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.

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.].

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.

Cu2+ ions modulate the interaction between α-synuclein and lipid membranes.

α-synuclein protein aggregates are the major constituent of Lewy bodies, which is a main pathogenic hallmark of Parkinson's disease. Both lipid membranes and Cu2+ ions can bind to α-synuclein and modulate its aggregation propensity and toxicity. However, the synergistic effect of copper ions and lipid membranes on α-synuclein remains to be explored. Here, we investigate how Cu2+ and α-synuclein simultaneously influence the lipidic structure of lipidic cubic phase(LCP) matrix by using small-angle X-ray scattering. α-Syn proteins destabilize the cubic-Pn3m phase of LCP that can be further recovered after the addition of Cu2 ions even at a low stoichiometric ratio. By using circular dichroism and nuclear magnetic resonance, we also study how lipid membranes and Cu2+ ions impact the secondary structures of α-synuclein at an atomic level. Although the secondary structure of α-synuclein with lipid membranes is not significantly changed to a large extent in the presence of Cu2+ ions, lipid membranes promote the interaction between α-synuclein C-terminus and Cu2+ ions. The modulation of Cu2+ ions and lipid membranes on α-synuclein dynamics and structure may play an important role in the molecular pathogenesis of Parkinson's disease.

Multivariate effects of pH, salt, and Zn2+ ions on Aß40 fibrillation.

Amyloid-ß (Aß) peptide aggregation plays a central role in the progress of Alzheimer's disease (AD), of which Aß-deposited extracellular amyloid plaques are a major hallmark. The brain micro-environmental variation in AD patients, like local acidification, increased ionic strength, or changed metal ion levels, cooperatively modulates the aggregation of the Aß peptides. Here, we investigate the multivariate effects of varied pH, ionic strength and Zn2+ on Aß40 fibrillation kinetics. Our results reveal that Aß fibrillation kinetics are strongly affected by pH and ionic strength suggesting the importance of electrostatic interactions in regulating Aß40 fibrillation. More interestingly, the presence of Zn2+ ions can further alter or even reserve the role of pH and ionic strength on the amyloid fibril kinetics, suggesting the importance of amino acids like Histidine that can interact with Zn2+ ions. Both pH and ionic strength regulate the secondary nucleation processes, however regardless of pH and Zn2+ ions, ionic strength can also modulate the morphology of Aß40 aggregates. These multivariate effects in bulk solution provide insights into the correlation of pH-, ionic strength- or Zn2+ ions changes with amyloid deposits in AD brain and will deepen our understanding of the molecular pathology in the local brain microenvironment.

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.