The C-terminal domain of the antiamyloid chaperone DNAJB6 binds to amyloid-ß peptide fibrils and inhibits secondary nucleation.

The DNAJB6 chaperone inhibits fibril formation of aggregation-prone client peptides through interaction with aggregated and oligomeric forms of the amyloid peptides. Here, we studied the role of its C-terminal domain (CTD) using constructs comprising either the entire CTD or the first two or all four of the CTD ß-strands grafted onto a scaffold protein. Each construct was expressed as WT and as a variant with alanines replacing five highly conserved and functionally important serine and threonine residues in the first ß-strand. We investigated the stability, oligomerization, antiamyloid activity, and affinity for amyloid-ß (Aß42) species using optical spectroscopy, native mass spectrometry, chemical crosslinking, and surface plasmon resonance technology. While DNAJB6 forms large and polydisperse oligomers, CTD was found to form only monomers, dimers, and tetramers of low affinity. Kinetic analyses showed a shift in inhibition mechanism. Whereas full-length DNAJB6 activity is dependent on the serine and threonine residues and efficiently inhibits primary and secondary nucleation, all CTD constructs inhibit secondary nucleation only, independently of the serine and threonine residues, although their dimerization and thermal stabilities are reduced by alanine substitution. While the full-length DNAJB6 inhibition of primary nucleation is related to its propensity to form coaggregates with Aß, the CTD constructs instead bind to Aß42 fibrils, which affects the nucleation events at the fibril surface. The retardation of secondary nucleation by DNAJB6 can thus be ascribed to the first two ß-strands of its CTD, whereas the inhibition of primary nucleation is dependent on the entire protein or regions outside the CTD.

13C- and 15N-labeling of amyloid-ß and inhibitory peptides to study their interaction via nanoscale infrared spectroscopy.

Interactions between molecules are fundamental in biology. They occur also between amyloidogenic peptides or proteins that are associated with different amyloid diseases, which makes it important to study the mutual influence of two polypeptides on each other's properties in mixed samples. However, addressing this research question with imaging techniques faces the challenge to distinguish different polypeptides without adding artificial probes for detection. Here, we show that nanoscale infrared spectroscopy in combination with 13C, 15N-labeling solves this problem. We studied aggregated amyloid-ß peptide (Aß) and its interaction with an inhibitory peptide (NCAM1-PrP) using scattering-type scanning near-field optical microscopy. Although having similar secondary structure, labeled and unlabeled peptides could be distinguished by comparing optical phase images taken at wavenumbers characteristic for either the labeled or the unlabeled peptide. NCAM1-PrP seems to be able to associate with or to dissolve existing Aß fibrils because pure Aß fibrils were not detected after mixing.

A Hairpin Motif in the Amyloid-ß Peptide Is Important for Formation of Disease-Related Oligomers.

The amyloid-ß (Aß) peptide is associated with the development of Alzheimer's disease and is known to form highly neurotoxic prefibrillar oligomeric aggregates, which are difficult to study due to their transient, low-abundance, and heterogeneous nature. To obtain high-resolution information about oligomer structure and dynamics as well as relative populations of assembly states, we here employ a combination of native ion mobility mass spectrometry and molecular dynamics simulations. We find that the formation of Aß oligomers is dependent on the presence of a specific ß-hairpin motif in the peptide sequence. Oligomers initially grow spherically but start to form extended linear aggregates at oligomeric states larger than those of the tetramer. The population of the extended oligomers could be notably increased by introducing an intramolecular disulfide bond, which prearranges the peptide in the hairpin conformation, thereby promoting oligomeric structures but preventing conversion into mature fibrils. Conversely, truncating one of the ß-strand-forming segments of Aß decreased the hairpin propensity of the peptide and thus decreased the oligomer population, removed the formation of extended oligomers entirely, and decreased the aggregation propensity of the peptide. We thus propose that the observed extended oligomer state is related to the formation of an antiparallel sheet state, which then nucleates into the amyloid state. These studies provide increased mechanistic understanding of the earliest steps in Aß aggregation and suggest that inhibition of Aß folding into the hairpin conformation could be a viable strategy for reducing the amount of toxic oligomers.

