# cis and trans isomers of phospho-tubulin-associated unit proteins differently affect amyloid aggregation

Mohammad J Hajipour, Sima Rezvantalab, Hossein Mohammad-Beigi, Reza Maleki, Ghazal Ghezelbashan, Esmael M Alyami, Ayyalusamy Ramamoorthy,

Lionel Maurizi

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## ► To cite this version:

Mohammad J Hajipour, Sima Rezvantalab, Hossein Mohammad-Beigi, Reza Maleki, Ghazal Ghezelbashan, et al., cis and trans isomers of phospho-tubulin-associated unit proteins differently affect amyloid aggregation. International Journal of Biological Macromolecules, 2025, 320 (1), pp.145500. 10.1016/j.ijbiomac.2025.145500. hal-05151795

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cis and *trans* isomers of phospho-tau proteins differently affect amyloid aggregation

Mohammad J. Hajipour<sup>a,b,c,*†</sup>, Sima Rezvantalab<sup>d,†</sup>, Hossein Mohammad-Beigi<sup>e</sup>, Reza Maleki<sup>f,**</sup>, Ghazal Ghezelbashan<sup>b,c</sup>, Esmael M. Alyami<sup>g</sup>, Ayyalusamy Ramamoorthy<sup>h,i</sup>, Lionel Maurizi<sup>j,k,l,**</sup>

ARTICLE INFO

Keywords:

Phosphorylated tau

Fibrillation

Amyloidogenesis

Alzheimer's disease

Parkinson's disease

ABSTRACT

The co-localization of phospho-tau proteins (p-tau) and amyloid beta (Aβ) or alpha-synuclein (α-Syn) aggregates and their combined pathological impacts have been recognized in the brains of both human and animal models of Alzheimer's disease (AD) and Parkinson's disease (PD). The fibrillation of amyloidogenic proteins is a dynamic process that can be affected by the presence of other proteins and therefore, it's essential to reveal how the cross-interaction between Aβ or α-Syn with p-tau affects their amyloidogenesis. Using simulation studies, we showed that *cis* and *trans* conformations of phosphorylated tau (phosphorylated at Thr231) show different affinities to Aβ and α-Syn. *Trans* p-tau showed more favorable and stronger interaction with Aβ and α-Syn and accelerated their aggregation. Consistently, the experimental approaches demonstrated that *trans* p-tau considerably enhances the fibrillation of both Aβ (from ~7 h to ~30 min) and α-Syn (from ~10 h to ~1 h) in a concentration-dependent manner, whereas *cis* p-tau, a neurotoxic isomer and early driver of tauopathy, exhibited a less pronounced acceleratory effect on amyloid aggregation (from 7 h to 5 h for Aβ and from 7 h to 5 h for α-Syn).

1. Introduction

Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, and amyotrophic lateral sclerosis (ALS) are strongly associated with the misfolding and aggregation of specific proteins. These disorders, collectively referred to as

protein misfolding diseases, are characterized by the accumulation of amyloid fibrils or toxic oligomers in neuronal tissues, ultimately leading to synaptic dysfunction, neuronal loss, and cognitive or motor impairment. Protein misfolding and disposal are typical events in mammalian cells. However, extra Protein misfolding overwhelms the protein quality control (PQC) systems responsible for disposing of misfolded proteins

<footnote><sup>a</sup> Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Stanford University, Stanford, CA 94304, USA</footnote>

<footnote><sup>b</sup> Center of Excellence in Inflammation, Infectious Disease and Immunity, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA</footnote>

<footnote><sup>c</sup> Department of Internal Medicine, Division of Infectious, Inflammatory and Immunologic Diseases, Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA</footnote>

<footnote><sup>d</sup> Department of Chemical Engineering, Urmia University of Technology, 57166-419 Urmia, Iran</footnote>

<footnote><sup>e</sup> Department of Biotechnology and Biomedicine, Technical University of Denmark, Kgs. Lyngby, Denmark</footnote>

<footnote><sup>f</sup> Department of Chemical Technologies, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran</footnote>

<footnote><sup>g</sup> Department of Biology, College of Science, King Khalid University, PO Box 960, Asir, Abha 61421, Saudi Arabia</footnote>

<footnote><sup>h</sup> Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Tallahassee, FL 32310, USA</footnote>

<footnote><sup>i</sup> National High Magnetic Field Laboratory, Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32310, USA</footnote>

<footnote><sup>j</sup> Université Bourgogne Europe, CNRS, Laboratoire Interdisciplinaire Carnot de Bourgogne ICB UMR 6303, 21000 Dijon, France</footnote>

<footnote><sup>k</sup> Université de Haute-Alsace, CNRS, IS2M UMR 7361, F-68100 Mulhouse, France</footnote>

<footnote><sup>l</sup> Université de Strasbourg, Strasbourg F-67081, France</footnote>

<footnote>* Correspondence to: M.J. Hajipour, Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Stanford University, Stanford, CA 94304, USA.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>*** Correspondence to: L. Maurizi, Université de Haute-Alsace, CNRS, IS2M UMR 7361, F-68100 Mulhouse, France.
E-mail addresses: mjh.hajipour@gmail.com (M.J. Hajipour), Rezamaleki96@gmail.com (R. Maleki), lionel.maurizi@uha.fr (L. Maurizi).</footnote>

