Proteotranscriptomic view on the role of spider silk glands during the silk spinning process
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ABSTRACT:
PROVIDER: PRJEB19361 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR1823513_1.fastq.gz | Fastqsanger.gz | |||
| ERR1823513_2.fastq.gz | Fastqsanger.gz |
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Project description:Spider silk proteins are synthesized in the silk-producing glands, where the spidroins are produced, stored and processed into a solid fiber from a crystalline liquid solution. Despite great interest in the spider silk properties, that make this material suitable for biomedical and biotechnological applications, the mechanism of formation and spinning of the silk fibers has not been fully elucidated; and no combination of proteomic and transcriptomic study has been carried out so far in the spider silk-producing glands. Nephila clavipes is an attractive orb-web spider to investigate the spinning process of silk production, given the properties of strength, elasticity and biocompatibility of their silk fibers. Thus, considering that the combination of proteomic and transcriptomic analysis may reveal an extensive repertoire of novel proteins involved in the silk spinning process, and in order to facilitate and enable proteomics in this non-model organism, the current study aims to construct a high quality reference mRNA-derived protein database that could be used to identify tissue specific expression patterns in spider silk glands. Next-generation sequencing has offered a powerful and cost-efficient technique for the generation of transcriptomic datasets in non-model species using diverse platforms such as the Illumina HiSeq, Roche 454, Pacific Biosystems, and Applied Biosystems SOLiD; In the current study, the Illumina HiSeq 2000 platform will be used to generate a N. clavipes spider silk glands transcriptome-based protein database. The transcriptome data generated in this study will provide a comprehensive and valuable genomic resource for future research of the group of spider silk-producing glands, in order to improve our understanding of the overall mechanism of action involved in production, secretion, storage, transport, protection and conformational changes of spidroins during the spinning process, and prey capture; and the results may be relevant for scientists in material Science, biology, biochemistry, and environmental scientists.
2018-12-30 | E-MTAB-5451 | biostudies-arrayexpress
Project description:Spiders are a highly diverse group of arthropods that occur in most habitats on land. Notably, spiders have significant ecological impact as predators because of their extraordinary prey capture adaptations, venom and silk. Spider venom is among the most heterogeneous animal venoms and has pharmacological applications, while spider silk is characterized by great toughness with potential for biomaterial application. We describe the genome sequences of two spiders representing two major taxonomic groups, the social velvet spider Stegodyphus mimosarum (Araneomorphae), and the Brazilian white-knee tarantula Acanthoscurria geniculata (Mygalomorphae). We annotate genes using a combination of transcriptomic and in-depth proteomic analyses. The genomes are large (2.6 Gb and 6 Gb, respectively) with short exons and long introns and approximately 50% repeats, reminiscent of typical mammalian genomes. Phylogenetic analyses show that spiders and ticks are sister groups outgrouped by mites, and phylogenetic dating using a molecular clock dates separation of velvet spider and tarantula at 270 my. Based on the genomes and proteomes, we characterize the genetic basis of venom and silk production of both species in detail. Venom protein composition differs markedly between the two spiders, with lipases as the most abundant protein in the velvet spider and present only at low concentration in tarantula. Venom in both spiders contains proteolytic enzymes, and our analyses suggest that these enzymes target and process precursor peptides that subsequently mediate the toxic effects of venom. Complete analysis of silk genes reveal a diverse suite of silk proteins in the velvet spider including novel types of spidroins, and dynamic evolution of major ampullate spidroin genes, whereas silk protein diversity in tarantula is far less complex. The difference in silk proteins between species is consistent with a more complex silk gland morpholgy and use of three-dimentional capture webs consisting of multiple silk types in aranomorph spiders.
