Binding of cell-penetrating penetratin peptides to plasma membrane vesicles correlates directly with cellular uptake

AbstractCell-penetrating peptides (CPPs) gain access to intracellular compartments mainly via endocytosis and have capacity to deliver macromolecular cargo into cells. Although the involvement of various endocytic routes has been described it is still unclear which interactions are involved in eliciting an uptake response and to what extent affinity for particular cell surface components may determine the efficiency of a particular CPP. Previous biophysical studies of the interaction between CPPs and either lipid vesicles or soluble sugar-mimics of cell surface proteoglycans, the two most commonly suggested CPP binding targets, have not allowed quantitative correlations to be established. We here explore the use of plasma membrane vesicles (PMVs) derived from cultured mammalian cells as cell surface models in biophysical experiments. Further, we examine the relationship between affinity for PMVs and uptake into live cells using the CPP penetratin and two analogs enriched in arginines and lysines respectively. We show, using centrifugation to sediment PMVs, that the amount of peptide in the pellet fraction correlates linearly with the degree of cell internalization and that the relative efficiency of all-arginine and all-lysine variants of penetratin can be ascribed to their respective cell surface affinities. Our data show differences between arginine- and lysine-rich variants of penetratin that has not been previously accounted for in studies using lipid vesicles. Our data also indicate greater differences in binding affinity to PMVs than to heparin, a commonly used cell surface proteoglycan mimic. Taken together, this suggests that the cell surface interactions of CPPs are dependent on several cell surface moieties and their molecular organization on the plasma membrane

Solvent exposure of Tyr10 as a probe of structural differences between monomeric and aggregated forms of the amyloid-β peptide.

Aggregation of amyloid-β (Aβ) peptides is a characteristic pathological feature of Alzheimer's disease. We have exploited the relationship between solvent exposure and intrinsic fluorescence of a single tyrosine residue, Tyr10, in the Aβ sequence to probe structural features of the monomeric, oligomeric and fibrillar forms of the 42-residue Aβ1-42. By monitoring the quenching of Tyr10 fluorescence upon addition of water-soluble acrylamide, we show that in Aβ1-42 oligomers this residue is solvent-exposed to a similar extent to that found in the unfolded monomer. By contrast, Tyr10 is significantly shielded from acrylamide quenching in Aβ1-42 fibrils, consistent with its proximity to the fibrillar cross-β core. Furthermore, circular dichroism measurements reveal that Aβ1-42 oligomers have a considerably lower β-sheet content than the Aβ1-42 fibrils, indicative of a less ordered molecular arrangement in the former. Taken together these findings suggest significant differences in the structural assembly of oligomers and fibrils that are consistent with differences in their biological effects.This work was funded by grants to E.K.E from the Wenner-Gren Foundations, the Hasselblad Foundation, and the Swedish Innovation Agency (Vinnova) and to C.M.D from the Wellcome Trust. The TEM imaging was carried out in the Multi-Imaging Unit in the Department of Physiology, Development and Neuroscience, University of Cambridge, UK and quantitative amino acid analysis was carried out at the Protein and Nucleic Acid Chemistry Facility, Department of Biochemistry, University of Cambridge, UK.This is the final version of the article. It was first available from Elsevier via http://dx.doi.org/10.1016/j.bbrc.2015.11.01

Direct observations of amyloid β self-assembly in live cells provide insights into differences in the kinetics of Aβ(1-40) and Aβ(1-42) aggregation.

Insight into how amyloid β (Aβ) aggregation occurs in vivo is vital for understanding the molecular pathways that underlie Alzheimer's disease and requires new techniques that provide detailed kinetic and mechanistic information. Using noninvasive fluorescence lifetime recordings, we imaged the formation of Aβ(1-40) and Aβ(1-42) aggregates in live cells. For both peptides, the cellular uptake via endocytosis is rapid and spontaneous. They are then retained in lysosomes, where their accumulation leads to aggregation. The kinetics of Aβ(1-42) aggregation are considerably faster than those of Aβ(1-40) and, unlike those of the latter peptide, show no detectable lag phase. We used superresolution fluorescence imaging to examine the resulting aggregates and could observe compact amyloid structures, likely because of spatial confinement within cellular compartments. Taken together, these findings provide clues as to how Aβ aggregation may occur within neurons

Using Tetracysteine-Tagged TDP-43 with a Biarsenical Dye To Monitor Real-Time Trafficking in a Cell Model of Amyotrophic Lateral Sclerosis.

