Identification of hybrid amyloid strains assembled from amyloid-β and human islet amyloid polypeptide

Cross-fibrillation of amyloid-β (Aβ) peptides and human islet amyloid polypeptides (hIAPP) has revealed a close correlation between Alzheimer’s disease and type 2 diabetes (T2D). Importantly, different amyloid strains are likely to lead to the clinical pathological heterogeneity of degenerative diseases due to toxicity. However, given the complicated cross-interactions between different amyloid peptides, it is still challenging to identify the polymorphism of the hybrid amyloid strains and reveal mechanistic insights into aggregation, but highly anticipated due to their significance. In this study, we investigated the cross-fibrillation of Aβ peptides and different hIAPP species (monomers, oligomers, and fibrils) using combined experimental and simulation approaches. Cross-seeding and propagation of different amyloid peptides monitored by experimental techniques proved that the three species of hIAPP aggregates have successively enhanced Aβ fibrillation, especially for hIAPP fibrils. Moreover, the polymorphism of these morphologically similar hybrid amyloid strains could be distinguished by testing their mechanical properties using quantitative nanomechanical mapping, where the assemblies of Aβ-hIAPP fibrils exhibited the high Young’s modulus. Furthermore, the enhanced internal molecular interactions and β-sheet structural transformation were proved by exploring the conformational ensembles of Aβ-hIAPP heterodimer and Aβ-hIAPP decamer using molecular dynamic simulations. Our findings pave the way for identifying different hybrid amyloid strains by quantitative nanomechanical mapping and molecular dynamic simulations, which is important not only for the precise classification of neurodegenerative disease subtypes but also for future molecular diagnosis and therapeutic treatment of multiple interrelated degenerative diseases.


Introduction
Emerging evidence from clinical and epidemiological studies indicates the close link between different degenerative diseases of Alzheimer's disease (AD) and type 2 diabetes (T2D) [1][2][3][4].Patients with diabetes have a higher incidence of cognitive decline and an increased risk of developing dementia [5,6].In essence, these two different diseases of AD and T2D have shared a similar pathological hallmark which is misfolding and aggregation of the causative amyloid peptides of β-amyloid (Aβ) for AD and human islet amyloid peptides (hIAPP) for T2D [7].Recent results provided initial evidence that the cross-interaction of Aβ and hIAPP strongly correlates with or even produces the mutually reinforcing disease development of AD and T2D [8].Aβ and hIAPP peptides hold the same nucleation and self-propagating pathway, and share a common structural characteristic of βsheet strands in the fibrils [9][10][11].Besides, the high degrees of sequence identity and similarity of Aβ and hIAPP [12] supported the formation of hybrid amyloid fibrils.Actually, recent studies have already confirmed the co-assembling between Aβ and hIAPP peptides.In vivo, it is founded on the early studies that Aβ and hIAPP coexist in the serum, cerebrospinal fluids, and pancreatic islet amyloid deposits of T2D patients [13].The cross-seeding and propagation of Aβ and hIAPP have been reported with aggregation efficiencies depending on the specific experimental conditions [14,15].The detailed cross-seeding interaction behavior between Aβ and hIAPP peptides has been revealed using computational simulation.The self-aggregation and co-aggregation of Aβ and hIAPP shared similar recognition sites [16][17][18].Thus, these investigations confirmed the interaction propensity between Aβ and hIAPP peptides, which might provide the molecular explanation for the potential link between AD and T2D.The polymorphic structure is a key characteristic of amyloid-like fibril strains, and it has been proven to be conserved in vivo and in vitro [19][20][21].Such as the Aβ peptide can form multiple amyloid conformations depending on the seeding variables [22].The Aβ fibril strains were anisotropic in different patients and even in the different tissues of the same patient [23].And recent observations determined that four fibril polymorphic atomic structures of hIAPP fibrils by cryo-EM [24].Essentially, polymorphism arises from the different fibril nuclei and their extension into different fibril morphologies controlled by kinetic factors [25][26][27].The growth conditions are undoubtedly responsible for the predominant fibril morphology since different amyloid fibril strains have different spontaneous nucleation rates, extension rates, and fragmentation rates under the specific environment.Moreover, another important factor hereof is the specific constitutes of seeding or these fibrils, the various interactions within the seeding and monomeric peptide in solution would further induce the particular ensembles of fibril architectures.And then, a different number of constituting protofilaments, and relative arrangement orientations would lead to the polymorphism of propagating amyloid fibril strains [28,29].Therefore, in addition to the variation of formation conditions, the different fibrils consist ensembles would also give rise to more diverse hybrid fibril morphologies at the microscopic level.For the cross-degenerative diseases of AD and T2D, the variations in clinical characteristics and neuropathology may be attributable to the hybrid-amyloid polymorphism [30,31], which is an analogy to self-propagating amyloid strains.
Undertaking efforts to elucidate strain-type polymorphism of amyloid fibrils is crucial for gaining deeper insights into obscure structural factors ruling the infectivity or toxicity of protein aggregates in AD and T2D diseases and also for finding new effective therapeutic strategies [32].Recently, the high-resolution structural studies of amyloid strains have revealed that there are differences at the nanoscale for these different strains, and they further contribute to neurodegeneration through different mechanisms [33][34][35].The experimental techniques including solid-state NMR spectroscopy [36], microfocus x-ray diffraction [37], and the cryo-EM technology [38], have recently been used to explore the highresolution structure of amyloid aggregates.However, how to identify the polymorphism in hybrid amyloid strains in a simple way is still challenged, because the high similarity of these strains increases the difficulty of identification.In this work, we proposed a way to identify different hybrid amyloid strains assembled from Aβ peptides and distinct species of hIAPP by testing the mechanical properties of different strains in experiment.It is indicated that a higher β-sheet structure content can be formed in the hybrid amyloid strains of Aβ and hIAPP, especially for hIAPP fibril, which led to the high Young's modulus of hybrid amyloid strain.Further, MD simulations were carried out to investigate the cross-interactions between Aβ peptide and hIAPP monomer (Aβ-hIAPP heterodimer) or hIAPP decamer (Aβ-hIAPP decamer).It was verified that the stronger interaction between hIAPP and Aβ, as well as the formation of α-helix structure in heterodimer and the higher β-sheet structure in decamer system, would contribute to the formation and aggregation of transient oligomers, as well as subsequent fibrillations.Collective experimental and computational results confirmed that the polymorphism of hybrid-amyloid fibril strains could be originated from different inter-molecular interactions and packing way of proteins with the specific nanomechanical property, which might be a fingerprint for identifying different amyloid strains.

