Article

ISSN 0006-2979, Biochemistry (Moscow), 2026, Vol. 91, No. 5, pp. 836-846 © Pleiades Publishing, Ltd., 2026.

836

Multidirectional Effects of SARS-CoV-2 Coronavirus

Proteins on Amyloid Transformation of Alpha-Synuclein

Yulia Y. Stroylova

1,a

, Anastasiya V. Konstantinova

, Denis V. Pozdyshev

Ivan A. Katrukha

2,3

, Reza Yousefi

, and Vladimir I. Muronetz

1,5

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University,

119991 Moscow, Russia

Faculty of Biology, Lomonosov Moscow State University, 119991 Moscow, Russia

Hytest Russia, 117105 Moscow, Russia

Protein Chemistry Laboratory (PCL), Institute of Biochemistry and Biophysics,

University of Tehran, 1417614335 Tehran, Iran

Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University,

119991 Moscow, Russia

e-mail: stroylovayy@my.msu.ru

Received March 3, 2026

Revised May 6, 2026

Accepted May 6, 2026

Abstract— Parkinson’s disease is associated with amyloid aggregation of alpha-synuclein, which could be af-

fected by the proteins of the SARS-CoV-2 coronavirus, possibly accelerating and provoking neurodegeneration.

The purpose of this work was to compare the effects of the N-protein and the receptor binding domain (RBD)

of the S  protein on fibrillization of the alpha-synuclein preparation produced using an original technique

that excludes presence of non-native forms of alpha-synuclein that alter kinetics of the process. Presence of

an elongated form of alpha-synuclein in the previously studied protein preparations is associated with the

erroneous reading of the rare for E.  coli TGA stop codon in the pET33b(+) expression plasmid as trypto-

phan, which led to the continued translation to the next stop codon. To prevent this effect, a new plasmid

design was suggested with replacement of the original stop codon with a double stop codon TAA, which

made it possible to obtain a homogeneous protein preparation without the admixture of alpha-synuclein

with increased molecular weight. It has been shown that the N-protein is able to accelerate alpha-synucle-

in fibrillization, while the RBD of the S  protein inhibits aggregation. According to the electron microscopy

data, structure of the fibrils formed in the presence of viral proteins is also different. The obtained data

are important for understanding the mechanisms of development of post-covid synucleinopathies, as well

as consequences of vaccination with the viral proteins.

DOI: 10.1134/S0006297926600559

Keywords: SARS-CoV-2, N  protein, RBD of the S  protein, alpha-synuclein, Parkinson’s disease, neurodegenera-

tion, amyloid fibrils, protein aggregation

* To whom correspondence should be addressed.

# These authors contributed equally to this study.

INTRODUCTION

Studying the mechanisms that drive pathologi-

cal transformation of amyloidogenic proteins is im-

portant for developing methods to prevent and treat

neurodegenerative diseases. A large body of research

has shown that many different factors could influ-

ence formation of amyloid structures by the pro-

teins involved in neurodegeneration. These factors

include, for example, small molecules (such as metal

ions and phospholipids) that stimulate amyloid for-

mation and induce such processes through broad

physiological changes in the body including metabol-

ic disorders, activity of chaperone systems, inflamma-

tion, changes in microbiota and immune status [1-8].

IMPACT OF SARS-CoV-2 PROTEINS ON ALPHA-SYNUCLEIN 837

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

For example, hyperglycemia in diabetes has been

linked to post-translational modifications like glyca-

tion, which could also affect amyloid transformation

of alpha-synuclein (α-Syn) [9, 10].

More recently, a new factor has been identified, it

was associated with the global spread of severe acute

respiratory syndrome coronavirus  2 (SARS-CoV-2) in-

fection. There are many reports, sometimes conflict-

ing, about how coronavirus infection and vaccination

might influence the development of neurodegenera-

tive diseases [11-18]. Some studies suggest that the

coronavirus infection disease 2019 (COVID-19) could

trigger or accelerate neurodegeneration [11, 13, 14].

The virus could promote neuroinflammation, disrupt

the blood–brain barrier, and activate astrocytes and

microglia [15-18], all of which are key processes in

the development of neurodegenerative disorders. In

addition, the SARS-CoV-2 has been shown to potential-

ly enter the central nervous system, as viral proteins

and RNA have been detected in the brain tissue (es-

pecially in the brainstem) in several autopsy studies

[19, 20].

