Identification of cytoskeletal proteins as binding partners of Bri2 BRICHOS domain.

Proteins must fold into three-dimensional structures to execute their biological functions. Therefore, maintenance of protein homeostasis, proteostasis, including prevention of protein misfolding is essential for cellular activity and health. Molecular chaperones are key actors in proteostasis. BRICHOS domain is an intramolecular chaperone that also interferes with several aggregation-prone proteins including amyloid ß (Aß), involved in Alzheimer's disease (AD). To extend the knowledge about Bri2 BRICHOS interactome we here used recombinant human (rh) Bri2 BRICHOS-mCherry fusion protein to probe for potential binding partners. Firstly, exogenously added Bri2 BRICHOS-mCherry was used to stain brain sections of wildtype and amyloid precursor protein (App) knock-in AD mice exhibiting robust Aß pathology. Unexpectedly, we found that rh Bri2 BRICHOS-mCherry stained the cytoplasm of neurons which are devoid of Aß deposits. To identify these intraneuronal proteins that bind to the rh Bri2 BRICHOS domain, we performed co-immunoprecipitation (co-IP) of mouse brain hippocampi homogenates using the Bri2 BRICHOS-mCherry probe and analyzed co-IP proteins by LC-MS/MS. This identified several cytoskeletal proteins including spectrin alpha and beta chain, drebrin, tubulin ß3, and ß-actin as binding partners. The interactions were confirmed by a second round of pulldown experiments using rh Bri2 BRICHOS linked to magnetic beads. The interaction of rh Bri2 BRICHOS and tubulin ß3 was further investigated by staining both mouse brain sections and SH-SY5Y neuroblastoma cells with rh Bri2 BRICHOS-mCherry and tubulin ß3 immunostaining, which revealed partial co-localization. These data suggest a possible interplay of extracellular chaperone Bri2 BRICHOS domain in the intracellular space including the cytoskeleton.

Metal ion coordination delays amyloid-ß peptide self-assembly by forming an aggregation-inert complex.

A detailed understanding of the molecular pathways for amyloid-ß (Aß) peptide aggregation from monomers into amyloid fibrils, a hallmark of Alzheimer's disease, is crucial for the development of diagnostic and therapeutic strategies. We investigate the molecular details of peptide fibrillization in vitro by perturbing this process through addition of differently charged metal ions. Here, we used a monovalent probe, the silver ion, that, similarly to divalent metal ions, binds to monomeric Aß peptide and efficiently modulates Aß fibrillization. On the basis of our findings, combined with our previous results on divalent zinc ions, we propose a model that links the microscopic metal-ion binding to Aß monomers to its macroscopic impact on the peptide self-assembly observed in bulk experiments. We found that substoichiometric concentrations of the investigated metal ions bind specifically to the N-terminal region of Aß, forming a dynamic, partially compact complex. The metal-ion bound state appears to be incapable of aggregation, effectively reducing the available monomeric Aß pool for incorporation into fibrils. This is especially reflected in a decreased fibril-end elongation rate. However, because the bound state is significantly less stable than the amyloid state, Aß peptides are only transiently redirected from fibril formation, and eventually almost all Aß monomers are integrated into fibrils. Taken together, these findings unravel the mechanistic consequences of delaying Aß aggregation via weak metal-ion binding, quantitatively linking the contributions of specific interactions of metal ions with monomeric Aß to their effects on bulk aggregation.

Blood-brain and blood-cerebrospinal fluid passage of BRICHOS domains from two molecular chaperones in mice.

Targeting toxicity associated with ß-amyloid (Aß) misfolding and aggregation is a promising therapeutic strategy for preventing or managing Alzheimer's disease. The BRICHOS domains from human prosurfactant protein C (proSP-C) and integral membrane protein 2B (Bri2) efficiently reduce neurotoxicity associated with Aß42 fibril formation both in vitro and in vivo In this study, we evaluated the serum half-lives and permeability into the brain and cerebrospinal fluid (CSF) of recombinant human (rh) proSP-C and Bri2 BRICHOS domains injected intravenously into WT mice. We found that rh proSP-C BRICHOS has a longer blood serum half-life compared with rh Bri2 BRICHOS and passed into the CSF but not into the brain parenchyma. As judged by Western blotting, immunohistochemistry, and ELISA, rh Bri2 BRICHOS passed into both the CSF and brain. Intracellular immunostaining for rh Bri2 BRICHOS was observed in the choroid plexus epithelium as well as in the cerebral cortex. Our results indicate that intravenously administered rh proSP-C and Bri2 BRICHOS domains have different pharmacokinetic properties and blood-brain/blood-CSF permeability in mice. The finding that rh Bri2 BRICHOS can reach the brain parenchyma after peripheral administration may be harnessed in the search for new therapeutic strategies for managing Alzheimer's disease.

