Jerry Yang

Controlling protein translocation through nanopores with bio-inspired fluid walls

Synthetic nanopores have been used to study individual biomolecules in high thoroughput but their performance as sensors does not match biological ion channels. Controlling the translocation times of single-molecule analytes and their non-specific interaction with pore walls remain a challenge. Inspired by the olfactory sensilla of the insect antenna, here we show that coating nanopores with fluid bilayer lipids allows the pore diameters to be fine-tuned in sub-nanometre increments. Incorporation of mobile ligands in the lipid conferred specificity and slowed down the translocation of targeted proteins sufficiently to time-resolve translocation events of individual proteins. The lipid coatings also prevented pores from clogging, eliminated non-specific binding and enabled the translocation of amyloid-beta (Aβ) oligomers and fibrils. Through combined analysis of translocation time, volume, charge, shape and ligand affinity, different proteins were identified.

Applications of biological pores in nanomedicine, sensing, and nanoelectronics.

Biological protein pores and pore-forming peptides can generate a pathway for the flux of ions and other charged or polar molecules across cellular membranes. In nature, these nanopores have diverse and essential functions that range from maintaining cell homeostasis and participating in cell signaling to activating or killing cells. The combination of the nanoscale dimensions and sophisticated - often regulated - functionality of these biological pores make them particularly attractive for the growing field of nanobiotechnology. Applications range from single-molecule sensing to drug delivery and targeted killing of malignant cells. Potential future applications may include the use of nanopores for single strand DNA sequencing and for generating bio-inspired, and possibly, biocompatible visual detection systems and batteries. This article reviews the current state of applications of pore-forming peptides and proteins in nanomedicine, sensing, and nanoelectronics.

Amyloid-β-Induced Ion Flux in Artificial Lipid Bilayers and Neuronal Cells: Resolving a Controversy

Understanding the pathogenicity of amyloid-beta (Aβ) peptides constitutes a major goal in research on Alzheimer’s disease (AD). One hypothesis entails that Aβ peptides induce uncontrolled, neurotoxic ion flux through cellular membranes. The exact biophysical mechanism of this ion flux is, however, a subject of an ongoing controversy which has attenuated progress toward understanding the importance of Aβ-induced ion flux in AD. The work presented here addresses two prevalent controversies regarding the nature of transmembrane ion flux induced by Αβ peptides. First, the results clarify that Αβ can induce stepwise ion flux across planar lipid bilayers as opposed to a gradual increase in transmembrane current; they show that the previously reported gradual thinning of membranes with concomitant increase in transmembrane current arises from residues of the solvent hexafluoroisopropanol, which is commonly used for the preparation of amyloid samples. Second, the results provide additional evidence suggesting that Aβ peptides can induce ion channel-like ion flux in cellular membranes that is independent from the postulated ability of Αβ to modulate intrinsic cellular ion channels or transporter proteins.

Inhibitors of Catalase-Amyloid Interactions Protect Cells from β-Amyloid-Induced Oxidative Stress and Toxicity

Compelling evidence shows a strong correlation between accumulation of neurotoxic β-amyloid (Aβ) peptides and oxidative stress in the brains of patients afflicted with Alzheimer disease (AD). One hypothesis for this correlation involves the direct and harmful interaction of aggregated Aβ peptides with enzymes responsible for maintaining normal, cellular levels of reactive oxygen species (ROS). Identification of specific, destructive interactions of Aβ peptides with cellular anti-oxidant enzymes would represent an important step toward understanding the pathogenicity of Aβ peptides in AD. This report demonstrates that exposure of human neuroblastoma cells to cytotoxic preparations of aggregated Aβ peptides results in significant intracellular co-localization of Aβ with catalase, an anti-oxidant enzyme responsible for catalyzing the degradation of the ROS intermediate hydrogen peroxide (H2O2). These catalase-Aβ interactions deactivate catalase, resulting in increased cellular levels of H2O2. Furthermore, small molecule inhibitors of catalase-amyloid interactions protect the hydrogen peroxide-degrading activity of catalase in Aβ-rich environments, leading to reduction of the co-localization of catalase and Aβ in cells, inhibition of Aβ-induced increases in cellular levels of H2O2, and reduction of the toxicity of Aβ peptides. These studies, thus, provide evidence for the important role of intracellular catalase-amyloid interactions in Aβ-induced oxidative stress and propose a novel molecular strategy to inhibit such harmful interactions in AD.

Amyloid-binding small molecules efficiently block SEVI (semen-derived enhancer of virus infection)- and semen-mediated enhancement of HIV-1 infection.

Semen was recently shown to contain amyloid fibrils formed from a self-assembling peptide fragment of the protein prostatic acid phosphatase. These amyloid fibrils, termed semen-derived enhancer of virus infection, or SEVI, have been shown to strongly enhance HIV infectivity and may play an important role in sexual transmission of HIV, making them a potential microbicide target. One novel approach to target these fibrils is the use of small molecules known to intercalate into the structure of amyloid fibrils, such as derivatives of thioflavin-T. Here, we show that the amyloid-binding small molecule BTA-EG6 (the hexa(ethylene glycol) derivative of benzothiazole aniline) is able to bind SEVI fibrils and effectively inhibit both SEVI-mediated and semen-mediated enhancement of HIV infection. BTA-EG6 also blocks the interactions of SEVI with HIV-1 virions and HIV-1 target cells but does not cause any inflammation or toxicity to cervical epithelial cells. These results suggest that an amyloid-binding small molecule may have utility as a microbicide, or microbicidal supplement, for HIV-1.