TAR DNA-binding protein 43 (TDP-43) has been identified as the major constituent of the proteinaceous inclusions that are characteristic of most forms of amyotrophic lateral sclerosis (ALS) and ubiquitin positive frontotemporal lobar degeneration (FTLD). Wild type TDP-43 inclusions are a pathological hallmark of >95% of patients with sporadic ALS and of the majority of familial ALS cases, and they are also found in a significant proportion of FTLD cases. ALS is the most common form of motor neuron disease, characterized by progressive weakness and muscular wasting, and typically leads to death within a few years of diagnosis. To determine how the translocation and misfolding of TDP-43 contribute to ALS pathogenicity, it is crucial to define the dynamic behavior of this protein within the cellular environment. It is therefore necessary to develop cell models that allow the location of the protein to be defined. We report the use of TDP-43 with a tetracysteine tag for visualization using fluorogenic biarsenical compounds and show that this model displays features of ALS observed in other cell models. We also demonstrate that this labeling procedure enables live-cell imaging of the translocation of the protein from the nucleus into the cytosol

Nanobodies Raised against Monomeric α-Synuclein Distinguish between Fibrils at Different Maturation Stages

AbstractNanobodies are single-domain fragments of camelid antibodies that are emerging as versatile tools in biotechnology. We describe here the interactions of a specific nanobody, NbSyn87, with the monomeric and fibrillar forms of α-synuclein (αSyn), a 140-residue protein whose aggregation is associated with Parkinson's disease. We have characterized these interactions using a range of biophysical techniques, including nuclear magnetic resonance and circular dichroism spectroscopy, isothermal titration calorimetry and quartz crystal microbalance measurements. In addition, we have compared the results with those that we have reported previously for a different nanobody, NbSyn2, also raised against monomeric αSyn. This comparison indicates that NbSyn87 and NbSyn2 bind with nanomolar affinity to distinctive epitopes within the C-terminal domain of soluble αSyn, comprising approximately amino acids 118–131 and 137–140, respectively. The calorimetric and quartz crystal microbalance data indicate that the epitopes of both nanobodies are still accessible when αSyn converts into its fibrillar structure. The apparent affinities and other thermodynamic parameters defining the binding between the nanobody and the fibrils, however, vary significantly with the length of time that the process of fibril formation has been allowed to progress and with the conditions under which formation occurs, indicating that the environment of the C-terminal domain of αSyn changes as fibril assembly takes place. These results demonstrate that nanobodies are able to target forms of potentially pathogenic aggregates that differ from each other in relatively minor details of their structure, such as those associated with fibril maturation

Membrane binding of pH-sensitive influenza fusion peptides. Positioning, configuration, and induced leakage in a lipid vesicle model

pH-sensitive HA2 fusion peptides from influenza virus hemagglutinin have potential as endosomal escape-inducing components in peptide-based drug delivery. Polarized light spectroscopy and tryptophan fluorescence were used to assess the conformation, orientation, effect on lipid order, and binding kinetics of wild-type peptide HA2(1-23) and a glutamic acid-enfiched analogue (INF7) in large unilamellar POPC or POPC/POPG (4: 1) lipid vesicles (LUVs). pH-sensitive membrane leakage was established for INF7 but not HA2(1-23) using an entrapped-dye assay. A correlation is indicated between leakage and a low degree of lipid chain order (assessed by linear dichroism, LD, of the membrane orientation probe retinoic acid). Both peptides display poor alignment in zwitterionic POPC LUVs compared to POPC/POPG (4: 1) LUVs, and it was found that peptide- lipid interactions display slow kinetics (hours), resulting in reduced lipid order and increased tryptophan shielding. At pH 7.4, INF7 displays tryptophan emission and LD features indicative of a surface-orientated peptide, suggesting that its N-terminal glutamic acid residues prevent deep penetration into the hydrocarbon core. At pH 5.0, INF7 displays weaker LD signals, indicating poor orientation, possibly due to aggregation. By contrast, the orientation of the HA2(1-23) peptide backbone supports previously reported oblique insertion (similar to 60-65 degrees relative to the membrane normal), and aromatic side-chain orientations are consistent with an interfacial (pH-independent) location of the C-terminus. We propose that a conformational change upon reduction of pH is limited to minor rearrangements of the peptide "hinge region" around Trp14 and repositioning of this residue