Preparation of co-assembly peptides
The peptides of hIAPP 1-37 (hIAPP) and Aβ 1-40 (Aβ) were purchased from Guoping Pharmaceutical Co., Shanghai, China.Firstly, the two kinds of lyophilized powders were separately dissolved in 1 ml 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP; Tokyo Chemical Industry, Japan), then placed it in a thermo-shaker for 12 h at 25 °C and 360 rpm, and dried under vacuum before use.As for the multiple hIAPP aggregates preparation, 0.2 mg hIAPP was dissolved in 100 μl pure deionized water to obtain the hIAPP monomer solution.While the hIAPP oligomer solution was obtained by incubating the aqueous hIAPP solution for 4 h at 37 °C and 350 rpm.Further, the hIAPP fibrils were obtained by prolonging the incubation time to 24 h under the same experimental conditions.In addition, for the co-assembly of Aβ and hIAPP aggregates, three kinds of mixed solutions were prepared by severally adding the three hIAPP solutions obtained above to a centrifuge tube equipped with Aβ peptide, with a concentration of 5 μM hIAPP aggregates and 40 μM Aβ peptide.Notably, at the concentration of 5 μM, the hIAPP peptide was insufficient for the normal conformational transformation process of amyloid fibrillation.Finally, these mixed solutions were incubated in the constant temperature oscillator at 37 °C, 350 rpm.

Thioflavin T (ThT) fluorescence assay
Fluorescence intensities were recorded by the microplate reader (synergyh1, biotek), with the ThT fluorescence emission wavelengths of 465-600 nm and an excitation wavelength at 450 nm.A total of 200 μl sample solution composed of peptides solution (25 μl), solvate (150 μl), and 1 M ThT solution (25 μl) was added into a 96-well plate for measurement.All the measurements were repeated in three times, and the data was averaged by the intensity value of every sample.

