# S100A9 Alters the Pathway of Alpha-Synuclein Amyloid Aggregation

^{1}

^{2}

^{*}

## Abstract

**:**

## 1. Introduction

## 2. Results

^{−1}(the second derivative main minimum at 1626 cm

^{−1}), while both A2 and A3 have maxima at 1624 cm

^{−1}(the second derivative minima at 1623 cm

^{−1}and 1624 cm

^{−1}respectively), indicating that the A1 population has the weakest hydrogen bonding in the $\beta $-sheet structure. The A1 spectrum also has a shoulder at 1636 cm

^{−1}reflected by a minimum in the second derivative at 1636 cm

^{−1}(weaker type of hydrogen bonding), which is not present in the other two populations. The A2 and A3 population fibrils also have some differences between their secondary structures. This is most evident by the existence of a minimum in the A2 second derivative spectrum at 1641 cm

^{−1}and a shoulder at 1614 cm

^{−1}, which are not present in the case of A3 and indicate the presence of different types of hydrogen bonding. There is also some distinction in the turn/loop motif region, where A2 has a minimum at 1673 cm

^{−1}, while for A3 it is at 1667 cm

^{−1}.

^{−1}, 1636 cm

^{−1}and the shoulder at 1619 cm

^{−1}, as well as turns-related minimum at 1667 cm

^{−1}(Figure 2A,B). Only at the lowest S100A9 concentration ($2.3$ $\mathsf{\mu}\mathrm{M}$) the spectrum is a bit different with only one broad minimum at 1625 cm

^{−1}, suggesting a mixture of conformations in the sample. It is important to mention, that only washed pellet, which contained only a trace amount of S100A9 as confirmed by SDS-PAGE (Appendix A Figure A2), was used to collect FTIR data. To determine that the observed effect is related to S100A9 and not to the presence of any similar size protein, the aggregation experiment was carried out with 70 $\mathsf{\mu}\mathrm{M}$ $\alpha $-syn and 70 $\mathsf{\mu}\mathrm{M}$ hen egg-white lysozyme ($14.4$ kDa). Unlike with 70 $\mathsf{\mu}\mathrm{M}$ S100A9, three populations with distinct ThT fluorescence intensity and FTIR spectra (Appendix A Figure A3) remain similar to the control sample without S100A9.

^{−1}and the more expressed shoulder at 1614 cm

^{−1}, indicating a minor change of hydrogen bond strength.

^{−1}and a shoulder appears at 1616 cm

^{−1}, indicating the formation of stronger hydrogen bonds. There is also a minimum at 1670 cm

^{−1}associated with the fibril turn/loop motifs. At the highest NaCl concentration, the main minimum remains at a similar position and we observe a loss of the 1616 cm

^{−1}shoulder. There is also a small variation in the turn/loop motif region, which becomes more similar to the lower NaCl concentration samples. The FTIR spectrum of $\alpha $-syn aggregated in the presence of S100A9 and 375 $\mathrm{m}\mathrm{M}$ NaCl is very similar to the spectrum of A3 population.

