Conformational Switch of a Peptide Provides a Novel Strategy to Design Peptide Loaded Porous Organic Polymer for Pyroptosis Pathway Mediated Cancer Therapy.

While peptide-based drug development is extensively explored, this strategy has limitations due to rapid excretion from the body (or shorter half-life in the body) and vulnerability to protease-mediated degradation. To overcome these limitations, a novel strategy for the development of a peptide-based anticancer agent is introduced, utilizing the conformation switch property of a chameleon sequence stretch (PEP1) derived from a mycobacterium secretory protein, MPT63. The selected peptide is then loaded into a new porous organic polymer (PG-DFC-POP) synthesized using phloroglucinol and a cresol derivative via a condensation reaction to deliver the peptide selectively to cancer cells. Utilizing ensemble and single-molecule approaches, this peptide undergoes a transition from a disordered to an alpha-helical conformation, triggered by the acidic environment within cancer cells that is demonstrated. This adopted alpha-helical conformation resulted in the formation of proteolysis-resistant oligomers, which showed efficient membrane pore-forming activity selectively for negatively charged phospholipids accumulated in cancer cell membranes. The experimental results demonstrated that the peptide-loaded PG-DFC-POP-PEP1 exhibited significant cytotoxicity in cancer cells, leading to cell death through the Pyroptosis pathway, which is established by monitoring numerous associated events starting from lysosome membrane damage to GSDMD-induced cell membrane demolition. This novel conformational switch-based drug design strategy is believed to have great potential in endogenous environment-responsive cancer therapy and the development of future drug candidates to mitigate cancers.

Quantitative DNA-PAINT imaging of AMPA receptors in live neurons.

DNA-point accumulation for imaging at nanoscale topography (DNA-PAINT) can image fixed biological specimens with nanometer resolution and absolute stoichiometry. In living systems, however, the usage of DNA-PAINT has been limited due to high salt concentration in the buffer required for specific binding of the imager to the docker attached to the target. Here, we used multiple binding motifs of the docker, from 2 to 16, to accelerate the binding speed of the imager under physiological buffer conditions without compromising spatial resolution and maintaining the basal level homeostasis during the measurement. We imaged endogenous α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) in cultured neurons-critical proteins involved in nerve communication-by DNA-PAINT in 3-dimensions using a monovalent single-chain variable fragment (scFv) to the GluA1 subunit of AMPAR. We found a heterogeneous distribution of synaptic AMPARs: ≈60% are immobile, primarily in nanodomains, defined as AMPARs that are within 0.3 µm of the Homer1 protein in the postsynaptic density; the other ∼40% of AMPARs have restricted mobility and trajectory.

Peptide-PAINT Using a Transfected-Docker Enables Live- and Fixed-Cell Super-Resolution Imaging.

Point accumulation for imaging in nanoscale topography (PAINT) is a single-molecule technique for super-resolution microscopy, which uses exchangeable single stranded DNA oligos or peptide-pairs to create blinking phenomenon and achieves ≈5-25 nanometer resolution. Here, it is shown that by transfecting the protein-of-interest with a docker-coil, rather than by adding the docker externally-as is the norm when using DNA tethers or antibodies as dockers-similar localization can be achieved, ≈10 nm. However, using a transfected docker has several experimental advances and simplifications. Most importantly, it allows Peptide-PAINT to be applied to transfected live cells for imaging surface proteins in mammalian cells and neurons under physiological conditions. The enhanced resolution of Peptide-PAINT is also shown for organelles in fixed cells to unravel structural details including ≈40-nm and ≈60-nm axial repeats in vimentin filaments in the cytoplasm, and fiber shapes of sub-100-nm histone-rich regions in the nucleus.

High-throughput force measurement of individual kinesin-1 motors during multi-motor transport.

Molecular motors often work in teams to move a cellular cargo. Yet measuring the forces exerted by each motor is challenging. Using a sensor made with denatured ssDNA and multi-color fluorescence, we measured picoNewtons of forces and nanometer distances exerted by individual constrained kinesin-1 motors acting together while driving a common microtubule in vitro. We find that kinesins primarily exerted less than 1 pN force, even while the microtubule is bypassing artificial obstacles of 20-100 nanometer size. Occasionally, individual forces increase upon encountering obstacles, although at other times they do not, with the cargo continuing in a directional manner. Our high-throughput technique, which can measure forces by many motors simultaneously, is expected to be useful for many different types of molecular motors.