2014-05-01 | PXD000318 | Pride
Project description:Porous membranes are frequently used as supports of cell monolayers in functional studies of epithelial and endothelial barriers. However, conventional polymer-based membranes such as those made of polycarbonate do not mimic the structural and biochemical properties of native basement membranes, which may limit cellular differentiation and function. Here, we use a nanofibrillar membrane made of recombinant spider silk functionalized with the integrin-binding RGD motif of fibronectin and coated with human kidney-specific laminin-521 (LN/FN-silk) as a novel substrate for renal epithelial monolayer cultures. Cellular morphology, mRNA expression, barrier properties and transporter activity were assessed using scanning and transmission electron microscopy, RNA-sequencing, lucifer yellow permeability assays, immunofluorescence staining and fluorescent probe transport assays. Immortalized renal proximal tubular epithelial cell (RPTEC/TERT1) monolayers cultured on LN/FN-silk membranes and in co-culture with human fibroblasts (fHDF/TERT166) exhibited cuboidal morphology, reduced cell death and maintained barrier integrity with tight junction expression compared to conventional polymer-based transwell membranes. In contrast to the LN/FN-silk membranes, the polycarbonate membranes released the endocrine disrupting toxicant bisphenol A, triggering estrogen-mediated signalling. Furthermore, cells on LN/FN-silk membranes showed enhanced directional anion and cation transport, compared to cells cultured on conventional membranes. In summary, the results indicate that LN/FN-silk membranes improve differentiation and functional maintenance of renal epithelial cell cultures, thereby allowing preservation of renal epithelial transport functions.
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Project description:This project contains proteomic (LC-MS/MS) data from 27 samples. The samples are as follows: (1) Major ampullate glands dissolved in 8M Urea (3 replicates); (2) Major ampullate silk fibers dissolved in 8M Urea (3 replicates); (3) Major ampullate silk fibers dissolved in Hexafluoroisopropanol (3 replicates); (4) Major ampullate silk fibers dissolved in 9M Lithium Bromide (3 replicates); (5) Major ampullate silk fibers dissolved in 2M Urea (3 replicates); (6) Major ampullate silk fibers dissolved in 4M Urea (3 replicates); (7) Major ampullate silk fibers dissolved in 8M Urea (3 replicates); (8) Major ampullate silk fibers first dissolved in Formic acid and then in 2M Urea (2 replicates); (9) Major ampullate silk fibers first dissolved in Formic acid and then in 4M Urea (2 replicates); (10) Major ampullate silk fibers first dissolved in Formic acid and then in 8M Urea (2 replicates);
2024-07-16 | PXD052116 | Pride
Project description:Spider silk research has largely focused on spidroins, proteins that are the primary components of spider silk fibers. Although a number of spidroins have been characterized, other types of proteins associated with silk synthesis are virtually unknown. Previous comparison of tissue-specific RNAseq libraries identified 647 predicted genes that were differentially expressed in silk glands of the Western black widow, Latrodectus hesperus. Only ~5% of these transcripts encode spidroins and the remaining predicted genes presumably encode other proteins associated with silk production. Here, we used proteomic analysis of multiple silk glands and dragline silk fiber to investigate the translation of the differentially expressed genes. We find 48 proteins encoded by the differentially expressed transcripts in L. hesperus major ampullate, minor ampullate, and tubuliform silk glands, and detect 16 SST encoded proteins in major ampullate silk fibers. The observed proteins include known silk-related proteins, but most are uncharacterized, with no annotation. These unannotated proteins likely include novel silk associated proteins. Major ampullate and minor ampullate glands have the highest overlap of identified proteins, consistent with their shared, distinctive ampullate shape and the overlapping functions of major ampullate and minor ampullate silks. Our study substantiates and prioritizes predictions from differential expression analysis of spider silk gland transcriptomes.
2015-08-25 | PXD002592 | Pride
Project description:To offer a robust and highly characterized three-dimensional (3D) model for anti-cancer drug discovery and basic research, we developed a 3D system in which the immortalized breast cancer cell lines MCF-7 and MDA-MB-231 were grown in recombinantly produced spider silk functionalized with the cell adhesion motif from fibronectin (FN-silk). The aim of this study is to evaluate whole-transcriptome changes driven by growth in such 3D model.
