection can lead to lethal disease in immunocompromised individuals including AIDS and organ‐transplant patients. No existing drugs are capable of eradicating the parasites from infected people. Opening new options, we recently reported that Toxoplasma parasites intercept host cytosolic proteins by funneling them into a lysosome‐equivalent organelle termed vacuolar compartment (VAC) where they are digested. Additional work revealed that the VAC harbors a homolog of the malaria chloroquine resistance transporter (CRT). A single point mutation in CRT confers resistance to chloroquine treatment for malaria, profoundly impacting fatalities. Knocking out the CRT gene in Toxoplasma results in an enlarged VAC and reduced fitness. Topology analysis of the CRT primary sequence showed that four cysteine residues localize to a loop oriented in the lumen of the VAC. We hypothesized that these conserved cysteine residues function to regulate redox potential of the VAC and/or bind to metal ions to optimize the activity of VAC proteases. We performed site‐directed mutagenesis to mutate these four cysteine amino acids to serine residues individually and introduce them into CRT‐deficient parasites. We found that ΔcrtCRTC585S and ΔcrtCRTC596S did not restore the size of the VAC, and that ΔcrtCRTC609S did restored it but about 50% of the parasites showed mistrafficking of CRT. By identifying important conserved residues in a therapeutically relevant transporter this work forms a basis for future mechanistic dissection of CRT function

Looking for the key to secrets of RNA under the lamp-post.

Looking for the key to secrets of RNA under the lamp-post.

Pseudomonas syringae pv. tabaci 에서 LuxR-type 전사조절자인 PsyR에 의한 병원성 유전자들의 조절

Pseudomonas syringae pathovar tabaci is a plant pathogenic bacterium that causes wildfire disease in tobacco plants. In P. syringae pv. tabaci, PsyI, a LuxI-type protein, acts as an AHL synthase, while primary and secondary sequence analysis of PsyR has revealed that it is a homolog of the LuxR-type transcriptional regulator that responds to AHL molecules. In this study, using phenotypic and genetic analyses in P. syringae pv. tabaci, we show the effect of PsyR protein as a quorum-sensing (QS) transcriptional regulator. Regulatory effects of PsyR on swarming motility and production of siderophores, tabtoxin, and N-acyl homoserine lactones were examined via phenotypic assays, and confirmed by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Further qRT-PCR showed that PsyR regulates expression of these virulence genes in response to environmental signals. However, an upstream region of the gene was not bound with purified MBP-PsyR protein; rather, PsyR was only able to shift the upstream region of psyI. These results suggested that PsyR may be indirectly controlled via intermediate-regulatory systems and that auto-regulation by PsyR does not occur.

Computational study of β-N-acetylhexosaminidase from Talaromyces flavus, a glycosidase with high substrate flexibility.

Backgroundβ-N-Acetylhexosaminidase (GH20) from the filamentous fungus Talaromyces flavus, previously identified as a prominent enzyme in the biosynthesis of modified glycosides, lacks a high resolution three-dimensional structure so far. Despite of high sequence identity to previously reported Aspergillus oryzae and Penicilluim oxalicum β-N-acetylhexosaminidases, this enzyme tolerates significantly better substrate modification. Understanding of key structural features, prediction of effective mutants and potential substrate characteristics prior to their synthesis are of general interest.ResultsComputational methods including homology modeling and molecular dynamics simulations were applied to shad light on the structure-activity relationship in the enzyme. Primary sequence analysis revealed some variable regions able to influence difference in substrate affinity of hexosaminidases. Moreover, docking in combination with consequent molecular dynamics simulations of C-6 modified glycosides enabled us to identify the structural features required for accommodation and processing of these bulky substrates in the active site of hexosaminidase from T. flavus. To access the reliability of predictions on basis of the reported model, all results were confronted with available experimental data that demonstrated the principal correctness of the predictions as well as the model.ConclusionsThe main variable regions in β-N-acetylhexosaminidases determining difference in modified substrate affinity are located close to the active site entrance and engage two loops. Differences in primary sequence and the spatial arrangement of these loops and their interplay with active site amino acids, reflected by interaction energies and dynamics, account for the different catalytic activity and substrate specificity of the various fungal and bacterial β-N-acetylhexosaminidases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0465-8) contains supplementary material, which is available to authorized users.

