Generation of novel anti-apoE monoclonal antibodies that selectively recognize apoE isoforms.

Apolipoprotein E (apoE) is a regulator of lipid metabolism, cholesterol transport, and the clearance and aggregation of amyloid ß in the brain. The three human apoE isoforms apoE2, apoE3, and apoE4 only differ in one or two residues. Nevertheless, the functions highly depend on the isoform types and lipidated states. Here, we generated novel anti-apoE monoclonal antibodies (mAbs) and obtained an apoE4-selective mAb whose epitope is within residues 110-117. ELISA and bio-layer interferometry measurements demonstrated that the dissociation constants of mAbs are within the nanomolar range. Using the generated antibodies, we successfully constructed sandwich ELISA systems, which can detect all apoE isoforms or selectively detect apoE4. These results suggest the usability of the generated anti-apoE mAbs for selective detection of apoE isoforms.

Host-to-graft propagation of inoculated α-synuclein into transplanted human induced pluripotent stem cell-derived midbrain dopaminergic neurons.

Introduction: Cell therapeutic clinical trials using fetal mesencephalic tissue provided a proof-of-concept for regenerative therapy in patients with Parkinson's disease. Postmortem studies of patients with fetal grafts revealed that α-synuclein+ Lewy body (LB)-like inclusions emerged in long-term transplantation and might worsen clinical outcomes even if the grafts survived and innervated in the recipients. Various studies aimed at addressing whether host-derived α-synuclein could be transferred to the grafted neurons to assess α-synuclein+ inclusion appearance in the grafts. However, determining whether α-synuclein in the grafted neurons has been propagated from the host is difficult due to the intrinsic α-synuclein expression. Methods: We induced midbrain dopaminergic (mDA) neurons from human induced pluripotent stem cells (hiPSCs) and transplanted them into the striatum of immunodeficient rats. The recombinant human α-synuclein preformed fibrils (PFFs) were inoculated into the cerebral cortex after transplantation of SNCA-/- hiPSC-derived mDA neural progenitors into the striatum of immunodeficient rats to evaluate the host-to-graft propagation of human α-synuclein PFFs. Additionally, we examined the incorporation of human α-synuclein PFFs into SNCA-/- hiPSC-derived mDA neurons using in vitro culture system. Results: We detected human α-synuclein-immunoreactivity in SNCA-/- hiPSC-derived mDA neurons that lacked endogenous α-synuclein expression in vitro. Additionally, we observed host-to-graft α-synuclein propagation into the grafted SNCA-/- hiPSC-derived mDA neurons. Conclusion: We have successfully proven that intracerebral inoculated α-synuclein PFFs are propagated and incorporated from the host into grafted SNCA-/- hiPSC-derived mDA neurons. Our results contribute toward the basic understanding of the molecular mechanisms related to LB-like α-synuclein deposit formation in grafted mDA neurons.

Structural flexibility of apolipoprotein E-derived arginine-rich peptides improves their cell penetration capability.

Amphipathic arginine-rich peptide, A2-17, exhibits moderate perturbation of lipid membranes and the highest cell penetration among its structural isomers. We investigated the direct cell-membrane penetration mechanism of the A2-17 peptide while focusing on structural flexibility. We designed conformationally constrained versions of A2-17, stapled (StpA2-17) and stitched (StchA2-17), whose α-helical conformations were stabilized by chemical crosslinking. Circular dichroism confirmed that StpA2-17 and StchA2-17 had higher α-helix content than A2-17 in aqueous solution. Upon liposome binding, only A2-17 exhibited a coil-to-helix transition. Confocal microscopy revealed that A2-17 had higher cell penetration efficiency than StpA2-17, whereas StchA2-17 remained on the cell membrane without cell penetration. Although the tryptophan fluorescence analysis suggested that A2-17 and its analogs had similar membrane-insertion positions between the interface and hydrophobic core, StchA2-17 exhibited a higher membrane affinity than A2-17 or StpA2-17. Atomic force microscopy demonstrated that A2-17 reduced the mechanical rigidity of liposomes to a greater extent than StpA2-17 and StchA2-17. Finally, electrophysiological analysis showed that A2-17 induced a higher charge influx through transient pores in a planer lipid bilayer than StpA2-17 and StchA2-17. These findings indicate that structural flexibility, which enables diverse conformations of A2-17, leads to a membrane perturbation mode that contributes to cell membrane penetration.