Circular dichroism spectra (CD)
The CD spectra of aggregates were performed by using the spectropolarimeter (JASCO, Hachioji City, Japan, No. PTC-348W1), with a spectra region of 190-250 nm at room temperature.A volume of 300 μl sample was held in the 0.1 cm quartz cuvette to be tested.The pure dd-water was subtracted as the baseline.All the data was collected with a side-width of 2 nm and a scan speed of 50 nm min −1 .Each CD experiment was repeated at least three times.

Atomic force microscopy (AFM)
First, the morphology of peptide aggregates was monitored by a tapping-Mode AFM. 10 μl peptide solution was taken and deposited onto a freshly cleaved mica substrate for 10 min, and further dried in air for AFM measurement.It was carried out by using a commercial AFM MultiMode VIII (Bruker, Santa Barbara, USA) with an ultra-sharp silicon probe with a spring constant of 26 N m −1 under ambient conditions.All images were acquired as 512 × 512 pixels images.
Besides, Young's modulus map was recorded in the PeakForce quantitative nanomechanical mapping (QNM) mode that primarily focused on the measurements of nanostructure and nanomechanical properties of the biological samples.It was performed under an atmospheric condition at a scan rate of 1 Hz (Multimode SPM and Nanoscope V controller, Bruker).The ultra-sharp silicon tips were used for morphology, modulus, and adhesion imaging.The standard spring constant was 200 N m −1 and a typical tip radius was 2 nm.The Derjaguin-Mueller-Toporov (DMT) modulus of these amyloid aggregates were analyzed by using the Nanoscope Analysis software.

Transmission electron microscopy (TEM)
The peptide solution of 10 μl was deposited on copper rhodium 230 mesh grids with continuous carbon films for 10 min and then stained with a drop of 10 μl freshly filtered 1% uranyl acetate for 4 min.The morphology and size of aggregates were characterized by using a Tecnai 12 transmission electron microscope (Philips, Netherlands).The acceleration voltage was set at 120 KV.

Chromatography methods
Size exclusion chromatography (SEC) was performed on an Agilent 1260 series system.150 mM sodium phosphate buffer (pH = 7.0) was used as the mobile phase.The chromatography was run at the temperature of 30 °C and pressure of 48 bar, with a 1.0 ml min −1 flow rate on a SRT SEC-300MK column (5 μm, 300 Å, 7.8 × 300 nm; S/N: 7F26216).These samples were prepared by adding 40 μl peptide solutions after incubation for 12 h and 10 μl fluorescent dye reagent.Elution was monitored by UV at 280 nm.

Molecular dynamics simulations (MD)
To investigate the mechanism of different promotion effects of the hIAPP monomers, oligomers, and fibrils on the fibrillation of Aβ peptides.We chose two structural models of hIAPP to study the interaction with Aβ monomer using molecular dynamic simulation, which is the hIAPP monomer model [39] (PDB: 2L86) and the hIAPP decamer model [40] (PDB: 6VW2).The Aβ structure model [41] was also obtained from the Protein Data Bank (PDB: 1AML).The initial distance between Aβ and hIAPP monomers was 2 nm.Meanwhile, two additional systems of Aβ homodimer system and hIAPP homodimer system were established for comparison.These dimer systems and the Aβ-hIAPP decamer system were immersed in TIP3P water boxes with the size of 6 × 6 × 6 nm 3 and 9 × 10 × 9 nm 3 , respectively.Ions were added into the systems to neutralize the entire system.Under periodic boundary conditions, the distance from any edge of the water box to any peptide atom is at least 15 Å.The force field of Aβ and hIAPP peptides were adopted as the AMBER99SB-ILDN force field [42].Three independent parallel simulations were carried out for each system.
The MD production runs of these systems of Aβ-hIAPP heterodimer, Aβ homodimer, hIAPP homodimer, and Aβ-hIAPP decamer were energy minimized by 10 000 steps of steepest descent minimization, and then three dimer systems were conducted for 300 ns, while the Aβ-hIAPP decamer system was run for 500 ns under the NPT ensemble (T = 310 K and P = 1 atm) respectively.The equations of motion were integrated using the Leapfrog integrator [43].Time step was set up to 2 fs.The long-range electrostatic and short-range van der Waals interactions were calculated by the Particle Mesh Ewald (PME) method [44].And the cutoff distance were 1.0 nm. the defined secondary structure of proteins (DSSP) algorithm [45] was used for the secondary structure analysis of protein.These simulations were conducted and visualized using the Gromacs 2020 program [46], and visual molecular dynamics (VMD) program [47].

