Extracellular-Vesicle Catch-and-Release Isolation System Using a Net-Charge Invertible Curvature-Sensing Peptide.

Extracellular vesicles (EVs) carry various informative components, including signaling proteins, transcriptional regulators, lipids, and nucleic acids. These components are utilized for cell-cell communication between donor and recipient cells. EVs have shown great promise as pharmaceutical-targeting vesicles and have attracted the attention of researchers in the fields of biological and medical science because of their importance as diagnostic and prognostic markers. However, the isolation and purification of EVs from cell-cultured media remain challenging. Ultracentrifugation is the most widely used method, but it requires specialized and expensive equipment. In the present study, we proposed a novel methodology to isolate EVs using a simple and convenient method, i.e., an EV catch-and-release isolation system (EV-CaRiS) using a net-charge invertible curvature-sensing peptide (NIC). Curvature-sensing peptides recognize vesicles by binding to lipid-packing defects on highly curved membranes regardless of the expression levels of biomarkers. NIC was newly designed to reversibly capture and release EVs in a pH-dependent manner. NIC allowed us to achieve reproducible EV isolation from three human cell lines on resin using a batch method and single-particle imaging of EVs containing the ubiquitous exosome markers CD63 and CD81 by total internal reflection fluorescence microscopy (TIRFM). EV-CaRiS was demonstrated as a simple and convenient methodology for EV isolation, and NIC is promising for applications in the single-particle analysis of EVs.

Structural factors governing binding of curvature-sensing peptides to bacterial extracellular vesicles covered with hydrophilic polysaccharide chains.

Extracellular vesicles (EVs) have attracted an attention as important targets in the fields of biology and medical science because they contain physiologically active molecules. Curvature-sensing peptides are currently used as novel tools for marker-independent EV detection techniques. A structure-activity correlation study demonstrated that the α-helicity of the peptides is prominently involved in peptide binding to vesicles. However, whether a flexible structure changing from a random coil to an α-helix upon binding to vesicles or a restricted α-helical structure is an important factor in the detection of biogenic vesicles is still unclear. To address this issue, we compared the binding affinities of stapled and unstapled peptides for bacterial EVs with different surface polysaccharide chains. We found that unstapled peptides showed similar binding affinities for bacterial EVs regardless of surface polysaccharide chains, whereas stapled peptides showed substantially decreased binding affinities for bacterial EVs covered with capsular polysaccharides. This is probably because curvature-sensing peptides must pass through the layer of hydrophilic polysaccharide chains prior to binding to the hydrophobic membrane surface. While stapled peptides with restricted structures cannot easily pass through the layer of polysaccharide chains, unstapled peptides with flexible structures can easily approach the membrane surface. Therefore, we concluded that the structural flexibility of curvature-sensing peptides is a key factor for governing the highly sensitive detection of bacterial EVs.

Molecular Mechanism of Apoptosis by Amyloid ß-Protein Fibrils Formed on Neuronal Cells.

Aggregational states of amyloid ß-protein (Aß) are critical for its neurotoxicity, although they are not well-characterized, particularly after binding to the cell membranes. This is one reason why the mechanisms of Aß neurotoxicity are controversial and elusive. In this study, the effects of toxic Aß-(1-42) fibrils formed in the membrane on cellular processes were investigated using human neuroblastoma SH-SY5Y cells. Consistent with previous observations, fibrillar Aßs formed on the membranes induced activation of caspase-3, the effector caspase for apoptosis. Knockdown analyses of the initiator caspases, caspase-8 and caspase-9, indicated that the apoptosis was induced via activation of caspase-8, followed by activation of caspase-9 and caspase-3. We also found that inflammation signaling pathways including Toll-like receptors and inflammasomes NOD-, LRR-, and pyrin domain-containing protein 3 are involved in the initiation of apoptosis by the Aß fibrils. These inflammation-related molecules are promising targets for the prevention of apoptotic cell death induced by Aß.

Meeting Peptides in Kyoto.

The 10th International Peptide Symposium was held in Kyoto last December in conjunction with the 55th Japanese Peptide Symposium. Around 800 peptide scientists from 31 different countries and regions enjoyed sessions covering various aspects of state-of-the-art peptide science, such as synthetic methodology, chemical biology, cell biology, biophysics, and medicinal/ medical applications.

