The lipid raft microenvironment is implicated in the generation of the pathological amyloid-β (Aβ) species in amyloid precursor protein (APP) that is associated with neurodegenerative diseases. Evidence shows that APP forms a transmembrane homodimer with changeable structures as a function of the membrane compositions. However, the molecular responsibility of the dimerization and structural alteration for the amyloidogenic process in segregated membranes remains largely unclear. Here, we performed multiple coarse grained (CG) simulations to explore the behavioral preference of the transmembrane domain of APP (called C99) that is affected by the lipid raft microenvironment. The results showed that C99 was anchored at the boundary of the lipid raft relying on the conserved hydrophobic motif of V710xxA713xxxV717xxxV721. Moreover, the dimerization of C99 was greatly destabilized by the lipid raft, which led to a susceptible switching packing conformation. The molecular driving forces were derived from the combined regulation of the saturated lipids and cholesterols rather than from the simple binding competition of cholesterol in the C99 dimerization. The molecular details of the differential dimerization in the raft-forming and bulk fluid bilayer environments were compared, and the structural information was helpful for further understanding the enzymolysis responsiveness of APP.

Cytolytic peptides (CPs) have long been employed as broad-spectrum antibiotic agents to overcome multidrug resistance. However, the development of novel peptide drugs is still limited by the elusive molecular understanding of the membrane-lysis mechanism and modeling of CPs, especially of the short helical species. In this study, a known anticancer CP named PTP-7b (FLGALFKALSHLL) in disrupting membranes via self-assembling approach was studied by combining experiments and time-extended coarse-grained dynamic simulations. Effective membrane disintegration was induced by aggregation of the membrane-bound peptide individuals, rather than the preassembled peptide clusters. The disturbance level of lipid bilayers depended on the peptide concentrations, which was responsible for the long time-costing of PTP-7b in killing cells. On the basis of lines of simulations and energy-landscape calculations, the dynamics of membrane deformation evolving toward preliminary leakage resulted from the aggregated PTP-7b was demonstrated, which was subjected to the spatiotemporal cooperation of the membrane-inserted and the periplasmic peptides. The molecular mechanism incorporated the 11th histidine interaction coupled with the peptide amphiphilicity in accelerating phospholipid migration outward. This study revealed elaborate modeling and dynamics information about the short helical CPs in membrane lysis, which would be helpful to understand the underlying mechanisms and rational design of CPs for drug application.

Many lytic peptides contain a heptad sequence with leucine or isoleucine residues at “a” and “d” positions. However, their roles in the peptide-induced cytolytic process remain unclear. We have recently reported an anticancer lytic peptide ZXR-2 (FKIGGFIKKLWRSLLA), which contains a shortened zipper-like sequence with Ile/Leu at “a” and “d” positions. To understand the roles of these Ile/Leu residues, a series of analogs were constructed by sequentially replacing the Ile or Leu residue with alanine (Ala). Significant reduction of the cytolytic activity was observed when the Ile (3rd and 7th) and Leu (10th and 14th) residues at the “a” and “d” positions were substituted, while the replacement of the separate Leu (15th) residue had less effect. Based on the quenching of the intrinsic fluorescence of the peptides and their induced surface pressure changes of lipid monolayer, it was conjectured that the peptide ZXR-2 might insert into cell membranes from the C-terminal and to a depth of the W11 position. Accordingly, I3, I7, and L10 residues which mainly exposed in aqueous solution were more responsible for the peptide self-association on cell membranes, while L14, together with L15, might help peptide insert and anchor to cell membranes. These results are significant to elucidate the crucial roles of such Ile/Leu residues at “a” and “d” positions in peptide–peptide and peptide–membrane interactions to exert the membrane disruption activity of lytic peptides. With further understanding about the structure–activity relationship of lytic peptides, it would be helpful for designing novel anticancer lytic peptides.