Lipid vesicle composition influences the incorporation and fluorescence properties of the lipophilic sulphonated carbocyanine dye SP-DiO

Lipophilic carbocyanine dyes are widely used as fluorescent cell membrane probes in studies ranging from biophysics to cell biology. While they are extremely useful for qualitative observation of lipid structures, a major problem impairing quantitative studies is that the chemical environment of the lipid bilayer affects both the dye's insertion efficiency and photophysical properties. We present a systematic investigation of the sulphonated carbocyanine dye 3,3 '-dioctadecyl-5,5 '-di(4-sulfophenyl) (SP-DiO) and demonstrate how its insertion efficiency into pre-formed lipid bilayers and its photophysical properties therein determine its apparent fluorescence intensity in different lipid environments. For this purpose, we use large unilamellar vesicles (LUVs) made of lipids with distinct chain unsaturation, acyl chain length, head group charge, and with variation in membrane cholesterol content as models. Using a combination of absorbance, fluorescence emission, and fluorescence lifetime measurements we reveal that SP-DiO incorporates more efficiently into liquid disordered phases compared to gel phases. Moreover, incorporation into the latter phase is most efficient when the mismatch between the length of the lipid and dye hydrocarbon chains is small. Furthermore, SP-DiO incorporation is less efficient in LUVs composed of negatively charged lipids. Lastly, when cholesterol was included in the LUV membranes, we observed significant spectral shifts, consistent with dye aggregation. Taken together, our study highlights the complex interplay between membrane composition and labeling efficiency with lipophilic dyes and advocates for careful assessment of fluorescence data when attempting a quantitative analysis of fluorescence data with such molecules

Cationic Nanoparticle Interactions with Catabolic Cartilage Modify Macrophage Cytokine Production

Cationic nanoparticles (NPs) have emerged as promising candidates for intra-articular drug delivery, showcasing their potential as efficient carriers. However, despite their evident utility, many studies neglect to investigate their interactions with the extracellular matrix (ECM). This oversight represents a significant gap in our understanding, particularly in the context of osteoarthritis (OA) treatment, where viable therapeutic options are limited. The clinical translation of functionalize drug carriers for OA has been hindered, in part, by our incomplete comprehension of how these materials interact with and respond to the pathological environment. This study aims to address this gap by examining how cationic NPs interact with ECM components in an ex vivo OA cartilage explant model. By comparing the behavior of smaller (<10nm) and larger (~270 nm) cationic NPs and subjecting them to explants preconditioned with OA-specific catabolic enzymes, we observed multifaceted effects on ECM integrity and biomolecule conformation. All NP-biomolecule complexes induced differential cytokine production from stimulated macrophages. Smaller polyamidoamine (PAMAM) nanocarriers reduced glycosaminoglycan (GAG) release from explants but increased proinflammatory cytokine stimulation under pathological conditions, while larger poly lactic(co-glycolic)acid(PLGA)/polyethylenimine(PEI)-based nanocarriers maintained GAG release but induced a significantly lower proinflammatory cytokine response. Collagenase-induced OA-mimicking preconditioning produced distinct profiles across the NP panel in GAG interactions and cytokine production. The findings from these studies underscore the significance of tailored nanocarrier approaches to achieve optimal therapeutic efficacy for OA and other complex diseases