Curcumin Reduces Amyloid Beta Oligomer Interactions with Anionic Membranes.

Many neurodegenerative diseases involve amyloidogenic proteins forming surface-bound aggregates on anionic membranes, and the peptide amyloid ß (Aß) in Alzheimer's disease is one prominent example of this. Curcumin is a small polyphenolic molecule that provides an interesting opportunity to understand the fundamental mechanisms of membrane-mediated aggregation because it embeds into membranes to alter their structure while also altering Aß aggregation in an aqueous environment. The purpose of this work was to understand interactions among curcumin, ß-sheet-rich Aß fibrillar oligomers (FO), and a model anionic membrane. From a combination of liquid surface X-ray scattering experiments and molecular dynamics simulations, we found that curcumin embedded into an anionic 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol (DMPG) membrane to rest between the lipid headgroups and the tails, causing disorder and membrane thinning. FO accumulation on the membrane was reduced by ∼66% in the presence of curcumin, likely influenced by membrane thinning. Simulation results suggested curcumin clusters near exposed phenylalanine residues on a membrane-embedded FO structure. Altogether, curcumin inhibited FO interactions with a DMPG membrane, likely through a combination of altered membrane structure and interactions with the FO surface. This work elucidates the mechanism of curcumin as a small molecule that inhibits amyloidogenesis through a combination of both membrane and protein interactions.

Structure of Galectin-3 bound to a model membrane containing ganglioside GM1.

Galectin-3 (Gal-3) is a ß-galactosidase-binding protein involved in various biological processes, including neuronal growth and adhesion. The pairing of Gal-3 with ganglioside GM1's pentasaccharide chain at the outer leaflet of the plasma membrane, which triggers downstream cell-signaling cascades, seems to be involved in these processes. A crucial feature of Gal-3 is its ability to form oligomers and supramolecular assemblies that connect various carbohydrate-decorated molecules. Although we know the atomistic structure of Gal-3 bound to small carbohydrate ligands, it remains unclear how Gal-3 binds GM1 in a membrane. Furthermore, the influence of this interaction on Gal-3's structure and oligomeric assembly has to be elucidated. In this study, we used X-ray reflectivity (XR) from a model membrane to determine the structure and surface coverage of Gal-3 bound to a membrane containing GM1. We observed that the carbohydrate recognition domain interacts with GM1's pentasaccharide, while the N-terminal domain is pointed away from the membrane, likely to facilitate protein-protein interactions. In a membrane containing 20 mol % GM1, Gal-3 covered ∼50% of the membrane surface with one Gal-3 molecule bound per 2130 Å2. We used molecular dynamics simulations and Voronoi tessellation algorithms to build an atomistic model of membrane-bound Gal-3, which is supported by the XR results. Overall, this work provides structural information describing how Gal-3 can bind GM1's pentasaccharide chain, a prerequisite for triggering regulatory processes in neuronal growth and adhesion.

Tau and Membranes: Interactions That Promote Folding and Condensation.

Tau misfolding and assembly is linked to a number of neurodegenerative diseases collectively described as tauopathies, including Alzheimer's disease (AD) and Parkinson's disease. Anionic cellular membranes, such as the cytosolic leaflet of the plasma membrane, are sites that concentrate and neutralize tau, primarily due to electrostatic interactions with tau's microtubule binding repeat domain (RD). In addition to electrostatic interactions with lipids, tau also has interactions with membrane proteins, which are important for tau's cellular functions. Tau also interacts with lipid tails to facilitate direct translocation across the membrane and can form stable protein-lipid complexes involved in cell-to-cell transport. Concentrated tau monomers at the membrane surface can form reversible condensates, change secondary structures, and induce oligomers, which may eventually undergo irreversible crosslinking and fibril formation. These ß-sheet rich tau structures are capable of disrupting membrane organization and are toxic in cell-based assays. Given the evidence for relevant membrane-based tau assembly, we review the emerging hypothesis that polyanionic membranes may serve as a site for phase-separated tau condensation. Membrane-mediated phase separation may have important implications for regulating tau folding/misfolding, and may be a powerful mechanism to spatially direct tau for native membrane-mediated functions.

Lipid membrane templated misfolding and self-assembly of intrinsically disordered tau protein.

The aggregation of the intrinsically disordered tau protein into highly ordered ß-sheet-rich fibrils is implicated in the pathogenesis of a range of neurodegenerative disorders. The mechanism of tau fibrillogenesis remains unresolved, particularly early events that trigger the misfolding and assembly of the otherwise soluble and stable tau. We investigated the role the lipid membrane plays in modulating the aggregation of three tau variants, the largest isoform hTau40, the truncated construct K18, and a hyperphosphorylation-mimicking mutant hTau40/3Epi. Despite being charged and soluble, the tau proteins were also highly surface active and favorably interacted with anionic lipid monolayers at the air/water interface. Membrane binding of tau also led to the formation of a macroscopic, gelatinous layer at the air/water interface, possibly related to tau phase separation. At the molecular level, tau assembled into oligomers composed of ~ 40 proteins misfolded in a ß-sheet conformation at the membrane surface, as detected by in situ synchrotron grazing-incidence X-ray diffraction. Concomitantly, membrane morphology and lipid packing became disrupted. Our findings support a general tau aggregation mechanism wherein tau's inherent surface activity and favorable interactions with anionic lipids drive tau-membrane association, inducing misfolding and self-assembly of the disordered tau into ß-sheet-rich oligomers that subsequently seed fibrillation and deposition into diseased tissues.