Endowment of pH Responsivity to Anticancer Peptides by Introducing 2,3-Diaminopropionic Acid Residues.

Endowment of pH responsivity to anticancer peptides is a promising approach to achieve better selectivity to cancer tissues. In this research, a template peptide was designed based on magainin 2, an antimicrobial peptide with anticancer activity, and a series of peptides were designed by replacing different numbers of lysine with the unnatural amino acid, 2,3diaminopropionic acid (Dap), which has a positive charge at weakly acidic pH in cancer tissues, but is neutral at physiological pH 7.4. These Dap-containing peptides are expected to interact more strongly with tumor cells than with normal cells because 1) weakly acidic conditions form in tumors, and 2) the membrane of tumor cells is more anionic than that of normal cells. Although all examined peptides showed potent cytotoxicities to multidrug-resistant cancer cells at a weakly acidic pH (ED50 ≈5 µm), the toxicity decreased with an increase in the number of Dap at pH 7.4 (8 Dap residues resulted in ED50 ≈60 µm). Furthermore, the introduction of Dap reduced cytotoxicity against normal cells. Thus, Dap led to significantly improved cancer targeting due to a pH-dependent charge shift. Fluorescence imaging and model membrane experiments supported this charge-shift model.

Toxic Amyloid Tape: A Novel Mixed Antiparallel/Parallel ß-Sheet Structure Formed by Amyloid ß-Protein on GM1 Clusters.

The abnormal aggregation of amyloid ß-protein (Aß) is considered central in the pathogenesis of Alzheimer's disease. We focused on membrane-mediated amyloidogenesis and found that amyloid fibrils formed on monosialoganglioside GM1 clusters were more toxic than those formed in aqueous solution. In this study, we investigated the structure of the toxic fibrils by Aß-(1-40) in detail in comparison with less-toxic fibrils formed in aqueous solution. The less-toxic fibrils contain in-resister parallel ß-sheets, whereas the structure of the toxic fibrils is unknown. Atomic force microscopy revealed that the toxic fibrils had a flat, tape-like morphology composed of a single ß-sheet layer. Isotope-edited infrared spectroscopy indicated that almost the entire sequence of Aß is included in the ß-sheet. Chemical cross-linking experiments using Cys-substituted Aßs suggested that the fibrils mainly contained both in-resister parallel and two-residue-shifted antiparallel ß-sheet structures. Solid-state NMR experiments also supported this conclusion. Thus, the toxic fibrils were found to possess a novel unique structure.

GXXXG-Mediated Parallel and Antiparallel Dimerization of Transmembrane Helices and Its Inhibition by Cholesterol: Single-Pair FRET and 2D IR Studies.

Small-residue-mediated interhelical packings are ubiquitously found in helical membrane proteins, although their interaction dynamics and lipid dependence remain mostly uncharacterized. We used a single-pair FRET technique to examine the effect of a GXXXG motif on the association of de novo designed (AALALAA)3 helices in liposomes. Dimerization occurred with sub-second lifetimes, which was abolished by cholesterol. Utilizing the nearly instantaneous time-resolution of 2D IR spectroscopy, parallel and antiparallel helix associations were identified by vibrational couplings across helices at their interface. Taken together, the data illustrate that the GXXXG motif controls helix packing but still allows for a dynamic and lipid-regulated oligomeric state.

A pH-dependent charge reversal peptide for cancer targeting.

Naturally occurring cationic antimicrobial peptides exhibit not only antimicrobial activity, but also anticancer activity and are expected to be new weapons in cancer treatment. The selectivity for cancer cells over normal cells is at least partly due to the more negative surface of cancer cells. A lower pH in tumor tissue (pH 6.2-6.9) than that in normal tissues (pH 7.3-7.4) has also been utilized to develop anticancer agents. However, cytotoxicity against normal cells at physiological pH is often an issue. Furthermore, acidic regions can be found in some normal tissues such as the kidneys. Therefore, existing approaches to cancer targeting are not fully satisfactory. In this study, we designed a peptide, HE (GIHHWLHSAHEFGEHFVHHIMNS-amide), with a charge that reverses from -1.5 at pH 7.4 to +6 at pH 5.5 for cancer targeting at low pH based on the antimicrobial peptide magainin 2 by introducing 6 His, an additional Glu, and an amidated terminal. HE interacted with cancer-mimicking negatively charged liposomes in a pH-dependent fashion with a midpoint with a pH of 6.5 just above the membrane surface. The peptide killed human renal adenocarcinoma ACHN cells at pH 6.0, but not at pH 7.4, and was nontoxic against human normal glomerular mesangial cells even at this low pH. Thus, the novel peptide may be a promising lead peptide for cancer therapy, although this derivatization resulted in weakened cytotoxicity.

