Comparing the structural properties of human and rat islet amyloid polypeptide by MD computer simulations

Conformational properties of the full-length human and rat islet amyloid polypeptide 1-37 (amyloidogenic hIAPP and non-amyloidogenic rIAPP, respectively) were studied at 310 and 330 K by MD simulations both for the cysteine (reduced IAPP) and cystine (oxidized IAPP) moieties. At all temperatures studied, IAPP does not adopt a well-defined conformation and is essentially random coil in solution, although transient helices appear forming along the peptide between residues 8 and 22, particularly in the reduced form. Above the water percolation transition (at 320 K), the reduced hIAPP moiety presents a considerably diminished helical content remaining unstructured, while the natural cystine moiety reaches a rather compact state, presenting a radius of gyration that is almost 10% smaller and characterized by intrapeptide H-bonds that form many β-bridges in the C-terminal region. This compact conformation presents a short end-to-end distance and seems to form through the formation of β-sheet conformations in the C-terminal region with a minimization of the Y/F distances in a two-step mechanism: the first step taking place when the Y37/F23 distance is ~ 1.1 nm, and subsequently Y37/F15 reaches its minimum of ~ 0.86 nm. rIAPP, which does not aggregate, also presents transient helical conformations. A particularly stable helix is located in proximity of the C-terminal region, starting from residues L27 and P28. Our MD simulations show that P28 in rIAPP influences the secondary structure of IAPP by stabilizing the peptide in helical conformations. When this helix is not present, the peptide presents bends or H-bonded turns at P28 that seem to inhibit the formation of the β-bridges seen in hIAPP. Conversely, hIAPP is highly disordered in the C-terminal region, presenting transient isolated β-strand conformations, particularly at higher temperatures and when the natural disulfide bond is present. Such conformational differences found in our simulations could be responsible for the different aggregational propensities of the two different homologues. In fact, the fragment 30-37, which is identical in both homologues, is known to aggregate in vitro, hence the overall sequence must be responsible for the amyloidogenicity of hIAPP. The increased helicity in rIAPP induced by the serine-to-proline variation at residue 28 seems to be a plausible inhibitor of its aggregation.     

Effect of Proline Mutations on the Monomer Conformations of Amylin

The formation of human islet amyloid polypeptide (hIAPP) is implicated in the loss of pancreatic β-cells in type II diabetes. Rat amylin, which differs from human amylin at six residues, does not lead to formation of amyloid fibrils. Pramlintide is a synthetic analog of human amylin that shares three proline substitutions with rat amylin. Pramlintide has a much smaller propensity to form amyloid aggregates and has been widely prescribed in amylin replacement treatment. It is known that the three prolines attenuate β-sheet formation. However, the detailed effects of these proline substitutions on full-length hIAPP remain poorly understood. In this work, we use molecular simulations and bias-exchange metadynamics to investigate the effect of proline substitutions on the conformation of the hIAPP monomer. Our results demonstrate that hIAPP can adopt various β-sheet conformations, some of which have been reported in experiments. The proline substitutions perturb the formation of long β-sheets and reduce their stability. More importantly, we find that all three proline substitutions of pramlintide are required to inhibit β conformations and stabilize the α-helical conformation. Fewer substitutions do not have a significant inhibiting effect.     

Cyclic N-Terminal Loop of Amylin Forms Non Amyloid Fibers

We report for the first time, to our knowledge, that the N-terminal loop (N_loop) of amylin (islet amyloid polypeptide (IAPP) residues 1–8) forms extremely long and stable non-β-sheet fibers in solution under the same conditions in which human amylin (hIAPP) forms amyloid fibers. This observation applies to the cyclic, oxidized form of the N_loop but not to the linear, reduced form, which does not form fibers. Our findings indicate a potential role of direct N_loop-N_loop interactions in hIAPP aggregation, which has not been previously explored, with important implications for the mechanism of hIAPP amyloid fiber formation, the inhibitory action of IAPP variants, and the competition between ordered and disordered aggregation in peptides of the calcitonin peptide family.     