Dementia-related Bri2 BRICHOS is a versatile molecular chaperone that efficiently inhibits Aß42 toxicity in Drosophila.

Formation of fibrils of the amyloid-ß peptide (Aß) is suggested to play a central role in neurodegeneration in Alzheimer's disease (AD), for which no effective treatment exists. The BRICHOS domain is a part of several disease-related proproteins, the most studied ones being Bri2 associated with familial dementia and prosurfactant protein C (proSP-C) associated with lung amyloid. BRICHOS from proSP-C has been found to be an efficient inhibitor of Aß aggregation and toxicity, but its lung-specific expression makes it unsuited to target in AD. Bri2 is expressed in the brain, affects processing of Aß precursor protein, and increased levels of Bri2 are found in AD brain, but the specific role of its BRICHOS domain has not been studied in vivo Here, we find that transgenic expression of the Bri2 BRICHOS domain in the Drosophila central nervous system (CNS) or eyes efficiently inhibits Aß42 toxicity. In the presence of Bri2 BRICHOS, Aß42 is diffusely distributed throughout the mushroom bodies, a brain region involved in learning and memory, whereas Aß42 expressed alone or together with proSP-C BRICHOS forms punctuate deposits outside the mushroom bodies. Recombinant Bri2 BRICHOS domain efficiently prevents Aß42-induced reduction in γ-oscillations in hippocampal slices. Finally, Bri2 BRICHOS inhibits several steps in the Aß42 fibrillation pathway and prevents aggregation of heat-denatured proteins, indicating that it is a more versatile chaperone than proSP-C BRICHOS. These findings suggest that Bri2 BRICHOS can be a physiologically relevant chaperone for Aß in the CNS and needs to be further investigated for its potential in AD treatment.

Dissociation of a BRICHOS trimer into monomers leads to increased inhibitory effect on Aß42 fibril formation.

The BRICHOS domain is associated with human amyloid disease, and it efficiently prevents amyloid fibril formation of the amyloid ß-peptide (Aß) in vitro and in vivo. Recombinant human prosurfactant protein C (proSP-C) BRICHOS domain forms a homotrimer as observed by x-ray crystallography, analytical ultracentrifugation, native polyacrylamide gel electrophoresis, analytical size exclusion chromatography and electrospray mass spectrometry. It was hypothesized that the trimer is an inactive storage form, as a putative substrate-binding site identified in the monomer, is buried in the subunit interface of the trimer. We show here increased dissociation of the BRICHOS trimer into monomers, by addition of detergents or of bis-ANS, known to bind to the putative substrate-binding site, or by introducing a Ser to Arg mutation expected to interfere with trimer formation. This leads to increased capacity to delay Aß(42) fibril formation. Cross-linking of the BRICHOS trimer with glutaraldehyde, in contrast, renders it unable to affect Aß(42) fibril formation. Moreover, proSP-C BRICHOS expressed in HEK293 cells is mainly monomeric, as detected by proximity ligation assay. Finally, proteolytic cleavage of BRICHOS in a loop region that is cleaved during proSP-C biosynthesis results in increased capacity to delay Aß(42) fibril formation. These results indicate that modulation of the accessibility of the substrate-binding site is a means to regulate BRICHOS activity.

Amyloid-ß-induced action potential desynchronization and degradation of hippocampal gamma oscillations is prevented by interference with peptide conformation change and aggregation.