Characterization of Uranyl (UO22+) Ion Binding to Amyloid Beta (Aß) Peptides: Effects on Aß Structure and Aggregation.

Uranium (U) is naturally present in ambient air, water, and soil, and depleted uranium (DU) is released into the environment via industrial and military activities. While the radiological damage from U is rather well understood, less is known about the chemical damage mechanisms, which dominate in DU. Heavy metal exposure is associated with numerous health conditions, including Alzheimer's disease (AD), the most prevalent age-related cause of dementia. The pathological hallmark of AD is the deposition of amyloid plaques, consisting mainly of amyloid-ß (Aß) peptides aggregated into amyloid fibrils in the brain. However, the toxic species in AD are likely oligomeric Aß aggregates. Exposure to heavy metals such as Cd, Hg, Mn, and Pb is known to increase Aß production, and these metals bind to Aß peptides and modulate their aggregation. The possible effects of U in AD pathology have been sparsely studied. Here, we use biophysical techniques to study in vitro interactions between Aß peptides and uranyl ions, UO22+, of DU. We show for the first time that uranyl ions bind to Aß peptides with affinities in the micromolar range, induce structural changes in Aß monomers and oligomers, and inhibit Aß fibrillization. This suggests a possible link between AD and U exposure, which could be further explored by cell, animal, and epidemiological studies. General toxic mechanisms of uranyl ions could be modulation of protein folding, misfolding, and aggregation.

Prion Protein Octarepeat Domain Forms Transient ß-Sheet Structures upon Residue-Specific Binding to Cu(II) and Zn(II) Ions.

Misfolding of the cellular prion protein (PrPC) is associated with the development of fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSEs). Metal ions appear to play a crucial role in PrPC misfolding. PrPC is a combined Cu(II) and Zn(II) metal-binding protein, where the main metal-binding site is located in the octarepeat (OR) region. Thus, the biological function of PrPC may involve the transport of divalent metal ions across membranes or buffering concentrations of divalent metal ions in the synaptic cleft. Recent studies have shown that an excess of Cu(II) ions can result in PrPC instability, oligomerization, and/or neuroinflammation. Here, we have used biophysical methods to characterize Cu(II) and Zn(II) binding to the isolated OR region of PrPC. Circular dichroism (CD) spectroscopy data suggest that the OR domain binds up to four Cu(II) ions or two Zn(II) ions. Binding of the first metal ion results in a structural transition from the polyproline II helix to the ß-turn structure, while the binding of additional metal ions induces the formation of ß-sheet structures. Fluorescence spectroscopy data indicate that the OR region can bind both Cu(II) and Zn(II) ions at neutral pH, but under acidic conditions, it binds only Cu(II) ions. Molecular dynamics simulations suggest that binding of either metal ion to the OR region results in the formation of ß-hairpin structures. As the formation of ß-sheet structures can be a first step toward amyloid formation, we propose that high concentrations of either Cu(II) or Zn(II) ions may have a pro-amyloid effect in TSE diseases.

Erratum: The amyloid-inhibiting NCAM-PrP peptide targets Aß peptide aggregation in membrane-mimetic environments.

[This corrects the article DOI: 10.1016/j.isci.2021.102852.].

Amino acid substitutions in human growth hormone affect secondary structure and receptor binding.