Amyloidogenic 60-71 deletion/ValThr insertion mutation of apolipoprotein A-I generates a new aggregation-prone segment that promotes nucleation through entropic effects.

The N-terminal fragment of apolipoprotein A-I (apoA-I), comprising residues 1-83, contains three segments prone to aggregation: residues 14-22, 53-58, and 67-72. We previously demonstrated that residues 14-22 are critical in apoA-I fibril formation while residues 53-58 entropically drove the nucleation process. Here, we investigated the impact of amyloidogenic mutations (Δ60-71/VT, Δ70-72, and F71Y) located around residues 67-72 on fibril formation by the apoA-I 1-83 fragment. Thioflavin T fluorescence assay demonstrated that the Δ60-71/VT mutation significantly enhances both nucleation and fibril elongation rates, whereas the Δ70-72 and F71Y mutations had minimal effects. Circular dichroism measurements and microscopic observations revealed that all variant fragments formed straight fibrils, transitioning from random coils to ß-sheet structures. Kinetic analysis demonstrated that primary nucleation is the dominant step in fibril formation, with fibril elongation reaching saturation at high protein concentrations. Thermodynamically, both nucleation and fibril elongation were enthalpically and entropically unfavorable in all apoA-I 1-83 variants, in which the entropic barrier of nucleation was almost eliminated for the Δ60-71/VT variant. Taken together, our results suggest the presence of new aggregation-prone segment in the Δ60-71/VT variant that promotes nucleation through entropic effects.

Intramolecular interaction kinetically regulates fibril formation by human and mouse α-synuclein.

Regulation of α-synuclein (αS) fibril formation is a potent therapeutic strategy for αS-related neurodegenerative disorders. αS, an intrinsically disordered 140-residue intraneural protein, comprises positively charged N-terminal, hydrophobic non-amyloid ß component (NAC), and negatively charged C-terminal regions. Although mouse and human αS share 95% sequence identity, mouse αS forms amyloid fibrils faster than human αS. To evaluate the kinetic regulation of αS fibrillation, we examined the effects of mismatched residues in human and mouse αS on fibril formation and intramolecular interactions. Thioflavin T fluorescence assay using domain-swapped or C-terminal-truncated αS variants revealed that mouse αS exhibited higher nucleation and fibril elongation than human αS. In mouse αS, S87N substitution in the NAC region rather than A53T substitution is dominant for enhanced fibril formation. FÓ§rester resonance energy transfer analysis demonstrated that the intramolecular interaction of the C-terminal region with the N-terminal and NAC regions observed in human αS is perturbed in mouse αS. In mouse αS, S87N substitution is responsible for the perturbed interaction. These results indicate that the interaction of the C-terminal region with the N-terminal and NAC regions suppresses αS fibril formation and that the human-to-mouse S87N substitution in the NAC region accelerates αS fibril formation by perturbing intramolecular interaction.