Results and discussion
To reveal the polymorphic effect on hybrid amyloid strains of Aβ and different hIAPP species, we investigated the cofibrillation of Aβ with hIAPP monomers, oligomers, and fibrils.Meanwhile, one group of Aβ peptides without hIAPP was taken into account for comparison.The addition of hIAPP monomers, oligomers, and even fibrils might save as seeds for the fibrillation of Aβ peptides.First of all, the aggregation kinetics of Aβ peptides in the presence of multiple hIAPP aggregates were detected and characterized by using thioflavin T (ThT) assay.As shown in figure 1(A), Aβ peptides would experience the normal fibrillation with the secondary structural formation of β-sheets within 12 h, and then remain stable at 12-120 h (figure S1, ESI †).Meanwhile, the kinetic process of hIAPP fiber formation was also shown in figure S1, which exhibited a typical growth kinetic curve and reached the highest plateau due to its higher concentration.However, there was no nucleation growth process for the hIAPP peptide at a concentration of 5 μM (figure S1), therefore it was used to investigate the cross-fibrillation process of Aβ and hIAPP monomer.In fact, the ThT results of pure Aβ and hIAPP were similar to the typical 'S-shaped' growth kinetics curve reported in previous studies [48,49], and our results here led to a shorter nucleation time and an earlier plateau due to the higher concentration.Importantly, the aggregation kinetic curves showed that the aggregations of Aβ and hIAPP oligomers or fibrils have a higher slope at the growth stage of aggregation kinetic curves than Aβ-hIAPP monomer or pure Aβ, suggesting a higher ratio of co-fibrillation.The fibrillation of Aβ peptides would be finially promoted by the additional hIAPP aggregates, which was reflected by the higher fluorescence intensity at the stable stage after 12 h.Besides, the fluorescence intensity of Aβ-hIAPP monomer was similar to that of Aβ-hIAPP oligomers at the stable stage, but the one of Aβ-hIAPP fibrils displayed the highest among these three hybrids.Further, the secondary structure of these aggregates were characterized by using CD spectra, it is proved that the formation of β-sheets structure was promoted mostly with adding the hIAPP aggregates.The intensity of the CD signal at 218 nm demonstrates the promotion effect of hIAPP aggregates on the fibrillation of Aβ with an order of hIAPP fibrils > oligomers > monomers.Meanwhile, the size exclusion chromatography (SEC)/gel-filtration was further used to characterize the co-fibrillation of Aβ and different hIAPP aggregates (figure 1(C)).As the principle of SEC measurement, the large protein molecules would first elute from the column, while the smaller one would be retained.
From the results, it can be found that there was a small peak at about 15 min for the aggregates of Aβ, Aβ-hIAPP monomer, and Aβ-hIAPP oligomers, while it disappeared in case of Aβ-hIAPP fibrils.This finding indicated a better co-fibrillation of Aβ and hIAPP fibrils than hIAPP monomers or oligomers.Besides, there was an obvious broad peak before the sharp peak in case of Aβ-hIAPP fibrils, which suggested the biggest aggregates were formed under this condition.
In addition, the intuitive structural morphologies of hybrid amyloid fibril strains were characterized by AFM.We monitored the kinetics of fibril formation and recorded the structural transitions during the fibrillation of Aβ and Aβ-hIAPP aggregates at 2, 5, and 12 h (figure 2(A)).Aβ could aggregate into oligomers after 2 h incubation and into fibrils with the height of 1.6 ± 0.3 nm after 5 h.With the addition of hIAPP aggregates, the co-fibrillation could happen (figure 2(B)), and the average heights of the hybrid amyloid strains of Aβ-hIAPP monomer, oligomer, and fibrils at 5 h were determined to be 2.1 ± 0.7 nm, 2.2 ± 0.3 nm, and 6.0 ± 0.9 nm, respectively.Subsequently, the mature fibrils were formed after incubation for 12 h (figure 2(C)).The height of pure Aβ fibril was 13.7 ± 1.3 nm, and it was slim than that of hybrid fibrils of Aβ-hIAPP monomers (14.5 ± 2.4 nm), Aβ-hIAPP oligomers (15.6 ± 0.6 nm), and Aβ-hIAPP fibrils (19.2 ± 1.8 nm).Overall, it is confirmed that the hIAPP fibrils have a better promotion effect on the fibrillation of Aβ than other species.In addition, TEM could further provide structural information on these hybrid amyloid strains (figure S2, ESI †), which is consistent with the results obtained by AFM.
Importantly, to identify and distinguish these hybrid amyloid strains with similar morphologies, quantitative nanomechanical mapping (QNM) measurements [50,51] were performed to obtain the nanomechanical properties of these strains as the possible fingerprint feature.As shown in figure 3, Young's modulus of hIAPP oligomers and fibrils were measured to be 0.5 ± 0.3 GPa and 0.8 ± 0.2 GPa, and pure Aβ fibrils were characterized to be 1.7 ± 0.3 Gpa.While for the hybrid amyloid strains, Young's modulus were determined to be 1.8 ± 0.2 GPa for Aβ-hIAPP monomer, 2.0 ± 0.5 GPa for Aβ-hIAPP oligomers, and 2.3 ± 0.3 GPa for Aβ-hIAPP fibrils.Young's modulus of hybrid amyloid strains was determined to be higher than those of pure Aβ and hIAPP aggregates.Especially, the species of hIAPP fibrils exhibit an obviously improved effect on Young's modulus of hybrid amyloid strain.In essence, the variation in Young's modulus might indicate the different internal packing densities and the intermolecular interactions between amyloid proteins [52][53][54].The low Young's modulus values infer a relatively lower packing density inside cross-fibrils, and vice versa.Therefore, Young's modulus of sequential enhancement illustrated the different co-assembling pathways during the cross-fibrillation.
Further, to deeply reveal the specific mechanism and identify the difference among these cross-fibrillation pathways of Aβ-hIAPP monomers, oligomers, and fibrils, the molecular dynamic simulations were performed to explore the interaction between Aβ and different hIAPP species.Considering the uncertainty of the secondary structure content in the oligomers and distinguishing the different aggregate forms of hIAPP, two models of hIAPP monomeric and decamer were chosen in the simulation, which represent the unfolded hIAPP monomers and the converted proto-filament with high ordered β-sheet structure, respectively.For the dimer system of Aβ and hIAPP monomer, there was a compact heterodimer structure formed at the end of simulation (figure 4(A)).Meanwhile, an Aβ homodimer system was established for comparison, which formed a relatively unconsolidated structure (figure 4(B)).In detail, the heterodimer has an asymmetric secondary structure distribution with the random coil structure of Aβ and the helical structure exist in hIAPP, which would lead to a more stable structure of the heterodimer than homodimer in accordance with the symmetry-breaking model [55].In detail, the values of radius of gyration (Rg) and solvent accessible surface area (SASA) of Aβ-hIAPP heterodimer and Aβ homodimer