Particular interest has been focused on the role

of coronavirus proteins in aggregation of α-syn. Accu-

mulation of this protein in the form of Lewy bodies

is directly associated with Parkinson’s disease and

other synucleinopathies [13]. However, whether the

spike (S) protein of SARS-CoV-2 could influence α-syn

aggregation remains unclear. Experimental studies in

both cellular and cell-free systems show that the full-

length S protein does not significantly affect amyloid

formation and may even slow it down at early stages

[21, 22]. At the same time, molecular docking studies

suggest that the receptor-binding domain (RBD) of the

S  protein could strongly interact with amyloidogenic

proteins, including α-syn  [23]. Although there is no

clear evidence that the S protein directly affects hu-

man amyloid proteins, it contains several amyloi-

dogenic peptide regions that could form fibrils and

show typical fibril formation behavior, as detected by

the thioflavin  T fluorescence  [24]. Sprotein is activat-

ed during the cell entry by cleavage with a furin-like

protease, which could produce additional peptides

capable of interacting with the amyloid proteins. In

our previous work, we showed that the RBD of the

S protein does interact with α-syn, but instead of ac-

celerating fibril formation, it slows it down mediating

formation of aggregates with weaker amyloid proper-

ties and lower toxicity  [25].

According to the hypothesis proposed by Tavasso-

ly etal., the RBD of the Sprotein contains regions that

bind heparin. α-Syn, like other amyloidogenic proteins,

such as tau and beta-amyloid, could also bind hepa-

rin and heparan sulfate  [26]. These common binding

properties could promote binding of α-syn to the sur-

face of viral particles or to the S protein itself  [13].

In contrast to the S  protein, the nucleocapsid

N-protein of the coronavirus has been shown to

strongly accelerate amyloid transformation of α-syn,

significantly shortening lag phase of the fibril forma-

tion  [21,  27]. It appears that the nucleocapsid protein

(N protein) could initiate aggregation process by in-

corporating into the fibrils at early stage thus de-

termining their initial structure. Later, these mixed

fibrils act as seeds for further growth, eventually

leading to formation of the fibrils that are indistin-

guishable from those formed by α-syn alone  [27].

The conflicting results regarding the effects of

the N protein and the RBD of the S protein on α-syn

aggregation may be related to the specific character-

istics of the preparation of this protein. The recom-

binant α-syn could contain variants with altered ami-

no acid sequences. For example, when expressed in

E.  coli, a form could appear in which tyrosine at po-

sition 136 is replaced with cysteine. This modification

is not always easy to detect and requires specific ex-

perimental conditions (SDS-PAGE without addition of

thiol reagents)  [28]. Such variants could form dimers,

presence of which slow down amyloid transformation

of the wild type protein. In addition, longer forms of

α-syn could arise due to translation errors, and ad-

dition of extra sequence tags added for purification

could also affect its behavior. Even small amounts

of these altered forms could influence experimental

results and lead to inconsistent findings. Therefore,

it is preferrable to use the α-syn preparations with

sequences as close as possible to the natural form,

when comparing the effects of different coronavirus

proteins.

In this study, we examined the effect of the

SARS-CoV-2 N-protein and the RBD of the S  protein

on transformation of α-syn, preparation of which was

obtained using an original method, in order to test

the hypothesis that amyloidogenesis of this protein is

enhanced upon exposure to the coronavirus proteins.

MATERIALS AND METHODS

Materials. In this study, we used isopropyl-β-D-1-

thiogalactopyranoside, Coomassie Brilliant Blue

G-250 (Helicon, Russia), thioflavin  T (ThT), 3-(4,5-di-

methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

(Sigma- Aldrich, USA), PMSF (Dia-M, Russia), inorgan-

ic salts (Panreac, Spain), and buffers (Amresco, USA).

We also used fetal bovine serum (FBS, HyClone,

USA), GlutaMAX (Gibco, USA), DMEM/F12 medium,

trypsin, and penicillin/streptomycin (PanEco, Rus-

sia). Human embryonic kidney cell line [HEK293T]

(CRL-3216, ATCC) were used, along with monoclonal

antibodies against α-syn (LB 509, Abcam, UK), N  pro-

tein (3706, Hytest, Finland), and GAPDH (6C5, Hytest).

STROYLOVA et al.838

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

Human neuroblastoma SH-SY5Y cell line was kindly

provided by Irina Naletova (University of Catania,

Italy).

Plasmid construction (pET33b  (+) SYN WT-TAA).