Structural adaptation of vertebrate endonuclease G for 5-hydroxymethylcytosine recognition and function.

Modified DNA bases functionally distinguish the taxonomic forms of life-5-methylcytosine separates prokaryotes from eukaryotes and 5-hydroxymethylcytosine (5hmC) invertebrates from vertebrates. We demonstrate here that mouse endonuclease G (mEndoG) shows specificity for both 5hmC and Holliday junctions. The enzyme has higher affinity (>50-fold) for junctions over duplex DNAs. A 5hmC-modification shifts the position of the cut site and increases the rate of DNA cleavage in modified versus unmodified junctions. The crystal structure of mEndoG shows that a cysteine (Cys69) is positioned to recognize 5hmC through a thiol-hydroxyl hydrogen bond. Although this Cys is conserved from worms to mammals, a two amino acid deletion in the vertebrate relative to the invertebrate sequence unwinds an α-helix, placing the thiol of Cys69 into the mEndoG active site. Mutations of Cys69 with alanine or serine show 5hmC-specificity that mirrors the hydrogen bonding potential of the side chain (C-H < S-H < O-H). A second orthogonal DNA binding site identified in the mEndoG structure accommodates a second arm of a junction. Thus, the specificity of mEndoG for 5hmC and junctions derives from structural adaptations that distinguish the vertebrate from the invertebrate enzyme, thereby thereby supporting a role for 5hmC in recombination processes.

Passive Immunotherapies Targeting Amyloid Beta and Tau Oligomers in Alzheimer's Disease.

Alzheimer's disease (AD) is historically difficult to treat, in part because of the inaccessible nature of brain pathology. Amyloid beta and tau proteins drive pathology by forming toxic oligomers that eventually deposit as insoluble amyloid plaques and neurofibrillary tangles. Recent clinical studies suggest that effective drugs must specifically target oligomers, not native monomers or insoluble fibrils. Passive immunotherapy is a promising pharmaceutical strategy used to specifically target these oligomers in situ. Using the specificity of antibodies coupled with the natural power of the body's immune response, this treatment provides an opportunity for safe clearance of pathogenic protein species from the brain. Passive immunotherapies against amyloid beta and tau oligomers have progressed to clinical trials, with many currently in progress. Biochemical studies of antibody-oligomer complexes have helped identify previously unknown toxic epitopes, thus providing knowledge to the AD field as a whole. This mini-review focuses on the efforts to develop passive immunotherapy treatments for AD and discusses the knowledge gained from recent failures and clinical trials in progress.

Fibrillar and Nonfibrillar Amyloid Beta Structures Drive Two Modes of Membrane-Mediated Toxicity.

In Alzheimer's disease, the amyloid-beta peptide (Aß) is implicated in neuronal toxicity via interactions with the cell membrane. Monomeric Aß (Aßm) is intrinsically disordered, but it can adopt a range of aggregated conformations with varying toxicities from short fibrillar oligomers (FO), to globular nonfibrillar oligomers (NFO), and full-length amyloid fibrils. NFO is considered to be the most toxic, followed by fibrils, and finally Aßm. To elucidate molecular-level membrane interactions that contribute to their different toxicities, we used liquid surface X-ray scattering and Langmuir trough insertion assays to compare Aßm, FO, and NFO surface activities and interactions with anionic DMPG lipid monolayers at the air/water interface. All Aß species were highly surface active and rapidly adopted ß-sheet rich structures upon adsorption to the air/water interface. Likewise, all Aß species had affinity for the anionic membrane. Aßm rapidly converted to ß-sheet rich assemblies upon binding the membrane, and these aggregated structures of Aßm and FO disrupted hexagonally packed lipid domains and resulted in membrane thinning and instability. In contrast, NFO perturbed membrane structure by extracting lipids from the air/water interface and causing macroscale membrane deformations. Altogether, our results support two models for membrane-mediated Aß toxicity: fibril-induced reorganization of lipid packing and NFO-induced membrane destabilization and lipid extraction. This work provides a structural understanding of Aß neurotoxicity via membrane interactions and aids the effort in understanding early events in Alzheimer's disease and other neurodegenerative diseases.

Membrane-mediated fibrillation and toxicity of the tau hexapeptide PHF6.