<footnote n="1">¹ Authors contributed equally.</footnote>
and triggering amyloid diseases over time [1–5]. Protein misfolding, fibrillation, and accumulation give rise to a cascade of neurotoxic events that damage neuron cells and disrupt synaptic connections [6–8]. AD and PD are known as multifactorial diseases with different factors contributing to their etiology. However, the fibrillar and oligomeric forms of amyloidogenic proteins (Amyloid beta (Aβ) and Alpha-synuclein (α-Syn)) are known as the main culprits in their progression [9–16]. In contrast to the amyloid hypothesis [17], which indicates that protein fibrils/oligomers play a critical role in neurodegenerative disease development, a recent study showed that reducing the level of a soluble form of Aβ (monomers) can be toxic [18,19]. They demonstrated that regardless of the level of amyloid plaque in the brain, people will be cognitively normal if the level of soluble Aβ in their brain is above 270 pg/mL [18]. People with low levels of soluble Aβ protein are more likely to show cognitive decline and behavioral abnormalities [19]. The transformation of soluble Aβ proteins into insoluble plaques is a natural reaction to different stimuli such as infectious stress [20,21]. Therefore, regardless of considering the soluble protein decrease or oligomer/plaques increase as the main causes of AD or PD, we should prevent the transformation of amyloidogenic proteins/monomers to oligomers/fibrils to maintain elevated levels of soluble proteins/monomers and minimize the presence of oligomeric/fibrillar plaques in the brain.

In contrast to Aβ, which mostly accumulates in the brain of AD patients, α-Syn and tau deposition occur in various neurodegenerative diseases, which are known as tauopathies and synucleinopathies [22–25]. The colocalization of two or more different kinds of protein fibrils/aggregates in neurodegenerative diseases has been extensively reported [26–28]. The pathological aggregation of tau or neurofibrillary tangles has been recognized as the hallmark of AD and PD [29,30]. Hyperphosphorylation of tau protein results in its dissociation from microtubules and aggregation. There are several phosphorylation sites on tau proteins, and they show different biological impacts depending on their phosphorylation site [31]. Tau phosphorylation at the Thr231 site leads to the formation of the two *cis* and *trans* conformations, among which *cis* is neurotoxic and *trans* has a protective role [32]. The *cis* p-tau isomer is more susceptible to aggregation and more resistant to dephosphorylation, unlike the *trans* p-tau isomer. The *cis-trans* isomerization of phosphorylated tau by proline-directed kinases is reported to regulate various signaling pathways and is involved in the pathogenesis of AD and PD [33–35]. Although the distinct biological roles of the cis and trans conformations of p-tau are well established, their specific and differential impacts on the aggregation kinetics and pathological interactions with Aβ and α-Syn have not been understood. It is crucial to investigate how *cis* p-tau and *trans* p-tau isomers affect the fibrillation of Aβ and α-Syn. Due to distinct structural and functional differences between the *cis* and *trans* conformations of phosphorylated tau (p-tau), particularly regarding their biological activity and interactions, we hypothesized that these two isomers might differentially affect the fibrillation of Aβ and α-Syn. The primary objective of this study was to investigate the mechanisms by which *cis*- and *trans*-p-tau interact with Aβ and α-Syn, and to determine how these interactions influence the fibrillation dynamics of both amyloidogenic proteins. Here, we combined both simulation and experimental approaches and revealed the mechanism of interactions of *cis* and *trans* isomers of p-tau with Aβ and α-Syn, and showed that *cis* p-tau and *trans* p-tau differently enhance the fibrillation of Aβ and α-Syn.

## 2. Materials and methods

### 2.1. Method

Molecular dynamics simulates atomistic interactions over time by numerically solving Newton's equations of motion. Newton's equations of motion describe the laws of motion of objects in classical mechanics. Molecular dynamics studies the dynamical evolution of molecular systems at the atomic level. The main steps of molecular dynamics

simulation are: defining initial conditions (specifying the positions, velocities, and other physical properties of atoms at the beginning of the simulation), calculating interatomic forces based on Newton's equations, and analyzing the results. Structures of cis and *trans* tau peptides were built using Avogadro software (using the build peptide module), and biotin was added manually to the end of each peptide. Next, the peptides were characterized using Ligand Reader & Modeler [36] from the charm-goi.org web interface [37]. Later, structures of β-Amyloid and α-synuclein were obtained from the Protein Data Bank with PDB code 1IYT [38] and 2KKW [39,40], respectively, which were transferred to the CHARMM-GUI PDB Manipulator [41]. Structures were used in the CHARMM-GUI Multicomponent Assembler [42] to prepare a simulation box including β-Amyloid and α-synuclein (Table 1). To achieve the biological condition as much as possible, the pH of all simulations was set to 7.4 (representing the physiological pH level of plasma) with 0.15 concentration of NaCl salt and TIP3P water molecules [43].

The time step of each MD simulation was 2 fs, and periodic boundary conditions (PBC) were applied in all three dimensions. The cut-off of the van der Waals (vdW) interaction was set at 1.4 nm, and the long-range electrostatic interaction was calculated via the particle mesh Ewald (PME) method [44]. The bond lengths were constrained by the LINCS algorithm [45]. All MD simulations were performed after energy minimization, with NVT ensemble and NPT simulation, and the temperature was maintained at 303.15 K for 50 ns. Finally, the main simulations were performed for 100 ns through 2 ps time steps. Next, an was run at 310 K and 1 atm for 30 ns. Snapshots from the simulations were created by the Visual Molecular Dynamics (VMD) software [46].