The original pET33b  (+) SYN WT plasmid was modi-

fied by replacing the TGA stop codon with a double

stop codon (TAATAA) using site-directed mutagenesis

(Phusion kit), following the manufacturer’s instruc-

tions. Specific primers were used for this step. The

resulting plasmid was verified by Sanger sequenc-

ing and then transformed from E.  coli XL1-Blue into

the E.  coli BL21(DE3). The final producer strain was

stored frozen in an LB medium containing kanamy-

cin (50  µg/mL) and 15% glycerol.

Expression of recombinant α-syn in E.  coli.

A 10-mL sample of overnight culture was added to

200  mL of an LB medium with kanamycin and grown

at 37°C with shaking (200 rpm) until OD600 reached

0.6-0.8. Protein expression was next induced by add-

ing IPTG to a final concentration of 1  mM. After

5  h of expression, the cells were collected by cen-

trifugation (20  min, 4400g, 4°C). A 10-fold volume of

a 30  mM potassium-phosphate buffer (pH  7.5) con-

taining 1  mM PMSF protease inhibitor was added to

the bacterial pellet, and the pellet was resuspended.

The bacteria were next disrupted by ultrasonication

(Branson Digital Sonifier, Marshall Scientific, USA) us-

ing 3 pulses of 20  s at 20% amplitude. The lysates

were centrifuged at 11,000g for 20  min at 4°C. The

supernatant containing α-syn and other proteins was

acidified to pH  2.8 with 9% hydrochloric acid fol-

lowed by centrifugation at 11,000g for 5  min at 4°C

to precipitate denatured proteins. After centrifuga-

tion, pH of the supernatant was quickly brought to

neutral values (7.0-7.5) with a 1  M sodium-phosphate

buffer (pH  11.0). α-Syn was precipitated by salting

out with addition of a dry ammonium sulfate to 40%

saturation, the sulfate suspension was incubated

at 4°C for 12  h, and next centrifuged at 11,000g for

15  min. The pellet was dissolved in a bidistilled wa-

ter, dialyzed overnight against bidistilled water, cen-

trifuged at 11,000  g (15  min), after which the protein

preparation was aliquoted and lyophilized.

Fibril formation of α-syn. Lyophilized α-syn was

dissolved in a PBS (pH  7.0), centrifuged, and its con-

centration was measured spectrophotometrically us-

ing specific absorption of 0.1% protein solution value

of 0.413. Samples containing α-syn alone or mixed

with N protein or RBD of the S protein in 1-ml glass

vials were incubated at 37°C with constant shaking

(600  rpm) for 48  h. Fibril formation was monitored

using ThT fluorescence.

ThT fluorescence measurements. To measure

fluorescence, ThT was added to protein samples (to

final concentration 3  µM) to provide a 10-fold molar

excess. After 10  min of incubation, fluorescence spec-

tra were recorded using a Fluoromax spectrofluo-

rimeter (Horiba Jobin Yvon, France) using excitation

wavelength 430  nm, emission wavelength 485  nm.

For kinetic measurements, samples were prepared

as described in 96-well plates and analyzed using

a CLARIOstar plate reader (BMG LABTECH GmbH,

Germany). Each experiment was repeated three times,

and representative curves are shown.

SDS-PAGE and Western blotting. SDS-PAGE was

performed according to the standard Laemmli meth-

od. After electrophoresis, proteins were transferred

onto a PVDF membrane (pre-activated with ethanol)

in a transfer buffer at a constant current of 150 mA

for 3  h under cooling conditions. Transfer efficiency

was assessed by Ponceau S staining. The membrane

was blocked in a PBST supplemented with a 10%

non-fat dry milk. Incubation with primary antibodies

was carried out at 4°C overnight (mouse monoclonal

antibodies specific to α-syn, 1  :  1000). The membrane

was next washed with PBST and incubated for 1  h

with secondary polyclonal anti-mouse goat antibodies

conjugated with horseradish peroxidase (1  :  10,000)

after washing with PBST. Signal enhancement was

performed using a WesternBright ECL kit (Advansta,

USA), and bands were visualized using a ChemiDoc

XRS+ system (Bio-Rad, USA) with variable exposure

(5-800  s). Subsequently, N-protein detection was per-

formed using a similar protocol with mouse an-

ti-N-protein antibodies (clone 3706, 1  :  500). For band

detection on the membrane, staining with a solution

containing 0.3  mg/mL diaminobenzidine, 0.03% hy-

drogen peroxide in 0.1  M Tris-HCl, pH  7.6 was used.

