Regionalization of cell types in silk glands of Larinioides sclopetarius suggest that spider silk fibers are complex layered structures.

In order to produce artificial silk fibers with properties that match the native spider silk we likely need to closely mimic the spinning process as well as fiber architecture and composition. To increase our understanding of the structure and function of the different silk glands of the orb weaver Larinioides sclopetarius, we used resin sections for detailed morphology, paraffin embedded sections for a variety of different histological stainings, and a histochemical method for localization of carbonic anhydrase activity. Our results show that all silk glands, except the tubuliform glands, are composed of two or more columnar epithelial cell types, some of which have not been described previously. We observed distinct regionalization of the cell types indicating sequential addition of secretory products during silk formation. This means that the major ampullate, minor ampullate, aciniform type II, and piriform silk fibers most likely are layered and that each layer has a specific composition. Furthermore, a substance that stains positive for polysaccharides may be added to the silk in all glands except in the type I aciniform glands. Active carbonic anhydrase was found in all silk glands and/or ducts except in the type I aciniform and tubuliform glands, with the strongest staining in aggregate glands and their ductal nodules. Carbonic anhydrase plays an important role in the generation of a pH gradient in the major ampullate glands, and our results suggest that some other glands may also harbor pH gradients.

Mass Spectrometry of RNA-Binding Proteins during Liquid-Liquid Phase Separation Reveals Distinct Assembly Mechanisms and Droplet Architectures.

Liquid-liquid phase separation (LLPS) of heterogeneous ribonucleoproteins (hnRNPs) drives the formation of membraneless organelles, but structural information about their assembled states is still lacking. Here, we address this challenge through a combination of protein engineering, native ion mobility mass spectrometry, and molecular dynamics simulations. We used an LLPS-compatible spider silk domain and pH changes to control the self-assembly of the hnRNPs FUS, TDP-43, and hCPEB3, which are implicated in neurodegeneration, cancer, and memory storage. By releasing the proteins inside the mass spectrometer from their native assemblies, we could monitor conformational changes associated with liquid-liquid phase separation. We find that FUS monomers undergo an unfolded-to-globular transition, whereas TDP-43 oligomerizes into partially disordered dimers and trimers. hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over LLPS. The divergent assembly mechanisms revealed by ion mobility mass spectrometry of soluble protein species that exist under LLPS conditions suggest structurally distinct complexes inside liquid droplets that may impact RNA processing and translation depending on biological context.

Liquid-Liquid Phase Separation Primes Spider Silk Proteins for Fiber Formation via a Conditional Sticker Domain.

Many protein condensates can convert to fibrillar aggregates, but the underlying mechanisms are unclear. Liquid-liquid phase separation (LLPS) of spider silk proteins, spidroins, suggests a regulatory switch between both states. Here, we combine microscopy and native mass spectrometry to investigate the influence of protein sequence, ions, and regulatory domains on spidroin LLPS. We find that salting out-effects drive LLPS via low-affinity stickers in the repeat domains. Interestingly, conditions that enable LLPS simultaneously cause dissociation of the dimeric C-terminal domain (CTD), priming it for aggregation. Since the CTD enhances LLPS of spidroins but is also required for their conversion into amyloid-like fibers, we expand the stickers and spacers-model of phase separation with the concept of folded domains as conditional stickers that represent regulatory units.

Biological Materials Processing: Time-Tested Tricks for Sustainable Fiber Fabrication.

There is an urgent need to improve the sustainability of the materials we produce and use. Here, we explore what humans can learn from nature about how to sustainably fabricate polymeric fibers with excellent material properties by reviewing the physical and chemical aspects of materials processing distilled from diverse model systems, including spider silk, mussel byssus, velvet worm slime, hagfish slime, and mistletoe viscin. We identify common and divergent strategies, highlighting the potential for bioinspired design and technology transfer. Despite the diversity of the biopolymeric fibers surveyed, we identify several common strategies across multiple systems, including: (1) use of stimuli-responsive biomolecular building blocks, (2) use of concentrated fluid precursor phases (e.g., coacervates and liquid crystals) stored under controlled chemical conditions, and (3) use of chemical (pH, salt concentration, redox chemistry) and physical (mechanical shear, extensional flow) stimuli to trigger the transition from fluid precursor to solid material. Importantly, because these materials largely form and function outside of the body of the organisms, these principles can more easily be transferred for bioinspired design in synthetic systems. We end the review by discussing ongoing efforts and challenges to mimic biological model systems, with a particular focus on artificial spider silks and mussel-inspired materials.