Isotope-edited vibrational circular dichroism study reveals a flexible N-terminal structure of islet amyloid peptide (NFGAIL) in amyloid fibril form: A site-specific local structural analysis

The short peptide fragment NFGAIL (IAPf) is a well-known amyloidogenic peptide (22–27), derived from human islet amyloid polypeptide(hIAPP), whose fibrillar structure is often used to better understand the wild-type hIAPP amyloid fibrils, associated with type II diabetes. Despite an extensive study, the fibrillar structure of IAPf at the amino acid residue level is still unclear. Herein, the vibrational circular dichroism(VCD) spectroscopic technique coupled with isotope labelling strategy has been used to study the site-specific local structure of IAPf amyloid fibrils. Two ¹³C labeled IAPfs were designed and used along with unlabelled IAPf to achieve this. The ¹³C labelled (on -C=O) glycine(IAPf-G) and phenylalanine (IAPf-F) residues were introduced into the IAPf sequence separately by replacing natural glycine (residue 24) and phenylalanine (residue 23), respectively. VCD spectral analysis on IAPf-G suggests that IAPf fibrils adopt parallel β-sheet conformation with glycine residues are part of β-sheet and in-register. Unlike IAPf-G, VCD analysis on IAPf-F reveals that phenylalanine residues exist in the turn/hairpin conformation rather than β-sheet region. Both VCD results thus suggest that IAPf amyloid fibril consists of a mixture of β-sheet as a major conformation involving GAIL and turn/hairpin as a minor conformation involving NF rather than an idealized β-sheet involving all the amino acids. While previous studies speculated that the full NFGAIL sequence could participate in the β-sheet formation, the present site-specific structural analysis of IAPf amyloid fibrils at residue level using isotope-edited VCD has gained significant attention. Such residue level information has important implications for understanding the role of NFGAIL sequence in the amyloid fibrillation of hIAPP.     

Effect of Proline Mutations on the Monomer Conformations of Amylin

The formation of human islet amyloid polypeptide (hIAPP) is implicated in the loss of pancreatic β-cells in type II diabetes. Rat amylin, which differs from human amylin at six residues, does not lead to formation of amyloid fibrils. Pramlintide is a synthetic analog of human amylin that shares three proline substitutions with rat amylin. Pramlintide has a much smaller propensity to form amyloid aggregates and has been widely prescribed in amylin replacement treatment. It is known that the three prolines attenuate β-sheet formation. However, the detailed effects of these proline substitutions on full-length hIAPP remain poorly understood. In this work, we use molecular simulations and bias-exchange metadynamics to investigate the effect of proline substitutions on the conformation of the hIAPP monomer. Our results demonstrate that hIAPP can adopt various β-sheet conformations, some of which have been reported in experiments. The proline substitutions perturb the formation of long β-sheets and reduce their stability. More importantly, we find that all three proline substitutions of pramlintide are required to inhibit β conformations and stabilize the α-helical conformation. Fewer substitutions do not have a significant inhibiting effect.     

All-atom structure ensembles of islet amyloid polypeptides determined by enhanced sampling and experiment data restraints

Exploring the accurate structure ensembles are critical to understand the functions of intrinsically disordered proteins (IDPs). As a well-known IDP, islet amyloid polypeptide (IAPP) plays important roles in the development of human type II diabetes (T2D). The toxicity of human IAPP (hIAPP) is induced by the amyloidosis of the peptide, however, its aggregation mechanism remains ambiguous. The characterization of structure ensemble of hIAPP, as well as the differences between hIAPP and its non-amyloidogenic homologous such as rat IAPP (rIAPP), would greatly help to illuminate the amyloidosis mechanism of IAPP. In this study, the atomic structure ensembles of hIAPP and rIAPP were characterized by all-atom molecular dynamics (MD) simulations combined with enhanced sampling technology and experiment data restraints. The obtained structure ensembles were firstly compared with those determined by the conventional MD (cMD) and enhanced sampling without experiment data restraints. The results showed that the enhanced sampling and experiment data restraints would improve the simulation accuracy. The transient N-terminal α-helix structures were adopted by the sub-states of both hIAPP and rIAPP, however, the C-terminal helical structures were only present on hIAPP. The hydrophobic residues in the amyloid-core region of hIAPP are exposed to the solvent. The structure ensemble differences between hIAPP and rIAPP revealed in this work provide potential explain to the amyloidogenic mechanism and would be helpful for the design of drugs to combat T2D.     