Altered Membrane Mechanics Provides a Receptor-Independent Pathway for Serotonin Action.

Serotonin, an important signaling molecule in humans, has an unexpectedly high lipid membrane affinity. The significance of this finding has evoked considerable speculation. Here we show that membrane binding by serotonin can directly modulate membrane properties and cellular function, providing an activity pathway completely independent of serotonin receptors. Atomic force microscopy shows that serotonin makes artificial lipid bilayers softer, and induces nucleation of liquid disordered domains inside the raft-like liquid-ordered domains. Solid-state NMR spectroscopy corroborates this data at the atomic level, revealing a homogeneous decrease in the order parameter of the lipid chains in the presence of serotonin. In the RN46A immortalized serotonergic neuronal cell line, extracellular serotonin enhances transferrin receptor endocytosis, even in the presence of broad-spectrum serotonin receptor and transporter inhibitors. Similarly, it increases the membrane binding and internalization of oligomeric peptides. Our results uncover a mode of serotonin-membrane interaction that can potentiate key cellular processes in a receptor-independent fashion.

Ordered and Disordered Segments of Amyloid-ß Drive Sequential Steps of the Toxic Pathway.

While the roles of intrinsically disordered protein domains in driving interprotein interactions are increasingly well-appreciated, the mechanism of toxicity of disease-causing disordered proteins remains poorly understood. A prime example is Alzheimer's disease (AD) associated amyloid beta (Aß). Aß oligomers are highly toxic partially structured peptide assemblies with a distinct ordered region (residues ∼10-40) and a shorter disordered region (residues ∼1-9). Here, we investigate the role of this disordered domain and its relation to the ordered domain in the manifestation of toxicity through a set of Aß fragments and stereoisomers designed for this purpose. We measure their effects on lipid membranes and cultured neurons, probing their toxicity, intracellular distributions, and specific molecular interactions using the techniques of confocal imaging, lattice light sheet imaging, fluorescence lifetime imaging, and fluorescence correlation spectroscopy. Remarkably, we find that neither part-Aß10-40 or Aß1-9, is toxic by itself. The ordered part (Aß10-40) is the major determinant of how Aß attaches to lipid bilayers, enters neuronal cells, and localizes primarily in the late endosomal compartments. However, once Aß enters the cell, it is the disordered part (only when it is connected to the rest of the peptide) that has a strong and stereospecific interaction with an unknown cellular component, as demonstrated by distinct changes in the fluorescence lifetime of a fluorophore attached to the N-terminal. This interaction appears to commit Aß to the toxic pathway. Our findings correlate well with Aß sites of familial AD mutations, a significant fraction of which cluster in the disordered region. We conclude that, while the ordered region dictates attachment and cellular entry, the key to toxicity lies in the ordered part presenting the disordered part for a specific cellular interaction.

Fluorescence quenching by lipid encased nanoparticles shows that amyloid-ß has a preferred orientation in the membrane.

Short range plasmonic fields around a nanoparticle can modulate fluorescence or Raman processes. In lipid encased nanoparticles, this can potentially measure the relative depths of different parts of a membrane protein from the surface. We employ this technique to discover that membrane inserted amyloid-ß oligomers have a preferred molecular orientation.

Spontaneous Fluctuations Can Guide Drug Design Strategies for Structurally Disordered Proteins.