Plantainoside B in Bacopa monniera Binds to Aß Aggregates Attenuating Neuronal Damage and Memory Deficits Induced by Aß.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by dementia. The most characteristic pathological changes in AD brain include extracellular amyloid-ß (Aß) accumulation and neuronal loss. Particularly, cholinergic neurons in the nucleus basalis of Meynert are some of the first neuronal groups to degenerate; accumulating evidence suggests that Aß oligomers are the primary form of neurotoxicity. Bacopa monniera is a traditional Indian memory enhancer whose extract has shown neuroprotective and Aß-reducing effects. In this study, we explored the low molecular weight compounds from B. monniera extracts with an affinity to Aß aggregates, including its oligomers, using Aß oligomer-conjugated beads and identified plantainoside B. Plantainoside B exhibited evident neuroprotective effects by preventing Aß attachment on the cell surface of human induced pluripotent stem cell (hiPSC)-derived cholinergic neurons. Moreover, it attenuated memory impairment in mice that received intrahippocampal Aß injections. Furthermore, radioisotope experiments revealed that plantainoside B has affinity to Aß aggregates including its oligomers and brain tissue from a mouse model of Aß pathology. In addition, plantainoside B could delay the Aß aggregation rate. Accordingly, plantainoside B may exert neuroprotective effects by binding to Aß oligomers, thus interrupting the binding of Aß oligomers to the cell surface. This suggests its potential application as a theranostics in AD, simultaneously diagnostic and therapeutic drugs.

Analysis of the interaction of cyclosporine congeners with cell membrane models.

Owing to the relatively high molecular weight of macrocyclic peptides, investigation of the cellular uptake mechanism is required for the efficient design of macrocyclic peptides as potential drugs. We have previously reported, using HPLC, that cyclosporine A, a model macrocyclic peptide, and its congeners B, C, and D had different lipophilicity despite differing by only one amino acid. In the present study, we investigated how this difference in lipophilicity affected the interaction of the congeners with cell membranes. The circular dichroism spectra showed that the secondary structures were similar between the four congeners even at high temperature. The molar ellipticity of the four congeners in the presence of liposomes, as a cell membrane model, differed, and cyclosporines D and A showed lower molar ellipticity, while cyclosporine C exhibited higher molar ellipticity. Fluorescent spectra analysis using Laurdan indicated that liposome hydration was decreased in the presence of the cyclosporines, especially cyclosporines D and A. HPLC analysis also quantitatively showed that the amount of cyclosporine molecules internalized in HpG2 cells was the largest for cyclosporine D. We determined, using spectroscopy and HPLC, that the intensity of the interaction of the congeners with cell membranes was overall correlated with the lipophilicity derived from the side chains of each congener. Our results will contribute to the design of new macrocyclic peptides with favorable drug properties.

Design and Synthesis of 6-O-Phosphorylated Heparan Sulfate Oligosaccharides to Inhibit Amyloid ß Aggregation.

Dysregulation of amyloidogenic proteins and their abnormal processing and deposition in tissues cause systemic and localized amyloidosis. Formation of amyloid ß (Aß) fibrils that deposit as amyloid plaques in Alzheimer's disease (AD) brains is an earliest pathological hallmark. The polysulfated heparan sulfate (HS)/heparin (HP) is one of the non-protein components of Aß deposits that not only modulates Aß aggregation, but also acts as a receptor for Aß fibrils to mediate their cytotoxicity. Interfering with the interaction between HS/HP and Aß could be a therapeutic strategy to arrest amyloidosis. Here we have synthesized the 6-O-phosphorylated HS/HP oligosaccharides and reported their competitive effects on the inhibition of HP-mediated Aß fibril formation in vitro using a thioflavin T fluorescence assay and a tapping mode atomic force microscopy.

Current Status and Challenges of Analytical Methods for Evaluation of Size and Surface Modification of Nanoparticle-Based Drug Formulations.

The present review discusses the current status and difficulties of the analytical methods used to evaluate size and surface modifications of nanoparticle-based pharmaceutical products (NPs) such as liposomal drugs and new SARS-CoV-2 vaccines. We identified the challenges in the development of methods for (1) measurement of a wide range of solid-state NPs, (2) evaluation of the sizes of polydisperse NPs, and (3) measurement of non-spherical NPs. Although a few methods have been established to analyze surface modifications of NPs, the feasibility of their application to NPs is unknown. The present review also examined the trends in standardization required to validate the size and surface measurements of NPs. It was determined that there is a lack of available reference materials and it is difficult to select appropriate ones for modified NP surface characterization. Research and development are in progress on innovative surface-modified NP-based cancer and gene therapies targeting cells, tissues, and organs. Next-generation nanomedicine should compile studies on the practice and standardization of the measurement methods for NPs to design surface modifications and ensure the quality of NPs.