Calcium-activated membrane interaction of the islet amyloid polypeptide: implications in the pathogenesis of type II diabetes mellitus

The role played by Ca²⁺ ions in the interaction of the human islet amyloid polypeptide (hIAPP) with model membranes has been investigated by differential scanning calorimetry (DSC) and circular dichroism (CD) experiments. In particular, the interaction of hIAPP and its rat isoform (rIAPP) with zwitterionic dipalmitoyl-phosphatidylcholine (DPPC), negatively charged dipalmitoyl-phosphatidylserine (DPPS) vesicles and with a 3:1 mixtures of them, has been studied in the presence of Ca²⁺ ions. The experiments have evidenced that amorphous, soluble hIAPP assemblies interact with the hydrophobic core of DPPC bilayers. Conversely, the presence of Ca²⁺ ions is necessary to activate a preferential interaction of hIAPP with the hydrophobic core of DPPS membranes. These findings support the hypothesis that an impaired cellular homeostasis of Ca²⁺ ions may promote the insertion of hIAPP into the hydrophobic core of carrier vesicles which is thought to contribute to an eventual intracellular accumulation of β-sheet rich hIAPP aggregates.     

Structural characteristics of the beta-sheet-like human and rat islet amyloid polypeptides as determined by scanning tunneling microscopy

We demonstrate in this work that scanning tunneling microscopy (STM) provides a useful approach to obtaining structural information about human islet amyloid polypeptide (hIAPP) and rat islet amyloid polypeptide (rIAPP) assembly on highly oriented pyrolytic graphite (HOPG) with sub-molecular resolution. The observed hIAPP and rIAPP lamellae consisted of parallel stripes. The STM images of hIAPPs show multiple molecular folding structures, with an average of 11 amino acid residues for the core regions. In addition, the STM images also reveal the assembly characteristics of rIAPP lamellae and may indicate a secondary structural conformation from random coil to beta-sheet-like on the graphite surface.     

A hIAPP-derived all-d-amino-acid inhibits hIAPP fibrillation efficiently at membrane surface by targeting α-helical oligomeric intermediates

A variety of peptides and peptide derivatives have been constructed using the “β-sheet core segment” of amyloid proteins as inhibitors of amyloidogenic fibrillation. A novel all-d-amino-acid from hIAPP β-sheet core segment (hIAPP 22–27) is demonstrated to inhibit hIAPP fibril formation efficiently both at the phospholipid membrane and in bulk solution. The inhibitor terminates hIAPP aggregation to the α-helical oligomeric intermediates at the membrane surface, whereas it stops the aggregation at the stage of β-sheet oligomeric intermediates in bulk solution. This is the first evidence that the inhibition mechanism of the inhibitor at membrane surface is significantly different from that in bulk solution.     

Conserved and cooperative assembly of membrane-bound alpha-helical states of islet amyloid polypeptide

The conversion of soluble protein into beta-sheet-rich amyloid fibers is the hallmark of a number of serious diseases. Precursors for many of these systems (e.g., Abeta from Alzheimer's disease) reside in close association with a biological membrane. Membrane bilayers are reported to accelerate the rate of amyloid assembly. Furthermore, membrane permeabilization by amyloidogenic peptides can lead to toxicity. Given the beta-sheet-rich nature of mature amyloid, it is seemingly paradoxical that many precursors are either intrinsically alpha-helical or transiently adopt an alpha-helical state upon association with membrane. In this work, we investigate these phenomena in islet amyloid polypeptide (IAPP). IAPP is a 37-residue peptide hormone which forms amyloid fibers in individuals with type II diabetes. Fiber formation by human IAPP (hIAPP) is markedly accelerated by lipid bilayers despite adopting an alpha-helical state on the membrane. We further show that IAPP partitions into monomeric and oligomeric helical assemblies. Importantly, it is this latter state which most strongly correlates to both membrane leakage and accelerated fiber formation. A sequence variant of IAPP from rodents (rIAPP) does not form fibers and is reputed not to permeabilize membranes. Here, we report that rIAPP is capable of permeabilizing membranes under conditions that permit rIAPP membrane binding. Sequence and spectroscopic comparisons of rIAPP and hIAPP enable us to propose a general mechanism for the helical acceleration of amyloid formation in vitro. As rIAPP cannot form amyloid fibers, our results show that fiber formation need not be directly coupled to toxicity.     