Oligomerization-function relationship of EGFR on living cells detected by the coiled-coil labeling and FRET microscopy.

The epidermal growth factor receptor (EGFR) is a well-studied receptor tyrosine kinase and an important anticancer therapeutic target. The activity of EGFR autophosphorylation and transphosphorylation, which induces several cell signaling pathways, has been suggested to be related to its oligomeric state. However, the oligomeric states of EGFRs induced by EGF binding and the receptor-ligand stoichiometry required for its activation are still controversial. In the present study, we performed Förster resonance energy transfer (FRET) measurements by combining the coiled-coil tag-probe labeling method and spectral imaging to quantitatively analyze EGFR oligomerization on living CHO-K1 cell membranes at physiological expression levels. In the absence of its ligands, EGFRs mainly existed as monomers with a small fraction of predimers (~10%), whereas ~70% of the EGFRs formed dimers after being stimulated with the ligand EGF. Ligand-induced dimerization was not significantly affected by the perturbation of membrane components (cholesterol or monosialoganglioside GM3). We also investigated both dose and time dependences of EGF-dependent EGFR dimerization and autophosphorylation. The formation of dimers occurred within 20s of the ligand stimulation and preceded its autophosphorylation, which reached a plateau 90 s after the stimulation. The EGF concentration needed to evoke half-maximum dimerization (~1 nM) was lower than that for half-maximum autophosphorylation (~8 nM), which suggested the presence of an inactive dimer binding a single EGF molecule.

How do membranes initiate Alzheimer's Disease? Formation of toxic amyloid fibrils by the amyloid ß-protein on ganglioside clusters.

Alzheimer's disease (AD), a severe neurodegenerative disorder, causes more than half of dementia cases. According to the popular "Aß hypothesis" to explain the mechanism of this disease, amyloid ß-peptides (Aß) of 39-43 amino acid residues aggregate and deposit onto neurons, igniting the neurotoxic cascade of the disease. Therefore, researchers studying AD would like to elucidate the mechanisms by which essentially water-soluble but hydrophobic Aß aggregates under pathological conditions. Most researchers have investigated the aggregation of Aß in aqueous solution, and they concluded that the final aggregation product, the amyloid fibrils, were less toxic than the component peptide oligomers. They consequently shifted their interests to more toxic "soluble oligomers", structures that form as intermediates or off-pathway products during the aggregation process. Some researchers have also investigated artificial oligomers prepared under nonphysiological conditions. In contrast to these "in solution" studies, we have focused on "membrane-mediated" amyloidogenesis. In an earlier study, other researchers identified a specific form of Aß that was bound to monosialoganglioside GM1, a sugar lipid, in brains of patients who exhibited the early pathological changes associated with AD. This Account summarizes 15 years of our research on this topic. We have found that Aß specifically binds to GM1 that occurs in clusters, but not when it is uniformly distributed. Clustering is facilitated by cholesterol. Upon binding, Aß changes its conformation from a random coil to an α-helix-rich structure. A CH-π interaction between the aromatic side chains of Aß and carbohydrate moieties appended to GM1 appears to be important for binding. In addition, as Aß accumulates and reaches its first threshold concentration (Aß/GM1 = ∼0.013), aggregated ß-sheets of ∼15 molecules appear and coexist with the helical form. However, this ß-structure is stable and does not form larger aggregates. When the disease progresses further and the Aß/GM1 ratio exceeds ∼0.044, the ß-structure converts to a second ß-structure that can seed aggregates. The seed recruits monomers from the aqueous phase to form toxic amyloid fibrils that have larger surface hydrophobicity and can contain antiparallel ß-sheets. In contrast, amyloid fibrils formed in aqueous solution are less toxic and have parallel ß-sheets. The less polar environments of GM1 clusters play an important role in the formation of these toxic fibrils. Membranes that contain GM1 clusters not only accelerate the aggregation of Aß by locally concentrating Aß molecules but also generate amyloid fibrils with unique structures and significant cytotoxicity. The inhibition of this aggregation cascade could be a promising strategy for the development of AD-modulating therapies.