Influence of experimental methods on the mechanical properties of silk fibers: A systematic literature review and future road map.

Spider silk fibers are of scientific and industrial interest because of their extraordinary mechanical properties. These properties are normally determined by tensile tests, but the values obtained are dependent on the morphology of the fibers, the test conditions, and the methods by which stress and strain are calculated. Because of this, results from many studies are not directly comparable, which has led to widespread misconceptions in the field. Here, we critically review most of the reports from the past 50 years on spider silk mechanical performance and use artificial spider silk and native silks as models to highlight the effect that different experimental setups have on the fibers' mechanical properties. The results clearly illustrate the importance of carefully evaluating the tensile test methods when comparing the results from different studies. Finally, we suggest a protocol for how to perform tensile tests on silk and biobased fibers.

Engineered Spider Silk Proteins for Biomimetic Spinning of Fibers with Toughness Equal to Dragline Silks.

Spider silk is the toughest fiber found in nature, and bulk production of artificial spider silk that matches its mechanical properties remains elusive. Development of miniature spider silk proteins (mini-spidroins) has made large-scale fiber production economically feasible, but the fibers' mechanical properties are inferior to native silk. The spider silk fiber's tensile strength is conferred by poly-alanine stretches that are zipped together by tight side chain packing in ß-sheet crystals. Spidroins are secreted so they must be void of long stretches of hydrophobic residues, since such segments get inserted into the endoplasmic reticulum membrane. At the same time, hydrophobic residues have high ß-strand propensity and can mediate tight inter-ß-sheet interactions, features that are attractive for generation of strong artificial silks. Protein production in prokaryotes can circumvent biological laws that spiders, being eukaryotic organisms, must obey, and the authors thus design mini-spidroins that are predicted to more avidly form stronger ß-sheets than the wildtype protein. Biomimetic spinning of the engineered mini-spidroins indeed results in fibers with increased tensile strength and two fiber types display toughness equal to native dragline silks. Bioreactor expression and purification result in a protein yield of ≈9 g L-1 which is in line with requirements for economically feasible bulk scale production.

Abilities of the BRICHOS domain to prevent neurotoxicity and fibril formation are dependent on a highly conserved Asp residue.

Proteins can self-assemble into amyloid fibrils or amorphous aggregates and thereby cause disease. Molecular chaperones can prevent both these types of protein aggregation, but to what extent the respective mechanisms are overlapping is not fully understood. The BRICHOS domain constitutes a disease-associated chaperone family, with activities against amyloid neurotoxicity, fibril formation, and amorphous protein aggregation. Here, we show that the activities of BRICHOS against amyloid-induced neurotoxicity and fibril formation, respectively, are oppositely dependent on a conserved aspartate residue, while the ability to suppress amorphous protein aggregation is unchanged by Asp to Asn mutations. The Asp is evolutionarily highly conserved in >3000 analysed BRICHOS domains but is replaced by Asn in some BRICHOS families. The conserved Asp in its ionized state promotes structural flexibility and has a pK a value between pH 6.0 and 7.0, suggesting that chaperone effects can be differently affected by physiological pH variations.

Spidroin N-terminal domain forms amyloid-like fibril based hydrogels and provides a protein immobilization platform.

Recombinant spider silk proteins (spidroins) have multiple potential applications in development of novel biomaterials, but their multimodal and aggregation-prone nature have complicated production and straightforward applications. Here, we report that recombinant miniature spidroins, and importantly also the N-terminal domain (NT) on its own, rapidly form self-supporting and transparent hydrogels at 37 °C. The gelation is caused by NT α-helix to ß-sheet conversion and formation of amyloid-like fibrils, and fusion proteins composed of NT and green fluorescent protein or purine nucleoside phosphorylase form hydrogels with intact functions of the fusion moieties. Our findings demonstrate that recombinant NT and fusion proteins give high expression yields and bestow attractive properties to hydrogels, e.g., transparency, cross-linker free gelation and straightforward immobilization of active proteins at high density.

Solution Structure of Tubuliform Spidroin N-Terminal Domain and Implications for pH Dependent Dimerization.