The aggregation of the tau protein into neurofibrillary tangles is believed to correlate with cognitive decline in several neurodegenerative disorders, including Alzheimer's disease. Recent studies suggest that tau's interactions with the cell membrane could serve as a toxicity pathway and also enhance fibrillation into paired helical filaments (PHFs). Conformational changes associated with tau-membrane interactions are poorly understood, and their characterization could improve our understanding of tau pathogenicity. In this study, we investigated the molecular level structural changes associated with the interaction of the tau hexapeptide PHF6 with model lipid membranes and characterized the effects of these interactions on membrane stability and peptide fibrillation. We used two PHF6 forms, the aggregation-prone PHF6 with N-terminal acetylation (Ac-PHF6) and the non-aggregation prone PHF6 with a standard N terminus (NH3+-PHF6). We found that both PHF6 peptides are neurotoxic and exhibit similar membrane-mediated changes, consisting of: 1) favorable interactions with anionic membranes, 2) membrane destabilization through lipid extraction, and 3) membrane-mediated fibrillation. The rate at which these changes occurred was the main difference between the two peptides. NH3+-PHF6 displayed slow membrane-mediated fibrillation after 6 days of incubation, whereas Ac-PHF6 adopted a ß-sheet conformation at the surface of the membrane within hours. Ac-PHF6 interactions with the membrane were also accompanied by membrane invagination and rapid membrane destabilization. Overall, our results reveal that membrane interactions could play a critical role in tau toxicity and fibrillation, and highlight that unraveling these interactions is important for significantly advancing the development of therapeutic strategies to manage tau-associated neurodegenerative diseases.

Effect of Hydroxymethylcytosine on the Structure and Stability of Holliday Junctions.

5-Hydroxymethylcytosine (5hmC) is an epigenetic marker that has recently been shown to promote homologous recombination (HR). In this study, we determine the effects of 5hmC on the structure, thermodynamics, and conformational dynamics of the Holliday junction (the four-stranded DNA intermediate associated with HR) in its native stacked-X form. The hydroxymethyl and the control methyl substituents are placed in the context of an amphimorphic GxCC trinucleotide core sequence (where xC is C, 5hmC, or the methylated 5mC), which is part of a sequence also recognized by endonuclease G to promote HR. The hydroxymethyl group of the 5hmC junction adopts two distinct rotational conformations, with an in-base-plane form being dominant over the competing out-of-plane rotamer that has typically been seen in duplex structures. The in-plane rotamer is seen to be stabilized by a more stable intramolecular hydrogen bond to the junction backbone. Stabilizing hydrogen bonds (H-bonds) formed by the hydroxyl substituent in 5hmC or from a bridging water in the 5mC structure provide approximately 1.5-2 kcal/mol per interaction of stability to the junction, which is mostly offset by entropy compensation, thereby leaving the overall stability of the G5hmCC and G5mCC constructs similar to that of the GCC core. Thus, both methyl and hydroxymethyl modifications are accommodated without disrupting the structure or stability of the Holliday junction. Both 5hmC and 5mC are shown to open the structure to make the junction core more accessible. The overall consequences of incorporating 5hmC into a DNA junction are thus discussed in the context of the specificity in protein recognition of the hydroxymethyl substituent through direct and indirect readout mechanisms.

Force Field Model of Periodic Trends in Biomolecular Halogen Bonds.

The study of the noncovalent interaction now defined as a halogen bond (X-bond) has become one of the fastest growing areas in experimental and theoretical chemistry--its applications as a design tool are highly extensive. The significance of the interaction in biology has only recently been recognized, but has now become important in medicinal chemistry. We had previously derived a set of empirical potential energy functions to model the structure-energy relationships for bromines in biomolecular X-bonds (BXBs). Here, we have extended this force field for BXBs (ffBXB) to the halogens (Cl, Br, and I) that are commonly seen to form stable X-bonds. The ffBXB calculated energies show a remarkable one-to-one linear relationship to explicit BXB energies determined from an experimental DNA junction system, thereby validating the approach and the model. The resulting parameters allow us to interpret the stabilizing effects of BXBs in terms of well-defined physical properties of the halogen atoms, including their size, shape, and charge, showing periodic trends that are predictable along the Group VII column of elements. Consequently, we have established the ffBXB as an accurate computational tool that can be applied, for example, for the design of new therapeutic compounds against clinically important targets and new biomolecular-based materials.

Determining thermodynamic properties of molecular interactions from single crystal studies.

The concept of single crystals of macromolecules as thermodynamic systems is not a common one. However, it should be possible to derive thermodynamic properties from single crystal structures, if the process of crystallization follows thermodynamic rules. We review here an example of how the stabilizing potentials of molecular interactions can be measured from studying the properties of DNA crystals. In this example, we describe an assay based on the four-stranded DNA junction to determine the stabilizing potentials of halogen bonds, a class of electrostatic interactions, analogous to hydrogen bonds, that are becoming increasing recognized as important for conferring specificity in protein-ligand complexes. The system demonstrates how crystallographic studies, when coupled with calorimetric methods, allow the geometries at the atomic level to be directly correlated with the stabilizing energies of molecular interactions. The approach can be generally applied to study the effects of DNA sequence and modifications of the thermodynamic stability of the Holliday junction and, by inference, on recombination and recombination dependent processes.