Human, vector and parasite Hsp90 proteins: A comparative bioinformatics analysis

The treatment of protozoan parasitic diseases is challenging, and thus identification and analysis of new drug targets is important. Parasites survive within host organisms, and some need intermediate hosts to complete their life cycle. Changing host environment puts stress on parasites, and often adaptation is accompanied by the expression of large amounts of heat shock proteins (Hsps). Among Hsps, Hsp90 proteins play an important role in stress environments. Yet, there has been little computational research on Hsp90 proteins to analyze them comparatively as potential parasitic drug targets. Here, an attempt was made to gain detailed insights into the differences between host, vector and parasitic Hsp90 proteins by large-scale bioinformatics analysis. A total of 104 Hsp90 sequences were divided into three groups based on their cellular localizations; namely cytosolic, mitochondrial and endoplasmic reticulum (ER). Further, the parasitic proteins were divided according to the type of parasite (protozoa, helminth and ectoparasite). Primary sequence analysis, phylogenetic tree calculations, motif analysis and physicochemical properties of Hsp90 proteins suggested that despite the overall structural conservation of these proteins, parasitic Hsp90 proteins have unique features which differentiate them from human ones, thus encouraging the idea that protozoan Hsp90 proteins should be further analyzed as potential drug targets.

De novo secondary structure motif discovery using RNAProfile.

RNA secondary structure plays critical roles in several biological processes. For example, many trans-acting noncoding RNA genes and cis-acting RNA regulatory elements present functional motifs, conserved both in structure and sequence, that can be hardly detected by primary sequence analysis alone. We describe here how conserved secondary structure motifs shared by functionally related RNA sequences can be detected through the software tool RNAProfile. RNAProfile takes as input a set of unaligned RNA sequences expected to share a common motif, and outputs the regions that are most conserved throughout the sequences, according to a similarity measure that takes into account both the sequence of the regions and the secondary structure they can form according to base-pairing and thermodynamic rules. The method is split into two parts. First, it identifies candidate regions within the input sequences, and associates with each region a locally optimal secondary structure. Then, it compares candidate regions to one another, both at sequence and structure level, and builds motifs exploring the search space through a greedy heuristic. We provide a detailed guide to the different parameters that can be employed, and usage examples showing the different software capabilities.

Identification, characterization, immobilization of a novel type hydrolase (LmH) from Listeria monocytogenes

A novel type of hydrolase (LmH) from Listeria monocytogenes was identified, characterized, and immobilized for biotechnological applications. Primary sequence analysis indicated that LmH had a catalytic triad (Ser91-Asp192-His222) with a molecular weight of 27.8kDa. Homologs of this enzyme are produced by many Gram-positive bacteria including Bacillus, Staphylococcus, and Enterococcus. Biochemical properties of LmH were investigated by performing mass spectrometry, dynamic light scattering (DLS), enzyme assays, enantioselective analysis, circular dichroism (CD) spectroscopy, fluorescence analysis, and macroscopic hydrogel formations. Interestingly, cross-linked enzyme aggregates (CLEAs) of LmH exhibited enhanced stability and good recycling abilities compared to free LmH. These molecular characteristics of LmH highlight its great potential for the pharmaceutical, biotechnological, and chemical industries.

Clinical impacts of genomic copy number gains at Xq28.

Duplications of the Xq28 region are the most frequent chromosomal aberrations observed in patients with intellectual disability (ID), especially in males. These duplications occur by variable mechanisms, including interstitial duplications mediated by segmental duplications in this region and terminal duplications (functional disomy) derived from translocation with other chromosomes. The most commonly duplicated region includes methyl CpG-binding protein 2 gene (MECP2), which has a minimal duplicated size of 0.2 Mb. Patients with MECP2 duplications show severe ID, intractable seizures and recurrent infections. Duplications in the telomeric neighboring regions, which include GDP dissociation inhibitor 1 gene (GDI1) and ras-associated protein RAB39B gene (RAB39B), are independently associated with ID, and many segmental duplications located in this region could mediate these frequently observed interstitial duplications. In addition, large duplications, including MECP2 and GDI1, induce hypoplasia of the corpus callosum. Abnormalities observed in the white matter, revealed by brain magnetic resonance imaging, are a common finding in patients with MECP2 duplications. As primary sequence analysis cannot be used to determine the region responsible for chromosomal duplication syndrome, finding this region relies on the collection of genotype–phenotype data from patients.