The interaction between human Growth Hormone (hGH) and hGH Receptor (hGHR) has basic relevance to cancer and growth disorders, and hGH is the scaffold for Pegvisomant, an anti-acromegaly therapeutic. For the latter reason, hGH has been extensively engineered by early workers to improve binding and other properties. We are particularly interested in E174 which belongs to the hGH zinc-binding triad; the substitution E174A is known to significantly increase binding, but to now no explanation has been offered. We generated this and several computationally-selected single-residue substitutions at the hGHR-binding site of hGH. We find that, while many successfully slow down dissociation of the hGH-hGHR complex once bound, they also slow down the association of hGH to hGHR. The E174A substitution induces a change in the Circular Dichroism spectrum that suggests the appearance of coiled-coiling. Here we show that E174A increases affinity of hGH against hGHR because the off-rate is slowed down more than the on-rate. For E174Y (and certain mutations at other sites) the slowdown in on-rate was greater than that of the off-rate, leading to decreased affinity. The results point to a link between structure, zinc binding, and hGHR-binding affinity in hGH.

Choosing an Optimal Solvent Is Crucial for Obtaining Cell-Penetrating Peptide Nanoparticles with Desired Properties and High Activity in Nucleic Acid Delivery.

Cell-penetrating peptides (CPPs) are highly promising transfection agents that can deliver various compounds into living cells, including nucleic acids (NAs). Positively charged CPPs can form non-covalent complexes with negatively charged NAs, enabling simple and time-efficient nanoparticle preparation. However, as CPPs have substantially different chemical and physical properties, their complexation with the cargo and characteristics of the resulting nanoparticles largely depends on the properties of the surrounding environment, i.e., solution. Here, we show that the solvent used for the initial dissolving of a CPP determines the properties of the resulting CPP particles formed in an aqueous solution, including the activity and toxicity of the CPP-NA complexes. Using different biophysical methods such as dynamic light scattering (DLS), atomic force microscopy (AFM), transmission and scanning electron microscopy (TEM and SEM), we show that PepFect14 (PF14), a cationic amphipathic CPP, forms spherical particles of uniform size when dissolved in organic solvents, such as ethanol and DMSO. Water-dissolved PF14, however, tends to form micelles and non-uniform aggregates. When dissolved in organic solvents, PF14 retains its α-helical conformation and biological activity in cell culture conditions without any increase in cytotoxicity. Altogether, our results indicate that by using a solvent that matches the chemical nature of the CPP, the properties of the peptide-cargo particles can be tuned in the desired way. This can be of critical importance for in vivo applications, where CPP particles that are too large, non-uniform, or prone to aggregation may induce severe consequences.

Residue-specific binding of Ni(II) ions influences the structure and aggregation of amyloid beta (Aß) peptides.

Alzheimer's disease (AD) is the most common cause of dementia worldwide. AD brains display deposits of insoluble amyloid plaques consisting mainly of aggregated amyloid-ß (Aß) peptides, and Aß oligomers are likely a toxic species in AD pathology. AD patients display altered metal homeostasis, and AD plaques show elevated concentrations of metals such as Cu, Fe, and Zn. Yet, the metal chemistry in AD pathology remains unclear. Ni(II) ions are known to interact with Aß peptides, but the nature and effects of such interactions are unknown. Here, we use numerous biophysical methods-mainly spectroscopy and imaging techniques-to characterize Aß/Ni(II) interactions in vitro, for different Aß variants: Aß(1-40), Aß(1-40)(H6A, H13A, H14A), Aß(4-40), and Aß(1-42). We show for the first time that Ni(II) ions display specific binding to the N-terminal segment of full-length Aß monomers. Equimolar amounts of Ni(II) ions retard Aß aggregation and direct it towards non-structured aggregates. The His6, His13, and His14 residues are implicated as binding ligands, and the Ni(II)·Aß binding affinity is in the low µM range. The redox-active Ni(II) ions induce formation of dityrosine cross-links via redox chemistry, thereby creating covalent Aß dimers. In aqueous buffer Ni(II) ions promote formation of beta sheet structure in Aß monomers, while in a membrane-mimicking environment (SDS micelles) coil-coil helix interactions appear to be induced. For SDS-stabilized Aß oligomers, Ni(II) ions direct the oligomers towards larger sizes and more diverse (heterogeneous) populations. All of these structural rearrangements may be relevant for the Aß aggregation processes that are involved in AD brain pathology.