To analyze the surface area in contact with proteins, the accessible surface area analysis (SASA) or solvent accessible surface area (SASA) has been calculated. SASA is the surface area of a biomolecule that is accessible to the solvent. With this analysis, it is possible to find out how much of the biomolecule's surface is in contact with the solvent and how much of its surface is in contact with other molecules (in aggregation). Therefore, SASA can be a measure of the degree of aggregation and density. The lower the SASA, the less the solvent-accessible surface area and the greater the degree of aggregation and density of molecules. Molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) has been used to calculate electrostatic, van der Waals, and total energies between biomolecules. The new tool G_MM-PBSA, which is an extended method of the MM-PBSA method, is used here [47]. MM-PBSA has found wide application in calculating the energies between biomolecules. However, there are limitations to its use. To overcome these limitations, an open-source tool called g_mmpbsa has been developed, which can be implemented in the GROMACS software. We performed binding free energy calculations using the MM/PBSA approach in CHARMM36, analyzing 100 snapshots extracted at 200 ps intervals from the last 20 ns of each trajectory. The total binding free energy (ΔGbind) was decomposed into molecular mechanics (ΔGMM: electrostatic + van der Waals interactions calculated with CHARMM36, 14 Å cutoff), solvation (ΔGsolv: polar contributions from Poisson-Boltzmann calculations using dielectric constants of 2 for protein and 80 for water; nonpolar terms from SASA with $γ = 0.00542$ kcal/mol/Å²).

The Definition of Secondary Structure of Proteins (DSSP) algorithm was used to analyze the secondary structure of the protein. The DSSP algorithm is implemented in the GROMACS software and can be used to analyze the secondary structure of proteins. The basis of this analysis is the analysis of hydrogen bonding patterns. To calculate the displacement of the structures, RMSF (Root Mean Square Fluctuation) analysis is used. RMSF stands for Root Mean Square Fluctuation, which is a similar analysis to RMSD, but it has an important advantage over RMSD because, instead of showing the displacement of the entire biomolecule over time, it can also calculate the displacement of individual amino acids and residues. This feature allows it to show the fluctuations and displacement of individual residues. In Fig. 2e, this analysis is used, and the displacement of each residue is shown.
<table><caption>**Table 1**

Components in the simulation boxes and the corresponding size of the simulation box.</caption><thead><tr><th></th><th>Cis-tau peptide</th><th>Trans-tau peptide</th><th>β-Amyloid</th><th>α-synuclein</th><th>Na<sup>+</sup> ions</th><th>Cl<sup>-</sup> ions</th><th>Water molecules</th><th>Box size (Å<sup>3</sup>)</th></tr></thead><tbody><tr><td rowspan="3">Phase 1</td><td>1</td><td>-</td><td>1</td><td>-</td><td>33</td><td>29</td><td>10,441</td><td>70 × 70 × 70</td></tr><tr><td>-</td><td>1</td><td>1</td><td>-</td><td>31</td><td>29</td><td>10,441</td><td>70 × 70 × 70</td></tr><tr><td>1</td><td>-</td><td>-</td><td>1</td><td>182</td><td>172</td><td>60,771</td><td>125 × 125 × 125</td></tr><tr><td rowspan="6">Phase 2</td><td>-</td><td>1</td><td>-</td><td>1</td><td>180</td><td>172</td><td>60,727</td><td>125 × 125 × 125</td></tr><tr><td>5</td><td>-</td><td>3</td><td>-</td><td>464</td><td>432</td><td>152,710</td><td>70 × 70 × 70</td></tr><tr><td>-</td><td>5</td><td>3</td><td>-</td><td>454</td><td>432</td><td>152,704</td><td>70 × 70 × 70</td></tr><tr><td>-</td><td>-</td><td>3</td><td>-</td><td>461</td><td>434</td><td>153,246</td><td>70 × 70 × 70</td></tr><tr><td>5</td><td>-</td><td>-</td><td>3</td><td>39</td><td>25</td><td>9513</td><td>175 × 175 × 175</td></tr><tr><td>-</td><td>5</td><td>-</td><td>3</td><td>29</td><td>25</td><td>9559</td><td>175 × 175 × 175</td></tr><tr><td>-</td><td>-</td><td>-</td><td>3</td><td>36</td><td>27</td><td>10,033</td><td>175 × 175 × 175</td></tr></tbody></table>

## 2.2. α-Syn production and purification

Recombinant human α-synuclein (α-syn) was expressed in *Escherichia coli* BL21(DE3) strain using the pET11-D plasmid vector, following the protocol outlined by Mohammad-Beigi et al. [15] Briefly, the harvested cells were resuspended in an osmotic shock buffer (30 mM Tris-HCl, 40 % sucrose, 2 mM EDTA, pH 7.2) and incubated for 10 min. After centrifugation (9000g, 20 °C, 30 min), the resulting pellet was resuspended in ice-cold deionized water. Subsequently, 40 µL of saturated MgCl2 was added, and the mixture was incubated on ice for 3 min before another round of centrifugation. The supernatant was acidified by titration with 1 M HCl to reach a pH of 3.5. Following this, the supernatant was collected by centrifugation and then neutralized to pH 7.5 using 1 M NaOH. The neutralized solution was loaded onto a Q-Sepharose column (HiTrap Q HP) pre-equilibrated with 20 mM Tris-HCl, pH 7.5. α-syn was subsequently eluted using a gradient of NaCl ranging from 0.1 M to 0.5 M. The fractions containing α-syn eluted with the NaCl gradient were assessed using SDS-PAGE. The purified α-syn was dialyzed extensively against deionized water, lyophilized, and stored at -20 °C.