The amyloid-ß hypothesis of Alzheimer's Disease (AD) focuses on accumulation of amyloid-ß peptide (Aß) as the main culprit for the myriad physiological changes seen during development and progression of AD including desynchronization of neuronal action potentials, consequent development of aberrant brain rhythms relevant for cognition, and final emergence of cognitive deficits. The aim of this study was to elucidate the cellular and synaptic mechanisms underlying the Aß-induced degradation of gamma oscillations in AD, to identify aggregation state(s) of Aß that mediate the peptides neurotoxicity, and to test ways to prevent the neurotoxic Aß effect. We show that Aß(1-42) in physiological concentrations acutely degrades mouse hippocampal gamma oscillations in a concentration- and time-dependent manner. The underlying cause is an Aß-induced desynchronization of action potential generation in pyramidal cells and a shift of the excitatory/inhibitory equilibrium in the hippocampal network. Using purified preparations containing different aggregation states of Aß, as well as a designed ligand and a BRICHOS chaperone domain, we provide evidence that the severity of Aß neurotoxicity increases with increasing concentration of fibrillar over monomeric Aß forms, and that Aß-induced degradation of gamma oscillations and excitatory/inhibitory equilibrium is prevented by compounds that interfere with Aß aggregation. Our study provides correlative evidence for a link between Aß-induced effects on synaptic currents and AD-relevant neuronal network oscillations, identifies the responsible aggregation state of Aß and proofs that strategies preventing peptide aggregation are able to prevent the deleterious action of Aß on the excitatory/inhibitory equilibrium and on the gamma rhythm.

Synthesis and evaluation of antineurotoxicity properties of an amyloid-ß peptide targeting ligand containing a triamino acid.

Peptide-like compounds containing an arginine have been shown to bind and stabilize the central helix of the Alzheimer's disease related amyloid-ß peptide (Aß) in an α-helical conformation, thereby delaying its aggregation into cytotoxic species. Here we study a novel Aß targeting ligand AEDabDab containing the triamino acid, N(γ)-(2-aminoethyl)-2,4-diaminobutanoic (AEDab) acid. The new AEDab triamino acid carries an extra positive charge in the side chain and is designed to be incorporated into a ligand AEDabDab where the AEDab replaces an arginine moiety in a previously developed ligand Pep1b. This is done in order to increase the Aß-ligand interaction, and molecular dynamics (MD) simulation of the stability of the Aß central helix in the presence of the AEDabDab ligand shows further stabilization of the helical conformation of Aß compared to the previously reported Pep1b as well as compared to the AEOrnDab ligand containing an N(δ)-(2-aminoethyl)-2,5-diaminopentanoic acid unit which has an additional methylene group. To evaluate the effect of the AEDabDab ligand on the Aß neurotoxicity the AEDab triamino acid building block is synthesized by reductive alkylation of N-protected-glycinal with α-amino-protected diaminobutanoic acid, and the Aß targeting ligand AEDabDab is prepared by solid-phase synthesis starting with attachment of glutarate to the Wang support. Replacement of the arginine residue by the AEDab triamino acid resulted in an improved capability of the ligand to prevent the Aß1-42 induced reduction of gamma (γ) oscillations in hippocampal slice preparation.

Identification of potential aggregation hotspots on Aß42 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-ß peptide (Aß) 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. Here, we identify a site on the Aß42 fibril surface, consisting of three C-terminal ß-strands and particularly the solvent-exposed ß-strand stretching from residues 26-28, which is efficiently sensed by a designed variant of Bri2 BRICHOS. Remarkably, while only a low amount of BRICHOS binds to Aß42 fibrils, fibril-catalyzed nucleation processes are effectively prevented, suggesting that the identified site acts as a catalytic aggregation hotspot, which can specifically be blocked by BRICHOS. Hence, these findings provide an understanding how toxic nucleation events can be targeted by molecular chaperones.

BRICHOS domains efficiently delay fibrillation of amyloid ß-peptide.

Amyloid diseases such as Alzheimer, Parkinson, and prion diseases are associated with a specific form of protein misfolding and aggregation into oligomers and fibrils rich in ß-sheet structure. The BRICHOS domain consisting of ∼100 residues is found in membrane proteins associated with degenerative and proliferative disease, including lung fibrosis (surfactant protein C precursor; pro-SP-C) and familial dementia (Bri2). We find that recombinant BRICHOS domains from Bri2 and pro-SP-C prevent fibril formation of amyloid ß-peptides (Aß(40) and Aß(42)) far below the stoichiometric ratio. Kinetic experiments show that a main effect of BRICHOS is to prolong the lag time in a concentration-dependent, quantitative, and reproducible manner. An ongoing aggregation process is retarded if BRICHOS is added at any time during the lag phase, but it is too late to interfere at the end of the process. Results from circular dichroism and NMR spectroscopy, as well as analytical size exclusion chromatography, imply that Aß is maintained as an unstructured monomer during the extended lag phase in the presence of BRICHOS. Electron microscopy shows that although the process is delayed, typical amyloid fibrils are eventually formed also when BRICHOS is present. Structural BRICHOS models display a conserved array of tyrosine rings on a five-stranded ß-sheet, with inter-hydroxyl distances suited for hydrogen-bonding peptides in an extended ß-conformation. Our data imply that the inhibitory mechanism is reliant on BRICHOS interfering with molecular events during the lag phase.

