Ravit Malishev

Toxicity inhibitors protect lipid membranes from disruption by Aβ42.

Although the precise molecular factors linking amyloid β-protein (Aβ) to Alzheimers disease (AD) have not been deciphered, interaction of Aβ with cellular membranes has an important role in the disease. However, most therapeutic strategies targeting Aβ have focused on interfering with Aβ self-assembly rather than with its membrane interactions. Here, we studied the impact of three toxicity inhibitors on membrane interactions of Aβ42, the longer form of Aβ, which is associated most strongly with AD. The inhibitors included the four-residue C-terminal fragment Aβ(39-42), the polyphenol (-)-epigallocatechin-3-gallate (EGCG), and the lysine-specific molecular tweezer, CLR01, all of which previously were shown to disrupt different steps in Aβ42 self-assembly. Biophysical experiments revealed that incubation of Aβ42 with each of the three modulators affected membrane interactions in a distinct manner. Interestingly, EGCG and CLR01 were found to have significant interaction with membranes themselves. However, membrane bilayer disruption was reduced when the compounds were preincubated with Aβ42, suggesting that binding of the assembly modulators to the peptide attenuated their membrane interactions. Importantly, our study reveals that even though the three tested compounds affect Aβ42 assembly differently, membrane interactions were significantly inhibited upon incubation of each compound with Aβ42, suggesting that preventing the interaction of Aβ42 with the membrane contributes substantially to inhibition of its toxicity by each compound. The data suggest that interference with membrane interactions is an important factor for Aβ42 toxicity inhibitors and should be taken into account in potential therapeutic strategies, in addition to disruption or remodeling of amyloid assembly.

Lipid-Bilayer Dynamics Probed by a Carbon Dot-Phospholipid Conjugate

Elucidating the dynamic properties of membranes is important for understanding fundamental cellular processes and for shedding light on the interactions of proteins, drugs, and viruses with the cell surface. Dynamic studies of lipid bilayers have been constrained, however, by the relatively small number of pertinent molecular probes and the limited physicochemical properties of the probes. We show that a lipid conjugate comprised of a fluorescent carbon dot (C-dot) covalently attached to a phospholipid constitutes a versatile and effective vehicle for studying bilayer dynamics. The C-dot-modified phospholipids readily incorporated within biomimetic membranes, including solid-supported bilayers and small and giant vesicles, and inserted into actual cellular membranes. We employed the C-dot-phospholipid probe to elucidate the effects of polymyxin-B (a cytolytic peptide), valproic acid (a lipophilic drug), and amyloid-β (a peptide associated with Alzheimers disease) upon bilayer fluidity and lipid dynamics through the application of various biophysical techniques.

Unilamellar Vesicles from Amphiphilic Graphene Quantum Dots

Graphene quantum dots (GQDs) have attracted considerable interest due to their unique physicochemical properties and various applications. For the first time it is shown that GQDs surface-functionalized with hydrocarbon chains (i.e., amphiphilic GQDs) self-assemble into unilamellar spherical vesicles in aqueous solution. The amphiphilic GQD vesicles exhibit multicolor luminescence that can be readily exploited for membrane studies by fluorescence spectroscopy and microscopy. The GQD vesicles were used for microscopic analysis of membrane interactions and disruption by the peptide beta-amyloid.

Bacoside-A, an anti-amyloid natural substance, inhibits membrane disruption by the amyloidogenic determinant of prion protein through accelerating fibril formation

Bacosides, class of compounds extracted from the Bacopa monniera plant, exhibit interesting therapeutic properties, particularly enhancing cognitive functions and putative anti-amyloid activity. We show that bacoside-A exerted significant effects upon fibrillation and membrane interactions of the amyloidogenic fragment of the prion protein [PrP(106-126)]. Specifically, when co-incubated with PrP(106-126), bacoside-A accelerated fibril formation in the presence of lipid bilayers and in parallel inhibited bilayer interactions of the peptide aggregates formed in solution. These interesting phenomena were studied by spectroscopic and microscopic techniques, which suggest that bacoside A-promoted fibrillation reduced the concentration of membrane-active pre-fibrillar species of the prion fragment. This study suggests that induction of fibril formation and corresponding inhibition of membrane interactions are likely the underlying factors for ameliorating amyloid protein toxicity by bacoside-A.

Carbon and Nitrogen Based Nanosheets as Fluorescent Probes with Tunable Emission

2D carbon and nitrogen based semiconductors (CN) have attracted widespread attention for their possible use as low-cost and environmentally friendly materials for various applications. However, their limited solution-dispersibility and the difficulty in preparing exfoliated sheets with tunable photophysical properties restrain their exploitation in imaging-related applications. Here, the synthesis of carbon and nitrogen organic scaffolds with highly tunable optical properties, excellent dispersion in water and DMSO, and good bioimaging properties is reported. Tailored photophysical and chemical properties are acquired by the synthesis of new starting monomers containing different substituent chemical groups with varying electronic properties. Upon monomer condensation at moderate temperature, 350 °C, the starting chemical groups are fully preserved in the final CN. The low condensation temperature and the effective molecular-level modification of the CN scaffold lead to well-dispersed photoluminescent CN thin sheets with a wide range of emission wavelengths. The good bioimaging properties and the tunable fluorescence properties are exemplified by in situ visualization of giant unilamellar vesicles in a buffered aqueous solution as a model system. This approach opens the possibility for the design of tailor-made CN materials with tunable photophysical and chemical properties toward their exploitation in various fields, such as photocatalysis, bioimaging, and sensing.