## 2.3. Plate reader fibril formation assays

The fibril formation experiments were conducted using a Genios Pro fluorescence plate reader (Tecan, Mänerdorf, Switzerland). In each well of a 96-well-plate (Nunc, Thermo Fischer Scientific, Roskilde, Denmark), 150 µL of a PBS solution was prepared, containing freshly dissolved and filtered through a 0.2 µm filter of 70 µM α-syn or Abeta peptide (from a 6 mM stock in DMSO based on A280), 40 µM ThT, and varying concentrations of *Cis_Tau* and *Trans_Tau* (0.1, 1, and 10 µM). A 3 mm diameter glass bead was added to each well, and the plate was sealed with Crystal Clear sealing tape (Hampton Research, Aliso Viejo, CA). Fibrillation was monitored at 37 °C with orbital shaking at 300 rpm for α-syn, while Abeta fibrillated without shaking. Excitation of the samples was performed at 448 nm, and the emitted fluorescence was measured at 485 nm.

The Finke-Watzky (F-W) equation was fitted to the ThT fibrillation data:

$$F(t) = \frac{ThT_{\text{end level}}}{1 + e^{-\theta(t-t_{\text{v}})}}$$

where $t_{v}$ is the time required to produce half the total product (ThT end level), and $v$ is the rate of growth at $t_{v}$. This two-step model describes the macroscopic evolution of the amyloid formation, as measured by ThT fluorescence. In this model, only primary nucleation and autocatalytic growth of fibrils were considered.

## 2.4. TEM

10 µL of each sample solution was carefully placed onto a copper grid coated with collodion and allowed to sit on the grid for 3 min at room temperature to ensure proper adhesion. The grid was gently rinsed three times with double-deionized water. This step helps remove any excess or

unbound material from the surface of the grid, ensuring a clean sample preparation. In the next step, the copper grid was incubated with 3 µL of a solution containing 2 % (w/v) uranyl acetate for 2 min. After staining, the grid underwent another round of rinsing with double deionized water, again repeated three times. Finally, the prepared sample grids were left to air-dry overnight under vacuum conditions. Once dried, the sample grids were used to obtain transmission electron microscopy (TEM) images using a HITACHI H-7650 transmission microscope (Hitachi, Tokyo, Japan) at 25 °C.

## 2.5. Tau purchase and sequence

Two custom-prepared peptide sequences Biotin-s-s-V-A-V-V-R-pT-(Pip)-P-K-S-P-COOH and Biotin-s-s-V-A-V-V-R-pT-A-P-K-S-P-COOH were purchased (biosynthesis, Texas, USA) and used as cis and trans forms of tau protein.

## 2.6. Statistical analysis

To test the statistical significance of differences, ANOVA analysis of the data was carried out.

# 3. Results and discussion

To investigate the mechanisms by which cis and trans p-tau peptides interact with proteins such as Aβ and α-Syn, we conducted simulations in two phases: first, focusing on one-on-one interactions, and then expanding to multi-component systems. In the initial phase, we used all-atom molecular dynamics (MD) simulations to study the interaction between a single protein and a peptide. A detailed analysis of the results with the structure of peptides and proteins (Fig. S1) is provided in the supporting material. Following the exploration of possible one-on-one interactions between peptides and proteins, we examined the behavior and interactions of multiple proteins and peptides to simulate the complexity of molecular interactions and gain a deeper understanding of wet-lab environments at the molecular level.

## 3.1. Tau peptide interaction with Aβ

To characterize the interactions within each simulation, we evaluated the solvent-accessible surface area (SASA) of the components. SASA values for Aβ proteins and peptides were calculated over time and plotted accordingly. Control groups were considered to investigate the effect of cis/trans tau on amyloid aggregation. For this purpose, the control group without the presence of cis/trans tau was considered. In this way, the accumulation of amyloid without the presence of cis/trans tau was investigated and considered as a comparative reference. After that, the presence of cis/trans tau and its effect on amyloid aggregation were studied. Amyloid aggregation in all other conditions was compared with the control groups to investigate the effect of tau protein isomers on amyloid aggregation. In the control simulation, the SASA of Aβ proteins gradually decreased, indicating self-assembly of the proteins. In the
presence of both *cis* and *trans* p-tau, the SASA of Aβ followed a similar trend to the control during the first 30 ns. However, after this point, the SASA of Aβ dropped significantly, suggesting enhanced self-assembly due to stronger interactions with *trans* p-tau (Fig. 1a–d). The SASA analysis of peptide structures showed that *cis-tau* peptides were more dispersed in the solvent compared to *trans* p-tau, indicating weaker interactions with the proteins.

In the first phase of simulations, similar analyses confirmed that Aβ forms compact structures in the presence of *trans* p-tau in one-on-one interactions (Fig. S1a). Additionally, we calculated the radius of gyration (Rg) for Aβ as further evidence of self-assembly when *trans* p-tau was present (Fig. S2b). This showed that Aβ proteins formed compact structures after approximately 70 ns in the presence of *trans* p-tau. The Rg distribution also appeared tighter when Aβ was exposed to *trans* p-tau, further supporting this observation (Fig. S2c).

Snapshots of each simulation at 100 ns were analyzed to provide further insights. These images revealed that *trans* p-tau promotes the assembly of all components, whereas *cis* p-tau allows some Aβ to interact with the peptides while others move freely, similar to the behavior in the control simulation without peptides.

