Molecular basis for different substrate-binding sites and chaperone functions of the BRICHOS domain.

Proteins can misfold into fibrillar or amorphous aggregates and molecular chaperones act as crucial guardians against these undesirable processes. The BRICHOS chaperone domain, found in several otherwise unrelated proproteins that contain amyloidogenic regions, effectively inhibits amyloid formation and toxicity but can in some cases also prevent non-fibrillar, amorphous protein aggregation. Here, we elucidate the molecular basis behind the multifaceted chaperone activities of the BRICHOS domain from the Bri2 proprotein. High-confidence AlphaFold2 and RoseTTAFold predictions suggest that the intramolecular amyloidogenic region (Bri23) is part of the hydrophobic core of the proprotein, where it occupies the proposed amyloid binding site, explaining the markedly reduced ability of the proprotein to prevent an exogenous amyloidogenic peptide from aggregating. However, the BRICHOS-Bri23 complex maintains its ability to form large polydisperse oligomers that prevent amorphous protein aggregation. A cryo-EM-derived model of the Bri2 BRICHOS oligomer is compatible with surface-exposed hydrophobic motifs that get exposed and come together during oligomerization, explaining its effects against amorphous aggregation. These findings provide a molecular basis for the BRICHOS chaperone domain function, where distinct surfaces are employed against different forms of protein aggregation.

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.

Metal Binding of Alzheimer's Amyloid-ß and Its Effect on Peptide Self-Assembly.

Metal ions have been identified as key factors modulating the aggregation of amyloid-ß peptide (Aß) implicated in Alzheimer's disease (AD). The presence of elevated levels of metal ions in the amyloid plaques in AD patients supports the notion that the dysfunction of metal homeostasis is connected to the development of AD pathology. Here, recent findings from high- and low-resolution biophysical techniques are put into perspective, providing detailed insights into the molecular structures and dynamics of metal-bound Aß complexes and the effect of metal ions on the Aß aggregation process. In particular, the development of theoretical kinetic models deducing different microscopic nucleation events from the macroscopic aggregation behavior has enabled deciphering of the effect of metal ions on specific nucleation processes. In addition to these macroscopic measurements of bulk aggregation to quantify microscopic rates, recent NMR studies have revealed details about the structures and dynamics of metal-Aß complexes, thereby linking structural events to bulk aggregation. Interestingly, transition-metal ions, such as copper, zinc, and silver ions, form a compact complex with the N-terminal part of monomeric Aß, respectively, where the metal-bound "folded" state is in dynamic equilibrium with an "unfolded" state. The rates and thermodynamic features of these exchange dynamics have been determined by using NMR relaxation dispersion experiments. Additionally, the application of specifically tailored paramagnetic NMR experiments on the Cu(II)-Aß complex has been fruitful in obtaining structural constraints within the blind sphere of conventional NMR experiments. This enables the determination of molecular structures of the "folded" Cu(II)-coordinated N-terminal region of Aß. Furthermore, the discussed transition-metal ions modulate Aß self-assembly in a concentration-dependent manner, where low metal ion concentrations inhibit Aß fibril formation, while at high metal ion concentrations other processes occur, resulting in amorphous aggregate formation. Remarkably, the metal-Aß interaction predominately reduces one specific nucleation step, the fibril-end elongation, whereas primary and surface-catalyzed secondary nucleation mechanisms are less affected. Specific inhibition of fibril-end elongation theoretically predicts an enhanced generation of Aß oligomers, which is an interesting contribution to understanding metal-Aß-associated neurotoxic effects. Taken together, the metal binding process creates a metal-bound Aß complex, which is seemingly inert to aggregation. This process hence efficiently reduces the aggregation-prone peptide pool, which on the macroscopic level is reflected as slower aggregation kinetics. Thus, the specific binding of metals to the Aß monomer can be linked to the macroscopic inhibitory effect on Aß bulk aggregation, providing a molecular understanding of the Aß aggregation mechanism in the presence of metal ions, where the metal ion can be seen as a minimalist agent against Aß self-assembly. These insights can help to target Aß aggregation in vivo, where metal ions are key factors modulating the Aß self-assembly and Aß-associated neurotoxicity.

Misfolded alpha-synuclein detection by RT-QuIC in dementia with lewy bodies: a systematic review and meta-analysis.