The spidroin N-terminal domain (NT) is responsible for high solubility and pH-dependent assembly of spider silk proteins during storage and fiber formation, respectively. It forms a monomeric five-helix bundle at neutral pH and dimerizes at lowered pH, thereby firmly interconnecting the spidroins. Mechanistic studies with the NTs from major ampullate, minor ampullate, and flagelliform spidroins (MaSp, MiSp, and FlSp) have shown that the pH dependency is conserved between different silk types, although the residues that mediate this process can differ. Here we study the tubuliform spidroin (TuSp) NT from Argiope argentata, which lacks several well conserved residues involved in the dimerization of other NTs. We solve its structure at low pH revealing an antiparallel dimer of two five-α-helix bundles, which contrasts with a previously determined Nephila antipodiana TuSp NT monomer structure. Further, we study a set of mutants and find that the residues participating in the protonation events during dimerization are different from MaSp and MiSp NT. Charge reversal of one of these residues (R117 in TuSp) results in significantly altered electrostatic interactions between monomer subunits. Altogether, the structure and mutant studies suggest that TuSp NT monomers assemble by elimination of intramolecular repulsive charge interactions, which could lead to slight tilting of α-helices.

The dimerization mechanism of the N-terminal domain of spider silk proteins is conserved despite extensive sequence divergence.

The N-terminal (NT) domain of spider silk proteins (spidroins) is crucial for their storage at high concentrations and also regulates silk assembly. NTs from the major ampullate spidroin (MaSp) and the minor ampullate spidroin are monomeric at neutral pH and confer solubility to spidroins, whereas at lower pH, they dimerize to interconnect spidroins in a fiber. This dimerization is known to result from modulation of electrostatic interactions by protonation of well-conserved glutamates, although it is undetermined if this mechanism applies to other spidroin types as well. Here, we determine the solution and crystal structures of the flagelliform spidroin NT, which shares only 35% identity with MaSp NT, and investigate the mechanisms of its dimerization. We show that flagelliform spidroin NT is structurally similar to MaSp NT and that the electrostatic intermolecular interaction between Asp 40 and Lys 65 residues is conserved. However, the protonation events involve a different set of residues than in MaSp, indicating that an overall mechanism of pH-dependent dimerization is conserved but can be mediated by different pathways in different silk types.

A "spindle and thread" mechanism unblocks p53 translation by modulating N-terminal disorder.

Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of "life on the edge of solubility." Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT∗). The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Biophysical characterization reveals a compact conformation, with the disordered transactivation domain of p53 wrapped around the NT∗ domain. We conclude that interactions with NT∗ help to unblock translation of the proline-rich disordered region of p53. Expression of partially disordered cancer targets is similarly enhanced by NT∗. In summary, we demonstrate that inducing co-translational folding via a molecular "spindle and thread" mechanism unblocks protein translation in vitro.

Artificial and natural silk materials have high mechanical property variability regardless of sample size.

Silk fibres attract great interest in materials science for their biological and mechanical properties. Hitherto, the mechanical properties of the silk fibres have been explored mainly by tensile tests, which provide information on their strength, Young's modulus, strain at break and toughness modulus. Several hypotheses have been based on these data, but the intrinsic and often overlooked variability of natural and artificial silk fibres makes it challenging to identify trends and correlations. In this work, we determined the mechanical properties of Bombyx mori cocoon and degummed silk, native spider silk, and artificial spider silk, and compared them with classical commercial carbon fibres using large sample sizes (from 10 to 100 fibres, in total 200 specimens per fibre type). The results confirm a substantial variability of the mechanical properties of silk fibres compared to commercial carbon fibres, as the relative standard deviation for strength and strain at break is 10-50%. Moreover, the variability does not decrease significantly when the number of tested fibres is increased, which was surprising considering the low variability frequently reported for silk fibres in the literature. Based on this, we prove that tensile testing of 10 fibres per type is representative of a silk fibre population. Finally, we show that the ideal shape of the stress-strain curve for spider silk, characterized by a pronounced exponential stiffening regime, occurs in only 25% of all tested spider silk fibres.

An Image-Analysis-Based Method for the Prediction of Recombinant Protein Fiber Tensile Strength.