immediately decreased after the simulation started, and then converged with small fluctuations after 200 ns (figure S3, ESI †), which indicated the convergence of these two simulation trajectories.The relatively lower Rg and SASA values of the Aβ-hIAPP heterodimer indicated a higher intramolecular interaction in the heterodimer than homodimer, which was consistent with the point proposed in previous work [16,18].The inter-chain contact number between Aβ and hIAPP increased and maintained stable at the final stage (t = 200-300 ns) with an average contact number of 159, which is higher than that of 87 in Aβ-Aβ homodimer (figure 4(C)).In addition, the stable interaction energies of Aβ-hIAPP heterodimer and Aβ homodimer were −878.7 kJ mol −1 and −489.3 kJ mol −1 , respectively (figure 4(D)).Besides, the analysis of hydrogen bond between the two chains revealed a significantly higher average count of 17 in the heterodimer compared to 4 in the homodimer (figure S4, ESI †).Therefore, these results provide further evidence suggesting that the inter-chain interactions in the Aβ-hIAPP heterodimer are stronger than those in the Aβ homodimer.
More importantly, the secondary structural conversions of both Aβ-hIAPP heterodimer and Aβ-Aβ homodimer were analyzed.As shown in figure 4(G), the secondary structure of peptides in the heterodimer and homodimer both predominantly presented to be random coils, β-bridge, bend, turn, and helix structures, with the average contents of 33.8%, 1.9%, 19.7%, 20.2%, 23.1% for heterodimer, and 35.6%, 1.7%, 21.7%, 26.6%, 14.3% for homodimer, respectively.It was obvious that the α-helix content in the Aβ-hIAPP heterodimer was higher than that in the Aβ-Aβ homodimer.Further, we have constructed a hIAPP-hIAPP homodimer system for comparison, the analysis of secondary structure contents of hIAPP homodimer showed a 27% helix structure (figure S5, ESI †), which indicated a higher ability to form the helical conformation of hIAPP.According to the previous studies [56,57], it was reported that the helical conformation formed in the early oligomerization stage might accelerate the hydrophobic aggregation with other interacting peptides through exposing critical non-polar residues for interaction, and further promote the β-sheet structure formation for amyloid fibrillation [58].Therefore, it's reasonable to assume that the α-helix content increase in the heterodimer system here might have a beneficial effect on the early aggregation and formation of the transient oligomers, as well as the further cross-fibrillation of Aβ and hIAPP.In addition, there was a certain β-sheet content of 1.24% formed in Aβ-hIAPP heterodimer, while it was less and negligible in Aβ-Aβ homodimer, and 0.16% formed in hIAPP-hIAPP homodimer.Besides, the respective secondary structure probability of both hIAPP and Aβ in the heterodimer (figures 4(H)-(I)) showed a higher β-sheet and α-helix structure contents occurred on hIAPP peptides, which suggested the more favorable conformation conversion tendency of hIAPP [59], and further lead to the quicker formation of well-ordered structure in the transient hetero-oligomer than homo-oligomer.Importantly, it can be found that the content of β-sheet formation in heterodimer was far less than the α-helix, which proved the importance of helix conformation in the oligomerization stage.Based on the analysis of intermolecular interactions discussed above, it suggested that the stronger interaction between Aβ and hIAPP peptides would accelerate the peptide aggregation and hetero-oligomer formation.Moreover, the dynamic conversion of flexible conformations to α-helix might significantly contribute to the transient oligomers those are closely related to further β-sheet transition and facilitate the cross-fibrillation.
On the other hand, the interaction between Aβ and hIAPP decamer further verify the mechanism of promotion effect of polymorphic hIAPP aggregates on the Aβ fibrillation.The hIAPP decamer model was selected from the recent work reported by David S Eisenberg [40], and this fibril structure supports the potential for cross-seeding between hIAPP and Aβ.It contains the amyloid core segments of hIAPP spanning the region 14-37 and not concludes the region 1-13, due to the N-terminus of residues 1-13 prefer a more flexible conformation and display a relatively weak role for hIAPP fibril formation.The initial conformation with a center of masses (COMs) distance between Aβ and hIAPP decamer was 2.0 nm (figure 5(A)).Similarly, they stably adhered with each other after a sufficient simulation for 500 ns (figure 5(B)).The hIAPP decamer maintained a conformation similar to the initial, indicating the high stability of hIAPP fibrils.Moreover, Aβ monomer adhered onto the surface of hIAPP decamer, accompanying with a typical secondary structure conversion from random coil into β-sheet, which formed at the segments of L17-A21 and G33-V36 (figure 5(C)).In addition, this adhesion mainly depended on the hydrophobic residue F4, charged residues E3, R5, D7, H13 and other residues S8, N27, S26, V39, V40 of Aβ.The β-sheet structure probability of Aβ increased into 6% (figure 5(D)).Furthermore, the residue regions with high βsheet probability of Aβ mainly located in the hydrophobic area of L17-E22, V24-G25, and the C-terminal hydrophobic residues G29-G38, especially for V18-F20 and L34-M35 (figure 5(E)).In general, the N-terminus of Aβ with random coil structure plays an important role in the interaction with hIAPP decamer, ripping itself to hIAPP surface through the key hydrophobic and charged amino acids, and further allow the relatively flexible C-terminus accelerate to form β-sheet folding.
Though comparing the relative amounts of β-sheet in the Aβ-hIAPP decamer system and the Aβ-hIAPP heterodimer system, it can be found that Aβ has a higher tendency to form β-sheet in the presence of hIAPP fibril than hIAPP monomer.In the case of Aβ-hIAPP heterodimer, the stronger interaction between Aβ and hIAPP peptide, along with a higher tendency to convert to helix structure, might promote the aggregation of the transient hetero-oligomer, which is beneficial for further β-sheet formation and fibrillation.While in the case of Aβ-hIAPP decamer, the high ordered β-sheet structure of hIAPP might stably adsorb Aβ monomers to its surface, and support the conformational transformation of the C-terminus of Aβ from a random coil or helical structure to the β-sheet structure.These findings in simulations also corroborate the experimental findings that hIAPP fibrils have a higher efficiency in promoting the fibrillation of Aβ peptides.Thus, based on the CD and ThT results from the experiment, as well as the higher β-sheet formation observed in the calculations, it can be reasonably inferred that the heterogeneous aggregates promoted by hIAPP fibrils would possess stronger mechanical properties, which is consistent with previous studies suggesting a correlation between higher β-sheet conformations and enhanced mechanical strength [47].