## 3. Discussion

## 4. Materials and Methods

#### 4.1. Protein Production

#### 4.2. ThT Assay

_{3}, PBS buffer (10 $\mathrm{m}\mathrm{M}$ Na

_{2}HPO

_{4}, $1.8$ $\mathrm{m}\mathrm{M}$ KH

_{2}PO

_{4}, 137 $\mathrm{m}\mathrm{M}$ NaCl, $2.7$ $\mathrm{m}\mathrm{M}$ KCl), pH 7.4. Other samples contained $2.3$ $\mathsf{\mu}\mathrm{M}$, $4.5$ $\mathsf{\mu}\mathrm{M}$, $9.0$ $\mathsf{\mu}\mathrm{M}$, 18 $\mathsf{\mu}\mathrm{M}$, 35 $\mathsf{\mu}\mathrm{M}$, 70 $\mathsf{\mu}\mathrm{M}$ of S100A9 (during experiments on how S100A9 influences aggregation of $\alpha $-syn). For a negative control sample, 70 $\mathsf{\mu}\mathrm{M}$ of hen egg-white lysozyme prepared from powders (Sigma-Aldrich, St. Louis, MO, USA, cat. No. L6876) was used in a place of S100A9. Samples with different NaCl concentrations contained 35 $\mathsf{\mu}\mathrm{M}$ S100A9 and additional 50 $\mathrm{m}\mathrm{M}$, 125 $\mathrm{m}\mathrm{M}$, 250 $\mathrm{m}\mathrm{M}$, 375 $\mathrm{m}\mathrm{M}$ NaCl. The ${t}_{50}$ was determined by fitting the sigmoidal curve to aggregation kinetic data.

#### 4.3. FTIR Spectroscopy

_{2}O (with 400 $\mathrm{m}\mathrm{M}$ NaCl to improve sedimentation [47]. After repeating the centrifugation and resuspension procedure three times, the fibril pellet was resuspended into a final volume of 150 μL. For each sample, 256 interferograms with 2 cm

^{−1}resolution were collected using Bruker Invenio S spectrometer equipped with a liquid-nitrogen-cooled mercury-cadmium-telluride detector at room temperature in a dry-air flow-through chamber. A D

_{2}O spectrum was subtracted from each sample spectrum, after which the spectra were baseline corrected in the region at 1595 cm

^{−1}to 1700 cm

^{−1}and normalized. All data processing was done using GRAMS software.

#### 4.4. Atomic Force Microscopy

#### 4.5. SDS-PAGE

## 5. Conclusions

## Author Contributions

## Funding

## Institutional Review Board Statement

## Informed Consent Statement

## Data Availability Statement

## Conflicts of Interest

## Appendix A

**Figure A1.**Aggregation kinetics of $\alpha $-syn without (

**A**–

**E**) and with S100A9 (

**F**–

**J**) in a presence of different NaCl concentrations: 0 $\mathrm{m}\mathrm{M}$, 50 $\mathrm{m}\mathrm{M}$, 125 $\mathrm{m}\mathrm{M}$, 250 $\mathrm{m}\mathrm{M}$, 375 $\mathrm{m}\mathrm{M}$. Each sample represented by 6 aggregation curves (6 repeats). Protein solution: 70 $\mathsf{\mu}\mathrm{M}$ $\alpha $-syn, 0 or 35 $\mathsf{\mu}\mathrm{M}$ S100A9, 50 $\mathsf{\mu}\mathrm{M}$ ThT, 0.02% NaN

_{3}, PBS buffer, pH 7.4.

**Figure A2.**SDS-PAGE gel of $\alpha $-syn, $\alpha $-syn–S100A9, S100A9 products after aggregation (samples after aggregation kinetic experiment, FTIR samples). Columns in the gel: (1) 70 $\mathsf{\mu}\mathrm{M}$ $\alpha $-syn before aggregation, (2) 70 $\mathsf{\mu}\mathrm{M}$ S100A9 before aggregation, (3) protein ladder (the molecular weights marked on the right), (4) 70 $\mathsf{\mu}\mathrm{M}$ $\alpha $-syn fibrils, (5) $\alpha $-syn with $2.3$ $\mathsf{\mu}\mathrm{M}$ S100A9 fibrils, (6) $\alpha $-syn with $4.5$ $\mathsf{\mu}\mathrm{M}$ S100A9 fibrils, (7) $\alpha $-syn with $9.0$ $\mathsf{\mu}\mathrm{M}$ S100A9 fibrils, (8) $\alpha $-syn with 18 $\mathsf{\mu}\mathrm{M}$ S100A9 fibrils, (9) $\alpha $-syn with 35 $\mathsf{\mu}\mathrm{M}$ S100A9 fibrils, (10) $\alpha $-syn with 70 $\mathsf{\mu}\mathrm{M}$ S100A9 fibrils. Columns of the soluble protein after aggregation $\alpha $-syn with different concentration of S100A9: (11) 0 $\mathsf{\mu}\mathrm{M}$, (12) $2.3$ $\mathsf{\mu}\mathrm{M}$, (13) $4.5$ $\mathsf{\mu}\mathrm{M}$, (14) $9.0$ $\mathsf{\mu}\mathrm{M}$, (15) 18 $\mathsf{\mu}\mathrm{M}$, (16) 35 $\mathsf{\mu}\mathrm{M}$, (17) 70 $\mathsf{\mu}\mathrm{M}$, (18) 70 $\mathsf{\mu}\mathrm{M}$ sample went through $0.22$ $\mathsf{\mu}\mathrm{m}$ PVDF membrane filter, (19) 35 $\mathsf{\mu}\mathrm{M}$ filtrate. The soluble protein after aggregation of 70 $\mathsf{\mu}\mathrm{M}$ S100A9 (20).