Mechanisms of enhanced aggregation and fibril formation of Parkinson's disease-related variants of α-synuclein.

Aggregation of α-synuclein (α-syn) into amyloid fibrils is closely associated with Parkinson's disease (PD). Familial mutations or posttranslational truncations in α-syn are known as risk factor for PD. Here, we examined the effects of the PD-related A30P or A53T point mutation and C-terminal 123-140 or 104-140 truncation on the aggregating property of α-syn based on the kinetic and thermodynamic analyses. Thioflavin T fluorescence measurements indicated that A53T, Δ123‒140, and Δ104-140 variants aggregated faster than WT α-syn, in which the A53T mutation markedly increases nucleation rate whereas the Δ123‒140 or Δ104‒140 truncation significantly increases both nucleation and fibril elongation rates. Ultracentrifugation and western blotting analyses demonstrated that these mutations or truncations promote the conversion of monomer to aggregated forms of α-syn. Analysis of the dependence of aggregation reaction of α-syn variants on the monomer concentration suggested that the A53T mutation enhances conversion of monomers to amyloid nuclei whereas the C-terminal truncations, especially the Δ104-140, enhance autocatalytic aggregation on existing fibrils. In addition, thermodynamic analysis of the kinetics of nucleation and fibril elongation of α-syn variants indicated that both nucleation and fibril elongation of WT α-syn are enthalpically and entropically unfavorable. Interestingly, the unfavorable activation enthalpy of nucleation greatly decreases for the A53T and becomes reversed in sign for the C-terminally truncated variants. Taken together, our results indicate that the A53T mutation and the C-terminal truncation enhance α-syn aggregation by reducing unfavorable activation enthalpy of nucleation, and the C-terminal truncation further triggers the autocatalytic fibril elongation on the fibril surfaces.

Effect of hydrophobic moment on membrane interaction and cell penetration of apolipoprotein E-derived arginine-rich amphipathic α-helical peptides.

We previously developed an amphipathic arginine-rich peptide, A2-17, which has high ability to directly penetrate across cell membranes. To understand the mechanism of the efficient cell-penetrating ability of the A2-17 peptide, we designed three structural isomers of A2-17 having different values of the hydrophobic moment and compared their membrane interaction and direct cell penetration. Confocal fluorescence microscopy revealed that cell penetration efficiency of peptides tends to increase with their hydrophobic moment, in which A2-17 L14R/R15L, an A2-17 isomer with the highest hydrophobic moment, predominantly remains on plasma cell membranes. Consistently, Trp fluorescence analysis indicated the deepest insertion of A2-17 L14R/R15L into lipid membranes among all A2-17 isomers. Electrophysiological analysis showed that the duration and charge flux of peptide-induced pores in lipid membranes were prominent for A2-17 L14R/R15L, indicating the formation of stable membrane pores. Indeed, the A2-17 L14R/R15L peptide exhibited the strongest membrane damage to CHO-K1 cells. Atomic force microscopy quantitatively defined the peptide-induced membrane perturbation as the decrease in the stiffness of lipid vesicles, which was correlated with the hydrophobic moment of all A2-17 isomers. These results indicate that optimal membrane perturbation by amphipathic A2-17 peptide is critical for its efficient penetration into cells without inducing stabilized membrane pores.

Sulfated glycosaminoglycans mediate prion-like behavior of p53 aggregates.

