A Relationship between the Structures and Neurotoxic Effects of Aß Oligomers Stabilized by Different Metal Ions.

Oligomeric assemblies of the amyloid ß peptide (Aß) have been investigated for over two decades as possible neurotoxic agents in Alzheimer's disease. However, due to their heterogeneous and transient nature, it is not yet fully established which of the structural features of these oligomers may generate cellular damage. Here, we study distinct oligomer species formed by Aß40 (the 40-residue form of Aß) in the presence of four different metal ions (Al3+, Cu2+, Fe2+, and Zn2+) and show that they differ in their structure and toxicity in human neuroblastoma cells. We then describe a correlation between the size of the oligomers and their neurotoxic activity, which provides a type of structure-toxicity relationship for these Aß40 oligomer species. These results provide insight into the possible role of metal ions in Alzheimer's disease by the stabilization of Aß oligomers.

Sequence-based prediction of the intrinsic solubility of peptides containing non-natural amino acids.

Non-natural amino acids are increasingly used as building blocks in the development of peptide-based drugs as they expand the available chemical space to tailor function, half-life and other key properties. However, while the chemical space of modified amino acids (mAAs) such as residues containing post-translational modifications (PTMs) is potentially vast, experimental methods for measuring the developability properties of mAA-containing peptides are expensive and time consuming. To facilitate developability programs through computational methods, we present CamSol-PTM, a method that enables the fast and reliable sequence-based prediction of the intrinsic solubility of mAA-containing peptides in aqueous solution at room temperature. From a computational screening of 50,000 mAA-containing variants of three peptides, we selected five different small-size mAAs for a total number of 37 peptide variants for experimental validation. We demonstrate the accuracy of the predictions by comparing the calculated and experimental solubility values. Our results indicate that the computational screening of mAA-containing peptides can extend by over four orders of magnitude the ability to explore the solubility chemical space of peptides and confirm that our method can accurately assess the solubility of peptides containing mAAs. This method is available as a web server at https://www-cohsoftware.ch.cam.ac.uk/index.php/camsolptm .

Determination of the Structure and Dynamics of the Fuzzy Coat of an Amyloid Fibril of IAPP Using Cryo-Electron Microscopy.

In recent years, major advances in cryo-electron microscopy (cryo-EM) have enabled the routine determination of complex biomolecular structures at atomistic resolution. An open challenge for this approach, however, concerns large systems that exhibit continuous dynamics. To address this problem, we developed the metadynamic electron microscopy metainference (MEMMI) method, which incorporates metadynamics, an enhanced conformational sampling approach, into the metainference method of integrative structural biology. MEMMI enables the simultaneous determination of the structure and dynamics of large heterogeneous systems by combining cryo-EM density maps with prior information through molecular dynamics, while at the same time modeling the different sources of error. To illustrate the method, we apply it to elucidate the dynamics of an amyloid fibril of the islet amyloid polypeptide (IAPP). The resulting conformational ensemble provides an accurate description of the structural variability of the disordered region of the amyloid fibril, known as fuzzy coat. The conformational ensemble also reveals that in nearly half of the structural core of this amyloid fibril, the side chains exhibit liquid-like dynamics despite the presence of the highly ordered network backbone of hydrogen bonds characteristic of the cross-ß structure of amyloid fibrils.

The amyloid-ß pathway in Alzheimer's disease: a plain language summary.

WHAT IS THIS SUMMARY ABOUT?: This plain language summary of an article published in Molecular Psychiatry, reviews the evidence supporting the role of the amyloid-ß (Aß) pathway and its dysregulation in Alzheimer's disease (AD), and highlights the rationale for drugs targeting the Aß pathway in the early stages of the disease. WHY IS THIS IMPORTANT?: Aß is a protein fragment (or peptide) that exists in several forms distinguished by their size, shape/structure, degree of solubility and disease relevance. The accumulation of Aß plaques is a hallmark of AD. However, smaller, soluble aggregates of Aß - including Aß protofibrils - also play a role in the disease. Because Aß-related disease mechanisms are complex, the diagnosis, treatment and management of AD should be reflective of and guided by up-to-date scientific knowledge and research findings in this area. This article describes the Aß protein and its role in AD, summarizing the evidence showing that altered Aß clearance from the brain may lead to the imbalance, toxic buildup and misfolding of the protein - triggering a cascade of cellular, molecular and systematic events that ultimately lead to AD. WHAT ARE THE KEY TAKEAWAYS?: The physiological balance of brain Aß levels in the context of AD is complex. Despite many unanswered questions, mounting evidence indicates that Aß has a central role in driving AD progression. A better understanding of the Aß pathway biology will help identify the best therapeutic targets for AD and inform treatment approaches.