Conclusions
In summary, our work firstly elucidated and identified the similar hybrid amyloid strains assembled from Aβ monomer and distinct species of hIAPP (monomers, oligomers, and fibrils).The addition of hIAPP species has significantly enhanced the fibrillation of Aβ peptides.Especially, it is found that a more sufficient β-sheet content in the presence of hIAPP fibrils than other species.Importantly, these hybrid amyloid strains with similar morphologies were distinguished by mechanical properties testing, where the Aβ-hIAPP fibrils showed a maximum of Young's modulus.Furthermore, MD simulation revealed the molecular mechanism that the interaction between Aβ and hIAPP is superior to that Aβ in between, and there is a favorable β-sheet structural transformation of Aβ in the presence of hIAPP fibril than monomer, due to the anchoring of N-terminus of Aβ peptide on the surface of hIAPP fibril core, and the preferred β-sheet conversion at the C-terminus of Aβ.In general, our works identified these similar hybrid amyloid strains by quantitative nanomechanical mapping, and molecular dynamics simulations would lay the foundation for the identification of different hybrid amyloid strains.Finally, it will be contributive to the precise classification of multiple interrelated degenerative diseases, clinical molecular diagnosis and therapeutic treatment in future.

Supporting informations
ThT fluorescence curves; TEM images; Radius of gyration and solvent accessible surface area analysis; The secondary structure of hIAPP homodimer; The Hbond number analysis.