Structure-based "rational" drug design strategies fail for diseases associated with intrinsically disordered proteins (IDPs). However, structural disorder allows large-amplitude spontaneous intramolecular dynamics in a protein. We demonstrate a method that exploits this dynamics to provide quantitative information about the degree of interaction of an IDP with other molecules. A candidate ligand molecule may not bind strongly, but even momentary interactions can be expected to perturb the fluctuations. We measure the amplitude and frequency of the equilibrium fluctuations of fluorescently labeled small oligomers of hIAPP (an IDP associated with type II diabetes) in a physiological solution, using nanosecond fluorescence cross-correlation spectroscopy. We show that the interterminal distance fluctuates at a characteristic time scale of 134 ± 10 ns, and 6.4 ± 0.2% of the population is in the "closed" (quenched) state at equilibrium. These fluctuations are affected in a dose-dependent manner by a series of small molecules known to reduce the toxicity of various amyloid peptides. The degree of interaction increases in the following order: resveratrol < epicatechin ∼ quercetin < Congo red < epigallocatechin 3-gallate. Such ordering can provide a direction for exploring the chemical space for finding stronger-binding ligands. We test the biological relevance of these measurements by measuring the effect of these molecules on the affinity of hIAPP for lipid vesicles and cell membranes. We find that the ability of a molecule to modulate intramolecular fluctuations correlates well with its ability to lower membrane affinity. We conclude that structural disorder may provide new avenues for rational drug design for IDPs.

Aggregation-induced conformation changes dictate islet amyloid polypeptide (IAPP) membrane affinity.

Islet amyloid polypeptide (IAPP) is a 37 residue intrinsically disordered protein whose aggregation is associated with Type II diabetes. Like most amyloids, it appears that the intermediate aggregates ("oligomers") of IAPP are more toxic than the mature fibrils, and interaction with the cell membrane is likely to be an integral component of the toxicity. Here we probe the membrane affinity and the conformation of the peptide as a function of its aggregation state. We find that the affinity of the peptide for artificial lipid bilayers is more than 15 times higher in the small oligomeric state (hydrodynamic radius ~ 1.6 nm) compared to the monomeric state (hydrodynamic radius ~ 0.7 nm). Binding with RIN-m5F cell membranes also shows qualitatively similar behavior. The monomeric state, as determined by Forster Resonance Energy Transfer, has a much larger end to end distance than the oligomeric state, suggesting conformational change between the monomers and the oligomers. Raman and Infrared spectroscopic measurements show the presence of considerable alpha helical content in the oligomers, whereas the larger aggregates have largely beta sheet character. Therefore, the conformation of the small oligomers is distinct from both the smaller monomers and the larger oligomers, and this is associated with an enhanced membrane affinity. This provides a possible structural basis for the enhanced toxicity of amyloid oligomers. Such change is also reminiscent of amyloid beta, another aggregation prone amyloidogenic peptide, though the nature of the conformational change is quite different in the two cases. We infer that conformational change underlying oligomer formation is a key factor in determining the enhanced membrane affinity of disease causing oligomers, but the toxic "oligomer fold" may not be universal.

Label-free imaging of neurotransmitters in live brain tissue by multi-photon ultraviolet microscopy.

Visualizing small biomolecules in living cells remains a difficult challenge. Neurotransmitters provide one of the most frustrating examples of this difficulty, as our understanding of signaling in the brain critically depends on our ability to follow the neurotransmitter traffic. Last two decades have seen considerable progress in probing some of the neurotransmitters, e.g. by using false neurotransmitter mimics, chemical labeling techniques, or direct fluorescence imaging. Direct imaging harnesses the weak UV fluorescence of monoamines, which are some of the most important neurotransmitters controlling mood, memory, appetite, and learning. Here we describe the progress in imaging of these molecules using the least toxic direct excitation route found so far, namely multi-photon (MP) imaging. MP imaging of serotonin, and more recently that of dopamine, has allowed researchers to determine the location of the vesicles, follow their intracellular dynamics, probe their content, and monitor their release. Recent developments have even allowed ratiometric quantitation of the vesicular content. This review shows that MP ultraviolet (MP-UV) microscopy is an effective but underutilized method for imaging monoamine neurotransmitters in neurones and brain tissue.

Fluorogenic Detection of Monoamine Neurotransmitters in Live Cells.

