Zhengjian Lv

Mechanism of amyloid β−protein dimerization determined using single−molecule AFM force spectroscopy

Aβ42 and Aβ40 are the two primary alloforms of human amyloid β−protein (Aβ). The two additional C−terminal residues of Aβ42 result in elevated neurotoxicity compared with Aβ40, but the molecular mechanism underlying this effect remains unclear. Here, we used single−molecule force microscopy to characterize interpeptide interactions for Aβ42 and Aβ40 and corresponding mutants. We discovered a dramatic difference in the interaction patterns of Aβ42 and Aβ40 monomers within dimers. Although the sequence difference between the two peptides is at the C−termini, the N−terminal segment plays a key role in the peptide interaction in the dimers. This is an unexpected finding as N−terminal was considered as disordered segment with no effect on the Aβ peptide aggregation. These novel properties of Aβ proteins suggests that the stabilization of N−terminal interactions is a switch in redirecting of amyloids form the neurotoxic aggregation pathway, opening a novel avenue for the disease preventions and treatments.

Nonnative SOD1 trimer is toxic to motor neurons in a model of amyotrophic lateral sclerosis

Significance Protein aggregation is a hallmark of neurodegenerative disease and is hypothesized to cause neuron death. Despite extensive study of disease-associated aggregating proteins, mechanisms of neuron death remain a mystery, and no cures or effective treatments yet exist. Here, we demonstrate the toxicity of a small aggregate of the Cu,Zn superoxide dismutase (SOD1) protein, associated with amyotrophic lateral sclerosis (ALS). We present an experimentally verified structural model of this toxic species and show that SOD1 mutants designed to promote formation of this aggregate increase cell death, providing a direct link between aggregate presence and neuron death. Knowledge of toxic species and the ability to manipulate their formation provides a valuable direction for pursuit of therapeutic strategies in ALS. Since the linking of mutations in the Cu,Zn superoxide dismutase gene (sod1) to amyotrophic lateral sclerosis (ALS) in 1993, researchers have sought the connection between SOD1 and motor neuron death. Disease-linked mutations tend to destabilize the native dimeric structure of SOD1, and plaques containing misfolded and aggregated SOD1 have been found in the motor neurons of patients with ALS. Despite advances in understanding of ALS disease progression and SOD1 folding and stability, cytotoxic species and mechanisms remain unknown, greatly impeding the search for and design of therapeutic interventions. Here, we definitively link cytotoxicity associated with SOD1 aggregation in ALS to a nonnative trimeric SOD1 species. We develop methodology for the incorporation of low-resolution experimental data into simulations toward the structural modeling of metastable, multidomain aggregation intermediates. We apply this methodology to derive the structure of a SOD1 trimer, which we validate in vitro and in hybridized motor neurons. We show that SOD1 mutants designed to promote trimerization increase cell death. Further, we demonstrate that the cytotoxicity of the designed mutants correlates with trimer stability, providing a direct link between the presence of misfolded oligomers and neuron death. Identification of cytotoxic species is the first and critical step in elucidating the molecular etiology of ALS, and the ability to manipulate formation of these species will provide an avenue for the development of future therapeutic strategies.

Nanoprobing of the Effect of Cu2+ Cations on Misfolding, Interaction and Aggregation of Amyloid β Peptide

Misfolding and aggregation of the amyloid β-protein (Aβ) are hallmarks of Alzheimer’s disease. Both processes are dependent on the environmental conditions, including the presence of divalent cations, such as Cu2+. Cu2+ cations regulate early stages of Aβ aggregation, but the molecular mechanism of Cu2+ regulation is unknown. In this study we applied single molecule AFM force spectroscopy to elucidate the role of Cu2+ cations on interpeptide interactions. By immobilizing one of two interacting Aβ42 molecules on a mica surface and tethering the counterpart molecule onto the tip, we were able to probe the interpeptide interactions in the presence and absence of Cu2+ cations at pH 7.4, 6.8, 6.0, 5.0, and 4.0. The results show that the presence of Cu2+ cations change the pattern of Aβ interactions for pH values between pH 7.4 and pH 5.0. Under these conditions, Cu2+ cations induce Aβ42 peptide structural changes resulting in N-termini interactions within the dimers. Cu2+ cations also stabilize the dimers. No effects of Cu2+ cations on Aβ-Aβ interactions were observed at pH 4.0, suggesting that peptide protonation changes the peptide-cation interaction. The effect of Cu2+ cations on later stages of Aβ aggregation was studied by AFM topographic images. The results demonstrate that substoichiometric Cu2+ cations accelerate the formation of fibrils at pH 7.4 and 5.0, whereas no effect of Cu2+ cations was observed at pH 4.0. Taken together, the combined AFM force spectroscopy and imaging analyses demonstrate that Cu2+ cations promote both the initial and the elongation stages of Aβ aggregation, but protein protonation diminishes the effect of Cu2+.