Peptides are considered as a new generation of drugs due to their high structural and functional diversity. However, the development of peptide drugs is always limited by their poor stability and short circulation time. Carriers are applied for peptide drug delivery, but there may be problems like poor loading efficiency and undesired xenobiotic toxicity. Peptide self-assembly is an effective approach to improve the stability and control the release of peptide drugs. In this study, two self-assembling anticancer peptides are designed by appending a pair of glutamic acid and asparagine to either the N-terminus or the C-terminus of a lytic peptide. This simple, yet rational sequence modification was made to change the amphiphilic pattern and secondary structural content of the parent peptide, thereby modulating its self-assembly process. It was found that the N-terminus modified peptide favors the formation of nanofibrils and the peptide with C-terminal modification formed micelles. Although both nanostructures showed prolonged action profiles and improved serum stability compared to the parent peptide, the morphology of the nanostructures is highly critical to manipulate the release profile of the free peptide from the assembly and regulate their bioactivity. We believe the self-assembly approach demonstrated in this study can be applied to a variety of therapeutic peptide drugs to improve their stability and therapeutic activity for the development of carrier-free drug delivery system.Download high-res image (139KB)Download full-size image

•The activity of AMP ZXR-2 against oral facultative anaerobic bacteria was tested.•ZXR-2 showed fast-killing activity and limited hemolytic activity.•ZXR-2 prevented the formation of S. mutans biofilms.Dental caries, a highly prevalent oral disease, is primarily caused by pathogenic bacteria infection, and most of them are anaerobic. Herein, we investigated the activity of a designed antimicrobial peptide ZXR-2, and found it showed broad-spectrum activity against a variety of Gram-positive and Gram-negative oral bacteria, particularly the caries-related taxa Streptococcus mutans. Time-course killing assays indicated that ZXR-2 killed most bacterial cells within 5 min at 4 × MIC. The mechanism of ZXR-2 involved disruption of cell membranes, as observed by scanning electron microscopy. Moreover, ZXR-2 inhibited the formation of S. mutans biofilm, but showed limited hemolytic effect. Based on its potent antimicrobial activity, rapid killing, and inhibition of S. mutans biofilm formation, ZXR-2 represents a potential therapeutic for the prevention and treatment of dental caries.

ε-Poly-L-lysine (ε-PL) is an unusual, naturally occurring homo-polyamide made of 25–35 L-lysines with antimicrobial activities. ε-PL is widely used in food and medical fields because of its high levels of safety and biodegradability. In our studies, the effects of different fermentation methods on ε-PL biosynthesis by Streptomyces sp. DES20 were determined. Without any control, 2.637 g L−1 ε-PL was produced in a 5 L fermentation tank in 120 h. When feeding glucose and ammonium sulfate during fermentation, the production yield increased to 6.687 g L−1. After adding 2 g L−1 sodium citrate, the ε-PL yield further increased to 8.351 g L−1 at the end of fermentation. The weak acid anion-exchange resin FPC3500 was used to separate and extract ε-PL from the fermentation liquid. ε-PL was eluted with NH4OH with 85.40% purity at a recovery rate of 80.99%. Whereas through hydrochloric acid elution, the recovery rate of ε-PL increased to 89.23% with 95.26% purity. The degree of polymerization was identified as 26–33 using MALDI-TOF-MS. The ε-PL structure was characterized by ultraviolet spectroscopy, infrared spectroscopy, 1H NMR, 13C NMR, and HMBC. ε-PL exhibits significant antimicrobial activities against four different kinds of bacteria.

Saponins and flavonoids are the main bioactive ingredients in Camellia oleifera Abel. In this study, the fragmentation pathways of triterpene saponins in Camellia oleifera were investigated and confirmed by electrospray ionization linear ion trap-orbitrap (LTQ-orbitrap), and a new strategy for rapid characterization of saponins by ultra-high-pressure liquid chromatography with LTQ-orbitrap mass spectrometry (UHPLC-LTQ-orbitrap-MSn) was developed. Based on the summarized fragmentation rules, 36 triterpene saponins from the testa of C. oleifera extracted by n-butanol were found, 23 of which were completely and tentatively characterized. Besides, 35 saponins were identified as novel saponins. Additionally, 8 flavonol-O-glycosides were separated from C. oleifera, with 7 being tentatively characterized but one unambiguously identified, and one of the 7 flavonol-O-glycosides is reported for the first time in genus Camellia. This study provides a systematic strategy for the identification of saponins by the UPLC-MSn method in genus Camellia and summarized essential data for phytochemical studies of C. oleifera.