**Figure A3.**Aggregation kinetics (

**A1**–

**A3**), FTIR spectra (

**B**) and second derivatives (

**C**) of $\alpha $-syn populations (indicated by

**A1**–

**A3**), when $\alpha $-syn was aggregated in the presence of 70 $\mathsf{\mu}\mathrm{M}$ lysozyme monomers. All aggregation conditions were identical to the ones during $\alpha $-syn aggregation with 70 $\mathsf{\mu}\mathrm{M}$ S100A9, as described in the Materials and Methods section.

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**Figure 1.**Aggregation kinetic curves of $\alpha $-syn (

**A**–

**G**) and the respective ${t}_{50}$ (

**H**) at different concentrations of S100A9: $0.0$ $\mathsf{\mu}\mathrm{M}$ (

**A**), $2.3$ $\mathsf{\mu}\mathrm{M}$ (

**B**), $4.5$ $\mathsf{\mu}\mathrm{M}$ (

**C**), $9.0$ $\mathsf{\mu}\mathrm{M}$ (

**D**), 18 $\mathsf{\mu}\mathrm{M}$ (

**E**), 35 $\mathsf{\mu}\mathrm{M}$ (

**F**), and 70 $\mathsf{\mu}\mathrm{M}$ (

**G**). A1, A2 and A3 denote different populations of $\alpha $-syn kinetic curves. Protein solution: 70 $\mathsf{\mu}\mathrm{M}$ $\alpha $-syn, PBS buffer, 0.02% NaN

_{3}, 50 $\mathsf{\mu}\mathrm{M}$ ThT, pH 7.4. Aggregation performed at 37 ${}^{\xb0}\mathrm{C}$ with constant agitation of 300 RPM. The error bars are the standard deviation from the mean (12 repeats).

**Figure 2.**FTIR spectra (

**A**) and the second derivatives (

**B**) of fibrils obtained after $\alpha $-syn aggregation with different concentration of S100A9: $0.0$ $\mathsf{\mu}\mathrm{M}$, $2.3$ $\mathsf{\mu}\mathrm{M}$, $4.5$ $\mathsf{\mu}\mathrm{M}$, $9.0$ $\mathsf{\mu}\mathrm{M}$, 18 $\mathsf{\mu}\mathrm{M}$, 35 $\mathsf{\mu}\mathrm{M}$, 70 $\mathsf{\mu}\mathrm{M}$. The spectra and second derivatives of $\alpha $-syn fibril populations are indicated by A1, A2 and A3. Concentration of S100A9 is color coded. Fibrils formed in PBS buffer, 50 $\mathsf{\mu}\mathrm{M}$ ThT, 0.02% NaN

_{3}, 300 RPM at 37 ${}^{\xb0}\mathrm{C}$ for 60 h.

**Figure 3.**AMF images of $\alpha $-syn (70 $\mathsf{\mu}\mathrm{M}$) aggregated without (

**A**–

**C**) or with different concentration of S100A9: $2.3$ $\mathsf{\mu}\mathrm{M}$ (

**D**), $4.5$ $\mathsf{\mu}\mathrm{M}$ (

**E**), 9 $\mathsf{\mu}\mathrm{M}$ (

**F**), 18 $\mathsf{\mu}\mathrm{M}$ (

**G**), 35 $\mathsf{\mu}\mathrm{M}$ (

**H**) and 70 $\mathsf{\mu}\mathrm{M}$ (

**I**). The cross-section height of fibrils (

**J**). The interquartile region (shown by the box), standard deviation (whiskers) and a median (horizontal line inside the box) were calculated from 50 fibrils.