Rational Design of Amyloid Binding Agents Based on the Molecular Rotor Motif

Alzheimer’s disease (AD) is characterized by a progressive loss of cognitive function and constitutes the most common and fatal neurodegenerative disorder.[1] Genetic and clinical evidence supports the hypothesis that accumulation of amyloid deposits in the brain plays an important role in the pathology of the disease. This event is associated with perturbations of biological functions in the surrounding tissue leading to neuronal cell death, thus contributing to the disease process. The deposits are comprised primarily of amyloid (Aβ) peptides, a 39–43 amino acid sequence that self aggregates into a fibrillar β-pleated sheet motif. While the exact three-dimensional structure of the aggregated Aβ peptides is not known, a model structure that sustains the property of aggregation has been proposed.[2] This creates opportunities for in vivo imaging of amyloid deposits that can not only help evaluate the time course and evolution of the disease, but can also allow the timely monitoring of therapeutic treatments.[3]

Multivariate analyses of amyloid-beta oligomer populations indicate a connection between pore formation and cytotoxicity.

Aggregates of amyloid-beta (Aβ) peptides are thought to be involved in the development of Alzheimer’s disease because they can change synaptic plasticity and induce neuronal cell death by inflammation, oxidative damage, and transmembrane pore formation. Exactly which oligomeric species underlie these cytotoxic effects remains unclear. The work presented here established well-controlled aggregation conditions of Aβ 1–40 or Aβ1–42 peptides over a 20-day period and characterized these preparations with regard to their β-sheet content, degree of fibril formation, relative abundance of various oligomer sizes, and propensity to induce membrane pore formation and cytotoxicity. Using this multivariate data set, a systematic and inherently unbiased partial least squares (PLS) approach showed that for both peptides the abundance of oligomers in the tetramer to 13-mer range contributed positively to both pore formation and cytotoxicity, while monomers, dimers, trimers, and the largest oligomers (>210 kDa) were negatively correlated to both phenomena. Multivariate PLS analysis is ideally suited to handle complex data sets and interdependent variables such as relative oligomer concentrations, making it possible to elucidate structure function relationships in complex mixtures. This approach, therefore, introduces an enabling tool to the field of amyloid research, in which it is often difficult to interpret the activity of individual species within a complex mixture of bioactive species.

Using Ion Channel-Forming Peptides to Quantify Protein−Ligand Interactions

This paper proposes a method for sensing affinity interactions by triggering disruption of self-assembly of ion channel-forming peptides in planar lipid bilayers. It shows that the binding of a derivative of alamethicin carrying a covalently attached sulfonamide ligand to carbonic anhydrase II (CA II) resulted in the inhibition of ion channel conductance through the bilayer. We propose that the binding of the bulky CA II protein (MW approximately 30 kD) to the ion channel-forming peptides (MW approximately 2.5 kD) either reduced the tendency of these peptides to self-assemble into a pore or extracted them from the bilayer altogether. In both outcomes, the interactions between the protein and the ligand lead to a disruption of self-assembled pores. Addition of a competitive inhibitor, 4-carboxybenzenesulfonamide, to the solution released CA II from the alamethicin-sulfonamide conjugate and restored the current flow across the bilayer by allowing reassembly of the ion channels in the bilayer. Time-averaged recordings of the current over discrete time intervals made it possible to quantify this monovalent ligand binding interaction. This method gave a dissociation constant of approximately 2 microM for the binding of CA II to alamethicin-sulfonamide in the bilayer recording chamber: this value is consistent with a value obtained independently with CA II and a related sulfonamide derivative by isothermal titration calorimetry.

Nanoscale Ionic Diodes with Tunable and Switchable Rectifying Behavior

Nanoscale ionic diodes have attracted interest as circuit elements for development of nanofluidic devices for a variety of applications, including biosensing, constructing artificial cells, and engineering biological batteries. This paper presents a bottom-up, self-assembly approach for constructing nanopores with rectified conductance behavior in a membrane using semisynthetic derivatives of the ion-channel-forming peptide gramicidin A. The capability to individually access each half of a dimeric gramicidin channel makes it possible to generate asymmetric channels in a membrane that exhibit diodelike conductance properties. The modular nature of these self-assembled channels affords the possibility of tuning their rectifying conductance properties by simple replacement of one peptide derivative with another in the membrane. Additionally, introduction of an external stimulus (here, an enzyme) to change the functional group attached to one side of the gramicidin pore induces diodelike conductance behavior in previously nonrectified channels, demonstrating the possibility of switching the conductance properties of these nanopores in situ in a controlled manner.

Aminonaphthalene 2-Cyanoacrylate (ANCA) Probes Fluorescently Discriminate between Amyloid-β and Prion Plaques in Brain

A major challenge for diagnosing and monitoring the progression of amyloid-based diseases is the capability to distinguish between amyloid deposits that are associated with related, but distinctly different, diseases. Here, we demonstrate that aminonaphthalenyl 2-cyanoacrylate-based probes can fluorescently discriminate between different types of amyloid deposits in brain. The discriminating capability of these molecular rotors is due to the stabilization of the ground versus excited states of these probes as a function of the polarity of their microenvironment (i.e., within the binding pocket on the amyloid). This property makes it possible, for the first time, to estimate the inherent static relative permittivity (ε(0)) of the binding pocket of each amyloid within tissue. The capability to selectively follow the deposition of specific amyloids in tissue may provide important information for therapeutic development that is not readily accessible from currently available technology.