A putative de-N-acetylase of the PIG-L superfamily affects fluoroquinolone tolerance inPseudomonas aeruginosa

A major cause of treatment failure of infections caused by Pseudomonas aeruginosa is the presence of antibiotic-insensitive persister cells. The mechanism of persister formation in P. aeruginosa is largely unknown, and so far, only few genetic determinants have been linked to P. aeruginosa persistence. Based on a previous high-throughput screening, we here present dnpA (de-N-acetylase involved in persistence; gene locus PA14_66140/PA5002) as a new gene involved in noninherited fluoroquinolone tolerance in P. aeruginosa. Fluoroquinolone tolerance of a dnpA mutant is strongly reduced both in planktonic culture and in a biofilm model, whereas overexpression of dnpA in the wild-type strain increases the persister fraction. In addition, the susceptibility of the dnpA mutant to different classes of antibiotics is not affected. dnpA is part of the conserved LPS core oligosaccharide biosynthesis gene cluster. Based on primary sequence analysis, we predict that DnpA is a de-N-acetylase, acting on an unidentified substrate. Site-directed mutagenesis suggests that this enzymatic activity is essential for DnpA-mediated persistence. A transcriptome analysis indicates that DnpA primarily affects the expression of genes involved in surface-associated processes. We discuss the implications of these findings for future antipersister therapies targeted at chronic P. aeruginosa infections.

Species-specific alternative splicing leads to unique expression of sno-lncRNAs.

BackgroundIntron-derived long noncoding RNAs with snoRNA ends (sno-lncRNAs) are highly expressed from the imprinted Prader-Willi syndrome (PWS) region on human chromosome 15. However, sno-lncRNAs from other regions of the human genome or from other genomes have not yet been documented.ResultsBy exploring non-polyadenylated transcriptomes from human, rhesus and mouse, we have systematically annotated sno-lncRNAs expressed in all three species. In total, using available data from a limited set of cell lines, 19 sno-lncRNAs have been identified with tissue- and species-specific expression patterns. Although primary sequence analysis revealed that snoRNAs themselves are conserved from human to mouse, sno-lncRNAs are not. PWS region sno-lncRNAs are highly expressed in human and rhesus monkey, but are undetectable in mouse. Importantly, the absence of PWS region sno-lncRNAs in mouse suggested a possible reason why current mouse models fail to fully recapitulate pathological features of human PWS. In addition, a RPL13A region sno-lncRNA was specifically revealed in mouse embryonic stem cells, and its snoRNA ends were reported to influence lipid metabolism. Interestingly, the RPL13A region sno-lncRNA is barely detectable in human. We further demonstrated that the formation of sno-lncRNAs is often associated with alternative splicing of exons within their parent genes, and species-specific alternative splicing leads to unique expression pattern of sno-lncRNAs in different animals.ConclusionsComparative transcriptomes of non-polyadenylated RNAs among human, rhesus and mouse revealed that the expression of sno-lncRNAs is species-specific and that their processing is closely linked to alternative splicing of their parent genes. This study thus further demonstrates a complex regulatory network of coding and noncoding parts of the mammalian genome.

Structural Proteins from Whelk Egg Capsule with Long Range Elasticity Associated with a Solid-State Phase Transition

The robust, proteinaceous egg capsules of marine prosobranch gastropods (genus Busycotypus ) exhibit unique biomechanical properties such as high elastic strain recovery and elastic energy dissipation capability. Capsule material possesses long-range extensibility that is fully recoverable and is the result of a secondary structure phase transition from α-helical coiled-coil to extended β-sheet rather than of entropic (rubber) elasticity. We report here the characterization of the precursor proteins that make up this material. Three different proteins have been purified and analyzed, and complete protein sequences deduced from messenger ribonucleic acid (mRNA) transcripts. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy indicate that the proteins are strongly α-helical in solution and primary sequence analysis suggests that these proteins have a propensity to form coiled-coils. This is in agreement with previous wide-angle X-ray scattering (WAXS) and solid-state Raman spectroscopic analysis of mature egg capsules.

Identification of two novel cytolysins from the hydrozoan Olindias sambaquiensis (Cnidaria)