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) are heteropolysaccharides implicated in the pathology of protein aggregation diseases including localized and systemic forms of amyloidosis. Among subdomains of sulfated GAGs, highly sulfated domains of HS, called HS S-domains, have been highlighted as being critical for HS function in amyloidoses. Recent studies suggest that the tumor suppressor p53 aggregates to form amyloid fibrils and propagates in a prion-like manner; however, molecules and mechanisms that are involved in the prion-like behavior of p53 aggregates have not been addressed. Here, we identified sulfated GAGs as molecules that mediate prion-like behavior of p53 aggregates. Sulfated GAGs at the cell surface were required for cellular uptake of recombinant and cancer cell-derived p53 aggregates and extracellular release of p53 from cancer cells. We further showed that HS S-domains accumulated within p53 deposits in human ovarian cancer tissues, and enzymatic remodeling of HS S-domains by Sulf-2 extracellular sulfatase down-regulated cellular uptake of p53 aggregates. Finally, sulfated GAG-dependent cellular uptake of p53 aggregates was critical for subsequent extracellular release of the aggregates and gain of oncogenic function in recipient cells. Our work provides a mechanism of prion-like behavior of p53 aggregates and will shed light on sulfated GAGs as a common mediator of prions.

Enhancement of direct membrane penetration of arginine-rich peptides by polyproline II helix structure.

The left-handed, extended polyproline II (PPII) helix is a unique secondary structure which potently modulates peptide/protein functions through its constraint conformation. To investigate the effect of PPII helix on the direct cell membrane penetration of arginine-rich peptides, we designed a polyproline-containing arginine-rich peptide P9R7W (PPPPPPPPPRRRRRRRW) by introducing nine proline residues into a linear R7W (RRRRRRRW) peptide. Circular dichroism spectroscopy showed that P9R7W has the PPII helix structure in solution whereas R7W is predominantly in random coil structure. Tryptophan fluorescence measurements demonstrated that P9R7W binds to negatively charged lipid vesicles with similar affinity to R7W, in which there was no significant change in the PPII helix structure. Flow cytometry and confocal laser scanning microscopy analyses showed that P9R7W has an ability to penetrate into CHO-K1 cells more efficiently compared to R7W with no cytotoxicity. Consistently, a channel current analysis unveiled that P9R7W penetrates planar lipid bilayer membranes more efficiently than R7W without significant membrane perturbation. Our results indicate that the PPII helix structure can enhance the membrane penetration efficiency of arginine-rich peptides without lipid membrane perturbation.

Phosphatidylethanolamine accelerates aggregation of the amyloidogenic N-terminal fragment of apoA-I.

Membrane lipid composition is known to influence aggregation and fibril formation of many amyloidogenic proteins. Here, we found that phosphatidylethanolamine (PE) accelerates aggregation of the N-terminal 1-83 fragment of an amyloidogenic G26R variant of apoA-I on lipid membranes. Circular dichroism and isothermal titration calorimetry measurements demonstrated that PE does not affect the α-helical structure and lipid binding property of apoA-I 1-83/G26R. Rather, fluorescence measurements indicated that PE induces more ordered lipid packing at the interfacial and acyl chain regions, providing more hydrophobic environments especially around the highly amyloidogenic regions in apoA-I on the membrane surface. These results suggest that PE promotes aggregation of the amyloidogenic N-terminal fragment of apoA-I on lipid membranes by inducing hydrophobic membrane environments.

Refining Calibration Procedures of Circular Dichroism Spectrometer to Improve Usability.

Circular dichroism (CD) is a technique used for conformational studies of peptides and proteins. We studied the specific calibration procedures of CD spectrometers based on procedures specified in the European Pharmacopoeia. We aimed to develop procedures to improve the usability of CD, in addition to reducing adverse effects on users' health. The use of ethanol instead of 1,4-dioxane as the solvent for isoandrosterone was examined. Both solvents yielded the same maximum value of +3.3 for molar CD. We also studied a two-point calibration method using (1S)-(+)-ammonium 10-camphorsulfonate instead of (1S)-(+)-10-camphorsulfonic acid, which is a hygroscopic compound. Both compounds yielded similar results and the values for (1S)-(+)-ammonium 10-camphorsulfonate of 2.39 ± 0.04 and -4.92 ± 0.06 at 290.5 and 192.5 nm, respectively, were within the criteria defined in the European Pharmacopoeia. The inter-laboratory repeatability was also acceptable. These studies provide specific procedures for calibrating CD spectrometers for drug development.