Evaluation of the stability of an SR398/GroES chaperonin complex.

The stability of an SR398/GroES chaperonin complex was examined. As was expected, based on the finding of previous studies, the SR398/GroES complex was extremely stable in the presence of an excess amount of free adenosine 5'-[γ-thio]triphosphate (ATPγS) or adenosine 5'-(ß,γ-imido)triphosphate (AMPPNP). However, the complex was not stable in the absence of nucleotides. These results indicate that ATPγS and AMPPNP repeatedly associated to and dissociated from the complex in a non-cooperative manner. This nucleotide exchange did not induce the dissociation of GroES and substrate from SR398, suggesting the importance of the cooperative dissociation of nucleotides from the cis-ring to release GroES and substrate proteins in the GroEL/GroES reaction cycle.

Stoichiometric analysis of oligomerization of membrane proteins on living cells using coiled-coil labeling and spectral imaging.

Many membrane proteins are proposed to work as oligomers; however, the conclusion is sometimes controversial, as for ß2-adorenergic receptor (ß2AR), which is one of the best-studied family A G-protein-coupled receptors. This is due to the lack of methods for easy and precise detection of the oligomeric state of membrane proteins on living cells. Here, we show that a combination of the coiled-coil tag-probe labeling method and spectral imaging enable a stoichiometric analysis of the oligomeric state of membrane proteins on living cells using monomeric, dimeric, and tetrameric standard membrane proteins. Using this method, we found that ß2ARs do not form constitutive homooligomers, while they exhibit their functions such as the cyclic adenosine 5'-monophosphate (cAMP) signaling and internalization upon agonist stimulation.

Interaction of antimicrobial peptide magainin 2 with gangliosides as a target for human cell binding.

Understanding how antimicrobial peptides (AMPs) interact with human cells is important to the development of antimicrobial agents as well as anticancer drugs. However, little is known about the mechanisms by which AMPs bind to cells and exert cytotoxicity. Negatively charged gangliosides on the cell surface are a potential target for cell binding. In this study, we investigated the interaction of F5W-magainin 2 (MG) with gangliosides in detail. MG was colocalized with gangliosides on HeLa cells, indicating that gangliosides act as a receptor for MG. MG also bound to gangliosides in model membranes. The affinity increased with the number of negatively charged sialic acid residues. Physicochemical studies revealed that MG interacts with the monosialoganglioside GM1 differently from the typical bacterial anionic phospholipid phosphatidylglycerol. MG bound to GM1 more strongly than to phosphatidylglycerol, and the binding isotherm for GM1 could be analyzed by the Langmuir equation assuming charge neutralization. This is in contrast to the binding of AMPs to phosphatidylglycerol-containing bilayers, which has been described by the electrostatic attraction-surface partitioning model. Fluorescence resonance energy transfer experiments supported the clustering of GM1, but not phosphatidylglycerol, by MG. Quenching data suggested that MG is bound to the sugar region of GM1. The bound peptide assumed a helical structure and induced the leakage of calcein and the coupled flip-flop of lipids, indicating the peptide also forms a toroidal pore in GM1-containing vesicles. However, the membrane permeabilizing activity was weaker against GM1-containing membranes than phosphatidylglycerol-doped liposomes in accordance with the trapping of the peptide in the sugar region. These results shed light on AMP-human cell interaction.

GM1 cluster mediates formation of toxic Aß fibrils by providing hydrophobic environments.