Introduction: Dementia with Lewy Bodies (DLB) is the second most common cause of neurodegenerative dementia after Alzheimer's disease (AD), but the field is still lacking a specific biomarker for its core pathology: alpha synuclein (α-syn). Realtime quaking induced conversion (RT-QuIC) has recently emerged as a strong biomarker candidate to detect misfolded α-syn in DLB. However, the variability in the parameters of the technique and the heterogeneity of DLB patients make the reproducibility of the results difficult. Here, we provide an overview of the state-of-the-art research of α-syn RT-QuIC in DLB focused on: (1) the capacity of α-syn RT-QuIC to discriminate DLB from controls, Parkinson's disease (PD) and AD; (2) the capacity of α-syn RT-QuIC to identify prodromal stages of DLB; and (3) the influence of co-pathologies on α-syn RT-QuIC's performance. We also assessed the influence of different factors, such as technical conditions (e.g., temperature, pH, shaking-rest cycles), sample type, and clinical diagnosis versus autopsy confirmation. Methods: We conducted a systematic review following the PRISMA guidelines in August 2022, without any limits in publication dates. Search terms were combinations of "RT-QuIC" and "Lewy Bodies," "DLB" or "LBD". Results: Our meta-analysis shows that α-syn RT-QuIC reaches very high diagnostic performance in discriminating DLB from both controls (pooled sensitivity and specificity of 0.94 and 0.96, respectively) and AD (pooled sensitivity and specificity of 0.95 and 0.88) and is promising for prodromal phases of DLB. However, the performance of α-syn RT-QuIC to discriminate DLB from PD is currently low due to low specificity (pooled sensitivity and specificity of 0.94 and 0.11). Our analysis showed that α-syn RT-QuIC's performance is not substantially influenced by sample type or clinical diagnosis versus autopsy confirmation. Co-pathologies did not influence the performance of α-syn RT-QuIC, but the number of such studies is currently limited. We observed technical variability across published articles. However, we could not find a clear effect of technical variability on the reported results. Conclusion: There is currently enough evidence to test misfolded α-syn by RT-QuIC for clinical use. We anticipate that harmonization of protocols across centres and advances in standardization will facilitate the clinical establishment of misfolded α-syn detection by RT-QuIC.

Amyloid inhibition by molecular chaperones in vitro can be translated to Alzheimer's pathology in vivo.

Molecular chaperones are important components in the cellular quality-control machinery and increasing evidence points to potential new roles for them as suppressors of amyloid formation in neurodegenerative diseases, such as Alzheimer's disease. Approaches to treat Alzheimer's disease have not yet resulted in an effective treatment, suggesting that alternative strategies may be useful. Here, we discuss new treatment approaches based on molecular chaperones that inhibit amyloid-ß (Aß) aggregation by different microscopic mechanisms of action. Molecular chaperones that specifically target secondary nucleation reactions during Aß aggregation in vitro - a process closely associated with Aß oligomer generation - have shown promising results in animal treatment studies. The inhibition of Aß oligomer generation in vitro seemingly correlates with the effects of treatment, giving indirect clues about the molecular mechanisms present in vivo. Interestingly, recent immunotherapy advances, which have demonstrated significant improvements in clinical phase III trials, have used antibodies that selectively act against Aß oligomer formation, supporting the notion that specific inhibition of Aß neurotoxicity is more rewarding than reducing overall amyloid fibril formation. Hence, specific modulation of chaperone activity represents a promising new strategy for treatment of neurodegenerative disorders.

Short hydrophobic loop motifs in BRICHOS domains determine chaperone activity against amorphous protein aggregation but not against amyloid formation.

ATP-independent molecular chaperones are important for maintaining cellular fitness but the molecular determinants for preventing aggregation of partly unfolded protein substrates remain unclear, particularly regarding assembly state and basis for substrate recognition. The BRICHOS domain can perform small heat shock (sHSP)-like chaperone functions to widely different degrees depending on its assembly state and sequence. Here, we observed three hydrophobic sequence motifs in chaperone-active domains, and found that they get surface-exposed when the BRICHOS domain assembles into larger oligomers. Studies of loop-swap variants and site-specific mutants further revealed that the biological hydrophobicities of the three short motifs linearly correlate with the efficiency to prevent amorphous protein aggregation. At the same time, they do not at all correlate with the ability to prevent ordered amyloid fibril formation. The linear correlations also accurately predict activities of chimeras containing short hydrophobic sequence motifs from a sHSP that is unrelated to BRICHOS. Our data indicate that short, exposed hydrophobic motifs brought together by oligomerisation are sufficient and necessary for efficient chaperone activity against amorphous protein aggregation.