Transmission electron microscopy (TEM). For

transmission electron microscopy, samples of WT

α-syn fibrils formed in the presence of N  protein or

RBD, or without them, were prepared as indicated

above. A copper grid (200 mesh) was placed in a drop

of the sample for 15 min, excess liquid was removed

with a filter paper, and next the grid was stained

with 1% uranyl acetate solution for 10  s, followed by

drying. The prepared specimens were examined us-

ing an LEO912 AB Omega electron microscope (Carl

Zeiss, Germany) at an accelerating voltage of 100  kV.

Proteinase  K digestion assay. Proteinase K

(0.2  μg/mL) was added to the obtained samples of

α-syn (35  μM) fibers formed in the presence of N-pro-

tein (20  μM) or RBD (20  μM) under conditions pro-

moting or not promoting fibrillization, and incubated

at 37°C for 1, 5, 10, 30  min. Proteolysis was stopped

by adding a 4×  sample buffer and heating at 95°C for

5  min. The samples were next analyzed by SDS-PAGE.

Control samples contained no proteinase  K. For quan-

titative analysis of proteinase K resistance, three in-

dependent experiments were performed; the data

were processed using ImageJ software followed by

calculation of a mean value and standard deviation.

IMPACT OF SARS-CoV-2 PROTEINS ON ALPHA-SYNUCLEIN 839

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

Statistical significance of differences was assessed us-

ing the Student’s t-test (significance level p  <  0.05).

Cell viability assay (MTT test). Wild-type human

neuroblastoma SH-SY5Y cells, as well as SH-SY5Y cells

stably expressing a WT α-syn and an A53T mutant

form  [29], were seeded into 96-well culture plates in

a DMEM/F12 medium supplemented with 10% fetal

bovine serum at a density of 15,000 cells per well.

After 24  h, N-protein, RBD, or a mixture of proteins

was added to the wells to a final concentration of

200  nM or 800  nM. Cells incubated in a DMEM/F12

medium with an appropriate volume of PBS were

used as a control. The cells were incubated at 37°C

and 5% CO

for 20  h, next an MTT reagent was add-

ed, the plate was incubated for 4  h, and the medi-

um in the wells was replaced with 100  μL of DMSO.

After a 10-min incubation with DMSO, optical den-

sity was measured at 570  nm and 630  nm using a

VersaMax microplate spectrophotometer (USA). The

ratio of absorbance in the wells with test samples to

absorbance in the control wells was used to assess

cell viability. The results were presented as a mean

± standard deviation of the mean. For data analysis,

one-way ANOVA with Bonferroni’s multiple compari-

son test was used. Statistical significance was set at

p  <  0.05. GraphPad Prism software was used for sta-

tistical analysis.

Fractionation of HEK293T cell lysates. HEK293T

cells were seeded into T25 culture flasks. After 24  h,

transfection with plasmids carrying the α-syn and

N-protein genes, separately or together, was per-

formed using a Lipofectamine 3000 reagent (USA) ac-

cording to the manufacturer’s instructions. Two days

after transfection, the cells were detached from the

flask surface using a trypsin-Versene solution, washed

with PBS containing 5%FBS, and centrifuged at500g.

The cell pellet was lysed using PBS supplemented

with a 1% Triton X-100, centrifuged at 14,000g at 4°C

for5min. The cell pellet was washed with a cold PBS

and re-centrifuged under the same conditions. The re-

sulting pellet was resuspended in a sample buffer and

sonicated for 10  s at 10% amplitude (Branson Digital

Sonifier, Marshall Scientific, USA). The samples were

analyzed by SDS-PAGE in a 15% gel followed by trans-

fer to a PVDF membrane and visualization with mono-

clonal antibodies against α-syn (LB 509, Abcam) and

against N-protein (clone 3706), and next with horse-

radish peroxidase-conjugated anti-mouse antibodies.

Anti-GAPDH antibodies (clone 6C5, 1  :  2000) were

used as a loading control. Signal was detected using

a WesternBright ECL kit (Advansta), and bands were

visualized with a ChemiDoc XRS+ system (Bio-Rad).

Mass spectrometry analysis. A detailed descrip-

tion of the sample preparation is provided in the

Online Resource  1. Mass spectra were acquired with

an UltrafleXtreme MALDI-TOF/TOF mass spectrometer

(Bruker Daltonics, Germany) equipped with a UV (Nd)

laser in positive ion reflector mode; accuracy of the

measured monoisotopic masses after recalibration us-

ing trypsin autolysis peaks was 40  ppm. Spectra were

acquired in the mass range of 600-6000  m/z, select-

ing the laser power optimal for achieving the best

resolution. For fragmentation spectra acquisition, the

tandem mode of the instrument was used, and accu-

racy of the fragment ion measurements was no worse

than 1  Da. Protein identification was performed using

the Mascot software (https://www.matrixscience.com/)

home ver.  2-3-02. Mass spectra were processed using

the FlexAnalysis  3.3 software package (Bruker Dal-

tonics), and HTM format peak lists were generated.