Receptor tyrosine kinases play an important role in mediating cell migration and adhesion associated with various biology processes. With a single-span transmembrane domain (TMD), the activities of the receptors are regulated by the definite packing configurations of the TMDs. For the EphA2 receptor, increasing studies have been conducted to investigate the packing domains that induce its switching TMD dimerization. However, the inherent transformation mechanisms including the interrelations among the involved packing domains remain unclear. Herein, we applied multiple simulation methods to explore the underlying packing mechanisms within the EphA2 TMD dimer. Our results demonstrated that the G540xxxG544 contributed to the formation of the right-handed configuration while the heptad repeat L535xxxG539xxA542xxxV546xxL549xxxG553 motif together with the FFxH559 region mediated the parallel mode. Furthermore, the FF557 residues packing mutually as rigid riveting structures were found comparable to the heptad repeat motif in maintaining the parallel configuration. In addition, the H559 residue associated definitely with the lower bilayer leaflet, which was proved to stabilize the parallel mode significantly. The simulations provide a full range of insights into the essential packing motifs or residues involved in the switching TMD dimer configurations, which can enrich our comprehension toward the EphA2 receptor.

•We developed an OTA aptamer-based surface plasmon resonance (SPR) biosensor.•The biosensor was able to detect OTA in concentration range of 0.094–100 ng/mL with a lower detection limit of 0.005 ng/mL.•The interaction between OTA and aptamer was screened in real time.•The approach was successfully applied to detect OTA in wine and peanut oil samples.Ochratoxin A (OTA), as a kind of chlorophenolic mycotoxin, exist widely in plant origin food and is harmful to human. Herein, a surface plasmon resonance (SPR) biosensor using an anti-OTA aptamer immobilized sensor chip was developed to measure ochratoxin A (OTA) quantificationally through a straightforward direct binding assay. The streptavidin protein as a crosslinker was immobilized onto the surface of a sensor chip and the biotin-aptamer was captured through streptavidin-biotin interaction. The biosensor exhibited a detection range from 0.094 to 100 ng/mL (linear range from 0.094 to 10 ng/mL) of OTA with a lower detection limit of 0.005 ng/mL. Detection of OTA in wine and peanut oil was further performed in the SPR biosensor using simple liquid–liquid extraction for sample pretreatments. Recoveries of ochratoxin A from spiked samples ranged from 86.9% to 116.5% and coefficients of variation (CVs) ranged from 0.2% to 6.9%. The developed methods in our studies showed good analytical performances with limits of detection much lower than the maximum residue limit, as well as a good reproducibility and stability.

E-cadherin is a transmembrane glycoprotein which is involved in the Ca2+-dependent cell–cell adhesion, and the adhesiveness is heavily dependent on the homodimerization of this molecule. Previous studies have shown that both the extracellular domain and cytoplasmic domain of E-cadherin contribute to its homodimerization. However, the roles of the transmembrane(TM) domain in the E-cadherin homodimerization have not been discussed in detail. In our experiments, SDS-PAGE showed higher molecular weight bands for the synthetic E-cadherin TM peptide, which indicated that the E-cadherin TM peptide is able to dimerize in the SDS micelle. The TOXCAT assay proved that the E-cadherin TM domain can form a moderate homo-oligomer in the Escherichia coli inner membrane. Furthermore, mutational analyses using the TOXCAT assays revealed that, instead of the common GxxxG dimerization motif, the leucine zipper motif is essential for the dimerization of the E-cadherin TM domain. Combining our experiment data and the computational simulation results, we provide insights for understanding the roles of the TM domain in the E-cadherin dimerization.

Although genetically modified (GM) food is becoming increasingly available, consumers are showing a growing awareness about the need to identify GM and non-GM foodstuff: the reliable identification of GM/non-GM food is therefore an important tool in the social, health and safety debates. The present research responds to this need (i) through developing a novel “single-pot” preparation of PAMAM magnetite nanoparticles (PMNPs) and by fully defining their specific characteristics; (ii) by demonstrating the capability of the PMNPs to isolate genomic DNA from different sample foods; and (iii) by experimentally demonstrating the identification of the isolated DNA by gel-electrophoresis, thus being capable of screening GM and non-GM food.Highlights► The reliable identification of GM/non-GM food is important. ► The research develops a novel “single-pot” preparation of PMNPs. ► PMNPs successfully isolate genomic DNA from different sample foods. ► Screening GM and non-GM food is achieved.