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.

A new kid in the folding funnel: Molecular chaperone activities of the BRICHOS domain.

The BRICHOS protein superfamily is a diverse group of proteins associated with a wide variety of human diseases, including respiratory distress, COVID-19, dementia, and cancer. A key characteristic of these proteins-besides their BRICHOS domain present in the ER lumen/extracellular part-is that they harbor an aggregation-prone region, which the BRICHOS domain is proposed to chaperone during biosynthesis. All so far studied BRICHOS domains modulate the aggregation pathway of various amyloid-forming substrates, but not all of them can keep denaturing proteins in a folding-competent state, in a similar manner as small heat shock proteins. Current evidence suggests that the ability to interfere with the aggregation pathways of substrates with entirely different end-point structures is dictated by BRICHOS quaternary structure as well as specific surface motifs. This review aims to provide an overview of the BRICHOS protein family and a perspective of the diverse molecular chaperone-like functions of various BRICHOS domains in relation to their structure and conformational plasticity. Furthermore, we speculate about the physiological implication of the diverse molecular chaperone functions and discuss the possibility to use the BRICHOS domain as a blood-brain barrier permeable molecular chaperone treatment of protein aggregation disorders.

Intravenous treatment with a molecular chaperone designed against ß-amyloid toxicity improves Alzheimer's disease pathology in mouse models.

Attempts to treat Alzheimer's disease with immunotherapy against the ß-amyloid (Aß) peptide or with enzyme inhibitors to reduce Aß production have not yet resulted in effective treatment, suggesting that alternative strategies may be useful. Here we explore the possibility of targeting the toxicity associated with Aß aggregation by using the recombinant human (rh) Bri2 BRICHOS chaperone domain, mutated to act selectively against Aß42 oligomer generation and neurotoxicity in vitro. We find that treatment of Aß precursor protein (App) knockin mice with repeated intravenous injections of rh Bri2 BRICHOS R221E, from an age close to the start of development of Alzheimer's disease-like pathology, improves recognition and working memory, as assessed using novel object recognition and Y maze tests, and reduces Aß plaque deposition and activation of astrocytes and microglia. When treatment was started about 4 months after Alzheimer's disease-like pathology was already established, memory improvement was not detected, but Aß plaque deposition and gliosis were reduced, and substantially reduced astrocyte accumulation in the vicinity of Aß plaques was observed. The degrees of treatment effects observed in the App knockin mouse models apparently correlate with the amounts of Bri2 BRICHOS detected in brain sections after the end of the treatment period.

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.

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.

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.

Increased CSF-decorin predicts brain pathological changes driven by Alzheimer's Aß amyloidosis.

Cerebrospinal fluid (CSF) biomarkers play an important role in diagnosing Alzheimer's disease (AD) which is characterized by amyloid-ß (Aß) amyloidosis. Here, we used two App knock-in mouse models, AppNL-F/NL-F and AppNL-G-F/NL-G-F, exhibiting AD-like Aß pathology to analyze how the brain pathologies translate to CSF proteomes by label-free mass spectrometry (MS). This identified several extracellular matrix (ECM) proteins as significantly altered in App knock-in mice. Next, we compared mouse CSF proteomes with previously reported human CSF MS results acquired from patients across the AD spectrum. Intriguingly, the ECM protein decorin was similarly and significantly increased in both AppNL-F/NL-F and AppNL-G-F/NL-G-F mice, strikingly already at three months of age in the AppNL-F/NL-F mice and preclinical AD subjects having abnormal CSF-Aß42 but normal cognition. Notably, in this group of subjects, CSF-decorin levels positively correlated with CSF-Aß42 levels indicating that the change in CSF-decorin is associated with early Aß amyloidosis. Importantly, receiver operating characteristic analysis revealed that CSF-decorin can predict a specific AD subtype having innate immune activation and potential choroid plexus dysfunction in the brain. Consistently, in AppNL-F/NL-F mice, increased CSF-decorin correlated with both Aß plaque load and with decorin levels in choroid plexus. In addition, a low concentration of human Aß42 induces decorin secretion from mouse primary neurons. Interestingly, we finally identify decorin to activate neuronal autophagy through enhancing lysosomal function. Altogether, the increased CSF-decorin levels occurring at an early stage of Aß amyloidosis in the brain may reflect pathological changes in choroid plexus, present in a subtype of AD subjects.

Autophagy Impairment in App Knock-in Alzheimer's Model Mice.

