Bioinformatic prediction of the stereoselectivity of modular polyketide synthase: an update of the sequence motifs in ketoreductase domain.

Polyketides are a major class of natural products, including bioactive medicines such as erythromycin and rapamycin. They are often rich in stereocenters biosynthesized by the ketoreductase (KR) domain within the polyketide synthase (PKS) assembly line. Previous studies have identified conserved motifs in KR sequences that enable the bioinformatic prediction of product stereochemistry. However, the reliability and applicability of these prediction methods have not been thoroughly assessed. In this study, we conducted a comprehensive bioinformatic analysis of 1,762 KR sequences from cis-AT PKSs to reevaluate the residues involved in conferring stereoselectivity. Our findings indicate that the previously identified fingerprint motifs remain valid for KRs in ß-modules from actinobacteria, but their reliability diminishes for KRs from other module types or taxonomic origins. Additionally, we have identified several new motifs that exhibit a strong correlation with the stereochemical outcomes of KRs. These updated fingerprint motifs for stereochemical prediction not only enhance our understanding of the enzymatic mechanisms governing stereocontrol but also facilitate accurate stereochemical prediction and genome mining of polyketides derived from modular cis-AT PKSs.

Mechanistic insights into sodium ion-mediated ligand binding affinity and modulation of 5-HT2B GPCR activity: implications for drug discovery and development.

PURPOSE: The G-protein coupled receptor (GPCR) family, implicated in neurological disorders and drug targets, includes the sensitive serotonin receptor subtype, 5-HT2B. The influence of sodium ions on ligand binding at the receptor's allosteric region is being increasingly studied for its impact on receptor structure. METHODS: High-throughput virtual screening of three libraries, specifically the Asinex-GPCR library, which contains 8,532 compounds and FDA-approved (2466 compounds) and investigational compounds (2731)) against the modeled receptor [4IB4-5HT2BRM] using the standard agonist/antagonist (Ergotamine/Methysergide), as previously selected from our studies based on ADMET profiling, and further on basis of binding free energy a single compound - dihydroergotamine is chosen. RESULTS: This compound displayed strong interactions with the conserved active site. Ions influence ligand binding, with stronger interactions (3-H-bonds and 1-π-bond around 3.35 Å) observed when an agonist and ions are present. Ions entry is guided by conserved motifs in helices III, IV, and VII, which regulate the receptor. Dihydroergotamine, the selected drug, showed binding variance based on ions presence/absence, affecting amino acid residues in these motifs. DCCM and PCA confirmed the stabilization of ligands, with a greater correlation (∼46.6%-PC1) observed with ions. Dihydroergotamine-modified interaction sites within the receptor necessary for activation, serving as a potential 5HT2BRM agonist. RDF analysis showed the sodium ions density around the active site during dihydroergotamine binding. CONCLUSION: Our study provides insights into sodium ion mobility's role in controlling ligand binding affinity in 5HT2BR, offering therapeutic development insights.

Evaluating conserved domains and motifs of decapod gonadotropin-releasing hormone G protein-coupled receptor superfamily.

G protein-coupled receptors (GPCRs) are an ancient family of signal transducers that are both abundant and consequential in metazoan endocrinology. The evolutionary history and function of the GPCRs of the decapod superfamilies of gonadotropin-releasing hormone (GnRH) are yet to be fully elucidated. As part of which, the use of traditional phylogenetics and the recycling of a diminutive set of mis-annotated databases has proven insufficient. To address this, we have collated and revised eight existing and three novel GPCR repertoires for GnRH of decapod species. We developed a novel bioinformatic workflow that included clustering analysis to capture likely GnRH receptor-like proteins, followed by phylogenetic analysis of the seven transmembrane-spanning domains. A high degree of conservation of the sequences and topology of the domains and motifs allowed the identification of species-specific variation (up to ~70%, especially in the extracellular loops) that is thought to be influential to ligand-binding and function. Given the key functional role of the DRY motif across GPCRs, the classification of receptors based on the variation of this motif can be universally applied to resolve cryptic GPCR families, as was achieved in this work. Our results contribute to the resolution of the evolutionary history of invertebrate GnRH receptors and inform the design of bioassays in their deorphanization and functional annotation.