Our goal was to identify the primary driver behind the self-assembly of Aβ proteins in the presence of *trans* p-tau peptides by performing an interaction energy analysis between the tau peptides and Aβ. The interaction patterns of tau peptides with Aβ vary based on the conformation of the tau peptides (*cis* or *trans*) (Fig. 1e and f). *Cis* p-tau peptides exhibited stronger electrostatic repulsion, which creates a repelling force between the peptides and proteins. This finding aligns with previous observations where certain amyloids appeared to float freely in the simulation box. In contrast, *trans* p-tau peptides interacted strongly with amyloids, leading to the formation of compact aggregates (see snapshot). Energy calculations for the one-on-one simulations revealed a similar trend, where *trans* p-tau showed stronger interactions with Aβ compared to *cis* p-tau (Fig. S3). Specifically, the interaction energy of *cis* p-tau (–78.4 kJ/mol) was significantly lower than that of *trans* p-tau (–149.5 kJ/mol). Larger-scale simulations, involving multiple proteins and peptides, further confirmed that *cis* p-tau peptides repel amyloids due to their higher electrostatic properties. Snapshots from the first and second phases of the simulations (Figs. S3 and S4) clearly show the tighter assemblies formed in the presence of *trans* p-tau.

To get a deep insight into the impact of p-tau isomers on the Aβ peptides, we performed an energy analysis for each residue, and the results for the three peptides are presented as average interaction energy in Fig. 1g, h. Lysine (LYS¹⁶, LYS²⁸) and arginine (ARG⁵) residues of Aβ peptides exhibited repulsion interactions with *trans* p-tau, while Glutamic acid (GLU), aspartic acid (ASP), and alanine (ALA) exhibited the highest affinity toward *trans* p-tau. Despite the shown repulsions, Aβ residues exhibit a –191.93 kJ/mole attraction toward *trans* p-tau.

Interestingly, the same residues exhibited a high affinity for *cis* p-tau as well. Glutamic acid (GLU³, GLU¹¹, GLU²²), aspartic acid (ASP⁷, ASP²³), glycine (GLY⁹, GLY²⁹, GLY³⁷, GLY³⁸), alanine (ALA⁴²), and isoleucine (ILE⁴¹) showed repulsion toward *cis* p-tau. A total of 99.98 kJ/mol of repulsion was observed between residues, which is mainly due to electrostatic interactions between charged residues and *cis* p-tau. The positively and negatively charged residues of Aβ peptides are mainly responsible for the attractive or repulsive interactions. The average interaction between three Aβ and five tau peptides was calculated and showed that similar trends exist in the interaction between individual residues and peptides (Fig. S5). ARG and LYS repel the *trans* p-tau and show affinity toward *cis* p-tau. In contrast, GLU, ASP, GLY, ILE, and ALA repel *cis-tau* and attract *trans* p-tau.

A similar analysis was conducted for one-on-one simulations (Fig. 2a, b) to confirm the determinant role of the same residues in interactions with tau peptides. Similarly, the same type of residue attracted and repelled tau peptides. The C-terminus residues demonstrated a higher binding affinity for *trans* p-tau peptide, while the N-terminus residues exhibited a greater affinity for *cis-tau* peptide. Another key aspect of Aβ

binding to tau peptides is the secondary structure adopted by the protein strands. The secondary structure of proteins in interaction with peptides in one-to-one simulations is outlined in Fig. 2c, d. At first glance, it can be observed that the Aβ strand with lower affinity toward *cis* p-tau and subsequently with lower aggregation has adopted mostly an α-helix structure. It was revealed that in the presence of *cis* p-tau peptides, Aβ's repellent residues mostly adopt α-helix (29.26 %) conformation, while in the presence of *trans* p-tau α-helix conformation drops to 26.5 %. In the presence of *trans* p-tau, coil/bend/turn structures can also be observed alongside the α-helix. The helical content increased when peptides were elongated rather than aggregated into structures.

As an additional analytical tool, root mean square fluctuation (RMSF) was used to gain insight into the interactions between Aβ and peptides by analyzing the fluctuations of their subunits (Fig. 2e). Given the presence of three Aβ in each simulation box, we calculated the average RMSF values for each of the 42 residues, along with their standard deviations. Interestingly, the RMSF of residues in both the control simulation and those with *cis* p-tau was lower than in the simulations with *trans* p-tau. These observations support the earlier findings and demonstrate that *trans* p-tau increases the strain on amyloids, thereby promoting their self-assembly. Similar results were also obtained in the one-on-one simulations (Fig. S3-B-i), where higher fluctuations in Aβ residues were observed in the presence of *trans* p-tau.

Finally, we examined the interactions between Aβ proteins and both *cis* and *trans* p-tau peptides by calculating the hydrogen bonding (Fig. 2f). Aβ formed more hydrogen bonds with *trans*-tau peptides than with *cis* peptides. However, the difference was modest, with Aβ forming approximately 9 hydrogen bonds with five *cis* p-tau peptides and 12 with five *trans* p-tau peptides. In the one-on-one simulations, a single Aβ formed about 2 and 3 hydrogen bonds with one *cis* p-tau and one *trans* p-tau, respectively.

## 3.2. Tau peptide interaction with α-syn

The MD simulation was performed to investigate the behavior of α-Syn proteins in the presence and absence of p-tau peptides. The first evaluation involved calculating the solvent-accessible surface area (SASA) for all components throughout the simulations, as shown in Fig. 3a–b. The SASA of α-Syn was lowest in the presence of *trans* p-tau, suggesting that *trans* p-tau promotes the self-assembly of α-Syn. In control case simulation (without tau peptides), the extended structure of α-Syn toward water molecules resulted in the highest SASA. Similar one-on-one simulations further confirmed that the SASA of α-syn decreased over 100 ns of interaction with *trans* p-tau (Fig. S7-A).