Alzheimer's disease (AD) is characterized by impaired protein homeostasis leading to amyloid-ß peptide (Aß) amyloidosis. Amyloid precursor protein (APP) knock-in mice exhibit robust Aß pathology, providing possibilities to determine its effect on protein homeostasis including autophagy. Here we compared human AD postmortem brain tissue with brains from two different types of App knock-in mice, App NL-F and App NL-G-F mice, exhibiting AD-like pathology. In AD postmortem brains, p62 levels are increased and p62-positive staining is detected in neurons, including potential axonal beadings, as well as in the vasculature and in corpora amylacea. Interestingly, p62 is also increased in the neurons in 12-month-old App NL-G-F mice. In brain homogenates from 12-month-old App NL-G-F mice, both p62 and light chain 3 (LC3)-II levels are increased as compared to wildtype (WT) mice, indicating inhibited autophagy. Double immunostaining for LC3 and Aß revealed LC3-positive puncta in hippocampus of 24-month-old App NL-F mice around the Aß plaques which was subsequently identified by electron microscopy imaging as an accumulation of autophagic vacuoles in dystrophic neurites around the Aß plaques. Taken together, autophagy is impaired in App knock-in mice upon increased Aß pathology, indicating that App knock-in mouse models provide a platform for understanding the correlation between Aß and autophagy.

Isotope-labeled amyloid-ß does not transmit to the brain in a prion-like manner after peripheral administration.

Findings of early cerebral amyloid-ß deposition in mice after peripheral injection of amyloid-ß-containing brain extracts, and in humans following cadaveric human growth hormone treatment raised concerns that amyloid-ß aggregates and possibly Alzheimer's disease may be transmissible between individuals. Yet, proof that Aß actually reaches the brain from the peripheral injection site is lacking. Here, we use a proteomic approach combining stable isotope labeling of mammals and targeted mass spectrometry. Specifically, we generate 13 C-isotope-labeled brain extracts from mice expressing human amyloid-ß and track 13 C-lysine-labeled amyloid-ß after intraperitoneal administration into young amyloid precursor protein-transgenic mice. We detect injected amyloid-ß in the liver and lymphoid tissues for up to 100 days. In contrast, injected 13 C-lysine-labeled amyloid-ß is not detectable in the brain whereas the mice incorporate 13 C-lysine from the donor brain extracts into endogenous amyloid-ß. Using a highly sensitive and specific proteomic approach, we demonstrate that amyloid-ß does not reach the brain from the periphery. Our study argues against potential transmissibility of Alzheimer's disease while opening new avenues to uncover mechanisms of pathophysiological protein deposition.

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.

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 Aß42 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 Aß42 fibril formation as free rh Bri2 BRICHOS, in combination with proteomic analysis on homogenates of SH-SY5Y cells. We identified 70 proteins that had more significant interactions with rh Bri2 BRICHOS relative to the corresponding control particles. Three previously identified Bri2 BRICHOS interacting proteins were also identified in our 'fishing' experiments. The binding affinity of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the top 'hit', was calculated and was identified as a strong interacting partner. Enrichment analysis of the retained proteins identified three biological pathways: Rho GTPase, heat stress response and pyruvate, cysteine and methionine metabolism.

Smallest Secondary Nucleation Competent Aß 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. The small ATP-independent molecular chaperone domain proSP-C BRICHOS, which is mainly trimeric, specifically inhibits fibril surface-catalyzed nucleation reactions that give rise to toxic oligomers during the aggregation of the Alzheimer's disease-related amyloid-ß peptide (Aß42). Here, we have created a stable proSP-C BRICHOS monomer mutant and show that it does not bind to monomeric Aß42 but has a high affinity for Aß42 fibrils, using surface plasmon resonance. Kinetic analysis of Aß42 aggregation profiles, measured by thioflavin T fluorescence, reveals that the proSP-C BRICHOS monomer mutant strongly inhibits secondary nucleation reactions and thereby reduces the level of catalytic formation of toxic Aß42 oligomers. To study binding between the proSP-C BRICHOS monomer mutant and small soluble Aß42 aggregates, we analyzed fluorescence cross-correlation spectroscopy measurements with the maximum entropy method for fluorescence correlation spectroscopy. We found that the proSP-C BRICHOS monomer mutant binds to the smallest emerging Aß42 aggregates that are comprised of eight or fewer Aß42 molecules, which are already secondary nucleation competent. Our approach can be used to provide molecular-level insights into the mechanisms of action of substances that interfere with protein aggregation.

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.