The conversion of soluble, nontoxic amyloid ß-proteins (Aß) to aggregated, toxic forms rich in ß-sheets is considered to be a key step in the development of Alzheimer's disease. Accumulating evidence suggests that lipid-protein interactions play a crucial role in the aggregation of amyloidogenic proteins like Aß. Our group has previously reported that amyloid fibrils of Aß formed on membranes containing clusters of GM1 ganglioside (M-fibrils) exhibit greater cytotoxicity than fibrils formed in aqueous solution (W-fibrils) [ Okada ( 2008 ) J. Mol. Biol. 382 , 1066 - 1074 ]. W-fibrils are considered to consist of in-register parallel ß-sheets. However, the precise molecular structure of M-fibrils and force driving the formation of toxic fibrils remain unclear. In this study, we hypothesized that low-polarity environments provided by GM1 clusters drive the formation of toxic fibrils and compared the structure and cytotoxicity of W-fibrils, M-fibrils, and aggregates formed in a low-polarity solution mimicking membrane environments. First, we determined solvent conditions which mimic the polarity of raftlike membranes using Aß-(1-40) labeled with the 7-diethylaminocoumarin-3-carbonyl dye. The polarity of a mixture of 80% 1,4-dioxane and 20% water (v/v) was found to be close to that of raftlike membranes. Aß-(1-40) formed amyloid fibrils within several hours in 80% dioxane (D-fibrils) or in the presence of raftlike membranes, whereas a much longer incubation time was required for fibril formation in a conventional buffer. D-fibrils were morphologically similar to M-fibrils. Fourier-transform infrared spectroscopy suggested that M-fibrils and D-fibrils contained antiparallel ß-sheets. These fibrils had greater surface hydrophobicity and exhibited significant toxicity against human neuroblastoma SH-SY5Y cells, whereas W-fibrils with less surface hydrophobicity were not cytotoxic. We concluded that ganglioside clusters mediate the formation of toxic amyloid fibrils of Aß with an antiparallel ß-sheet structure by providing less polar environments.

Improvement of probe peptides for coiled-coil labeling by introducing phosphoserines.

We have developed a method of rapidly labeling membrane proteins in living cells using a high-affinity heterodimeric coiled-coil construct containing an E3 tag (EIAALEK)(3) genetically fused to the target protein and a K4 probe (KIAALKE)(4) labeled with a fluorophore such as tetramethylrhodamine (TMR) at its N-terminus (TMR-K4). However, coiled-coil labeling cannot be applied to highly negatively charged cell lines such as HEK293, because of the nonspecific adsorption of the positively charged K4 probes to cell membranes. To reduce the net positive charge, we synthesized new probes that include phosphoserine residues (pSer) between the K4 sequence and TMR fluorophore (TMR-(pSer)(n)-K4, [n = 1-3]). The affinity of the pSer-introduced probes was comparable to that of the TMR-K4 probe. However, the TMR-(pSer)(2)-K4 and TMR-(pSer)(3)-K4 probes tended to aggregate during labeling. In contrast, TMR-pSer-K4, which was as soluble as TMR-K4, achieved higher signal/background ratios (30-100) for four host cell lines (HEK293, HeLa, SH-SY5Y, and PC12) than did TMR-K4 (~10 for HEK293 cells), demonstrating that the improved probe can be used for various types of cells.

Paradoxical downregulation of CXC chemokine receptor 4 induced by polyphemusin II-derived antagonists.

CXC chemokine receptor 4 (CXCR4) is a G protein-coupled receptor implicated in cell entry of T-cell line-tropic HIV-1 strains. CXCR4 and its ligand stromal cell derived factor-1 (SDF-1)/CXCL12 play pivotal parts in many physiological processes and pathogenetic conditions (e.g., immune cell-homing and cancer metastasis). We previously developed the potent CXCR4 antagonist T140 from structure-activity relationship studies of the antimicrobial peptide polyphemusin II. T140 and its derivatives have been exploited in biological and biomedical studies for the SDF-1/CXCR4 axis. We investigated receptor localization upon ligand stimulation using fluorescent SDF-1 and T140 derivatives as well as a specific labeling technique for cellular-membrane CXCR4. Fluorescent T140 derivatives induced translocation of CXCR4 into the perinuclear region as observed by treatment with fluorescent SDF-1. T140 derivative-mediated internalization of CXCR4 was also monitored by the coiled-coil tag-probe system. These findings demonstrated that the CXCR4 antagonistic activity and anti-HIV activity of T140 derivatives were derived (at least in part) from antagonist-mediated receptor internalization.

Coiled-coil tag-probe labeling methods for live-cell imaging of membrane receptors.