Reciprocal Interactions between Membrane Bilayers and S. aureus PSMα3 Cross-α Amyloid Fibrils Account for Species-Specific Cytotoxicity

Phenol-soluble modulin α3 (PSMα3) is a functional amyloid secreted by the pathogenic bacterium Staphylococcus aureus. This 22-residue peptide serves as a key virulence determinant, toxic to human cells via the formation of unique cross-α amyloid-like fibrils. We demonstrate that bilayer vesicles accelerated PSMα3 fibril formation, and the fibrils, in turn, inserted deeply into bilayers mimicking mammalian cell membranes, accounting for PSMα3 cellular toxicity. Importantly, a mere amphipathic helical conformation was not a sufficient determinant for membrane-activity of PSMα3, pointing to the functional role of cross-α fibrils. In contrast to deep insertion of PSMα3 into mammalian membrane bilayers, the peptide only interacted with the surface of bilayers mimicking bacterial membranes, which might be related to its lack of antibacterial activity. Together, our data provide mechanistic insight into species-specific toxicity of a key bacterial amyloid virulence factor via reciprocal interactions with membranes, and open new perspectives into amyloid-related cytotoxicity mediated by helical fibril structures.

Nanoparticles modulate membrane interactions of human Islet amyloid polypeptide (hIAPP)

The dramatic expansion of nanotechnology applications, particularly the advent of nanomaterials and nanoparticles (NPs) into the consumer economy, have led to heightened awareness of their potential health risks. This study examines the impact of several NPs upon membrane-induced aggregation and bilayer interactions of the human Islet amyloid polypeptide (hIAPP). We report that several NPs - polymeric NPs, TiO2 NPs, and Au NPs displaying coating layers exhibiting different electrostatic charges - did not significantly interfere with the fibrillation process and fibril morphology of hIAPP, both in buffer or in biomimetic DMPC:DMPG vesicle solutions. Spectroscopic and microscopic analyses suggest, in fact, that the NPs promoted membrane-induced fibrillation. Importantly, we find that all the NPs examined, regardless of composition or surface properties, gave rise to more pronounced, synergistic bilayer interactions when co-incubated with hIAPP. NP-enhanced bilayer interactions of hIAPP might point to possible toxicity and pathogenicity risks of amyloidogenic peptides in the presence of NPs.

Bacoside-A, an Indian Traditional-Medicine Substance, Inhibits β-Amyloid Cytotoxicity, Fibrillation, and Membrane Interactions

Bacoside-A, a family of compounds extracted from the Bacopa monniera plant, is a folk-medicinal substance believed to exhibit therapeutic properties, particularly enhancing cognitive functions and improving memory. We show that bacoside-A exerted significant inhibitory effects upon cytotoxicity, fibrillation, and particularly membrane interactions of amyloid-beta (1-42) (Aβ42), the peptide playing a prominent role in Alzeheimers disease progression and toxicity. Specifically, preincubation of bacoside-A with Aβ42 significantly reduced cell toxicity and inhibited fibril formation both in buffer solution and, more significantly, in the presence of membrane vesicles. In parallel, spectroscopic and microscopic analyses reveal that bacoside-A blocked membrane interactions of Aβ42, while formation of Aβ42 oligomers was not disrupted. These interesting phenomena suggest that inhibition of Aβ42 oligomer assembly into mature fibrils, and blocking membrane interactions of the oligomers are likely the underlying factors for ameliorating amyloid toxicity by bacoside-A and its putative physiological benefits.

Vesicle-Based Assays to Study Membrane Interactions of Amyloid Peptides

The growing interest in membrane interactions of amyloidogenic peptides and proteins emanates from the realization that lipid bilayers and membranes play central roles in the toxicity and pathological pathways of amyloid diseases. This chapter presents experimental schemes designed to study membrane interactions and membrane-induced fibrillation of amyloid peptides.

Bacterial Model Membranes Reshape Fibrillation of a Functional Amyloid Protein

Biofilms are aggregates of cells that form surface-associated communities. The cells in biofilms are interconnected with an extracellular matrix, a network that is made mostly of polysaccharides, proteins, and sometimes nucleic acids. Some extracellular matrix proteins form fibers, termed functional amyloid or amyloid-like, to differentiate their constructive function from disease-related amyloid fibers. Recent functional amyloid assembly studies have neglected their interaction with membranes, despite their native formation in a cellular environment. Here, we use TasA, a major matrix protein in biofilms of the soil bacterium Bacillus subtilis, as a model functional amyloid protein and ask whether the bacterial functional amyloid interacts with membranes. Using biochemical, spectroscopic, and microscopic tools, we show that TasA interacts distinctively with bacterial model membranes and that this interaction mutually influences the morphology and structure of the protein and the membranes. At the protein level, fibers of similar structure and morphology are formed in the absence of membranes and in the presence of eukaryotic model membranes. However, in the presence of bacterial model membranes, TasA forms disordered aggregates with a different β sheet signature. At the membrane level, fluorescence microscopy and anisotropy measurements indicate that bacterial membranes deform more considerably than eukaryotic membranes upon interaction with TasA. Our findings suggest that TasA penetrates bacterial more than eukaryotic model membranes and that this leads to membrane disruption and to reshaping the TasA fiber formation pathway. Considering the important role of TasA in providing integrity to biofilms, our study may direct the design of antibiofilm drugs to the protein-membrane interface.