For the one-on-one interactions, the radius of gyration (Rg) of the α-syn protein was calculated and plotted over time, as well as the overall Rg distribution, in Fig. S7b and c, respectively. After the initial 20–30 ns, the Rg of α-Syn stabilized for both *trans* and *cis* p-tau interactions, with a smaller Rg in the presence of *trans* p-tau, indicating a more compact protein structure. The narrower Rg distribution, particularly in the presence of *trans* p-tau, highlights a protein size approximately five Å smaller than that observed with *cis* p-tau.

Snapshots from the simulations (Fig. 3c–d) confirm that the control case simulation (α-Syn without tau) exhibited the lowest level of interactions among α-syn proteins. A comparison of snapshots from the *trans* and *cis* p-tau simulations showed that *trans* p-tau promotes the formation of interwoven protein and peptide structures, while such organization was less evident with *cis* p-tau.

The energy of interaction between proteins and peptides was calculated over 100 ns to ensure the accuracy of the results. Fig. 3e–f displays the average energy of interaction during this time. It is clear that in the presence of *trans* p-tau, α-Syn proteins tightly bind to each other and *trans* p-tau, resulting in dense self-assemblies within the simulation box. However, due to the strong electrostatic repulsion between *cis* p-tau and α-Syn, their interaction tendency is greatly reduced, which may explain the similarities observed between the control case
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** a–d) Solvent accessible surface area (SASA) of all Aβ proteins in the simulations. Snapshots of final stages for the interaction of Aβ with or without tau peptides. e, f) Energy of the interactions between Aβ and (trans and cis) p-tau peptides. g, h) average energy of interacting residues with (trans and cis) p-tau peptides with Aβ.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** a, b) Energy of residues interacting with (*trans* and *cis*) p-tau peptide with Aβ, respectively. c, d) Secondary structures of Aβ strands during interactions with *trans* and *cis* p-tau peptides, respectively. e) RMSF (nm) of Aβ residues in simulations. f) Formation of Hbonds between tau peptides and Aβ during 100 ns interactions.</figcaption></figure>

and α-Syn-*cis* p-tau interaction. The interactions between peptides and α-Syn proteins are primarily governed by electrostatic forces, which dictate whether the total energy favors attraction or repulsion. In the one-on-one simulations, *trans* p-tau showed stronger interactions with α-Syn (Fig. S8), while *cis* p-tau exhibited significant repulsion (+42.5 kJ/mol). The *trans* conformation of tau displayed strong electrostatic attraction (−101.5 kJ/mol) with α-Syn. Additional snapshots from the simulations are provided in Fig. S9, offering further insight into the molecular behavior of α-Syn in the presence and absence of p-tau peptides.

The energy of interactions was analyzed to determine the residues that interact with peptides. As shown in Fig. 3g–h, GLU (GLU²⁸, GLU³⁵, GLU¹¹⁴, GLU²⁰, GLU¹³, GLU⁸³, GLU⁵⁷, GLU⁶¹, GLU⁴⁶) and ASP (ASP¹¹⁵, ASP², ASP⁹⁸) have a stronger affinity toward *trans* p-tau and a high level of repletion was observed for LYS residues (LYS³², LYS²¹, LYS¹², LYS³⁴, LYS⁵⁸, LYS²³, LYS¹⁰, LYS⁸⁰, LYS⁶, LYS⁶⁰). In the case of *cis* p-tau, different behavior was observed, where amine-containing residues tend to attract the peptide, while acidic residues (ASP and GLU) tend to repel it. In the presence of five *trans* p-tau and five *cis* p-tau peptides, the

overall interacting energy of the residues from three α-Syn strands was −236.3 and 75.76 kJ/mol, respectively (The details of all proteins are provided in Fig. S10). Due to the high number of residues (140) in α-Syn, a part of them is listed in the diagram.

For one-on-one simulations, similar analysis revealed that again the residues with the amine group bind the *cis* p-tau peptide while acidic residues attract the *trans* p-tau peptide (Fig. 4a, b). In Fig. 4c, d, secondary structures of α-Syn residues are shown within one-on-one simulations. As can be seen, α-Syn with less affinity toward *cis* peptides, resulting in a lower tendency for self-assembly, mostly adopts an α-helix structure. To further explore the simulation results, we calculated the RMSF for each α-Syn residue. The average RMSF and standard deviations for the residues of the three α-syn proteins are shown in Fig. 4e. Compared to *trans* p-tau, simulations with *cis* p-tau and the control exhibited lower residue fluctuations. In the presence of *trans* p-tau, α-Syn proteins showed a higher fluctuation level. These findings suggest that α-Syn proteins self-assemble/aggregate and form compact structures in the presence of *trans* p-tau. The one-on-one simulations also revealed that *trans* p-tau nearly doubled the RMSF of α-Syn residues,
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** a–d) SASA of the components in the simulations, including α-Syn proteins and tau peptides. Snapshots of simulations at the end of 100 ns are provided to better understand the interactions between components. e, f) Energy of interactions between α-Syn proteins and *cis* and *trans* p-tau peptides. g, h) hydrogen bond formation between *cis* and *trans* p-tau peptides and α-Syn proteins.</figcaption></figure>

further highlighting the effects imposed by *trans* p-tau during the adsorption and aggregation process (Fig. S11-a).