Silk fibers derived from the cocoon of silk moths and the wide range of silks produced by spiders exhibit an array of features, such as extraordinary tensile strength, elasticity, and adhesive properties. The functional features and mechanical properties can be derived from the structural composition and organization of the silk fibers. Artificial recombinant protein fibers based on engineered spider silk proteins have been successfully made previously and represent a promising way towards the large-scale production of fibers with predesigned features. However, for the production and use of protein fibers, there is a need for reliable objective quality control procedures that could be automated and that do not destroy the fibers in the process. Furthermore, there is still a lack of understanding the specifics of how the structural composition and organization relate to the ultimate function of silk-like fibers. In this study, we develop a new method for the categorization of protein fibers that enabled a highly accurate prediction of fiber tensile strength. Based on the use of a common light microscope equipped with polarizers together with image analysis for the precise determination of fiber morphology and optical properties, this represents an easy-to-use, objective non-destructive quality control process for protein fiber manufacturing and provides further insights into the link between the supramolecular organization and mechanical functionality of protein fibers.

A Novel Approach for the Production of Aggregation-Prone Proteins Using the Spidroin-Derived NT* Tag.

Spiders have evolved proteins that can be kept in a highly concentrated soluble form in the silk gland yet rapidly assemble into stable silk fibers under certain environmental conditions. The transition between soluble and fibrillar states is partly regulated by the pH-sensitive N-terminal (NT) domain which has emerged as nature's own solubility-enhancing domain. NT has an inherent capacity to keep the silk proteins' partly hydrophobic and very aggregation-prone regions from premature fibrillation in spite of storage at enormous concentrations. The genetically engineered double-mutant NT* shows increased solubility and stability and has arisen as a powerful tool for the production of aggregation-prone as well as other recombinant proteins. Here we describe a robust and highly efficient protocol for improved soluble expression of peptides and proteins by fusion to the NT* tag.

Treatment of Respiratory Distress Syndrome with Single Recombinant Polypeptides that Combine Features of SP-B and SP-C.

Treatment of respiratory distress syndrome (RDS) with surfactant replacement therapy in prematurely born infants was introduced more than 30 years ago; however, the surfactant preparations currently in clinical use are extracts from animal lungs. A synthetic surfactant that matches the currently used nature-derived surfactant preparations and can be produced in a cost-efficient manner would enable worldwide treatment of neonatal RDS and could also be tested against lung diseases in adults. The major challenge in developing fully functional synthetic surfactant preparations is to recapitulate the properties of the hydrophobic lung surfactant proteins B (SP-B) and SP-C. Here, we have designed single polypeptides that combine properties of SP-B and SP-C and produced them recombinantly using a novel solubility tag based on spider silk production. These Combo peptides mixed with phospholipids are as efficient as nature-derived surfactant preparations against neonatal RDS in premature rabbit fetuses.

Artificial spider silk supports and guides neurite extension in vitro.

Surgical intervention with the use of autografts is considered the gold standard to treat peripheral nerve injuries. However, a biomaterial that supports and guides nerve growth would be an attractive alternative to overcome problems with limited availability, morbidity at the site of harvest, and nerve mismatches related to autografts. Native spider silk is a promising material for construction of nerve guidance conduit (NGC), as it enables regeneration of cm-long nerve injuries in sheep, but regulatory requirements for medical devices demand synthetic materials. Here, we use a recombinant spider silk protein (NT2RepCT) and a functionalized variant carrying a peptide derived from vitronectin (VN-NT2RepCT) as substrates for nerve growth support and neurite extension, using a dorsal root ganglion cell line, ND7/23. Two-dimensional coatings were benchmarked against poly-d-lysine and recombinant laminins. Both spider silk coatings performed as the control substrates with regards to proliferation, survival, and neurite growth. Furthermore, NT2RepCT and VN-NT2RepCT spun into continuous fibers in a biomimetic spinning set-up support cell survival, neurite growth, and guidance to an even larger extent than native spider silk. Thus, artificial spider silk is a promising biomaterial for development of NGCs.

AA amyloid in human food chain is a possible biohazard.

AA amyloidosis can be transmitted experimentally in several mammalian and avian species as well as spontaneously between captive animals, even by oral intake of amyloid seeds. Amyloid seeding can cross species boundaries, and fibrils of one kind of amyloid protein may also seed other types. Here we show that meat from Swedish and Italian cattle for consumption by humans often contains AA amyloid and that bovine AA fibrils efficiently cross-seed human amyloid ß peptide, associated with Alzheimer's disease.

In Vitro Study of Human Immune Responses to Hyaluronic Acid Hydrogels, Recombinant Spidroins and Human Neural Progenitor Cells of Relevance to Spinal Cord Injury Repair.