An Updated Evolutionary and Structural Study of TBK1 Reveals Highly Conserved Motifs as Potential Pharmacological Targets in Neurodegenerative Diseases.

TANK-binding kinase 1 protein (TBK1) is a kinase that belongs to the IκB (IKK) family. TBK1, also known as T2K, FTDALS4, NAK, IIAE8, and NF-κB, is responsible for the phosphorylation of the amino acid residues, serine and threonine. This enzyme is involved in various key biological processes, including interferon activation and production, homeostasis, cell growth, autophagy, insulin production, and the regulation of TNF-α, IFN-ß, and IL-6. Mutations in the TBK1 gene alter the protein's normal function and may lead to an array of pathological conditions, including disorders of the central nervous system. The present study sought to elucidate the role of the TBK1 protein in amyotrophic lateral sclerosis (ALS), a human neurodegenerative disorder. A broad evolutionary and phylogenetic analysis of TBK1 was performed across numerous organisms to distinguish conserved regions important for the protein's function. Subsequently, mutations and SNPs were explored, and their potential effect on the enzyme's function was investigated. These analytical steps, in combination with the study of the secondary, tertiary, and quaternary structure of TBK1, enabled the identification of conserved motifs, which can function as novel pharmacological targets and inform therapeutic strategies for amyotrophic lateral sclerosis.

Allosteric modulation of conserved motifs and helices in 5HT2BR: Advances drug discovery and therapeutic approach towards drug resistant epilepsy.

The 5HT2BR, class-A GPCR is a new target, and its significance for seizure reduction in Dravet syndrome is just now gaining interest, suggesting its specific role in epileptic seizure management. Homology modeling of human 5HT2BR (P41595), was performed using a template 4IB4, the modeled structure was cross-validated (stereo chemical hindrance, Ramachandran plot, enrichment analysis) to mimic a closer native structure. Virtual screening (8532 compounds), drug-likeliness, mutagenicity, and carcinogenicity profiling prioritized six compounds for molecular dynamics (500 ns), Rgyr, DCCM. The receptor's C-alpha fluctuation upon bound agonist (6.91 Å), known antagonist (7.03 Å), and LAS 52115629 (5.83 Å) binding varies, leading to receptor stabilization. The residues C-alpha side-chain in active site strongly interacts (hydrogen bonds) with bound agonist (100% interaction: ASP135), known antagonist (95%:ASP135), and LAS 52115629 (100%:ASP135). The Rgyr for receptor-ligand complex, LAS 52115629 (25.68 Å), lies close to bound agonist-Ergotamine, and DCCM analysis also shows strong positive correlations for LAS 52115629 as compared to known drugs. LAS 52115629 is less likely to cause toxicity than known drugs. The structural parameters in the modeled receptor's conserved motifs (DRY, PIF, NPY) were altered for receptor activation upon ligand-binding, which otherwise was in the in-activated state. The ligand (LAS 52115629)-binding further alters the helices-III, V, VI (G-protein bound), and VII, which form potential interacting sites with the receptor and are proven necessary for activating the receptor. Therefore, LAS 52115629 can act as a potential 5HT2BR agonist, targeting drug-resistant epilepsy.Communicated by Ramaswamy H. Sarma.

Conserved GxxxG and WN motifs of MIC13 are essential for bridging two MICOS subcomplexes.

Mitochondrial ultrastructure is highly adaptable and undergoes dynamic changes upon physiological and energetic cues. MICOS (mitochondrial contact site and cristae organizing system), a large oligomeric protein complex, maintains mitochondrial ultrastructure as it is required for formation of crista junctions (CJs) and contact sites. MIC13 acts as a critical bridge between two MICOS subcomplexes. Deletion of MIC13 causes loss of CJs resulting in cristae accumulating as concentric rings and specific destabilization of the MIC10-subcomplex. Mutations in MIC13 are associated with infantile lethal mitochondrial hepato-encephalopathy, yet functional regions within MIC13 were not known. To identify and characterize such regions, we systemically generated 20 amino-acids deletion variants across the length of MIC13. While deletion of many of these regions of MIC13 is dispensable for its stability, the N-terminal region and a stretch between amino acid residues 84 and 103 are necessary for the stability and functionality of MIC13. We could further locate conserved motifs within these regions and found that a GxxxG motif in the N-terminal transmembrane segment and an internal WN motif are essential for stability of MIC13, formation of the MIC10-subcomplex, interaction with MIC10- and MIC60-subcomplexes and maintenance of cristae morphology. The GxxxG motif is required for membrane insertion of MIC13. Overall, we systematically found important conserved residues of MIC13 that are required to perform the bridging between the two MICOS subcomplexes. The study improves our understanding of the basic molecular function of MIC13 and has implications for its role in the pathogenesis of a severe mitochondrial disease.

