Dataset Information

Confirmation of Bioinformatics Predictions of the Structural Domains in Honeybee Silk.

ABSTRACT: Honeybee larvae produce a silk made up of proteins in predominantly a coiled coil molecular structure. These proteins can be produced in recombinant systems, making them desirable templates for the design of advanced materials. However, the atomic level structure of these proteins is proving difficult to determine: firstly, because coiled coils are difficult to crystalize; and secondly, fibrous proteins crystalize as fibres rather than as discrete protein units. In this study, we synthesised peptides from the central structural domain, as well as the N- and C-terminal domains, of the honeybee silk. We used circular dichroism spectroscopy, infrared spectroscopy, and molecular dynamics to investigate the folding behaviour of the central domain peptides. We found that they folded as predicted by bioinformatics analysis, giving the protein engineer confidence in bioinformatics predictions to guide the design of new functionality into these protein templates. These results, along with the infrared structural analysis of the N- and C-terminal domain peptides and the comparison of peptide film properties with those of the full-length AmelF3 protein, provided significant insight into the structural elements required for honeybee silk protein to form into stable materials.

SUBMITTER: Woodhead AL

PROVIDER: S-EPMC6403662 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Confirmation of Bioinformatics Predictions of the Structural Domains in Honeybee Silk.

Woodhead Andrea L AL Church Andrew T AT Rapson Trevor D TD Trueman Holly E HE Church Jeffrey S JS Sutherland Tara D TD

Polymers 20180716 7

Honeybee larvae produce a silk made up of proteins in predominantly a coiled coil molecular structure. These proteins can be produced in recombinant systems, making them desirable templates for the design of advanced materials. However, the atomic level structure of these proteins is proving difficult to determine: firstly, because coiled coils are difficult to crystalize; and secondly, fibrous proteins crystalize as fibres rather than as discrete protein units. In this study, we synthesised pep ...[more]

PMID: 30960701

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Project description:BACKGROUND: Spidroins are a unique family of large, structural proteins that make up the bulk of spider silk fibers. Due to the highly variable nature of their repetitive sequences, spidroin evolutionary relationships have principally been determined from their non-repetitive carboxy (C)-terminal domains, though they offer limited character data. The few known spidroin amino (N)-terminal domains have been difficult to obtain, but potentially contain critical phylogenetic information for reconstructing the diversification of spider silks. Here we used silk gland expression data (ESTs) from highly divergent species to evaluate the functional significance and phylogenetic utility of spidroin N-terminal domains. RESULTS: We report 11 additional spidroin N-termini found by sequencing approximately 1,900 silk gland cDNAs from nine spider species that shared a common ancestor > 240 million years ago. In contrast to their hyper-variable repetitive regions, spidroin N-terminal domains have retained striking similarities in sequence identity, predicted secondary structure, and hydrophobicity. Through separate and combined phylogenetic analyses of N-terminal domains and their corresponding C-termini, we find that combined analysis produces the most resolved trees and that N-termini contribute more support and less conflict than the C-termini. These analyses show that paralogs largely group by silk gland type, except for the major ampullate spidroins. Moreover, spidroin structural motifs associated with superior tensile strength arose early in the history of this gene family, whereas a motif conferring greater extensibility convergently evolved in two distantly related paralogs. CONCLUSIONS: A non-repetitive N-terminal domain appears to be a universal attribute of spidroin proteins, likely retained from the origin of spider silk production. Since this time, spidroin N-termini have maintained several features, consistent with this domain playing a key role in silk assembly. Phylogenetic analyses of the conserved N- and C-terminal domains illustrate dramatic radiation of the spidroin gene family, involving extensive duplications, shifts in expression patterns and extreme diversification of repetitive structural sequences that endow spider silks with an unparalleled range of mechanical properties.

Project description:BackgroundHypertrophy ligamentum flavum is a prevalent chronic spinal condition that affects middle-aged and older adults. However, the molecular pathways behind this disease are not well comprehended.ObjectiveThe objective of this work is to implement bioinformatics techniques in order to identify crucial biological markers and immune infiltration that are linked to hypertrophy ligamentum flavum. Further, the study aims to experimentally confirm the molecular mechanisms that underlie the hypertrophy ligamentum flavum.MethodsThe corresponding gene expression profiles (GSE113212) were selected from a comprehensive gene expression database. The gene dataset for hypertrophy ligamentum flavum was acquired from GeneCards. A network of interactions between proteins was created, and an analysis of functional enrichment was conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases. An study of hub genes was performed to evaluate the infiltration of immune cells in patient samples compared to tissues from the control group. Finally, samples of the ligamentum flavum were taken with the purpose of validating the expression of important genes in a clinical setting.ResultsOverall, 27 hub genes that were differently expressed were found through molecular biology. The hub genes were found to be enriched in immune response, chemokine-mediated signaling pathways, inflammation, ossification, and fibrosis processes, as demonstrated by GO and KEGG studies. The main signaling pathways involved include the TNF signaling pathway, cytokine-cytokine receptor interaction, and TGF-β signaling pathway. An examination of immunocell infiltration showed notable disparities in B cells (naïve and memory) and activated T cells (CD4 memory) between patients with hypertrophic ligamentum flavum and the control group of healthy individuals. The in vitro validation revealed markedly elevated levels of ossification and fibrosis-related components in the hypertrophy ligamentum flavum group, as compared to the normal group.ConclusionThe TGF-β signaling pathway, TNF signaling pathway, and related hub genes play crucial roles in the progression of ligamentum flavum hypertrophic. Our study may guide future research on fibrosis of the ligamentum flavum.

Dataset Information

Confirmation of Bioinformatics Predictions of the Structural Domains in Honeybee Silk.

Publications

Confirmation of Bioinformatics Predictions of the Structural Domains in Honeybee Silk.

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