EGCG inactivates a pore-forming toxin by promoting its oligomerization and decreasing its solvent-exposed hydrophobicity.

Natural proteinaceous pore-forming agents can bind and permeabilize cell membranes, leading to ion dyshomeostasis and cell death. In the search for antidotes that can protect cells from peptide toxins, we discovered that the polyphenol epigallocatechin gallate (EGCG) interacts directly with melittin from honeybee venom, resulting in the elimination of its binding to the cell membrane and toxicity by markedly lowering the extent of its solvent-exposed hydrophobicity and promoting its oligomerization into larger species. These physicochemical parameters have also been shown to play a key role in the binding to cells of misfolded protein oligomers in a host of neurodegenerative diseases, where oligomer-membrane binding and associated toxicity have been shown to correlate negatively with oligomer size and positively with solvent-exposed hydrophobicity. For melittin, which is not an amyloid-forming protein and has a very distinct mechanism of toxicity compared to misfolded oligomers, we find that the size-hydrophobicity-toxicity relationship also rationalizes the pharmacological attenuation of melittin toxicity by EGCG. These results highlight the importance of the physicochemical properties of pore forming agents in mediating their interactions with cell membranes and suggest a possible therapeutic approach based on compounds with a similar mechanism of action as EGCG.

Characterization of full-length p53 aggregates and their kinetics of formation.

Mutations in the TP53 gene are common in cancer with the R248Q missense mutation conferring an increased propensity to aggregate. Previous p53 aggregation studies showed that, at micromolar concentrations, protein unfolding to produce aggregation-prone species is the rate-determining step. Here we show that, at physiological concentrations, aggregation kinetics of insect cell-derived full-length wild-type p53 and p53R248Q are determined by a nucleation-growth model, rather than formation of aggregation-prone monomeric species. Self-seeding, but not cross-seeding, increases aggregation rate, confirming the aggregation process as rate determining. p53R248Q displays enhanced aggregation propensity due to decreased solubility and increased aggregation rate, forming greater numbers of larger amorphous aggregates that disrupt lipid bilayers and invokes an inflammatory response. These results suggest that p53 aggregation can occur under physiological conditions, a rate enhanced by R248Q mutation, and that aggregates formed can cause membrane damage and inflammation that may influence tumorigenesis.

Misfolded protein oligomers induce an increase of intracellular Ca2+ causing an escalation of reactive oxidative species.

Alzheimer's disease is characterized by the accumulation in the brain of the amyloid ß (Aß) peptide in the form of senile plaques. According to the amyloid hypothesis, the aggregation process of Aß also generates smaller soluble misfolded oligomers that contribute to disease progression. One of the mechanisms of Aß oligomer cytotoxicity is the aberrant interaction of these species with the phospholipid bilayer of cell membranes, with a consequent increase in cytosolic Ca2+ levels, flowing from the extracellular space, and production of reactive oxygen species (ROS). Here we investigated the relationship between the increase in Ca2+ and ROS levels immediately after the exposure to misfolded protein oligomers, asking whether they are simultaneous or instead one precedes the other. Using Aß42-derived diffusible ligands (ADDLs) and type A HypF-N model oligomers (OAs), we followed the kinetics of ROS production and Ca2+ influx in human neuroblastoma SH-SY5Y cells and rat primary cortical neurons in a variety of conditions. In all cases we found a faster increase of intracellular Ca2+ than ROS levels, and a lag phase in the latter process. A Ca2+-deprived cell medium prevented the increase of intracellular Ca2+ ions and abolished ROS production. By contrast, treatment with antioxidant agents prevented ROS formation, did not prevent the initial Ca2+ flux, but allowed the cells to react to the initial calcium dyshomeostasis, restoring later the normal levels of the ions. These results reveal a mechanism in which the entry of Ca2+ causes the production of ROS in cells challenged by aberrant protein oligomers.

π-Clamp-Mediated Homo- and Heterodimerization of Single-Domain Antibodies via Site-Specific Homobifunctional Conjugation.