**Figure 4.**Aggregation kinetics of $\alpha $-syn without (

**A**–

**C**) and with S100A9 (

**D**–

**F**) in a presence of different NaCl concentrations: 0 $\mathrm{m}\mathrm{M}$ (

**A**,

**D**), 50 $\mathrm{m}\mathrm{M}$ (

**B**,

**E**), 250 $\mathrm{m}\mathrm{M}$ (

**C**,

**F**). Each sample represented by 3 mostly recurring curves. The ${t}_{50}$ value (determined from 12 repeats) of $\alpha $-syn (orange) and $\alpha $-syn aggregated with S100A9 (olive) at different concentrations of NaCl (

**G**). Protein solution: 70 $\mathsf{\mu}\mathrm{M}$ $\alpha $-syn, 0 $\mathsf{\mu}\mathrm{M}$ or 35 $\mathsf{\mu}\mathrm{M}$ S100A9, 50 $\mathsf{\mu}\mathrm{M}$ ThT, 0.02% NaN

_{3}, PBS buffer, pH 7.40.

**Figure 5.**FTIR spectra (

**A**,

**C**) and second derivative (

**B**,

**D**) of $\alpha $-syn (70 $\mathsf{\mu}\mathrm{M}$) without or with 35 $\mathsf{\mu}\mathrm{M}$ S100A9 in PBS buffer with different concentration of NaCl.

**Figure 6.**AMF images of $\alpha $-syn aggregated without (

**A**–

**D**) and with (

**E**–

**H**) S100A9 in the presence of different concentrations of NaCl (additional to PBS): 0 $\mathrm{m}\mathrm{M}$ (

**A**,

**E**), 125 $\mathrm{m}\mathrm{M}$ (

**B**,

**F**), 250 $\mathrm{m}\mathrm{M}$ (

**C**,

**G**), and 375 $\mathrm{m}\mathrm{M}$ (

**D**,

**H**). Panel (

**I**) shows the heights of fibrils in the mentioned conditions from (

**A**) to (

**H**). The boxes, lines inside them, and whiskers correspond to interquartiles, medians, and standard deviations of the height distribution, respectively. Protein solution: 70 $\mathsf{\mu}\mathrm{M}$ $\alpha $-syn, 0 or 35 $\mathsf{\mu}\mathrm{M}$ S100A9, 50 $\mathsf{\mu}\mathrm{M}$ ThT, PBS, additional NaCl as mentioned, pH 7.4.

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Toleikis, Z.; Ziaunys, M.; Baranauskiene, L.; Petrauskas, V.; Jaudzems, K.; Smirnovas, V.
S100A9 Alters the Pathway of Alpha-Synuclein Amyloid Aggregation. *Int. J. Mol. Sci.* **2021**, *22*, 7972.
https://doi.org/10.3390/ijms22157972

**AMA Style**

Toleikis Z, Ziaunys M, Baranauskiene L, Petrauskas V, Jaudzems K, Smirnovas V.
S100A9 Alters the Pathway of Alpha-Synuclein Amyloid Aggregation. *International Journal of Molecular Sciences*. 2021; 22(15):7972.
https://doi.org/10.3390/ijms22157972

**Chicago/Turabian Style**

Toleikis, Zigmantas, Mantas Ziaunys, Lina Baranauskiene, Vytautas Petrauskas, Kristaps Jaudzems, and Vytautas Smirnovas.
2021. "S100A9 Alters the Pathway of Alpha-Synuclein Amyloid Aggregation" *International Journal of Molecular Sciences* 22, no. 15: 7972.
https://doi.org/10.3390/ijms22157972