2023-09-13 | GSE209570 | GEO
Project description:Spider silk research has largely focused on spidroins, proteins that are the primary components of spider silk fibers. Although a number of spidroins have been characterized, other types of proteins associated with silk synthesis are virtually unknown. Previous comparison of tissue-specific RNAseq libraries identified 647 predicted genes that were differentially expressed in silk glands of the Western black widow, Latrodectus hesperus. Only ~5% of these transcripts encode spidroins and the remaining predicted genes presumably encode other proteins associated with silk production. Here, we used proteomic analysis of multiple silk glands and dragline silk fiber to investigate the translation of the differentially expressed genes. We find 48 proteins encoded by the differentially expressed transcripts in L. hesperus major ampullate, minor ampullate, and tubuliform silk glands, and detect 16 SST encoded proteins in major ampullate silk fibers. The observed proteins include known silk-related proteins, but most are uncharacterized, with no annotation. These unannotated proteins likely include novel silk associated proteins. Major ampullate and minor ampullate glands have the highest overlap of identified proteins, consistent with their shared, distinctive ampullate shape and the overlapping functions of major ampullate and minor ampullate silks. Our study substantiates and prioritizes predictions from differential expression analysis of spider silk gland transcriptomes.
2015-08-25 | PXD002595 | Pride
Project description:The anterior silk gland in the silkworm plays an important role in the process of liquid fibroin to solid silk fiber .In view of this,the proteomics analysis was applied to to study the relationship between the function of proteins in the anterior silk gland and the mechanism of spinning. The anterior silk glands on the 3rd day of fifth instar were dissected.Aftter 1D SDS-PAGE ,one gel lane was cut into 10 bands and each band further sliced into small pieces was subjected to in-gel tryptic digestion for 20 hours.The digested peptides were separated by RP nanoscale capillary liquid chromatography and analyzed using a surveyor LC system (Thermo Figgigan, San Jose, CA).The eluate from the RP column was analyzed by Finnigan LTQ(Thermo Electron Corporation）linear ion trap Mass equipped with a nanospray souce in the positive ion mode. The MS analysis was performed with one full MS scan followed by three MS/MS scans on the most intense ions from the MS spectrum with the dynamic exclusion settings: repeat count 2, repeat duration 30s, exclusion duration 90s. Data were acquired in data-dependent mode using Xcalibur software.Ten raw datasets from LC-MS/MS were searched against the predicted silkworm database by Xia.et al which consists of 21312 silkworm proteins.The searching was carried out with the Turbo SEQUEST(Bioworks version 3.2, Thermo Electron).
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Project description:Recombinant Spider Silk Membranes Promote Human Renal Epithelial Differentiation and Function
| PRJNA1314116 | ENA
Project description:Spider mites are leaf cell-sucking herbivores that spin nanoscale silk fibers released from their second mouthpart appendages. Previous studies computationally predicted 17 silk protein genes by whole genome sequencing in the two-spotted spider mite, Tetranychus urticae, while two novel candidates, Fibroin-1 and Fibroin-2, were detected in the silk proteome. In addition, Fibroin-1, sFibroin-1 (high sequence similarity to Fibroin-1), and Fibroin-2 proteins were also detected in the saliva proteome. We investigated whether these three proteins function as silk, saliva, or both. The mRNA for Fibroin-1, sFibroin-1, and Fibroin-2 were all expressed in the salivary glands. Micro-CT scanning confirmed no direct connection between salivary glands and silk glands, i.e., saliva and silk meet each other during simultaneous secretion and spinning. Silk proteomics performed in the present study detected proteins encoded by 8 of the 17 originally predicted genes, as well as Fibroin-1, sFibroin-1, and Fibroin-2 proteins. In addition, cryo-SEM imaging visualized a bead-on-a-string pattern formed by a fluid on the silk fiber, as well as a fluid patch that remains at the piercing site on the leaf surface after mite feeding. Furthermore, RNAi-mediated silencing of Fibroin-1 and sFibroin-1 reduced the feeding duration of mites, resulting in low survival and fecundity. Fibroin-2 RNAi reduced the thickness of single filaments of silk fibers. These results suggest that Fibroin-1, sFibroin-1, and Fibroin-2 proteins are secreted from the salivary glands and then adhere to silk fibers. Fibroin-1 and sFibroin-1 proteins support mites establishing a stable piercing site by adhering the tip of the mouthparts to the leaf surface to suck leaf cell contents with their stylets, while Fibroin-2 coats silk fibers. Eight originally predicted genes are still candidates for silk protein genes. This study revealed the two roles of saliva in leaf cell-sucking herbivorous mites, which include stabilizing mouthparts during feeding and coating silk fibers, providing them with adhesive properties.
| PXD059809 | JPOST Repository