Big dynorphin is a neuroprotector scaffold against amyloid ß-peptide aggregation and cell toxicity.

Amyloid ß-peptide (Aß) misfolding into ß-sheet structures triggers neurotoxicity inducing Alzheimer's disease (AD). Molecules able to reduce or to impair Aß aggregation are highly relevant as possible AD treatments since they should protect against Aß neurotoxicity. We have studied the effects of the interaction of dynorphins, a family of opioid neuropeptides, with Aß40 the most abundant species of Aß. Biophysical measurements indicate that Aß40 interacts with Big Dynorphin (BigDyn), lowering the amount of hydrophobic aggregates, and slowing down the aggregation kinetics. As expected, we found that BigDyn protects against Aß40 aggregates when studied in human neuroblastoma cells by cell survival assays. The cross-interaction between BigDyn and Aß40 provides insight into the mechanism of amyloid pathophysiology and may open up new therapy possibilities.

Mass Spectrometry and Machine Learning Reveal Determinants of Client Recognition by Antiamyloid Chaperones.

The assembly of proteins and peptides into amyloid fibrils is causally linked to serious disorders such as Alzheimer's disease. Multiple proteins have been shown to prevent amyloid formation in vitro and in vivo, ranging from highly specific chaperone-client pairs to completely nonspecific binding of aggregation-prone peptides. The underlying interactions remain elusive. Here, we turn to the machine learning-based structure prediction algorithm AlphaFold2 to obtain models for the nonspecific interactions of ß-lactoglobulin, transthyretin, or thioredoxin 80 with the model amyloid peptide amyloid ß and the highly specific complex between the BRICHOS chaperone domain of C-terminal region of lung surfactant protein C and its polyvaline target. Using a combination of native mass spectrometry (MS) and ion mobility MS, we show that nonspecific chaperoning is driven predominantly by hydrophobic interactions of amyloid ß with hydrophobic surfaces in ß-lactoglobulin, transthyretin, and thioredoxin 80, and in part regulated by oligomer stability. For C-terminal region of lung surfactant protein C, native MS and hydrogen-deuterium exchange MS reveal that a disordered region recognizes the polyvaline target by forming a complementary ß-strand. Hence, we show that AlphaFold2 and MS can yield atomistic models of hard-to-capture protein interactions that reveal different chaperoning mechanisms based on separate ligand properties and may provide possible clues for specific therapeutic intervention.

Mercury Ion Binding to Apolipoprotein E Variants ApoE2, ApoE3, and ApoE4: Similar Binding Affinities but Different Structure Induction Effects.

Mercury intoxication typically produces more severe outcomes in people with the APOE-ε4 gene, which codes for the ApoE4 variant of apolipoprotein E, compared to individuals with the APOE-ε2 and APOE-ε3 genes. Why the APOE-ε4 allele is a risk factor in mercury exposure remains unknown. One proposed possibility is that the ApoE protein could be involved in clearing of heavy metals, where the ApoE4 protein might perform this task worse than the ApoE2 and ApoE3 variants. Here, we used fluorescence and circular dichroism spectroscopies to characterize the in vitro interactions of the three different ApoE variants with Hg(I) and Hg(II) ions. Hg(I) ions displayed weak binding to all ApoE variants and induced virtually no structural changes. Thus, Hg(I) ions appear to have no biologically relevant interactions with the ApoE protein. Hg(II) ions displayed stronger and very similar binding affinities for all three ApoE isoforms, with K D values of 4.6 µM for ApoE2, 4.9 µM for ApoE3, and 4.3 µM for ApoE4. Binding of Hg(II) ions also induced changes in ApoE superhelicity, that is, altered coil-coil interactions, which might modify the protein function. As these structural changes were most pronounced in the ApoE4 protein, they could be related to the APOE-ε4 gene being a risk factor in mercury toxicity.