Mechanisms of aggregation and fibril formation of the amyloidogenic N-terminal fragment of apolipoprotein A-I.

The N-terminal (1-83) fragment of the major constituent of plasma high-density lipoprotein, apolipoprotein A-I (apoA-I), strongly tends to form amyloid fibrils, leading to systemic amyloidosis. Here, using a series of deletion variants, we examined the roles of two major amyloidogenic segments (residues 14-22 and 50-58) in the aggregation and fibril formation of an amyloidogenic G26R variant of the apoA-I 1-83 fragment (apoA-I 1-83/G26R). Thioflavin T fluorescence assays and atomic force microscopy revealed that elimination of residues 14-22 completely inhibits fibril formation of apoA-I 1-83/G26R, whereas Δ32-40 and Δ50-58 variants formed fibrils with markedly reduced nucleation and fibril growth rates. CD measurements revealed structural transitions from random coil to ß-sheet structures in all deletion variants except for the Δ14-22 variant, indicating that residues 14-22 are critical for the ß-transition and fibril formation. Thermodynamic analysis of the kinetics of fibril formation by apoA-I 1-83/G26R indicated that both nucleation and fibril growth are enthalpically unfavorable, whereas entropically, nucleation is favorable, but fibril growth is unfavorable. Interestingly, the nucleation of the Δ50-58 variant was entropically unfavorable, indicating that residues 50-58 entropically promote the nucleation step in fibril formation of apoA-I 1-83/G26R. Moreover, a residue-level structural investigation of apoA-I 1-83/G26R fibrils with site-specific pyrene labeling indicated that the two amyloidogenic segments are in close proximity to form an amyloid core structure, whereas the N- and C-terminal tail regions are excluded from the amyloid core. These results provide critical insights into the aggregation mechanism and fibril structure of the amyloidogenic N-terminal fragment of apoA-I.

Biophysical Mechanism of Protein Export by Bacterial Type III Secretion System.

Type III secretion system (T3SS) is a protein injection nano-machine consisting of syringe and needle-like structure spanning bacterial inner and outer membranes. Bacteria insert the tip of T3SS needle to host cell membranes, and deliver effector proteins directly into host cells via T3SS to prime the host cell environment for infection. Thus inhibition of T3SS would be a potent strategy for suppressing bacterial infection. We previously demonstrated that T3SS needle rotates by proton-motive force (PMF) with the same mechanism as two evolutionally related rotary protein motors, flagellum and ATP synthase (FASEB J., 27, 2013, Ohgita et al.). Inhibition of needle rotation resulted in suppression of effector secretion, indicating the requirement of needle rotation for effector export. Simulation analysis of protein export by the T3SS needle suggests the importance of a hydrophobic helical groove formed by the conserved aromatic residue in the needle components. Based on these results, we have proposed a novel model of protein export by the T3SS needle, in which effector proteins are exported by PMF-dependent needle rotation oppositely to the hydrophobic helical groove in the needle. Quantitative examinations of the correlation between the speeds of T3SS rotation and the amount of effector export support this model. In this review, we summarize our current understanding of T3SS, and discuss our novel model of the protein export mechanism of T3SS based on the needle rotation.

The Accumulation of Heparan Sulfate S-Domains in Kidney Transthyretin Deposits Accelerates Fibril Formation and Promotes Cytotoxicity.