The increased fluctuations are likely due to the higher number of hydrogen bonds formed between the proteins and peptides. As shown in Fig. 4e, *trans* p-tau forms significantly more hydrogen bonds than *cis* p-tau, approximately 14 compared to 3. A similar trend was observed in the one-on-one systems, where *trans* p-tau established a greater number of hydrogen bonds with α-Syn (Fig. S11b). The data clearly shows that

*trans* p-tau peptides interact tightly with α-Syn proteins, wrapping around the strands and promoting tighter assemblies.

### 3.3. *cis* and *trans* p-tau isomers differently affected the fibrillation of Aβ and α-syn

To confirm the results of simulation studies, we studied the effects of *cis* p-tau and *trans* p-tau isomers on the fibrillation kinetics of Aβ and
<figure><img src="image_7.png" /><figcaption>Fig. 3. (continued).</figcaption></figure>

α-Syn in vitro. The fibrillation kinetics of amyloidogenic proteins are characterized by a sigmoidal curve, depicting a distinctive pattern in the temporal evolution of protein aggregation. This curve signifies three fundamental phases in the fibrillation process. Initially, there is a lag phase during which nucleation events occur, setting the stage for subsequent protein aggregation. This is followed by a steep elongation phase, marked by rapid protein aggregation and the formation of fibrillar structures. Finally, the system reaches a plateau phase, indicating a state of equilibrium where the rate of aggregation balances the dissolution or degradation of fibrils [48,49]. In the absence of p-tau, the fibrillation kinetics of both Aβ and α-Syn were fit by a sigmoidal curve, a pattern commonly observed in amyloidogenic proteins (Fig. 5a–d).

Transmission electron microscopy (TEM) images also confirmed the results of the ThT test and showed that mature Aβ and α-Syn form over time (Fig. 6k, l). The kinetics of Aβ and α-Syn fibrillation were also studied in the presence of increasing concentrations of the *cis* p-tau and *trans*-p-tau isomers using both ThT and TEM imaging (Figs. 5a–d and 6m–p). *trans*-p-tau enhanced the fibrillation of both Aβ and α-Syn in a concentration-dependent manner, exerting more pronounced acceleration effects on the fibrillation of α-Syn, clearly evidenced by the shift of the sigmoidal curve toward shorter lag times and elevated steep elongation phases (Figs. 5a–d and 6m, n). *Cis* p-tau demonstrated a mild accelerative effect on the Aβ fibrillation while exhibiting a negligible effect on the rate of α-syn fibrillation. These results were also confirmed by TEM images and a big skein of mature fibrils of Aβ and α-Syn formed in the presence of *trans* p-tau (Figs. 5a–d and 6o, p). Although *cis* p-tau

slightly accelerated the fibrillation process, the morphology of fibrils formed in the presence of *cis* p-tau was like a writhen skein that was completely different from that of control cases.

To quantify the acceleratory effects of *cis* and *trans* isomers of p-tau on different phases of fibrillation and to measure the total amount of fibrils formed after Aβ and α-Syn incubation with *cis* and *trans* isomers of p-tau, the ThT kinetic data were analyzed using the Finke–Watzky (F – W) model (Seq 1 and Seq 2) [15] (Fig. 5e–j). Applying this model to ThT kinetic data yields essential quantitative insights into the time needed to achieve half of the total products (t₁/₂) (Fig. 5e and i), the rate of fibril growth (v) (Fig. 5f and h), and the amount of end-level fibril (Fig. 5g and j). As seen in Fig. 5e, i, t₁/₂ of α-Syn fibrils decreased with increasing concentration of *trans* p-tau, and the shortest t₁/₂ was obtained at 10 µg of *trans* p-tau, while increasing concentration of *cis* p-tau did not change the t₁/₂ length. We also calculated the amount of fibril created at the end of the fibrillation process using ThT data. The highest level of fibril was formed after incubation of α-Syn with 10 µg of *trans* p-tau (Fig. 5g and j). To explain the accelerative effects of *cis* and *trans* isomers of p-tau on α-Syn fibrillation, the fibril growth rate step (elongation phase) of the fibrillation process was calculated, and it was shown that both *cis* p-tau and *trans* p-tau enhance the fibril growth rate (Fig. 5f and h).

Traditionally, *cis* p-tau has been considered highly toxic due to its tendency to misfold, promoting tau aggregation, neuronal damage, and cognitive decline [31]. It is often viewed as a hallmark of neurodegenerative diseases. In contrast, *trans* p-tau has been thought of as a relatively protective isomer, with a less significant role in pathological
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** a, b) Energy of residues interacting with (trans and cis) p-tau peptide with α-Syn, respectively. c, d) Secondary structures of α-Syn strands during interactions with trans and cis p-tau peptides, respectively. e) RMSF (nm) of α-syn residues in the presence and absence of cis and trans p-tau. f) hydrogen bond formation between tau peptides and α-Syn proteins.</figcaption></figure>

aggregation, as it typically adopts a conformation less susceptible to fibrillation [31,33]. However, this study challenges these established roles. Surprisingly, *cis* p-tau, despite its reputation as the toxic isomer, showed minimal effects in promoting *Aβ* and *α*-Syn aggregation, suggesting its role in fibril formation may be more complex than previously believed. Conversely, *trans* p-tau, often seen as the “safe” isomer, significantly enhanced tau fibrillation, a finding that contrasts sharply with previous assumptions [31,33]. This unexpected result suggests that *trans* p-tau may play a more active role in pathological aggregation than previously understood. These observations raise critical questions about the mechanisms underlying tauopathy progression. The findings indicate that under certain conditions, the *trans* isomer, once thought to be protective, could actively participate in the aggregation process. This highlights the need for further research to elucidate how isomerization

impacts tau dynamics and the contributions of both *cis* and *trans* p-tau in forming aggregates.