Cell-Penetrating Peptides with Unexpected Anti-Amyloid Properties.

Cell-penetrating peptides (CPPs) with sequences derived originally from a prion protein (PrP) have been shown to exhibit both anti-prion and anti-amyloid properties particularly against prion proteins and the amyloid-ß (Aß) peptide active in Alzheimer's disease. These disease-modifying properties are so far observed in cell cultures and in vitro. The CPP sequences are composed of a hydrophobic signal sequence followed by a highly positively charged hexapeptide segment. The original signal sequence of the prion protein can be changed to the signal sequence of the NCAM1 protein without losing the anti-prion activity. Although the detailed molecular mechanisms of these CPP peptides are not fully understood, they do form amyloid aggregates by themselves, and molecular interactions between the CPPs and PrP/Aß can be observed in vitro using various spectroscopic techniques. These initial intermolecular interactions appear to re-direct the aggregation pathways for prion/amyloid formation to less cell-toxic molecular structures (i.e., co-aggregates), which likely is why the disease-inducing PrP/Aß aggregation is counteracted in vivo.

The engineered peptide construct NCAM1-Aß inhibits fibrillization of the human prion protein (PrP).

In prion diseases, the prion protein (PrP) becomes misfolded and forms fibrillar aggregates that are responsible for prion infectivity and pathology. So far, no drug or treatment procedures have been approved for prion disease treatment. We have previously shown that engineered cell-penetrating peptide constructs can reduce the amount of prion aggregates in infected cells. However, the molecular mechanism underlying this effect is unknown. Here, we use atomic force microscopy (AFM) imaging to show that the amyloid aggregation and fibrillization of the human PrP protein can be inhibited by equimolar amounts of the 25 residues long engineered peptide construct NCAM1-Aß.

Membrane Molecular Interactions and Induced Structures of CPPs.

Cell penetrating peptides (CPPs) are generally defined as short positively charged peptides, containing 5-30 amino acids. Based on their physicochemical properties, they are classified as three main groups, namely hydrophobic, amphipathic, and hydrophilic. They are capable of interacting with the cell membrane without inducing serious toxicity, and they can carry cargo molecules across the membrane. Cargo molecules could be different therapeutics which makes CPPs valuable in the field of drug delivery into living cells. Nowadays, CPPs are considered as potential parts of therapeutics against several diseases.Despite similarities in their primary structure, the interactions of CPPs with a cell membrane may vary a lot. This is even more complicated when the CPP is bound to the cargo molecule. The mechanism(s) of their cellular uptake and endosomal escape have not been completely resolved. Understanding the mechanism of membrane interaction will help us designing a CPP with enhanced, selective cargo delivery, hopefully resulting in better disease treatments. So far energy independent direct membrane penetration and energy-dependent endocytosis have been suggested as two main mechanisms of cellular entry for CPPs, and both may be applicable for the same CPP-complex, depending on the conditions.In order to understand which mechanism is associated with a particular CPP 's cellular uptake in a particular cell (sometimes including endosomal escape), different biological and biophysical methods and strategies have been applied. In this chapter, we will address several biophysical methods, such as fluorescence spectroscopy, circular dichroism (CD) spectroscopy, dynamic light scattering, and NMR .We also review different membrane model systems which are suitable for the biophysical studies. These include large unilamellar phospholipid vesicles (LUVs ), which are the most commonly used in the lipid-peptide interaction studies. Detergent micelles and mixed micelles (bicelles) are also suitable membrane model systems, particularly in high-resolution NMR studies.

Zn(II) binding causes interdomain changes in the structure and flexibility of the human prion protein.