Genome-wide identification and expression analysis of the trehalose-6-phosphate synthase (TPS) gene family in cucumber (Cucumis sativus L.).

Trehalose-6-phosphate synthase (TPS) is significant in the growth, development and stress resistance of plants. We identified the cucumber TPS family and its physicochemical properties, domains, gene structures, evolutionary relationships, gene locations, cis-acting elements, conserved motifs, and expression patterns using bioinformatics. Our results uncovered seven CsTPS genes in the cucumber genome and named CsTPS1-CsTPS7 according to their locations in the chromosomes. Seven CsTPS genes were randomly distributed in six cucumber chromosomes. Domain analysis showed that the TPS and TPP domains exist in all CsTPSs, and an additional hydrolase-3 domain exist in CsTPS3, CsTPS5 and CsTPS6. Phylogenetic analysis showed that TPS proteins from Arabidopsis, rice, soybean, and cucumber were divided into two subfamilies (Class I and Class II) and they were further divided into seven subgroups. TPS proteins from Arabidopsis and cucumber were grouped together, suggesting a close evolutionary relationship. Gene structure analysis indicated that most Class I genes contained 16-17 introns, while Class II genes (except CsTPS7) had two introns. Motif analysis showed that Class II genes had 10 complete conserved motifs, while Class I genes lacked motif 8 and motif 9. Furthermore, CsTPS genes possessed numerous cis-acting elements related to stress, hormone, and light response in the promoter regions. GO analysis indicated multiple functions for the CsTPS proteins. Expression analysis of CsTPS genes in different tissues found that they were expressed in roots, stems and leaves, with the highest expression levels in roots. The expression analysis of CsTPSs under different treatments showed that CsTPS genes may participate in the response to abiotic stress, plant hormones and sugar treatments.

Conserved Structural Motifs of Two Distant IAV Subtypes in Genomic Segment 5 RNA.

The functionality of RNA is fully dependent on its structure. For the influenza A virus (IAV), there are confirmed structural motifs mediating processes which are important for the viral replication cycle, including genome assembly and viral packaging. Although the RNA of strains originating from distant IAV subtypes might fold differently, some structural motifs are conserved, and thus, are functionally important. Nowadays, NGS-based structure modeling is a source of new in vivo data helping to understand RNA biology. However, for accurate modeling of in vivo RNA structures, these high-throughput methods should be supported with other analyses facilitating data interpretation. In vitro RNA structural models complement such approaches and offer RNA structures based on experimental data obtained in a simplified environment, which are needed for proper optimization and analysis. Herein, we present the secondary structure of the influenza A virus segment 5 vRNA of A/California/04/2009 (H1N1) strain, based on experimental data from DMS chemical mapping and SHAPE using NMIA, supported by base-pairing probability calculations and bioinformatic analyses. A comparison of the available vRNA5 structures among distant IAV strains revealed that a number of motifs present in the A/California/04/2009 (H1N1) vRNA5 model are highly conserved despite sequence differences, located within previously identified packaging signals, and the formation of which in in virio conditions has been confirmed. These results support functional roles of the RNA secondary structure motifs, which may serve as candidates for universal RNA-targeting inhibitory methods.

Sequence and structure-based method to predict diacylglycerol lipases in protein sequence.