Cyclic N-Terminal Loop of Amylin Forms Non Amyloid Fibers

We report for the first time, to our knowledge, that the N-terminal loop (N_loop) of amylin (islet amyloid polypeptide (IAPP) residues 1–8) forms extremely long and stable non-β-sheet fibers in solution under the same conditions in which human amylin (hIAPP) forms amyloid fibers. This observation applies to the cyclic, oxidized form of the N_loop but not to the linear, reduced form, which does not form fibers. Our findings indicate a potential role of direct N_loop-N_loop interactions in hIAPP aggregation, which has not been previously explored, with important implications for the mechanism of hIAPP amyloid fiber formation, the inhibitory action of IAPP variants, and the competition between ordered and disordered aggregation in peptides of the calcitonin peptide family.     

Membrane Permeation Induced by Aggregates of Human Islet Amyloid Polypeptides

Several neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases as well as nonneuropathic diseases such as type II diabetes and atrial amyloidosis are associated with aggregation of amyloid polypeptides into fibrillar structures, or plaques. In this study, we use molecular dynamics simulations to test the stability and orientation of membrane-embedded aggregates of the human islet amyloid polypeptide (hIAPP) implicated in type II diabetes. We find that in both monolayers and bilayers of dipalmitoylphosphatidylglycerol (DPPG) hIAPP trimers and tetramers remain inside the membranes and preserve their β-sheet secondary structure. Lipid bilayer-inserted hIAPP trimers and tetramers orient inside DPPG at 60° relative to the membrane/water interface and lead to water permeation and Na⁺ intrusion, consistent with ion-toxicity in islet β-cells. In particular, hIAPP trimers form a water-filled β-sandwich that induce water permeability comparable with channel-forming proteins, such as aquaporins and gramicidin-A. The predicted disruptive orientation is consistent with the amphiphilic properties of the hIAPP aggregates and could be probed by chiral sum frequency generation (SFG) spectroscopy, as predicted by the simulated SFG spectra.     

Thermodynamics of β-Sheet Formation in Polyglutamine

The role of β-sheets in the early stages of protein aggregation, specifically amyloid formation, remains unclear. Interpretations of kinetic data have led to a specific model for the role of β-sheets in polyglutamine aggregation. According to this model, monomeric polyglutamine, which is intrinsically disordered, goes through a rare conversion into an ordered, metastable, β-sheeted state that nucleates aggregation. It has also been proposed that the probability of forming the critical nucleus, a specific β-sheet conformation for the monomer, increases with increasing chain length. Here, we test this model using molecular simulations. We quantified free energy profiles in terms of β-content for monomeric polyglutamine as a function of chain length. In accord with estimates from experimental data, the free energy penalties for forming β-rich states are in the 10-20 kcal/mol range. However, the length dependence of these free energy penalties does not mirror interpretations of kinetic data. In addition, although homodimerization of disordered molecules is spontaneous, the imposition of conformational restraints on polyglutamine molecules does not enhance the spontaneity of intermolecular associations. Our data lead to the proposal that β-sheet formation is an attribute of peptide-rich phases such as high molecular weight aggregates rather than monomers or oligomers.     

Molecular simulation uncovers the conformational space of the λ Cro dimer in solution

The significant variation among solved structures of the λ Cro dimer suggests its flexibility. However, contacts in the crystal lattice could have stabilized a conformation which is unrepresentative of its dominant solution form. Here we report on the conformational space of the Cro dimer in solution using replica exchange molecular dynamics in explicit solvent. The simulated ensemble shows remarkable correlation with available x-ray structures. Network analysis and a free energy surface reveal the predominance of closed and semi-open dimers, with a modest barrier separating these two states. The fully open conformation lies higher in free energy, indicating that it requires stabilization by DNA or crystal contacts. Most NMR models are found to be unstable conformations in solution. Intersubunit salt bridging between Arg⁴ and Glu⁵³ during simulation stabilizes closed conformations. Because a semi-open state is among the low-energy conformations sampled in simulation, we propose that Cro-DNA binding may not entail a large conformational change relative to the dominant dimer forms in solution.     