4. Conclusion

This study provides significant insights into the differential effects of *cis* and *trans* isomers of p-tau on amyloidogenesis, specifically the fibrillation of *Aβ* and *α*-Syn. We demonstrated that the *trans* isomer of p-tau strongly promotes the aggregation of both *Aβ* and *α*-Syn, leading to accelerated amyloid formation and tighter assemblies, as observed in both simulations and experimental data. In contrast, the *cis* isomer exhibited a more limited effect, particularly on *α*-Syn fibrillation, where its influence was minimal. These findings highlight the distinct roles that *cis* and *trans* p-tau play in amyloidogenic pathways, shedding light on
<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Sigmoidal kinetics profile of (a, b) α-Syn and (c, d) Aβ and in the absence and presence of increasing concentrations of *cis* and *trans* p-tau monitored by ThT fluorescence. Kinetic parameters (e) relative half time ($t_{1/2}/t_{1/2}$ control), (f) relative growth rate ($v/v_{control}$), and (g) relative ThT end level (ThT end level/ThT end level *control*) calculated for α-Syn fibrillation. Kinetic parameters (h) relative half time ($t_{1/2}/t_{1/2}$ control), (i) relative growth rate ($v/v_{control}$), and (j) relative ThT end level calculated (ThT end level/ThT end level *control*) for Aβ fibrillation. For the multiple comparison in (e–j), the P value was calculated by one-way ANOVA with Tukey post hoc test without any adjustment. n.s., not significant (P > 0.05).</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** TEM image of mature (k) α-Syn and (l) Aβ fibril formed in the absence of p-tau (control case) (scale bar 1 µm). TEM image of (m) α-Syn and (n) Aβ fibril formed in the presence of *trans* p-tau peptides (scale bar 1 µm). TEM image of (o) α-Syn and (p) Aβ fibril formed in the presence of *cis* p-tau peptides (scale bar 1 µm).</figcaption></figure>

their potential contributions to neurodegenerative processes such as AD and PD. Understanding the mechanistic interactions between tau iso- mers and amyloid proteins may pave the way for the development of more targeted therapeutic interventions aimed at modulating protein aggregation in neurodegenerative diseases.

### 4.1. Limitations

While these findings highlight the distinct modulatory roles of p-tau isomers in amyloidogenic pathways and their potential relevance to neurodegenerative processes such as AD and PD, it is important to note the limitations of this study. We used short synthetic peptides rather than full-length tau protein, and our findings have not yet been validated
in cellular or animal models.

Although our 100 ns MD simulations effectively revealed the distinct binding preferences of cis- and trans-p-tau (Thr231) with Aβ and α-Syn - findings corroborated by experimental fibrillation kinetics showing trans-p-tau's dramatic acceleration of aggregation (Aβ: ~7 h → 30 min; α-Syn: ~10 h → 1 h) - we recognize several inherent constraints. The simulation timescale, while sufficient to identify initial interaction affinities, cannot capture the complete amyloid maturation process, particularly the later stages of fibril formation and stabilization observed in experiments. Additionally, while we demonstrated trans-p-tau's stronger binding and pro-aggregatory effects compared to the cis-isomer (which showed more modest acceleration: Aβ/α-Syn ~7 h → 5 h), our model does not address potential formation of heterotypic p-tau/Aβ or p-tau/α-Syn oligomeric species that might emerge during prolonged co-incubation. Furthermore, the study focused specifically on Thr231-phosphorylated tau, leaving open questions about how other phosphosites or post-translational modifications might influence these cross-interactions. These limitations highlight opportunities for future studies employing enhanced sampling techniques to probe longer-timescale aggregation events to investigate larger oligomeric complexes, and systematic investigations of additional phospho-tau variants and their synergistic effects on amyloidogenesis.

### CRediT authorship contribution statement

**Mohammad J. Hajipour:** Writing – review & editing, Writing – original draft, Validation, Supervision, Methodology, Investigation, Conceptualization.
**Sima Rezvantalab:** Writing – review & editing, Writing – original draft, Validation, Supervision, Methodology, Investigation, Conceptualization.
**Hossein Mohammad-Beigi:** Writing – review & editing, Validation, Supervision.
**Reza Maleki:** Writing – review & editing, Validation, Supervision.
**Ghazal Ghezelbashan:** Writing – review & editing, Validation, Supervision.
**Esmael M. Alyami:** Writing – review & editing, Validation, Supervision.
**Ayyalusamy Ramamoorthy:** Writing – review & editing, Validation, Supervision.
**Lionel Maurizi:** Writing – review & editing, Validation, Supervision, Funding acquisition.

### Declaration of competing interest

The authors declare that they have no conflicts of interest related to this work. This research was conducted independently, without any financial, commercial, or personal relationships that could be perceived as a conflict of interest. All authors have approved the final manuscript and confirm that there are no competing financial or non-financial interests that could have influenced the outcomes of this study. For transparency, any relevant funding sources and institutional affiliations have been fully disclosed within the manuscript.

### Acknowledgment

The authors would like to acknowledge the support of EIPHI Graduate School (contract ANR-17-EURE-0002) and the ANR Nanoblorona (contract ANR 21 CE18 0015). This work has also been funded by the European Union through the PO FEDER-FSE Bourgogne 2021/2027 program. We also acknowledge Prof. Daniel Otzen (iNANO, Aarhus University) for providing us with α-Syn and Aβ.

### Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijbiomac.2025.145500.

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