Stabilization of neurotoxic Alzheimer amyloid-beta oligomers by protein engineering.

Soluble oligomeric aggregates of the amyloid-beta peptide (Abeta) have been implicated in the pathogenesis of Alzheimer's disease (AD). Although the conformation adopted by Abeta within these aggregates is not known, a beta-hairpin conformation is known to be accessible to monomeric Abeta. Here we show that this beta-hairpin is a building block of toxic Abeta oligomers by engineering a double-cysteine mutant (called Abetacc) in which the beta-hairpin is stabilized by an intramolecular disulfide bond. Abeta(40)cc and Abeta(42)cc both spontaneously form stable oligomeric species with distinct molecular weights and secondary-structure content, but both are unable to convert into amyloid fibrils. Biochemical and biophysical experiments and assays with conformation-specific antibodies used to detect Abeta aggregates in vivo indicate that the wild-type oligomer structure is preserved and stabilized in Abetacc oligomers. Stable oligomers are expected to become highly toxic and, accordingly, we find that beta-sheet-containing Abeta(42)cc oligomers or protofibrillar species formed by these oligomers are 50 times more potent inducers of neuronal apoptosis than amyloid fibrils or samples of monomeric wild-type Abeta(42), in which toxic aggregates are only transiently formed. The possibility of obtaining completely stable and physiologically relevant neurotoxic Abeta oligomer preparations will facilitate studies of their structure and role in the pathogenesis of AD. For example, here we show how kinetic partitioning into different aggregation pathways can explain why Abeta(42) is more toxic than the shorter Abeta(40), and why certain inherited mutations are linked to protofibril formation and early-onset AD.

Food protein-derived amyloids do not accelerate amyloid ß aggregation.

The deposition of proteins in the form of amyloid fibrils is closely associated with several serious diseases. The events that trigger the conversion from soluble functional proteins into insoluble amyloid are not fully understood. Many proteins that are not associated with disease can form amyloid with similar structural characteristics as the disease-associated fibrils, which highlights the potential risk of cross-seeding of disease amyloid by amyloid-like structures encountered in our surrounding. Of particular interest are common food proteins that can be transformed into amyloid under conditions similar to cooking. We here investigate cross-seeding of amyloid-ß (Aß), a peptide known to form amyloid during the development of Alzheimer's disease, by 16 types of amyloid fibrils derived from food proteins or peptides. Kinetic studies using thioflavin T fluorescence as output show that none of the investigated protein fibrils accelerates the aggregation of Aß. In at least two cases (hen egg lysozyme and oat protein isolate) we observe retardation of the aggregation, which appears to originate from interactions between the food protein seeds and Aß in aggregated form. The results support the view that food-derived amyloid is not a risk factor for development of Aß pathology and Alzheimer's disease.

Molecular Mechanisms of Amyloid-ß Self-Assembly Seeded by In Vivo-Derived Fibrils and Inhibitory Effects of the BRICHOS Chaperone.

Self-replication of amyloid-ß-peptide (Aß) fibril formation is a hallmark in Alzheimer's disease (AD). Detailed insights have been obtained in Aß self-assembly in vitro, yet whether similar mechanisms are relevant in vivo has remained elusive. Here, we investigated the ability of in vivo-derived Aß fibrils from two different amyloid precursor protein knock-in AD mouse models to seed Aß42 aggregation, where we quantified the microscopic rate constants. We found that the nucleation mechanism of in vivo-derived fibril-seeded Aß42 aggregation can be described with the same kinetic model as that in vitro. Further, we identified the inhibitory mechanism of the anti-amyloid BRICHOS chaperone on seeded Aß42 fibrillization, revealing a suppression of secondary nucleation and fibril elongation, which is strikingly similar as observed in vitro. These findings hence provide a molecular understanding of the Aß42 nucleation process triggered by in vivo-derived Aß42 propagons, providing a framework for the search for new AD therapeutics.

Prosaposin maintains lipid homeostasis in dopamine neurons and counteracts experimental parkinsonism in rodents.