Lipase enzymes play a central role in biotechnology and the food industry. Diacylglyceride lipases (DAG) have received considerable attention due to their physiological significance and potential industrial usage. However, compared to the wide application of triacylglycerol (TAG) lipases, DAG lipases have a limited application due to their low thermostability and specific activity. The molecular basis of substrate specificity of DAG lipases remains elusive, making structure-guided engineering of TAG to DAG lipase difficult. Besides, the number of available DAG lipases is limited compared to TAG lipases. In the current study, we identified structural consensus motifs of DAG lipases that contribute to their DAG specificity on a structural comparison of DAG and TAG lipases. To find potential DAG lipases, sequence motifs and predicted secondary structures were used to screen millions of protein sequences and predict new DAG lipases. In total, 83 new putative DAG lipases were identified. The predicted DAG lipases were validated by expression of randomly chosen putative DAG lipases followed by functional assay for their DAG and TAG specific activity. The reported method is efficient and cost-effective for discovering new DAG lipases used in the food industry after the required characterization to meet potential application needs.

Genome-wide analysis of AP2/ERF transcription factors family in Brassica napus.

The AP2/ERF transcription factor family plays an important role in different biological processes such as growth, development and response to abiotic and biotic stresses in plants. The genome-wide analysis identified 531 AP2/ERF genes in Brassica napus (oilseed rape or canola) that ranged from 333 to 6440 bp in genomic and 273-2493 bp in coding DNA sequence length. We classified BnAP2/ERF proteins into five subfamilies including AP2 (58 genes), ERF (250 genes), DREB/CBF (194 genes), RAV (26 genes), and Soloist (3 genes). Furthermore, AP2/ERF proteins were subdivided into 15 groups according to the AP2/ERF classification in Arabidopsis. The number of exons in BnAP2/ERF genes was from one to eleven and most of these genes in the same subfamily had the same exon-intron pattern. The results also indicated that the composition of conserved motifs in most proteins in each group was similar. The intron-exon patterns and the composition of conserved motifs validated the BnAP2/ERF transcription factors phylogenetic classification. Based on the results of genome distribution, BnAP2/ERF genes were located unevenly on the 19 B. napus chromosomes. The results indicated that gene duplication may play an important role in genome expansion of B. napus. Furthermore, genome evolution of B. napus using orthologous and paralogous identification was studied. We found 278, 380 and 366 orthologous gene pairs between B. napus with A. thaliana, B. rapa and B. oleracea, respectively. The results of this study will be useful in investigation of functional role and molecular mechanisms of BnAP2/ERF transcription factors genes in response to different stresses.

Diversity and evolution of cytochrome P450s of Jacobaea vulgaris and Jacobaea aquatica.

BACKGROUND: Collectively, plants produce a huge variety of secondary metabolites (SMs) which are involved in the adaptation of plants to biotic and abiotic stresses. The most characteristic feature of SMs is their striking inter- and intraspecific chemical diversity. Cytochrome P450 monooxygenases (CYPs) often play an important role in the biosynthesis of SMs and thus in the evolution of chemical diversity. Here we studied the diversity and evolution of CYPs of two Jacobaea species which contain a characteristic group of SMs namely the pyrrolizidine alkaloids (PAs). RESULTS: We retrieved CYPs from RNA-seq data of J. vulgaris and J. aquatica, resulting in 221 and 157 full-length CYP genes, respectively. The analyses of conserved motifs confirmed that Jacobaea CYP proteins share conserved motifs including the heme-binding signature, the PERF motif, the K-helix and the I-helix. KEGG annotation revealed that the CYPs assigned as being SM metabolic pathway genes were all from the CYP71 clan but no CYPs were assigned as being involved in alkaloid pathways. Phylogenetic analyses of full-length CYPs were conducted for the six largest CYP families of Jacobaea (CYP71, CYP76, CYP706, CYP82, CYP93 and CYP72) and were compared with CYPs of two other members of the Asteraceae, Helianthus annuus and Lactuca sativa, and with Arabidopsis thaliana. The phylogenetic trees showed strong lineage specific diversification of CYPs, implying that the evolution of CYPs has been very fast even within the Asteraceae family. Only in the closely related species J. vulgaris and J. aquatica, CYPs were found often in pairs, confirming a close relationship in the evolutionary history. CONCLUSIONS: This study discovered 378 full-length CYPs in Jacobaea species, which can be used for future exploration of their functions, including possible involvement in PA biosynthesis and PA diversity.

Identification of WRKY gene family and characterization of cold stress-responsive WRKY genes in eggplant.

