Identification of potential aggregation hotspots on Aβ42 fibrils blocked by the anti-amyloid chaperone-like BRICHOS domain.

Protein misfolding can generate toxic intermediates, which underlies several devastating diseases, such as Alzheimer's disease (AD). The surface of AD-associated amyloid-β peptide (Aβ) fibrils has been suggested to act as a catalyzer for self-replication and generation of potentially toxic species. Specifically tailored molecular chaperones, such as the BRICHOS protein domain, were shown to bind to amyloid fibrils and break this autocatalytic cycle.

Synthetic surfactant with a combined SP-B and SP-C analogue is efficient in rabbit models of adult and neonatal respiratory distress syndrome.

Respiratory distress syndrome (RDS) in premature infants is caused by insufficient amounts of endogenous lung surfactant and is efficiently treated with replacement therapy using animal-derived surfactant preparations. On the other hand, adult/acute RDS (ARDS) occurs secondary to for example, sepsis, aspiration of gastric contents, and multitrauma and is caused by alveolar endothelial damage, leakage of plasma components into the airspaces and inhibition of surfactant activity. Instillation of surfactant preparations in ARDS has so far resulted in very limited treatment effects, partly due to inactivation of the delivered surfactants in the airspace.

Display of a naïve affibody library on staphylococci for selection of binders by means of flow cytometry sorting.

Within the field of combinatorial protein engineering there is a great demand for robust high-throughput selection platforms that allow for unbiased protein library display, affinity-based screening, and amplification of selected clones. We have previously described the development of a staphylococcal display system used for displaying both alternative-scaffolds and antibody-derived proteins. In this study, the objective was to generate an improved expression vector for displaying and screening a high-complexity naïve affibody library, and to facilitate downstream validation of isolated clones.

Cooperative action of SP-A and its trimeric recombinant fragment with polymyxins against Gram-negative respiratory bacteria.

The exploration of therapies combining antimicrobial lung proteins and conventional antibiotics is important due to the growing problem of multidrug-resistant bacteria. The aim of this study was to investigate whether human SP-A and a recombinant trimeric fragment (rfhSP-A) have cooperative antimicrobial activity with antibiotics against pathogenic Gram-negative bacteria. We found that SP-A bound the cationic peptide polymyxin B (PMB) with an apparent dissociation constant ( ) of 0.

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 β-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.

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.

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.

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.

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.

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.

The synthesis and characterization of Bri2 BRICHOS coated magnetic particles and their application to protein fishing: Identification of novel binding proteins.

Human integral membrane protein 2B (ITM2B or Bri2) is a member of the BRICHOS family, proteins that efficiently prevent Aβ42 aggregation via a unique mechanism. The identification of novel Bri2 BRICHOS client proteins could help elucidate signaling pathways and determine novel targets to prevent or cure amyloid diseases. To identify Bri2 BRICHOS interacting partners, we carried out a 'protein fishing' experiment using recombinant human (rh) Bri2 BRICHOS-coated magnetic particles, which exhibit essentially identical ability to inhibit Aβ42 fibril formation as free rh Bri2 BRICHOS, in combination with proteomic analysis on homogenates of SH-SY5Y cells.

Smallest Secondary Nucleation Competent Aβ Aggregates Probed by an ATP-Independent Molecular Chaperone Domain.

Protein oligomerization is a commonly encountered strategy by which the functional repertoire of proteins is increased. This, however, is a double-edged sword strategy because protein oligomerization is notoriously difficult to control. Living organisms have therefore developed a number of chaperones that prevent protein aggregation.

High-yield Production of Amyloid-β Peptide Enabled by a Customized Spider Silk Domain.

During storage in the silk gland, the N-terminal domain (NT) of spider silk proteins (spidroins) keeps the aggregation-prone repetitive region in solution at extreme concentrations. We observe that NTs from different spidroins have co-evolved with their respective repeat region, and now use an NT that is distantly related to previously used NTs, for efficient recombinant production of the amyloid-β peptide (Aβ) implicated in Alzheimer's disease. A designed variant of NT from Nephila clavipes flagelliform spidroin, which in nature allows production and storage of β-hairpin repeat segments, gives exceptionally high yields of different human Aβ variants as a solubility tag.

High intracellular stability of the spidroin N-terminal domain in spite of abundant amyloidogenic segments revealed by in-cell hydrogen/deuterium exchange mass spectrometry.

Proteins require an optimal balance of conformational flexibility and stability in their native environment to ensure their biological functions. A striking example is spidroins, spider silk proteins, which are stored at extremely high concentrations in soluble form, yet undergo amyloid-like aggregation during spinning. Here, we elucidate the stability of the highly soluble N-terminal domain (NT) of major ampullate spidroin 1 in the Escherichia coli cytosol as well as in inclusion bodies containing fibrillar aggregates.