BackgroundAlthough the hydrozoan Olindias sambaquiensis is the most common jellyfish associated with human envenomation in southeastern and southern Brazil, information about the composition of its venom is rare. Thus, the present study aimed to analyze pharmacological aspects of O. sambaquiensis venom as well as clinical manifestations observed in affected patients. Crude protein extracts were prepared from the tentacles of animals; peptides and proteins were sequenced and submitted to circular dichroism spectroscopy. Creatine kinase, cytotoxicity and hemolytic activity were evaluated by specific methods.ResultsWe identified two novel cytolysins denominated oshem 1 and oshem 2 from the tentacles of this jellyfish. The cytolysins presented the amino acid sequences NEGKAKCGNTAGSKLTFKSADECTKTGQK (oshem 1) and NNSKAKCGDLAGWSKLTFKSADECTKTGQKS (oshem 2) with respective molecular masses of 3.013 kDa and 3.375 kDa. Circular dichroism revealed that oshem 1 has random coils and small α-helix conformation as main secondary structure whereas oshem 2 presents mainly random coils as its main secondary structure probably due to the presence of W (13) in oshem 2. The hemolysis levels induced by oshem 1 and oshem 2 using a peptide concentration of 0.2 mg/mL were, respectively, 51.7 ± 6.5% and 32.9 ± 8.7% (n = 12 and p ≤ 0.05). Oshem 1 and oshem 2 showed significant myonecrotic activity, evaluated by respective CK level measurements of 1890.4 ± 89 and 1212.5 ± 103 (n = 4 and p ≤ 0.05). In addition, myonecrosis was also evaluated by cell survival, which was measured at 72.4 ± 8.6% and 83.5 ± 6.7% (n = 12 and p ≤ 0.05), respectively. The structural analysis showed that both oshem 1 and oshem 2 should be classified as a small basic hemolytic peptide.ConclusionThe amino acid sequences of two peptides were highly similar while the primary amino acid sequence analysis revealed W (22th) as the most important mutation. Finally oshem 1 and oshem 2 are the first cytolytic peptides isolated from the Olindias sambaquiensis and should probably represent a novel class of cytolytic peptides.

The RNA helicase RHAU (DHX36) suppresses expression of the transcription factor PITX1

RNA Helicase associated with AU-rich element (RHAU) (DHX36) is a DEAH (Aspartic acid, Glumatic Acid, Alanine, Histidine)-box RNA helicase that can bind and unwind G4-quadruplexes in DNA and RNA. To detect novel RNA targets of RHAU, we performed an RNA co-immunoprecipitation screen and identified the PITX1 messenger RNA (mRNA) as specifically and highly enriched. PITX1 is a homeobox transcription factor with roles in both development and cancer. Primary sequence analysis identified three probable quadruplexes within the 3′-untranslated region of the PITX1 mRNA. Each of these sequences, when isolated, forms stable quadruplex structures that interact with RHAU. We provide evidence that these quadruplexes exist in the endogenous mRNA; however, we discovered that RHAU is tethered to the mRNA via an alternative non–quadruplex-forming region. RHAU knockdown by small interfering RNA results in significant increases in PITX1 protein levels with only marginal changes in mRNA, suggesting a role for RHAU in translational regulation. Involvement of components of the microRNA machinery is supported by similar and non-additive increases in PITX1 protein expression on Dicer and combined RHAU/Dicer knockdown. We also demonstrate a requirement of argonaute-2, a key RNA-induced silencing complex component, to mediate RHAU-dependent changes in PITX1 protein levels. These results demonstrate a novel role for RHAU in microRNA-mediated translational regulation at a quadruplex-containing 3′-untranslated region.

Mining Immune Epitopes in the Inner Ear

Etiologies for many inner ear disorders, including autoimmune inner ear disease, sudden sensorineural hearing loss, and Meniere's disease, remain unknown. Indirect evidence suggests an immune-mediated process involving an allergic or autoimmune mechanism. We examined whether known immunogenic proteins share sequence similarity with inner ear proteins, which may lead to cross-reactivity and detrimental immune activation. Comprehensive bioinformatic analyses of primary sequences of intact and mutated proteins associated with human hearing loss and all proteins known to be expressed in the human inner ear were compared with all immune epitopes in the Immune Epitope Database. The exact match and basic local alignment search tool computational algorithms identified 3036 and 106 unique epitope matches, respectively, the majority of which were infectious epitopes. If validated in future clinical trials, these candidate immune epitopes in the inner ear would be potential novel targets for diagnosis and treatment of some inner ear disorders and the resulting hearing loss.

Cloning and molecular characterization of BumaMPs1, a novel metalloproteinases from the venom of scorpion Buthus martensi Karsch

Scorpion venoms metalloproteinase is involved in a number of important biological, physiological and pathophysiological processes. In this work, a complete sequence of metalloproteinase was first obtained from venom of scorpion Buthus martensi and named as BumaMPs1. BumaMPs1 has 393 amino acid residues containing with a molecular mass of 44.53 kDa, showing an isoelectric point of 5.66. The primary sequence analysis indicated that the BumaMPs1 contains a zinc-binding motif (HELGHNLGISH), methionine-turn motif (YIM), disintegrin-like domain (ETCD) and N-glycosylation site. The multiple alignment of its deduced amino acid sequence and those of other metalloproteinase showed a high structural similarly, mainly among class reprolysin proteases. The phylogenetic analysis showed early divergence and independent evolution of BumaMPs1 from other metalloproteinase.