Post-translational protein-protein conjugation produces bioconjugates that are unavailable via genetic fusion approaches. A method for preparing protein-protein conjugates using π-clamp-mediated cysteine arylation with pentafluorophenyl sulfonamide functional groups is described. Two computationally designed antibodies targeting the SARS-CoV-2 receptor binding domain were produced (KD = 146, 581 nM) with a π-clamp sequence near the C-terminus and dimerized using this method to provide a 10-60-fold increase in binding (KD = 8-15 nM). When two solvent-exposed cysteine residues were present on the second protein domain, the π-clamp cysteine residue was selectively modified over an Asp-Cys-Glu cysteine residue, allowing for subsequent small-molecule conjugation. With this strategy, we build molecule-protein-protein conjugates with complete chemical control over the sites of modification.

Adsorption free energy predicts amyloid protein nucleation rates.

Primary nucleation is the fundamental event that initiates the conversion of proteins from their normal physiological forms into pathological amyloid aggregates associated with the onset and development of disorders including systemic amyloidosis, as well as the neurodegenerative conditions Alzheimer's and Parkinson's diseases. It has become apparent that the presence of surfaces can dramatically modulate nucleation. However, the underlying physicochemical parameters governing this process have been challenging to elucidate, with interfaces in some cases having been found to accelerate aggregation, while in others they can inhibit the kinetics of this process. Here we show through kinetic analysis that for three different fibril-forming proteins, interfaces affect the aggregation reaction mainly through modulating the primary nucleation step. Moreover, we show through direct measurements of the Gibbs free energy of adsorption, combined with theory and coarse-grained computer simulations, that overall nucleation rates are suppressed at high and at low surface interaction strengths but significantly enhanced at intermediate strengths, and we verify these regimes experimentally. Taken together, these results provide a quantitative description of the fundamental process which triggers amyloid formation and shed light on the key factors that control this process.

Identification of a Thyroid Hormone Derivative as a Pleiotropic Agent for the Treatment of Alzheimer's Disease.

The identification of effective pharmacological tools for Alzheimer's disease (AD) represents one of the main challenges for therapeutic discovery. Due to the variety of pathological processes associated with AD, a promising route for pharmacological intervention involves the development of new chemical entities that can restore cellular homeostasis. To investigate this strategy, we designed and synthetized SG2, a compound related to the thyroid hormone thyroxine, that shares a pleiotropic activity with its endogenous parent compound, including autophagic flux promotion, neuroprotection, and metabolic reprogramming. We demonstrate herein that SG2 acts in a pleiotropic manner to induce recovery in a C. elegans model of AD based on the overexpression of Aß42 and improves learning abilities in the 5XFAD mouse model of AD. Further, in vitro ADME-Tox profiling and toxicological studies in zebrafish confirmed the low toxicity of this compound, which represents a chemical starting point for AD drug development.

Common sequence motifs of nascent chains engage the ribosome surface and trigger factor.

In the cell, the conformations of nascent polypeptide chains during translation are modulated by both the ribosome and its associated molecular chaperone, trigger factor. The specific interactions that underlie these modulations, however, are still not known in detail. Here, we combine protein engineering, in-cell and in vitro NMR spectroscopy, and molecular dynamics simulations to explore how proteins interact with the ribosome during their biosynthesis before folding occurs. Our observations of α-synuclein nascent chains in living Escherichia coli cells reveal that ribosome surface interactions dictate the dynamics of emerging disordered polypeptides in the crowded cytosol. We show that specific basic and aromatic motifs drive such interactions and directly compete with trigger factor binding while biasing the direction of the nascent chain during its exit out of the tunnel. These results reveal a structural basis for the functional role of the ribosome as a scaffold with holdase characteristics and explain how handover of the nascent chain to specific auxiliary proteins occurs among a host of other factors in the cytosol.

Surface-Catalyzed Secondary Nucleation Dominates the Generation of Toxic IAPP Aggregates.

The aggregation of the human islet amyloid polypeptide (IAPP) is associated with diabetes type II. A quantitative understanding of this connection at the molecular level requires that the aggregation mechanism of IAPP is resolved in terms of the underlying microscopic steps. Here we have systematically studied recombinant IAPP, with amidated C-terminus in oxidised form with a disulphide bond between residues 3 and 7, using thioflavin T fluorescence to monitor the formation of amyloid fibrils as a function of time and IAPP concentration. We used global kinetic analyses to connect the macroscopic measurements of aggregation to the microscopic mechanisms, and show that the generation of new aggregates is dominated by the secondary nucleation of monomers on the fibril surface. We then exposed insulinoma cells to aliquots extracted from different time points of the aggregation process, finding the highest toxicity at the midpoint of the reaction, when the secondary nucleation rate reaches its maximum. These results identify IAPP oligomers as the most cytotoxic species generated during IAPP aggregation, and suggest that compounds that target secondary nucleation of IAPP could be most effective as therapeutic candidates for diabetes type II.