Gas-Phase Collisions with Trimethylamine-N-Oxide Enable Activation-Controlled Protein Ion Charge Reduction.

Modulating protein ion charge is a useful tool for the study of protein folding and interactions by electrospray ionization mass spectrometry. Here, we investigate activation-dependent charge reduction of protein ions with the chemical chaperone trimethylamine-N-oxide (TMAO). Based on experiments carried out on proteins ranging from 4.

Mass Spectrometry Reveals the Direct Action of a Chemical Chaperone.

Despite their fundamental biological importance and therapeutic potential, the interactions between chemical chaperones and proteins remain difficult to capture due to their transient and nonspecific nature. Using a simple mass spectrometric assay, we are able to follow the interactions between proteins and the chemical chaperone trimethylamine- N-oxide (TMAO). In this manner, we directly observe that the counteraction of TMAO and the denaturant urea is driven by the exclusion of TMAO from the protein surface, whereas the surfactant lauryl dimethylamine- N-oxide cannot be displaced.

A spidroin-derived solubility tag enables controlled aggregation of a designed amyloid protein.

Amyloidogenesis is associated with more than 30 diseases, but the molecular mechanisms involved in cell toxicity and fibril formation remain largely unknown. The inherent tendency of amyloid-forming proteins to aggregate renders expression, purification, and experimental studies challenging. NT* is a solubility tag derived from a spider silk protein that was recently introduced for the production of several aggregation-prone peptides and proteins at high yields.

Efficient protein production inspired by how spiders make silk.

Membrane proteins are targets of most available pharmaceuticals, but they are difficult to produce recombinantly, like many other aggregation-prone proteins. Spiders can produce silk proteins at huge concentrations by sequestering their aggregation-prone regions in micellar structures, where the very soluble N-terminal domain (NT) forms the shell. We hypothesize that fusion to NT could similarly solubilize non-spidroin proteins, and design a charge-reversed mutant (NT*) that is pH insensitive, stabilized and hypersoluble compared to wild-type NT.

Novel expression of a functional trimeric fragment of human SP-A with efficacy in neutralisation of RSV.

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and hospitalisation of infants in developed countries. Surfactant protein A (SP-A) is an important innate immune molecule, localized in pulmonary surfactant. SP-A binds to carbohydrates on the surface of pathogens in a calcium-dependent manner to enable neutralisation, agglutination and clearance of pathogens including RSV.

Diversified Structural Basis of a Conserved Molecular Mechanism for pH-Dependent Dimerization in Spider Silk N-Terminal Domains.

Conversion of spider silk proteins from soluble dope to insoluble fibers involves pH-dependent dimerization of the N-terminal domain (NT). This conversion is tightly regulated to prevent premature precipitation and enable rapid silk formation at the end of the duct. Three glutamic acid residues that mediate this process in the NT from Euprosthenops australis major ampullate spidroin 1 are well conserved among spidroins.

Carbonic anhydrase generates CO2 and H+ that drive spider silk formation via opposite effects on the terminal domains.

Spider silk fibers are produced from soluble proteins (spidroins) under ambient conditions in a complex but poorly understood process. Spidroins are highly repetitive in sequence but capped by nonrepetitive N- and C-terminal domains (NT and CT) that are suggested to regulate fiber conversion in similar manners. By using ion selective microelectrodes we found that the pH gradient in the silk gland is much broader than previously known.

Sequential pH-driven dimerization and stabilization of the N-terminal domain enables rapid spider silk formation.

The mechanisms controlling the conversion of spider silk proteins into insoluble fibres, which happens in a fraction of a second and in a defined region of the silk glands, are still unresolved. The N-terminal domain changes conformation and forms a homodimer when pH is lowered from 7 to 6; however, the molecular details still remain to be determined. Here we investigate site-directed mutants of the N-terminal domain from Euprosthenops australis major ampullate spidroin 1 and find that the charged residues D40, R60 and K65 mediate intersubunit electrostatic interactions.

Inhibiting HER3-mediated tumor cell growth with affibody molecules engineered to low picomolar affinity by position-directed error-prone PCR-like diversification.

The HER3 receptor is implicated in the progression of various cancers as well as in resistance to several currently used drugs, and is hence a potential target for development of new therapies. We have previously generated Affibody molecules that inhibit heregulin-induced signaling of the HER3 pathways. The aim of this study was to improve the affinity of the binders to hopefully increase receptor inhibition efficacy and enable a high receptor-mediated uptake in tumors.