Tag-probe labeling methods have advantages over conventional fusion with fluorescent proteins in terms of smaller labels, surface specificity, availability of pulse labeling, and ease of multicolor labeling. With this method, the gene of the target protein is fused with a short tag sequence, expressed in cells, and the protein is labeled with exogenous fluorescent probes that specifically bind to the tag. Various labeling principles, such as protein-ligand interaction, peptide-peptide interaction, peptide-metal interaction, and enzymatic reactions, have been applied to the tag-probe labeling of membrane receptors. We describe our coiled-coil tag-probe method in detail, including the design and synthesis of the tag and probe, labeling procedures, and observations by confocal microscopy. Applications to the analysis of receptor internalization and oligomerization are also introduced.

Fluorescence ratiometric detection of ligand-induced receptor internalization using extracellular coiled-coil tag-probe labeling.

We report a new method for the detection of ligand-induced receptor internalization by fluorescence ratiometric imaging of pH in endosomes in combination with a recently developed posttranslational labeling system based on the formation of a heterodimeric coiled-coil structure. The N-terminus of the ß2-adrenergic receptor expressed on the cell surface was doubly labeled with pH-sensitive fluorescein and pH-insensitive tetramethylrhodamine. A significant increase in the tetramethylrhodamine-to-fluorescein fluorescence intensity ratio was observed after incubation with agonists in a concentration-dependent manner. This simple and accurate method of detecting the agonistic activity of receptors will be useful for high-throughput screening of drug candidates.

Ganglioside-mediated aggregation of amyloid ß-proteins (Aß): comparison between Aß-(1-42) and Aß-(1-40).

Conversion of the soluble, non-toxic amyloid ß-protein (Aß) into an aggregated, toxic form rich in ß-sheets is considered a key step in the development of Alzheimer's disease. Accumulating evidence suggests that lipid rafts in membranes play a pivotal role in this process. We have proposed that Aß-(1-40) specifically bound to a ganglioside cluster forms cytotoxic fibrils via a conformational transition from an α-helix-rich structure to a ß-sheet-rich one. In the present study, we compared the interaction of Aß-(1-40) and Aß-(1-42) with both model and living cell membranes. Aß-(1-42) exhibited lipid specificity and affinity similar to Aß-(1-40), though its amyloidogenic activity was more than 10-fold that of Aß-(1-40). Antibody staining experiments, using the A11 antibody specific to Aß oligomers, demonstrated that oligomers were not detected during the aggregation process, and cell death was observed only after significant accumulation of the proteins, suggesting that the fibril-induced disruption of cell membranes leads to the cytotoxicity. Furthermore, we succeeded in visualizing fibrils formed on cell membranes using total internal reflection fluorescence microscopy. Aß-(1-40) formed long fibrils extruding to the aqueous phase, whereas Aß-(1-42) fibrils appeared to be laterally co-assembled and short.

A novel hybrid peptide targeting EGFR-expressing cancers.

Several potential molecular-targeted anticancer drugs focus on the inhibition of receptor tyrosine kinase and tumour growth, but these tyrosine kinase inhibitors (TKI) have been reported that the mutations of kinase-related signal molecule genes in cancer cells lead to the drug resistance. To overcome this issue, we have designed a novel targeting anticancer 'hybrid-peptide' EGFR-lytic peptide, in which epidermal growth factor receptor (EGFR) binding peptide is conjugated with a newly designed lytic-type peptide containing cationic-rich amino acids that disintegrates the cell membrane to kill cancer cells. In this report, cytotoxic activity of EGFR-lytic peptide was investigated in various human cancer and normal cell lines. It was found that the resulting conformational change in the novel lytic peptide enabled it to bind selectively to the membrane of cancer cells, and due to its acquired synergistic action, hybrid peptide demonstrated selective destruction of cancer cells as swiftly as 10 min after exposure. Treatment with EGFR-lytic peptide exerted a sufficient in vitro cytotoxic activity against TKI-resistant cancer cells with K-ras mutations. Moreover, in vivo analyses revealed that this peptide displayed significant antitumour activity in mouse xenograft models of both human K-ras mutation negative and positive cancers. Thus, hybrid peptide can be a unique and powerful tool for a new cancer-targeted therapy.