Induction of endoplasmic reticulum stress-induced beta-cell apoptosis and accumulation of polyubiquitinated proteins by human islet amyloid polypeptide

The islet in type 2 diabetes is characterized by an approximately 60% beta-cell deficit, increased beta-cell apoptosis, and islet amyloid derived from islet amyloid polypeptide (IAPP). Human IAPP (hIAPP) but not rodent IAPP (rIAPP) forms toxic oligomers and amyloid fibrils in an aqueous environment. We previously reported that overexpression of hIAPP in transgenic rats triggered endoplasmic reticulum (ER) stress-induced apoptosis in beta-cells. In the present study, we sought to establish whether the cytotoxic effects of hIAPP depend on its propensity to oligomerize, rather than as a consequence of protein overexpression. To accomplish this, we established a novel homozygous mouse model overexpressing rIAPP at a comparable expression rate and, on the same background, as a homozygous transgenic hIAPP mouse model previously reported to develop diabetes associated with beta-cell loss. We report that by 10 wk of age hIAPP mice develop diabetes with a deficit in beta-cell mass due to increased beta-cell apoptosis. The rIAPP transgenic mice counterparts do not develop diabetes or have decreased beta-cell mass. Both rIAPP and hIAPP transgenic mice have increased expression of BiP, but only hIAPP transgenic mice have elevated ER stress markers (X-box-binding protein-1, nuclear localized CCAAT/enhancer binding-protein homologous protein, active caspase-12, and accumulation of ubiquitinated proteins). These findings indicate that the beta-cell toxic effects of hIAPP depend on the propensity of IAPP to aggregate, but not on the consequence of protein overexpression.     

A two-site mechanism for the inhibition of IAPP amyloidogenesis by zinc

Human islet amyloid polypeptide (hIAPP) is a highly amyloidogenic protein co-secreted with insulin in response to glucose levels. The formation of hIAPP amyloid plaques near islet cells has been linked to the death of insulin-secreting β-cells in humans and the progression of type II diabetes. Since both healthy individuals and those with type II diabetes produce and secrete hIAPP, it is reasonable to look for factors involved in storing hIAPP and preventing amyloidosis. We have previously shown that zinc inhibits the formation of insoluble amyloid plaques of hIAPP; however, there remains significant ambiguity in the underlying mechanisms. In this study, we show that zinc binds unaggregated hIAPP at micromolar concentrations similar to those found in the extracellular environment. By contrast, the fibrillar amyloid form of hIAPP has low affinity for zinc. The binding stoichiometry obtained from isothermal titration calorimetry experiments indicates that zinc favors the formation of hIAPP hexamers. High-resolution NMR structures of hIAPP bound to zinc reveal changes in the electron environment along residues that would be located along one face of the amphipathic hIAPP α-helix proposed as an intermediate for amyloid formation. Results from electrospray ionization mass spectroscopy investigations showed that a single zinc atom is predominantly bound to hIAPP and revealed that zinc inhibits the formation of the dimer. At higher concentrations of zinc, a second zinc atom binds to hIAPP, suggesting the presence of a low-affinity secondary binding site. Combined, these results suggest that zinc promotes the formation of oligomers while creating an energetic barrier for the formation of amyloid fibers.     

A beta-hairpin comprising the nuclear localization sequence sustains the self-associated states of nucleosome assembly protein 1

The histone chaperone nucleosome assembly protein 1 (NAP1) is implicated in histone shuttling as well as nucleosome assembly and disassembly. Under physiological conditions, NAP1 dimers exist in a mixture of various high-molecular-weight oligomers whose size may be regulated by the cell cycle-dependent concentration of NAP1. Both the functional and structural significance of the observed oligomers are unknown. We have resolved the molecular mechanism by which yeast NAP1 (yNAP1) dimers oligomerize by applying x-ray crystallographic, hydrodynamic, and functional approaches. We found that an extended β-hairpin that protrudes from the compact core of the yNAP1 dimer forms a stable β-sheet with β-hairpins of neighboring yNAP1 dimers. Disruption of the β-hairpin (whose sequence is conserved among NAP1 proteins in various species) by the replacement of one or more amino acids with proline results in complete loss of yNAP1 dimer oligomerization. The in vitro functions of yNAP1 remain unaffected by the mutations. We have thus identified a conserved structural feature of NAP1 whose function, in addition to presenting the nuclear localization sequence, appears to be the formation of higher-order oligomers.     