The major modification of the c-Fos protein involves serine phosphoesterification at the sites located at the extreme C-terminus. Several transcriptionally important protein–protein interactions involving c-Fos are mediated by the C-terminus domain phosphorylation. A series of peptides and phosphopeptides corresponding to C-terminus (359–380) of c-Fos protein were synthesized and their conformations were determined by CD spectroscopy and 1H-NMR spectroscopy. The nonphosphorylated peptide (Fos-C) adopts an essentially random-coil conformation in aqueous solution as shown by CD spectroscopy, but becomes slightly more ordered into a type I turn by adding up to 80% trifluoroethanol (TFE) to increase the solvent hydrophobicity. There is tentative NMR evidence for a small population of species containing a type I turn at residues SPTL in the Fos-C. Phosphorylation at S362 induces local conformational changes that can be attributed to the formation of local hydrogen bonds between the phosphate group and nearby amide protons, including additional medium-range (ε-HN of R359) effects. Upon phosphorylation at S374, a hydrogen bond between the amide proton and phosphate group of pS374 is formed. However, the most significant changes upon phosphorylation at S374 are the destabilization of this turn structure, which is strongly supported by CD and NMR spectroscopy.

Lytic and cell-penetrating peptides (CPPs) are both membrane-active peptides sharing similar physicochemical properties. Although their respective functions have been intensively investigated, the difference of intrinsic properties between these two types of peptides is rarely discussed. In this study, we designed a series of analogs of a recently discovered CPP ZXR-1 (FKIGGFIKKLWRSKLA) by varying the charge distributions both on the helical wheel projection and along the sequence. These peptides showed different functions on cell membranes, including membrane lytic (peptide Z1), cell-penetrating (peptide ZXR-1, Z2 and Z3), and inactive (peptide Z4) peptides. The three groups of peptides displayed different interactions with model lipid monolayer, and found that peptide insertion might be an important dynamic step to distinguish lytic and cell penetrating functions. Based on the analysis of charge distribution patterns, it was proposed that the charge distributions on the helical wheel and along the sequence are both able to influence the functions of the membrane-active peptides. This finding provides a further understanding about the effect of charge distribution on the functions of membrane-active peptides, and will be helpful for the design of functional peptides.Download high-res image (115KB)Download full-size image

Dimerization of the transmembrane (TM) adaptor protein DAP12 plays a key role in mediating activation signals through TM-TM association with cell-surface receptors. Herein, we apply the TOXCAT assay and molecular dynamics simulation to analyze dynamics and dimerization of the TM helix of DAP12 in the membrane bilayer. In the TOXCAT assay, we performed site-specific mutagenesis of potential dimerization motifs in the DAP12 TM domain. Instead of the common GxxxG dimerization motif, mutating either of the polar residues Asp-50 and Thr-54 significantly decreased the TOXCAT signal for the dimerization of DAP12 TM domain. Furthermore, through the conformational difference between wild-type and mutant DAP12 TM homodimers, a combined coarse-grained and atomistic molecular dynamics simulation has identified both Asp-50 and Thr-54 at the dimerization interface. The experimental and computational results of the DAP12 TM dimer are in excellent agreement with the previously reported NMR structure obtained in detergent micelles. Such a combination of dynamics simulation and cell-based experiments can be applied to produce insights at the molecular level into the TM-TM association of many other transmembrane proteins.

Saponins and flavonoids are the main bioactive ingredients in Camellia oleifera Abel. In this study, the fragmentation pathways of triterpene saponins in Camellia oleifera were investigated and confirmed by electrospray ionization linear ion trap-orbitrap (LTQ-orbitrap), and a new strategy for rapid characterization of saponins by ultra-high-pressure liquid chromatography with LTQ-orbitrap mass spectrometry (UHPLC-LTQ-orbitrap-MSn) was developed. Based on the summarized fragmentation rules, 36 triterpene saponins from the testa of C. oleifera extracted by n-butanol were found, 23 of which were completely and tentatively characterized. Besides, 35 saponins were identified as novel saponins. Additionally, 8 flavonol-O-glycosides were separated from C. oleifera, with 7 being tentatively characterized but one unambiguously identified, and one of the 7 flavonol-O-glycosides is reported for the first time in genus Camellia. This study provides a systematic strategy for the identification of saponins by the UPLC-MSn method in genus Camellia and summarized essential data for phytochemical studies of C. oleifera.