Prosaposin (PSAP) modulates glycosphingolipid metabolism and variants have been linked to Parkinson's disease (PD). Here, we find altered PSAP levels in the plasma, CSF and post-mortem brain of PD patients. Altered plasma and CSF PSAP levels correlate with PD-related motor impairments. Dopaminergic PSAP-deficient (cPSAPDAT) mice display hypolocomotion and depression/anxiety-like symptoms with mildly impaired dopaminergic neurotransmission, while serotonergic PSAP-deficient (cPSAPSERT) mice behave normally. Spatial lipidomics revealed an accumulation of highly unsaturated and shortened lipids and reduction of sphingolipids throughout the brains of cPSAPDAT mice. The overexpression of α-synuclein via AAV lead to more severe dopaminergic degeneration and higher p-Ser129 α-synuclein levels in cPSAPDAT mice compared to WT mice. Overexpression of PSAP via AAV and encapsulated cell biodelivery protected against 6-OHDA and α-synuclein toxicity in wild-type rodents. Thus, these findings suggest PSAP may maintain dopaminergic lipid homeostasis, which is dysregulated in PD, and counteract experimental parkinsonism.

Molecular Chaperone BRICHOS Inhibits CADASIL-Mutated NOTCH3 Aggregation In Vitro.

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is the most common familial form of stroke, which is caused by mutations located in the epidermal growth factor (EGF)-like repeats of the NOTCH3 gene. Mutations cause the NOTCH3 (N3) protein to misfold and aggregate. These aggregates will be a component of granular osmiophilic material, which when accumulated around the arteries and arterioles is believed to cause the degradation of vascular smooth muscle cells (VSMC). VSMC degradation affects blood flow regulation and leads to white matter and neuronal death. Currently, there is no treatment for CADASIL. The dementia-relevant BRICHOS domain is a small multitalented protein with functions that include ATP-independent chaperone-like properties. BRICHOS has been shown to prevent the aggregation of both fibrillar and non-fibrillar structures. Therefore, the objective of this study is to investigate whether BRICHOS exhibits anti-aggregating properties on a recombinant CADASIL-mutated N3 protein consisting of the first five repeats of EGF (EGF1-5), harboring a cysteine instead of an arginine in the position 133, (R133C). We found that the N3 EGF1-5 R133C mutant is more prone to aggregate, while the wildtype is more stable. Recombinant human Bri2 BRICHOS is able to interact and stabilize the R133C-mutated N3 protein in a dose-dependent manner. These results suggest an anti-aggregating impact of BRICHOS on the N3 EGF1-5 R133C protein, which could be a potential treatment for CADASIL.

Molecular Structure of Cu(II)-Bound Amyloid-ß Monomer Implicated in Inhibition of Peptide Self-Assembly in Alzheimer's Disease.

Metal ions, such as copper and zinc ions, have been shown to strongly modulate the self-assembly of the amyloid-ß (Aß) peptide into insoluble fibrils, and elevated concentrations of metal ions have been found in amyloid plaques of Alzheimer's patients. Among the physiological transition metal ions, Cu(II) ions play an outstanding role since they can trigger production of neurotoxic reactive oxygen species. In contrast, structural insights into Cu(II) coordination of Aß have been challenging due to the paramagnetic nature of Cu(II). Here, we employed specifically tailored paramagnetic NMR experiments to determine NMR structures of Cu(II) bound to monomeric Aß. We found that monomeric Aß binds Cu(II) in the N-terminus and combined with molecular dynamics simulations, we could identify two prevalent coordination modes of Cu(II). For these, we report here the NMR structures of the Cu(II)-bound Aß complex, exhibiting heavy backbone RMSD values of 1.9 and 2.1 Å, respectively. Further, applying aggregation kinetics assays, we identified the specific effect of Cu(II) binding on the Aß nucleation process. Our results show that Cu(II) efficiently retards Aß fibrillization by predominately reducing the rate of fibril-end elongation at substoichiometric ratios. A detailed kinetic analysis suggests that this specific effect results in enhanced Aß oligomer generation promoted by Cu(II). These results can quantitatively be understood by Cu(II) interaction with the Aß monomer, forming an aggregation inert complex. In fact, this mechanism is strikingly similar to other transition metal ions, suggesting a common mechanism of action of retarding Aß self-assembly, where the metal ion binding to monomeric Aß is a key determinant.

Amyloid Fibril Formation of Arctic Amyloid-ß 1-42 Peptide is Efficiently Inhibited by the BRICHOS Domain.