Scaffolds of recombinant spider silk protein (spidroin) and hyaluronic acid (HA) hydrogel hold promise in combination with cell therapy for spinal cord injury. However, little is known concerning the human immune response to these biomaterials and grafted human neural stem/progenitor cells (hNPCs). Here, we analyzed short- and long-term in vitro activation of immune cells in human peripheral blood mononuclear cells (hPBMCs) cultured with/without recombinant spidroins, HA hydrogels, and/or allogeneic hNPCs to assess potential host-donor interactions. Viability, proliferation and phenotype of hPBMCs were analyzed using NucleoCounter and flow cytometry. hPBMC viability was confirmed after exposure to the different biomaterials. Short-term (15 h) co-cultures of hPBMCs with spidroins, but not with HA hydrogel, resulted in a significant increase in the proportion of activated CD69+ CD4+ T cells, CD8+ T cells, B cells and NK cells, which likely was caused by residual endotoxins from the Escherichia coli expression system. The observed spidroin-induced hPBMC activation was not altered by hNPCs. It is resource-effective to evaluate human compatibility of novel biomaterials early in development of the production process to, when necessary, make alterations to minimize rejection risk. Here, we present a method to evaluate biomaterials and hPBMC compatibility in conjunction with allogeneic human cells.

Expression of the human molecular chaperone domain Bri2 BRICHOS on a gram per liter scale with an E. coli fed-batch culture.

BACKGROUND: The human Bri2 BRICHOS domain inhibits amyloid formation and toxicity and could be used as a therapeutic agent against amyloid diseases. For translation into clinical use, large quantities of correctly folded recombinant human (rh) Bri2 BRICHOS are required. To increase the expression and solubility levels of rh Bri2 BRICHOS it was fused to NT*, a solubility tag derived from the N-terminal domain of a spider silk protein, which significantly increases expression levels and solubility of target proteins. To increase the expression levels even further and reach the g/L range, which is a prerequisite for an economical production on an industrial scale, we developed a fed-batch expression protocol for Escherichia coli. RESULTS: A fed-batch production method for NT*-Bri2 BRICHOS was set up and systematically optimized. This gradual improvement resulted in expression levels of up to 18.8 g/L. Following expression, NT*-Bri2 BRICHOS was purified by chromatographic methods to a final yield of up to 6.5 g/L. After removal of the NT*-tag and separation into different oligomeric species, activity assays verified that different assembly states of the fed-batch produced rh Bri2 BRICHOS have the same ability to inhibit fibrillar and non-fibrillar protein aggregation as the reference protein isolated from shake flask cultures. CONCLUSIONS: The protocol developed in this work allows the production of large quantities of rh Bri2 BRICHOS using the solubility enhancing NT*-tag as a fusion partner, which is required to effectively conduct pre-clinical research.

Citrullination Alters the Antibacterial and Anti-Inflammatory Functions of the Host Defense Peptide Canine Cathelicidin K9CATH In Vitro.

K9CATH is the sole cathelicidin in canines (dogs) and exhibits broad antimicrobial activity against both Gram-positive and Gram-negative bacteria. K9CATH also modulates inflammatory responses and binds to LPS. These activities depend on the secondary structure and a net-positive charge of the peptide. Peptidylarginine deiminases (PAD) convert cationic peptidyl arginine to neutral citrulline. Thus, we hypothesized that citrullination is a biologically relevant modification of the peptide that would reduce the antibacterial and LPS-binding activities of K9CATH. Recombinant PAD2 and PAD4 citrullinated K9CATH to various extents and circular dichroism spectroscopy revealed that both native and citrullinated K9CATH exhibited similar α-helical secondary structures. Notably, citrullination of K9CATH reduced its bactericidal activity, abolished its ability to permeabilize the membrane of Gram-negative bacteria and reduced the hemolytic capacity. Electron microscopy showed that citrullinated K9CATH did not cause any morphological changes of Gram-negative bacteria, whereas the native peptide caused clear alterations of membrane integrity, concordant with a rapid bactericidal effect. Finally, citrullination of K9CATH impaired its capacity to inhibit LPS-mediated release of proinflammatory molecules from mouse and canine macrophages. In conclusion, citrullination attenuates the antibacterial and the LPS-binding properties of K9CATH, demonstrating the importance of a net positive charge for antibacterial lysis of bacteria and LPS-binding effects and suggests that citrullination is a means to regulate cathelicidin activities.