Divergent evolution of protein conformational dynamics in dihydrofolate reductase

Molecular evolution is driven by mutations, which may affect the fitness of an organism and are then subject to natural selection or genetic drift. Analysis of primary protein sequences and tertiary structures has yielded valuable insights into the evolution of protein function, but little is known about evolution of functional mechanisms, protein dynamics and conformational plasticity essential for activity. We characterized the atomic-level motions across divergent members of the dihydrofolate reductase (DHFR) family. Despite structural similarity, E. coli and human DHFRs use different dynamic mechanisms to perform the same function, and human DHFR cannot complement DHFR-deficient E. coli cells. Identification of the primary sequence determinants of flexibility in DHFRs from several species allowed us to propose a likely scenario for the evolution of functionally important DHFR dynamics, following a pattern of divergent evolution that is tuned by the cellular environment.

Correlation of thermostability and conformational changes of catechol 2, 3-dioxygenases from two disparate micro-organisms

We have investigated the structure of recombinant catechol 2, 3-dioxygenase (C23O) purified from two species in which the enzyme has evolved to function at different temperature. The two species are mesophilic bacterium Pseudomonas putida strain mt-2 and thermophilic archaea Sulfolobus acidocaldariusDSM639. Using the primary sequence analysis, we show that both C23Os have only 30% identity and 48% similarity but contain conserved amino acid residues forming an active site area around the iron ion. The corresponding differences in homology, but structural similarity in active area residues, appear to provide completely different responses to heating the two enzymes. We confirm this by small angle X-ray scattering and demonstrate that the overall structure of C23O from P. putida is slightly different from its crystalline form whereas the solution scattering of C23O from S. acidocaldarius at temperatures between 4 and 85°C ideally fits the calculated scattering from the single crystal structure. The thermostability of C23O from S. acidocaldarius correlates well with conformation in solution during thermal treatment. The similarity of the two enzymes in primary and tertiary structure may be taken as a confirmation that two enzymes have evolved from a common ancestor.

In silico analysis of the fucosylation-associated genome of the human blood fluke Schistosoma mansoni: cloning and characterization of the enzymes involved in GDP-L-fucose synthesis and Golgi import.

BackgroundCarbohydrate structures of surface-expressed and secreted/excreted glycoconjugates of the human blood fluke Schistosoma mansoni are key determinants that mediate host-parasite interactions in both snail and mammalian hosts. Fucose is a major constituent of these immunologically important glycans, and recent studies have sought to characterize fucosylation-associated enzymes, including the Golgi-localized fucosyltransferases that catalyze the transfer of L-fucose from a GDP-L-fucose donor to an oligosaccharide acceptor. Importantly, GDP-L-fucose is the only nucleotide-sugar donor used by fucosyltransferases and its availability represents a bottleneck in fucosyl-glycotope expression.MethodsA homology-based genome-wide bioinformatics approach was used to identify and molecularly characterize the enzymes that contribute to GDP-L-fucose synthesis and Golgi import in S. mansoni. Putative functions were further investigated through molecular phylogenetic and immunocytochemical analyses.ResultsWe identified homologs of GDP-D-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase (GMER), which constitute a de novo pathway for GDP-L-fucose synthesis, in addition to a GDP-L-fucose transporter (GFT) that putatively imports cytosolic GDP-L-fucose into the Golgi. In silico primary sequence analyses identified characteristic Rossman loop and short-chain dehydrogenase/reductase motifs in GMD and GMER as well as 10 transmembrane domains in GFT. All genes are alternatively spliced, generating variants of unknown function. Observed quantitative differences in steady-state transcript levels between miracidia and primary sporocysts may contribute to differential glycotope expression in early larval development. Additionally, analyses of protein expression suggest the occurrence of cytosolic GMD and GMER in the ciliated epidermal plates and tegument of miracidia and primary sporocysts, respectively, which is consistent with previous localization of highly fucosylated glycotopes.ConclusionsThis study is the first to identify and characterize three key genes that are putatively involved in the synthesis and Golgi import of GDP-L-fucose in S. mansoni and provides fundamental information regarding their genomic organization, genetic variation, molecular phylogenetics, and developmental expression in intramolluscan larval stages.