BACKGROUND: WRKY proteins play a vital role in the plants response to different stresses, growth and development. Studies of WRKY proteins have been mainly focused on model plant Arabidopsis and a few other vegetable plants. However, the systematical study of eggplant WRKY transcription factor superfamily is scarce. METHODS: Bioinformatics has been used to identify and characterize the eggplant WRKY gene family. For the exploration of the differentially expressed WRKY genes, two cultivars with different cold-tolerance were used. Finally, we performed a virus-induced gene silencing (VIGS) experiment to verify the functions of SmWRKY26 and SmWRKY32. RESULTS: Fifty eight (58) genes encoding eggplant WRKY proteins were identified through searching the eggplant genome. Eggplant WRKY proteins could be classified into three groups or seven subgroups in accordance with other plants. WRKY variants were identified from the eggplant. Gene structure analysis showed that the number of intron in eggplant WRKY family was from 0 to 11, with an average of 4.4. Conserved motif analysis suggested that WRKY DNA-binding domain was conserved in eggplant WRKY proteins. Furthermore, RNA-seq data showed that WRKY genes were differentially expressed in eggplant response to cold stress. By using VIGS, the two differentially expressed genes-SmWRKY26 and SmWRKY32 were verified in response to cold stress. DISCUSSIONS: This study provides a foundation for further exploring the functions of WRKY proteins in eggplant response to stresses and eggplant genetic improvement in stresses.

Genome-wide expression analysis suggests glutaredoxin genes response to various stresses in cotton.

Oxidative stress reflects an imbalance between the systemic manifestation of reactive oxygen species (ROS) and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage. Glutaredoxins (GRXs) are ubiquitous oxidoreductase enzymes involved in diverse cellular processes and play a key role in oxidative stress responsive mechanisms. This study was aimed to explore the structure-function relationship and to provide a framework for functional validation and biochemical characterization of various GRX members. In this study, our analysis revealed the presence of 127 genes encoding GRX proteins in G. hirsutum. A total of 758 genes from two typical monocot and nine dicot species were naturally divided into four classes based on phylogenetic analysis. The classification was supported with organization of conserved protein motifs and sequence logos comparison between cotton, rice and Arabidopsis. Cotton GRX gene family has underwent strong purifying selection with limited functional divergence. A good collinearity was observed in the synteny analysis of four Gossypium species. Majority of cotton GRXs were influenced by various phytohormones and abiotic stress conditions during expression analysis, suggesting an important role of GRX proteins in response to oxidative stress. Cis-regulatory elements, gene enrichments and co-expression network analysis also support their predicted role against various abiotic stresses. Whole genome and segmental duplication were determined to be the two major impetuses for the expansion of gene numbers during the evolution. The identification of GRX genes showing differential expression in specific tissues or in response to environmental stimuli provides a new avenue for in-depth characterization of selected genes of importance. This study will further broaden our insights into the evolution and functional elucidation of GRX gene family in cotton.

Comparative phylogenetic analysis of aquaporins provides insight into the gene family expansion and evolution in plants and their role in drought tolerant and susceptible chickpea cultivars.

Aquaporins (AQPs) are water channel proteins that play a significant role in drought stress. Although the AQPs identified in multiple plant species, there is no detailed evolutionary and comparative study of AQPs regarding chickpea plant. The current study involved evolutionary analyses coupled with promoter and expression analyses of chickpea AQPs (CaAQPs). A total of 924 non-redundant AQPs were studied in 24 plant species including algae, mosses, lycophytes, monocots and dicots. Phylogenetic analysis demonstrated a clear divergence of eight AQP subfamilies (LIPs, SIPs, GIPs, NIPs, XIPs, PIPs, HIPs and TIPs). The comparative phylogenetic trees of AQP subfamilies among Arabidopsis, soybean, common bean, maize and chickpea demonstrated that the AQPs were highly species-specific. Interestingly, the dual NPA motif was conserved in all species. However, the ar/R selectivity filter signatures [W/T/S/N/G/A]-[V/S/L/I/A]-[S/G/A]-R (in NIPs), F-H-T-R (in PIPs), [H/N/Q/S]-[A/I/L/S/V]-[A/G]-[A/C/L/M/R/V] (in TIPs) and [V/I/L/M]-[V/I/A/F/M]-[A/S/F/C]-[N/F/L/I/A/S (in SIPs) were found in five species. Moreover, the Froger's positions (P1-P5) were found as [F/L/Y]-[S/T]-A-Y-[L/I/M/V/F] (in NIPs), [Q/E/M]-S-A-F-W (in PIPs), [A/L/S/T/V]-[A/C/N/S/T/V]-[P/R/S]-[Y/N/F]-[W/Q] (in TIPs) and [I/M/F]-[A/V]-[A/V]-Y-W (in SIPs). The MEME motif analyses showed that most of the motifs were specific to subfamily and subgroups. Tissue-specific expression profiling of CaAQPs revealed that CaTIPs and CaPIPs are highly expressed in most of the tissues, while CaNIPs and CaSIPs have low expression. In promoter analysis of CaAQPs, multiple stress-related cis-acting elements e.g. MYB, MYC, ABRE, etc. were found. Semi-quantitative RT-PCR analysis showed that CaPIP2;3 and CaNIP3;1 are positive regulator, while CaSIP1;1 and CaPIP2;1 have a negative role in drought tolerance. The findings and implications of this study are discussed in detail.