The crystal structure of an esterase from the hyperthermophilic microorganism <Emphasis Type="Italic">Pyrobaculum calidifontis</Emphasis> VA1 explains its enantioselectivity

The highly thermostable esterase from the hyperthermophilic archaeon Pyrobaculum calidifontis VA1 (PestE) shows high enantioselectivity (E?>?100) in the kinetic resolution of racemic chiral carboxylic acids, but little selectivity towards acetates of tertiary alcohols (E?=?2–4). To explain these unique properties, its crystal structure has been determined at 2.0 Å resolution. The enzyme is a member of the hormone-sensitive lipase group (group H) of the esterase/lipase superfamily on the basis of the amino acid sequence identity. The PestE structure shows a canonical α/β-hydrolase fold as core domain with a cap structure at the C-terminal end of the β-sheet. A tetramer in the crystal packing is formed of two dimers; the dimeric form is observed in solution. Conserved dimers and even tetramers are found in other group H proteins. The amino acid residues Ser157, His284, and Asp254 form the catalytic triad, which is typically found in α/β-hydrolases. The oxyanion hole is composed of Gly85 and Gly86 within the conserved sequence motif HGGG(M,F,W) (amino acid residues 83–87) and Ala158. With the elucidated structure, experimental results about enantioselectivity towards the two model substrate classes (as exemplified for 3-phenylbutanoic acid ethyl ester and 1,1,1-trifluoro-2-phenylbut-3-yn-2-yl acetate) could be explained by molecular modeling. For both enantiomers of the tertiary alcohol, orientations in two binding pockets were obtained without significant energy differences corresponding to the observed low enantioselectivity due to missing steric repulsions. In contrast, for the carboxylic acid ester, two different orientations with significant energy differences for each enantiomer were found matching the high E values.     

Conformational structure of bombesin as studied by vibrational and circular dichroism spectroscopy

Raman and Fourier transform infrared (FTIR) spectroscopies and circular dichroism (CD) have been applied to investigate the secondary structure of bombesin in the solid state and in phosphate buffer solution (pH 3.8). At concentrations around 10⁻⁵ M, circular dichroism reveals that bombesin exists as an irregular or disordered conformation. However, the secondary structure of the peptide appears to be a mixture of disordered structure and intermolecular β-sheets in 0.01 M sodium phosphate buffer when the peptide concentrations are higher than around 6.5 mM. The tendency of bombesin to form aggregated β-sheet species seems to be originated mainly in the sequence of the residues 7–14, as supported by the Raman spectra and β-sheet propensities (P_β) of the amino-acid residues. It is the hydrophobic force of this amino-acid sequence, and not a salt bridge effect, that is the factor responsible for the formation of peptide aggregates.     

Structural polymorphism of human islet amyloid polypeptide (hIAPP) oligomers highlights the importance of interfacial residue interactions

A 37-residue of human islet amyloid polypeptide (hIAPP or amylin) is a main component of amyloid plaques found in the pancreas of ～90% of type II diabetes patients. It is reported that hIAPP oligomers, rather than mature fibrils, are major toxic species responsible for pancreatic islet β-cell dysfunction and even cell death, but molecular structures of these oligomers remain elusive. In this work, on the basis of recent solid-state NMR and mass-per-length (MPL) data, we model a series of hIAPP oligomers with different β-layers (one, two, and three layers), symmetries (symmetry and asymmetry), and associated interfaces using molecular dynamics simulations. Three distinct interfaces formed by C-terminal β-sheet and C-terminal β-sheet (CC), N-terminal β-sheet and N-terminal β-sheet (NN), and C-terminal β-sheet and N-terminal β-sheet (CN) are identified to drive multiple cross-β-layers laterally associated together to form different amyloid organizations via different intermolecular interactions, in which the CC interface is dominated by polar interactions, the NN interface is dominated by hydrophobic interactions, and the CN interface is dominated by mixed polar and hydrophobic interactions. Overall, the structural stability of the proposed hIAPP oligomers is a result of delicate balance between maximization of favorable peptide-peptide interactions at the interfaces and optimization of solvation energy with globular structure. Different hIAPP oligomeric models indicate a general and intrinsic nature of amyloid polymorphism, driven by different interfacial side-chain interactions. The proposed models are compatible with recent experimental data in overall size, cross-section area, and molecular weight. A general hIAPP aggregation mechanism is proposed on the basis of our simulated models and experimental data.