The cellular modifier MOAG-4/SERF drives amyloid formation through charge complementation.

While aggregation-prone proteins are known to accelerate aging and cause age-related diseases, the cellular mechanisms that drive their cytotoxicity remain unresolved. The orthologous proteins MOAG-4, SERF1A, and SERF2 have recently been identified as cellular modifiers of such proteotoxicity. Using a peptide array screening approach on human amyloidogenic proteins, we found that SERF2 interacted with protein segments enriched in negatively charged and hydrophobic, aromatic amino acids. The absence of such segments, or the neutralization of the positive charge in SERF2, prevented these interactions and abolished the amyloid-promoting activity of SERF2. In protein aggregation models in the nematode worm Caenorhabditis elegans, protein aggregation and toxicity were suppressed by mutating the endogenous locus of MOAG-4 to neutralize charge. Our data indicate that MOAG-4 and SERF2 drive protein aggregation and toxicity by interactions with negatively charged segments in aggregation-prone proteins. Such charge interactions might accelerate primary nucleation of amyloid by initiating structural changes and by decreasing colloidal stability. Our study points at charge interactions between cellular modifiers and amyloidogenic proteins as potential targets for interventions to reduce age-related protein toxicity.

Squalamine and Its Derivatives Modulate the Aggregation of Amyloid-ß and α-Synuclein and Suppress the Toxicity of Their Oligomers.

The aberrant aggregation of proteins is a key molecular event in the development and progression of a wide range of neurodegenerative disorders. We have shown previously that squalamine and trodusquemine, two natural products in the aminosterol class, can modulate the aggregation of the amyloid-ß peptide (Aß) and of α-synuclein (αS), which are associated with Alzheimer's and Parkinson's diseases. In this work, we expand our previous analyses to two squalamine derivatives, des-squalamine and α-squalamine, obtaining further insights into the mechanism by which aminosterols modulate Aß and αS aggregation. We then characterize the ability of these small molecules to alter the physicochemical properties of stabilized oligomeric species in vitro and to suppress the toxicity of these aggregates to varying degrees toward human neuroblastoma cells. We found that, despite the fact that these aminosterols exert opposing effects on Aß and αS aggregation under the conditions that we tested, the modifications that they induced to the toxicity of oligomers were similar. Our results indicate that the suppression of toxicity is mediated by the displacement of toxic oligomeric species from cellular membranes by the aminosterols. This study, thus, provides evidence that aminosterols could be rationally optimized in drug discovery programs to target oligomer toxicity in Alzheimer's and Parkinson's diseases.

Kinetic analysis reveals that independent nucleation events determine the progression of polyglutamine aggregation in C. elegans.

Protein aggregation is associated with a wide range of degenerative human diseases with devastating consequences, as exemplified by Alzheimer's, Parkinson's, and Huntington's diseases. In vitro kinetic studies have provided a mechanistic understanding of the aggregation process at the molecular level. However, it has so far remained largely unclear to what extent the biophysical principles of amyloid formation learned in vitro translate to the complex environment of living organisms. Here, we take advantage of the unique properties of a Caenorhabditis elegans model expressing a fluorescently tagged polyglutamine (polyQ) protein, which aggregates into discrete micrometer-sized inclusions that can be directly visualized in real time. We provide a quantitative analysis of protein aggregation in this system and show that the data are described by a molecular model where stochastic nucleation occurs independently in each cell, followed by rapid aggregate growth. Global fitting of the image-based aggregation kinetics reveals a nucleation rate corresponding to 0.01 h-1 per cell at 1 mM intracellular protein concentration, and shows that the intrinsic molecular stochasticity of nucleation accounts for a significant fraction of the observed animal-to-animal variation. Our results highlight how independent, stochastic nucleation events in individual cells control the overall progression of polyQ aggregation in a living animal. The key finding that the biophysical principles associated with protein aggregation in small volumes remain the governing factors, even in the complex environment of a living organism, will be critical for the interpretation of in vivo data from a wide range of protein aggregation diseases.