Genome-Wide Comprehensive Analysis of the SABATH Gene Family in Arabidopsis and Rice.

Low molecular weight metabolites are important plant hormones and signaling molecules, and play an important part among the processes of plant development. Their activities may also be affected by the chemical modifications of methylation performed by SABATH. In this study, a total of 24 and 21 SABATH genes in Arabidopsis and rice, respectively, were identified and taken a comprehensive study. Phylogenetic analysis showed that AtSABATH and OsSABATH genes could be classified into 4 major groups and 6 subgroups. Gene expansion analysis showed that the main expansion mechanism of SABATH gene family in Arabidopsis and rice was tandem duplication and segmental duplication. The ratios of nonsynonymous (Ka) and synonymous (Ks) substitution rates of 12 pairs paralogous of AtSABATH and OsSABATH genes indicated that the SABATH gene family in Arabidopsis and rice had gone through purifying selection. Positive selection analysis with site models and branch-site models revealed that AtSABATH and OsSABATH genes had undergone selective pressure for adaptive evolution. Motif analysis showed that certain motifs only existed in specific subgroups or species, which indicated that the SABATH proteins of Arabidopsis and rice appear divergence in different species and subgroups. Functional divergence analysis also suggested that the AtSABATH and OsSABATH subgroup genes had functional differences, and the positive selection sites which contributed to functional divergence among subgroups were detected. These results provide insights into functional conservation and diversification of SABATH gene family, and are useful information for further elucidating SABATH gene family functions.

Identification, expression, and phylogenetic analyses of terpenoid biosynthesis-related genes in secondary xylem of loblolly pine (Pinus taeda L.) based on transcriptome analyses.

Loblolly pine (Pinus taeda L.) is one of the most important species for oleoresin (a mixture of terpenoids) in South China. The high oleoresin content of loblolly pine is associated with resistance to bark beetles and other economic benefits. In this study, we conducted transcriptome analyses of loblolly pine secondary xylem to gain insight into the genes involved in terpenoid biosynthesis. A total of 372 unigenes were identified as being critical for oleoresin production, including genes for ATP-binding cassette (ABC) transporters, the cytochrome P450 (CYP) protein family, and terpenoid backbone biosynthesis enzymes. Six key genes involved in terpenoid biosynthetic pathways were selected for multiple sequence alignment, conserved motif prediction, and phylogenetic and expression profile analyses. The protein sequences of all six genes exhibited a higher degree of sequence conservation, and upstream genes were relatively more conserved than downstream genes in terpenoid biosynthetic pathways. The N-terminal regions of these sequences were less conserved than the C-terminal ends, as the N-terminals were quite diverse in both length and composition. The phylogenetic analyses revealed that most genes originated from gene duplication after species divergence, and partial genes exhibited incomplete lineage sorting. In addition, the expression profile analyses showed that all six genes exhibited high expression levels during the high-oleoresin-yielding phase.

Asymmetric protein design from conserved supersecondary structures.