The cellular prion protein (PrPC) is a mainly α-helical 208-residue protein located in the pre- and postsynaptic membranes. For unknown reasons, PrPC can undergo a structural transition into a toxic, ß-sheet rich scrapie isoform (PrPSc) that is responsible for transmissible spongiform encephalopathies (TSEs). Metal ions seem to play an important role in the structural conversion. PrPC binds Zn(II) ions and may be involved in metal ion transport and zinc homeostasis. Here, we use multiple biophysical techniques including optical and NMR spectroscopy, molecular dynamics simulations, and small angle X-ray scattering to characterize interactions between human PrPC and Zn(II) ions. Binding of a single Zn(II) ion to the PrPC N-terminal domain via four His residues from the octarepeat region induces a structural transition in the C-terminal α-helices 2 and 3, promotes interaction between the N-terminal and C-terminal domains, reduces the folded protein size, and modifies the internal structural dynamics. As our results suggest that PrPC can bind Zn(II) under physiological conditions, these effects could be important for the physiological function of PrPC.

The amyloid-inhibiting NCAM-PrP peptide targets Aß peptide aggregation in membrane-mimetic environments.

Substantial research efforts have gone into elucidating the role of protein misfolding and self-assembly in the onset and progression of Alzheimer's disease (AD). Aggregation of the Amyloid-ß (Aß) peptide into insoluble fibrils is closely associated with AD. Here, we use biophysical techniques to study a peptide-based approach to target Aß amyloid aggregation. A peptide construct, NCAM-PrP, consists of a largely hydrophobic signal sequence linked to a positively charged hexapeptide. The NCAM-PrP peptide inhibits Aß amyloid formation by forming aggregates which are unavailable for further amyloid aggregation. In a membrane-mimetic environment, Aß and NCAM-PrP form specific heterooligomeric complexes, which are of lower aggregation states compared to Aß homooligomers. The Aß:NCAM-PrP interaction appears to take place on different aggregation states depending on the absence or presence of a membrane-mimicking environment. These insights can be useful for the development of potential future therapeutic strategies targeting Aß at several aggregation states.

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

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

Lithium ions display weak interaction with amyloid-beta (Aß) peptides and have minor effects on their aggregation.

Alzheimer's disease (AD) is an incurable disease and the main cause of age-related dementia worldwide, despite decades of research. Treatment of AD with lithium (Li) has showed promising results, but the underlying mechanism is unclear. The pathological hallmark of AD brains is deposition of amyloid plaques, consisting mainly of amyloid-ß (Aß) peptides aggregated into amyloid fibrils. The plaques contain also metal ions of e.g. Cu, Fe, and Zn, and such ions are known to interact with Aß peptides and modulate their aggregation and toxicity. The interactions between Aß peptides and Li+ ions have however not been well investigated. Here, we use a range of biophysical techniques to characterize in vitro interactions between Aß peptides and Li+ ions. We show that Li+ ions display weak and non-specific interactions with Aß peptides, and have minor effects on Aß aggregation. These results indicate that possible beneficial effects of Li on AD pathology are not likely caused by direct interactions between Aß peptides and Li+ ions.

Amyotrophic Lateral Sclerosis After Exposure to Manganese from Traditional Medicine Procedures in Kenya.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron loss and widespread muscular atrophy. Despite intensive investigations on genetic and environmental factors, the cause of ALS remains unknown. Recent data suggest a role for metal exposures in ALS causation. In this study we present a patient who developed ALS after a traditional medical procedure in Kenya. The procedure involved insertion of a black metal powder into several subcutaneous cuts in the lower back. Four months later, general muscle weakness developed. Clinical and electrophysiological examinations detected widespread denervation consistent with ALS. The patient died from respiratory failure less than a year after the procedure. Scanning electron microscopy and X-ray diffraction analyses identified the black powder as potassium permanganate (KMnO4). A causative relationship between the systemic exposure to KMnO4 and ALS development can be suspected, especially as manganese is a well-known neurotoxicant previously found to be elevated in cerebrospinal fluid from ALS patients. Manganese neurotoxicity and exposure routes conveying this toxicity deserve further attention.