The highly sulfated domains of heparan sulfate (HS), alias HS S-domains, are made up of repeated trisulfated disaccharide units [iduronic acid (2S)-glucosamine (NS, 6S)] and are selectively remodeled by extracellular endoglucosamine 6-sulfatases (Sulfs). Although HS S-domains are critical for signal transduction of several growth factors, their roles in amyloidoses are not yet fully understood. Herein, we found HS S-domains in the kidney of a patient with transthyretin amyloidosis. In in vitro assays with cells stably expressing human Sulfs, heparin, a structural analog of HS S-domains, promoted aggregation of transthyretin in an HS S-domain-dependent manner. Interactions of cells with transthyretin fibrils and cytotoxicity of these fibrils also depended on HS S-domains at the cell surface. Furthermore, glypican-5, encoded by the susceptibility gene for nephrotic syndrome GPC5, was found to be accumulated in the transthyretin amyloidosis kidney. Our study, thus, provides a novel insight into the pathologic roles of HS S-domains in amyloidoses, and we propose that enzymatic remodeling of HS chains by Sulfs may offer an effective approach to inhibiting formation and cytotoxicity of amyloid fibrils.

A novel amphipathic cell-penetrating peptide based on the N-terminal glycosaminoglycan binding region of human apolipoprotein E.

In the direct cell membrane penetration, arginine-rich cell-penetrating peptides are thought to penetrate into cells across the hydrophobic lipid membranes. To investigate the effect of the amphipathic property of arginine-rich peptide on the cell-penetrating ability, we designed a novel amphipathic cell-penetrating peptide, A2-17, and its derivative, A2-17KR, in which all lysine residues are substituted with arginine residues, based on the glycosaminoglycan binding region in the N-terminal α-helix bundle of human apolipoprotein E. Isothermal titration calorimetry showed that A2-17 variants have a strong ability to bind to heparin with high affinity. Circular dichroism and tryptophan fluorescence measurements demonstrated that A2-17 variants bind to lipid vesicles with a structural change from random coil to amphipathic α-helix, being inserted into the hydrophobic membrane interiors. Flow cytometric analysis and confocal laser scanning microscopy demonstrated the great cell penetration efficiency of A2-17 variants into CHO-K1 cells when incubated at low peptide concentrations (2 µM or less), suggesting that the increased amphipathicity with α-helix formation enhances the cell membrane penetration ability of arginine-rich peptides. Interestingly, A2-17KR exhibited lower efficiency of cell membrane penetration compared to A2-17 despite of their similar binding affinity to lipid membranes. Since high peptide concentrations (typically >10 µM) are usually prerequisite for efficient cell penetration of arginine-rich peptides, A2-17 is a unique amphipathic cell-penetrating peptide that exhibits an efficient cell penetration ability even at low peptide concentrations.

Effect of Phosphatidylserine and Cholesterol on Membrane-mediated Fibril Formation by the N-terminal Amyloidogenic Fragment of Apolipoprotein A-I.

Here, we examined the effects of phosphatidylserine (PS) and cholesterol on the fibril-forming properties of the N-terminal 1‒83 fragment of an amyloidogenic G26R variant of apoA-I bound to small unilamellar vesicles. A thioflavin T fluorescence assay together with microscopic observations showed that PS significantly retards the nucleation step in fibril formation by apoA-I 1‒83/G26R, whereas cholesterol slightly enhances fibril formation. Circular dichroism analyses demonstrated that PS facilitates a structural transition from random coil to α-helix in apoA-I 1‒83/G26R with great stabilization of the α-helical structure upon lipid binding. Isothermal titration calorimetry measurements revealed that PS induces a marked increase in capacity for binding of apoA-I 1‒83/G26R to the membrane surface, perhaps due to electrostatic interactions of positively charged amino acids in apoA-I with PS. Such effects of PS to enhance lipid interactions and inhibit fibril formation of apoA-I were also observed for the amyloidogenic region-containing apoA-I 8‒33/G26R peptide. Fluorescence measurements using environment-sensitive probes indicated that PS induces a more solvent-exposed, membrane-bound conformation in the amyloidogenic region of apoA-I without affecting membrane fluidity. Since cell membranes have highly heterogeneous lipid compositions, our findings may provide a molecular basis for the preferential deposition of apoA-I amyloid fibrils in tissues and organs.