Novel polymer linkers for single molecule AFM force spectroscopy

Flexible polymer linkers play an important role in various imaging and probing techniques that require surface immobilization, including atomic force microscopy (AFM). In AFM force spectroscopy, polymer linkers are necessary for the covalent attachment of molecules of interest to the AFM tip and the surface. The polymer linkers tether the molecules and provide their proper orientation in probing experiments. Additionally, the linkers separate specific interactions from nonspecific short-range adhesion and serve as a reference point for the quantitative analysis of single molecule probing events. In this report, we present our results on the synthesis and testing of a novel polymer linker and the identification of a number of potential applications for its use in AFM force spectroscopy experiments. The synthesis of the linker is based on the well-developed phosphoramidate (PA) chemistry that allows the routine synthesis of linkers with predetermined lengths and PA composition. These linkers are homogeneous in length and can be terminated with various functional groups. PA linkers with different functional groups were synthesized and tested in experimental systems utilizing different immobilization chemistries. We probed interactions between complementary DNA oligonucleotides; DNA and protein complexes formed by the site-specific binding protein SfiI; and interactions between amyloid peptide (Aβ42). The results of the AFM force spectroscopy experiments validated the feasibility of the proposed approach for the linker design and synthesis. Furthermore, the properties of the tether (length, functional groups) can be adjusted to meet the specific requirements for different force spectroscopy experiments and system characteristics, suggesting that it could be used for a large number of various applications.

Self-assembly of the full-length amyloid Aβ42 protein in dimers

The self-assembly of amyloid (Aβ) proteins into nano-aggregates is a hallmark of Alzheimers disease (AD) development, yet the mechanism of how disordered monomers assemble into aggregates remains elusive. Here, we applied long-time molecular dynamics simulations to fully characterize the assembly of Aβ42 monomers into dimers. Monomers undergo conformational changes during their interaction, but the resulting dimer structures do not resemble those found in fibril structures. To identify natural conformations of dimers among a set of simulated ones, validation approaches were developed and applied, and a subset of dimer conformations were characterized. These dimers do not contain long β-strands that are usually found in fibrils. The dimers are stabilized primarily by interactions within the central hydrophobic regions and the C-terminal regions, with a contribution from local hydrogen bonding. The dimers are dynamic, as evidenced by the existence of a set of conformations and by the quantitative analyses of the dimer dissociation process.

A novel pathway for amyloids self-assembly in aggregates at nanomolar concentration mediated by the interaction with surfaces.

A limitation of the amyloid hypothesis in explaining the development of neurodegenerative diseases is that the level of amyloidogenic polypeptide in vivo is below the critical concentration required to form the aggregates observed in post-mortem brains. We discovered a novel, on-surface aggregation pathway of amyloidogenic polypeptide that eliminates this long-standing controversy. We applied atomic force microscope (AFM) to demonstrate directly that on-surface aggregation takes place at a concentration at which no aggregation in solution is observed. The experiments were performed with the full-size Aβ protein (Aβ42), a decapeptide Aβ(14-23) and α-synuclein; all three systems demonstrate a dramatic preference of the on-surface aggregation pathway compared to the aggregation in the bulk solution. Time-lapse AFM imaging, in solution, show that over time, oligomers increase in size and number and release in solution, suggesting that assembled aggregates can serve as nuclei for aggregation in bulk solution. Computational modeling performed with the all-atom MD simulations for Aβ(14-23) peptide shows that surface interactions induce conformational transitions of the monomer, which facilitate interactions with another monomer that undergoes conformational changes stabilizing the dimer assembly. Our findings suggest that interactions of amyloidogenic polypeptides with cellular surfaces play a major role in determining disease onset.

Effect of acidic pH on the stability of α-synuclein dimers.

Environmental factors, such as acidic pH, facilitate the assembly of α‐synuclein (α‐Syn) in aggregates, but the impact of pH on the very first step of α‐Syn aggregation remains elusive. Recently, we developed a single‐molecule approach that enabled us to measure directly the stability of α‐Syn dimers. Unlabeled α‐Syn monomers were immobilized on a substrate, and fluorophore‐labeled monomers were added to the solution to allow them to form dimers with immobilized α‐Syn monomers. The dimer lifetimes were measured directly from the fluorescence bursts on the time trajectories. Herein, we applied the single‐molecule tethered approach for probing of intermolecular interaction to characterize the effect of acidic pH on the lifetimes of α‐Syn dimers. The experiments were performed at pH 5 and 7 for wild‐type α−Syn and for two mutants containing familial type mutations E46K and A53T. We demonstrate that a decrease of pH resulted in more than threefold increase in the α‐Syn dimers lifetimes with some variability between the α‐Syn species. We hypothesize that the stabilization effect is explained by neutralization of residues 96–140 of α‐Syn and this electrostatic effect facilitates the association of the two monomers. Given that dimerization is the first step of α‐Syn aggregation, we posit that the electrostatic effect thereby contributes to accelerating α‐Syn aggregation at acidic pH.

Phospholipid membranes promote the early stage assembly of α-synuclein aggregates

Development of Parkinson’s disease is associated with spontaneous self-assembly of α-synuclein (α-syn). Efforts aimed at understanding this process have produced little clarity and the mechanism remains elusive. We report a novel effect of phospholipid bilayers on the catalysis of α-syn aggregation from monomers. We directly visualized α-syn aggregation on supported lipid bilayers using time-lapse atomic force microscopy. We discovered that α-syn assemble in aggregates on bilayer surfaces even at the nanomolar concentration of monomers in solution. The efficiency of the aggregation process depends on the membrane composition, being highest for a negatively charged bilayer. Furthermore, assembled aggregates can dissociate from the surface, suggesting that on-surface aggregation can be a mechanism by which pathological aggregates are produced. Computational modeling revealed that interaction of α-syn with bilayer surface changes the protein conformation and its affinity to assemble into dimers, and these properties depend on the bilayer composition. A model of the membrane-mediated aggregation triggering the assembly of neurotoxic aggregates is proposed.