A dopamine metabolite stabilizes neurotoxic amyloid-ß oligomers.

Aberrant soluble oligomers formed by the amyloid-ß peptide (Aß) are major pathogenic agents in the onset and progression of Alzheimer's disease. A variety of biomolecules can influence the formation of these oligomers in the brain, although their mechanisms of action are still largely unknown. Here, we studied the effects on Aß aggregation of DOPAL, a reactive catecholaldehyde intermediate of dopamine metabolism. We found that DOPAL is able to stabilize Aß oligomeric species, including dimers and trimers, that exert toxic effects on human neuroblastoma cells, in particular increasing cytosolic calcium levels and promoting the generation of reactive oxygen species. These results reveal an interplay between Aß aggregation and key biochemical processes regulating cellular homeostasis in the brain.

Making biological membrane resistant to the toxicity of misfolded protein oligomers: a lesson from trodusquemine.

Trodusquemine is an aminosterol known to prevent the binding of misfolded protein oligomers to cell membranes and to reduce their toxicity in a wide range of neurodegenerative diseases. Its precise mechanism of action, however, remains unclear. To investigate this mechanism, we performed confocal microscopy, fluorescence resonance energy transfer (FRET) and nuclear magnetic resonance (NMR) measurements, which revealed a strong binding of trodusquemine to large unilamellar vesicles (LUVs) and neuroblastoma cell membranes. Then, by combining quartz crystal microbalance (QCM), fluorescence quenching and anisotropy, and molecular dynamics (MD) simulations, we found that trodusquemine localises within, and penetrates, the polar region of lipid bilayer. This binding behaviour causes a decrease of the negative charge of the bilayer, as observed through ζ potential measurements, an increment in the mechanical resistance of the bilayer, as revealed by measurements of the breakthrough force applied with AFM and ζ potential measurements at high temperature, and a rearrangement of the spatial distances between ganglioside and cholesterol molecules in the LUVs, as determined by FRET measurements. These physicochemical changes are all known to impair the interaction of misfolded oligomers with cell membranes, protecting them from their toxicity. Taken together, our results illustrate how the incorporation in cell membranes of sterol molecules modified by the addition of polyamine tails leads to the modulation of physicochemical properties of the cell membranes themselves, making them more resistant to protein aggregates associated with neurodegeneration. More generally, they suggest that therapeutic strategies can be developed to reinforce cell membranes against protein misfolded assemblies.

An intrinsically disordered motif regulates the interaction between the p47 adaptor and the p97 AAA+ ATPase.

VCP/p97, an enzyme critical to proteostasis, is regulated through interactions with protein adaptors targeting it to specific cellular tasks. One such adaptor, p47, forms a complex with p97 to direct lipid membrane remodeling. Here, we use NMR and other biophysical methods to study the structural dynamics of p47 and p47-p97 complexes. Disordered regions in p47 are shown to be critical in directing intra-p47 and p47-p97 interactions via a pair of previously unidentified linear motifs. One of these, an SHP domain, regulates p47 binding to p97 in a manner that depends on the nucleotide state of p97. NMR and electron cryomicroscopy data have been used as restraints in molecular dynamics trajectories to develop structural ensembles for p47-p97 complexes in adenosine diphosphate (ADP)- and adenosine triphosphate (ATP)-bound conformations, highlighting differences in interactions in the two states. Our study establishes the importance of intrinsically disordered regions in p47 for the formation of functional p47-p97 complexes.

Trodusquemine displaces protein misfolded oligomers from cell membranes and abrogates their cytotoxicity through a generic mechanism.

The onset and progression of numerous protein misfolding diseases are associated with the presence of oligomers formed during the aberrant aggregation of several different proteins, including amyloid-ß (Aß) in Alzheimer's disease and α-synuclein (αS) in Parkinson's disease. These small, soluble aggregates are currently major targets for drug discovery. In this study, we show that trodusquemine, a naturally-occurring aminosterol, markedly reduces the cytotoxicity of αS, Aß and HypF-N oligomers to human neuroblastoma cells by displacing the oligomers from cell membranes in the absence of any substantial morphological and structural changes to the oligomers. These results indicate that the reduced toxicity results from a mechanism that is common to oligomers from different proteins, shed light on the origin of the toxicity of the most deleterious species associated with protein aggregation and suggest that aminosterols have the therapeutically-relevant potential to protect cells from the oligomer-induced cytotoxicity associated with numerous protein misfolding diseases.