Monoamine neurotransmission is key to neuromodulation, but imaging monoamines in live neurons has remained a challenge. Here we show that externally added ortho-phthalaldehyde (OPA) can permeate live cells and form bright fluorogenic adducts with intracellular monoamines (e.g., serotonin, dopamine, and norepinephrine) and with L-DOPA, which can be imaged sensitively using conventional single-photon excitation in a fluorescence microscope. The peak excitation and emission wavelengths (λex = 401 nm and λem = 490 nm for serotonin; λex = 446 nm and λem = 557 nm for dopamine; and λex = 446 nm and λem = 544 nm for norepinephrine, respectively) are accessible to most modern confocal imaging instruments. The identity of monoamine containing structures (possibly neurotransmitter vesicles) in serotonergic RN46A cells is established by quasi-simultaneous imaging of serotonin using three-photon excitation microscopy. Mass spectrometry of cell extracts and of in vitro solutions helps us identify the chemical nature of the adducts and establishes the reaction mechanisms. Our method has low toxicity, high selectivity, and the ability to directly report the location and concentration of monoamines in live cells.

Major Reaction Coordinates Linking Transient Amyloid-ß Oligomers to Fibrils Measured at Atomic Level.

The structural underpinnings for the higher toxicity of the oligomeric intermediates of amyloidogenic peptides, compared to the mature fibrils, remain unknown at present. The transient nature and heterogeneity of the oligomers make it difficult to follow their structure. Here, using vibrational and solid-state nuclear magnetic resonance spectroscopy, and molecular dynamics simulations, we show that freely aggregating Aß40 oligomers in physiological solutions have an intramolecular antiparallel configuration that is distinct from the intermolecular parallel ß-sheet structure observed in mature fibrils. The intramolecular hydrogen-bonding network flips nearly 90°, and the two ß-strands of each monomeric unit move apart, to give rise to the well-known intermolecular in-register parallel ß-sheet structure in the mature fibrils. Solid-state nuclear magnetic resonance distance measurements capture the interstrand separation within monomer units during the transition from the oligomer to the fibril form. We further find that the D23-K28 salt-bridge, a major feature of the Aß40 fibrils and a focal point of mutations linked to early onset Alzheimer's disease, is not detectable in the small oligomers. Molecular dynamics simulations capture the correlation between changes in the D23-K28 distance and the flipping of the monomer secondary structure between antiparallel and parallel ß-sheet architectures. Overall, we propose interstrand separation and salt-bridge formation as key reaction coordinates describing the structural transition of the small Aß40 oligomers to fibrils.

Label-Free Ratiometric Imaging of Serotonin in Live Cells.

Ratiometric imaging can quantitatively measure changes in cellular analyte concentrations using specially designed fluorescent labels. We describe a label-free ratiometric imaging technique for direct detection of changes in intravesicular serotonin concentration in live cells. At higher concentrations, serotonin forms transient oligomers whose ultraviolet emission is shifted to longer wavelengths. We access the ultraviolet/blue emission using relatively benign three-photon excitation and split it into two imaging channels, whose ratio reports the concentration. The technique is sensitive at a physiologically relevant concentration range (10-150 mM serotonin). As a proof of principle, we measure the increase of intravesicular serotonin concentration with the addition of external serotonin. In general, since emission spectra of molecules are often sensitive to concentration, our method may be applicable to other natively fluorescent intracellular molecules which are present at high concentrations.

Stereoisomers Probe Steric Zippers in Amyloid-ß.

Shape complementarity between close-packed residues plays a critical role in the amyloid aggregation process. Here, we probe such "steric zipper" interactions in amyloid-ß (Aß40), whose aggregation is linked to Alzheimer's disease, by replacing natural residues by their stereoisomers. Such mutations are expected to specifically destabilize the shape sensitive "packing" interactions, which may potentially increase their solubility and change other properties. We study the stereomutants DF19 and DL34 and also the DA2/DF4/DH6/DS8 mutant of Aß40. F19-L34 is a critical contact in a tightly packed region of Aß, while residues 1-9 are known to be disordered. While both DF19 and DL34 slow down the kinetics of aggregation and form amyloid fibrils efficiently, only DL34 increases the final solubility. DF19 gives rise to additional off-pathway aggregation which results in large, kinetically stable aggregates, and has lower net solubility. DA2/DF4/DH6/DS8 does not have an effect on the kinetics or the solubility. Notably, both DF19 and DL34 oligomers have a significantly lower level of interactions with lipid vesicles and live cells. We conclude that stereoisomers can cause complex site dependent changes in amyloid properties, and provide an effective tool to determine the role of individual residues in shaping the packed interiors of amyloid aggregates.