Amyloid-ß peptide (Aß) aggregation is one of the hallmarks of Alzheimer's disease (AD). Mutations in Aß are associated with early onset familial AD, and the Arctic mutant E22G (Aßarc) is an extremely aggregation-prone variant. Here, we show that BRICHOS, a natural anti-amyloid chaperone domain, from Bri2 efficiently inhibits aggregation of Aßarc by mainly interfering with secondary nucleation. This is qualitatively different from the microscopic inhibition mechanism for the wild-type Aß, against which Bri2 BRICHOS has a major effect on both secondary nucleation and fibril end elongation. The monomeric Aß42arc peptide aggregates into amyloid fibrils significantly faster than wild-type Aß (Aß42wt), as monitored by thioflavin T (ThT) binding, but the final ThT intensity was strikingly lower for Aß42arc compared to Aß42wt fibrils. The Aß42arc peptide formed large aggregates, single-filament fibrils, and multiple-filament fibrils without obvious twists, while Aß42wt fibrils displayed a polymorphic pattern with typical twisted fibril architecture. Recombinant human Bri2 BRICHOS binds to the Aß42arc fibril surface and interferes with the macroscopic fibril arrangement by promoting single-filament fibril formation. This study provides mechanistic insights on how BRICHOS efficiently affects the aggressive Aß42arc aggregation, resulting in both delayed fibril formation kinetics and altered fibril structure.

Chaperones mainly suppress primary nucleation during formation of functional amyloid required for bacterial biofilm formation.

Unlike misfolding in neurodegenerative diseases, aggregation of functional amyloids involved in bacterial biofilm, e.g. CsgA (E. coli) and FapC (Pseudomonas), is carefully regulated. However, it is unclear whether functional aggregation is inhibited by chaperones targeting pathological misfolding and if so by what mechanism. Here we analyze how four entirely different human chaperones or protein modulators (transthyretin, S100A9, Bri2 BRICHOS and DNAJB6) and bacterial CsgC affect CsgA and FapC fibrillation. CsgA is more susceptible to inhibition than FapC and the chaperones vary considerably in the efficiency of their inhibition. However, mechanistic analysis reveals that all predominantly target primary nucleation rather than elongation or secondary nucleation, while stoichiometric considerations suggest that DNAJB6 and CsgC target nuclei rather than monomers. Inhibition efficiency broadly scales with the chaperones' affinity for monomeric CsgA and FapC. The chaperones tend to target the most aggregation-prone regions of CsgA, but do not display such tendencies towards the more complex FapC sequence. Importantly, the most efficient inhibitors (Bri2 BRICHOS and DNAJB6) significantly reduce bacterial biofilm formation. This commonality of chaperone action may reflect the simplicity of functional amyloid formation, driven largely by primary nucleation, as well as the ability of non-bacterial chaperones to deploy their proteostatic capacities across biological kingdoms.

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.

Functionalization of amyloid fibrils via the Bri2 BRICHOS domain.

Amyloid fibrils are mechanically robust and partly resistant to proteolytic degradation, making them potential candidates for scaffold materials in cell culture, tissue engineering, drug delivery and other applications. Such applications of amyloids would benefit from the possibility to functionalize the fibrils, for example by adding growth factors or cell attachment sites. The BRICHOS domain is found in a family of human proteins that harbor particularly amyloid-prone regions and can reduce aggregation as well as toxicity of several different amyloidogenic peptides. Recombinant human (rh) BRICHOS domains have been shown to bind to the surface of amyloid-ß (Aß) fibrils by immune electron microscopy. Here we produce fusion proteins between mCherry and rh Bri2 BRICHOS and show that they can bind to different amyloid fibrils with retained fluorescence of mCherry in vitro as well as in cultured cells. This suggests a "generic" ability of the BRICHOS domain to bind fibrillar surfaces that can be used to synthesize amyloid decorated with different protein functionalities.

High-yield Production of Amyloid-ß 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-ß peptide (Aß) implicated in Alzheimer's disease. A designed variant of NT from Nephila clavipes flagelliform spidroin, which in nature allows production and storage of ß-hairpin repeat segments, gives exceptionally high yields of different human Aß variants as a solubility tag. This tool enables efficient production of target peptides also in minimal medium and gives up to 10 times more isotope-labeled monomeric Aß peptides per liter bacterial culture than previously reported.