Figure 4 .
Figure 4. (A)-(B) The representative conformation images of Aβ-hIAPP and Aβ-Aβ dimers at the end of the simulation (t = 300 ns).Aβ chain was colored yellow, hIAPP chain was colored green.(C)-(D) The analyses of contact number (C) and interaction energy (D) between two chains of these two dimers.The contact number calculations were calculated by counting the number of contact atoms on one single Aβ chain.(E)-(F) Inter-molucular residue contact map of hIAPP-Aβ and Aβ-Aβ.The color bar indicates the average contact distance between two single chains at the last 10 ns (t = 290-300 ns).The closer the contact distance, the stronger the interaction between residues.(G) The average secondary structure probability of peptide dimer in the Aβ-hIAPP heterodimer and Aβ homodimer.(H)-(I) Each secondary structure probability of hIAPP (H) and Aβ (I) in the Aβ-hIAPP heterodimer.The secondary structural probabilities were averaged from the last 100 ns of the three independent simulations in the homodimer system or heterodimer system.

Figure 5 .
Figure 5. (A)-(B) The initial (A) and final (B) conformation of the Aβ-hIAPP decamer system (t = 0 ns and t = 500 ns).(C) The detailed binding distribution and specific binding sites of Aβ on the surface of hIAPP decamer.(D) Each secondary structure probability of Aβ in the Aβ-hIAPP decamer system.(E) The average β-sheet probability of each residue of Aβ in the Aβ-hIAPP decamer system.The average secondary structure probabilities were also obtained from the last 100 ns of the three independment simulations.