Computational design with supersecondary structures as building blocks has proven effective in the construction of new proteins with controlled geometries. So far, this approach has primarily exploited amplification, effectively harnessing the internal folding propensity of self-compatible fragments to achieve sufficient enthalpy for folding. Here we exploit an interface-driven strategy to depart from the repeat design realm, constructing an asymmetric, globular domain from heterologous supersecondary structures. We report the successful design of a dRP lyase domain fold, which agrees with the experimental NMR structure at atomic accuracy (backbone RMSD of 0.94 Å). Our results show that the residual folding information within conserved fragments, combined with efficient interface-directed sampling, can effectively yield globular proteins with novel sequences and biophysical properties.

Cloning and characterization of trehalase: a conserved glycosidase from oriental midge, Chironomus ramosus.

Insect trehalase is a multiferous enzyme, crucial for normal physiological functions as well as under stress conditions. In this report, we present a fundamental study of the trehalase gene segment (1587 bp) from Chironomus ramosus (CrTre) encoding for 529 amino acids, using appropriate bioinformatics tools. C. ramosus, a tropical midge is an emerging animal model to investigate the consequences of environmental stresses. We observed that CrTre belongs to GH family 37 in the CAZy database and possess 57-92% identity to dipteran trehalases. In silico characterization provided information regarding the structural, functional and evolutionary aspects of midge trehalase. In the phylogenetic tree, CrTre clustered with the soluble dipteran trehalases. Moreover, domain functional characterization of the deduced protein sequence by InterProScan (IPR001661), ProSite (PS00927 and PS00928) and Pfam (PF01204) indicated presence of highly conserved signature motifs which are important for the identification of trehalase superfamily. Furthermore, the instability index of CrTre was predicted to be < 40 suggesting its in vivo stability while, the high aliphatic index indicated towards its thermal stability (index value 71-81). The modelled 3D tertiary structure of CrTre depicts a (α/α)6 barrel toroidal core. The catalytic domain of the enzyme comprised Glu424 and Asp226 as the putative active site residues. Interestingly, the conserved motifs were observed to be formed by the flexible loopy regions in the tertiary structure. This study revealed essential sequence features of the midge trehalase and offers better insights into the structural aspects of this enzyme which can be correlated with its function.

Optimisation of peptides that actively cross the tympanic membrane by random amino acid extension: a phage display study.

Local treatment of middle ear (ME) disease currently requires surgical penetration of the tympanic membrane (TM). We previously discovered 12-mer peptides that are actively transported across the intact TM, a process that could be used for non-invasive drug delivery into the ME. To optimise transport and provide further understanding of the peptides transport mechanism, we extended two of the candidate peptides by six additional amino acids at random, and screened the resulting 18-mers libraries on TMs of rats with active bacterial otitis media (OM) for transport efficiency using phage display. Six identified peptides were individually tested in vivo for trans-TM transport to verify the tissue specificity. Three exhibited enhanced transport compared to their parent 12-mer scaffold, with the best showing an approximately nine-fold increase. Sequence analysis revealed anchor residues and structural features associated with enhanced transport. This included the prominent display of conserved sequence motifs at the extended free ends of the predicted peptide structures.

Genome-Wide Comprehensive Analysis the Molecular Phylogenetic Evaluation and Tissue-Specific Expression of SABATH Gene Family in Salvia miltiorrhiza.

The plant SABATH gene family is a group of O-methyltransferases (O-MTs), which belongs to the S-adenosyl-l-methionine-dependent methyltransferases (SAM-MTs). The resulting reaction products of SABATH genes play an important role in various processes of plant development. In this study, a total of 30 SABATH genes were detected in Salvia miltiorrhiza, which is an important medicinal plant, widely used to treat cardiovascular disease. Multiple sequence alignment and phylogenetic analyses showed that SmSABATH genes could be classified into three groups. The ratios of non-synonymous (Ka) and synonymous (Ks) substitution rates of 11 pairs paralogous of SmSABATH genes revealed that the SmSABATH genes had gone through purifying selection. Positive selection analyses using site models and branch-site models indicated that SmSABATH genes had undergone selective pressure for adaptive evolution. Functional divergence analyses suggested that the SmSABATH subgroup genes were divergent in terms of functions and positive selection sites that contributed to a functional divergence among the subgroups that were detected. Tissue-specific expression showed that the SABATH gene family in S. miltiorrhiza was primarily expressed in stems and leaves.