A search was performed in a home database using

the Mascot software (peptide fingerprint option) with

the above-mentioned accuracy, considering possible

oxidation of methionine residues by air oxygen. Frag-

mentation spectra of individual peptides were also

obtained. Using the Biotools3.3 software (Bruker Dal-

tonics), a search was performed based on combined

MS  +  MS/MS results.

RESULTS

Obtaining α-syn preparations without unwant-

ed peptide fragments. In our previous studies, in-

cluding work on the interaction of this protein with

the RBD of the S protein  [25], we used the recombi-

nant α-syn with the Y136Y substitution (TAC → TAT).

This change helps to prevent formation of variants

containing cysteine at position 136, which could sig-

nificantly affect fibril formation  [28]. The used protein

preparations also did not contain any additional tags,

which are commonly used to simplify purification.

However, we found that these preparations contained

not only the main monomer form (~18  kDa) but also

a longer form with size of about 26  kDa (Fig.  1a,

lane  3), which was clearly detected both by electro-

phoresis and by the antibodies against α-syn (Fig.  1b).

After sequencing the plasmid, we confirmed that

there were no mutations and that the gene sequence

was correct. Coding sequence of the SNCA gene end-

ed with the stop codon TGA, which is one of the rare

stop codons for E.  coli, but is a native stop codon for

the expression plasmid pET33b(+) (Novagen, USA).

Analysis of the literature revealed that when express-

ing proteins in this system, in some cases, the TGA

codon can be mistakenly read as tryptophan, and

translation of the protein from the plasmid continues

until the next stop codon [30].

In our case, this led to addition of 21 extra

amino acids at the C-terminus of the protein –

WDPAANKARKEAELAAATAEQ (Fig.  2a), explaining

appearance of the elongated form.

STROYLOVA et al.840

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

Fig.  1. Analysis of the original α-syn preparation by SDS electrophoresis and immunochemical staining. Lanes: 1)  lysates

of E. coli BL21(DE3) cells before IPTG induction; 2)  lysates of E. coli BL21(DE3) cells after induction of expression with

IPTG, M – molecular weight markers; 3)  obtained α-syn preparation  (a). Staining of the α-syn preparation with antibodies

specific to α-syn  (b).

Fig.  2. Analysis of the elongated form of recombinant α-syn. a)  Nucleotide sequence of the pET33b (+)_SYN Y136Y plasmid

fragment located after the SNCA gene coding sequence up to the TAA stop codon, and the corresponding amino acid se-

quence. Stop codons are highlighted in red; seven amino acid residues determined by mass spectrometry are shown in

bold. b)  Mass spectrometry analysis of the peptides after trypsin digestion of two forms of α-syn. The results of analysis

of the elongated form of α-syn are shown at the top; the results of analysis of the normal form of α-syn are shown at the

bottom. C-terminal peptides in the spectra are marked as C-term long and C-term.

IMPACT OF SARS-CoV-2 PROTEINS ON ALPHA-SYNUCLEIN 841

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

In parallel with sequencing, we performed

MALDI-TOF analysis of the major (18  kDa) and elongat-

ed (26  kDa) α-syn after trypsin digestion. Two prepa-

rations differed in the sequence of the peptide at the

protein’s C-terminus. For the major α-syn, the C-termi-

nal peptide had molecular mass of 4286  Da (Fig.  2b,

Cterm), while molecular mass of the C-terminus of

the elongated protein was 5069  Da (Fig.  2b, Cnew).

Difference between the two peptides was 783  Da.

This corresponded to a sequence of a tryptophan

residue and six amino acid residues that were pres-

ent in the plasmid after the SNCA gene stop codon:

WDPAANK (Fig.  2a). When we recalculated molecu-

lar mass of this peptide and subtracted the mass of

water, we obtained a value matching the difference

between the peptides. Thus, we discovered that the

TGA stop codon was read as tryptophan, and protein

translation continued further.

To prevent appearance of such elongated protein

forms, we generated a construct with replacement of

the TGA stop codon with the double TAA stop codon

(detailed description of the plasmid is given in the

“Materials and methods” section). This allowed us to

obtain and use a homogeneous preparations of α-syn

without the presence of the elongated protein forms

in the further study (Fig.  S1, lanes  4 and  5, Online

Resource  1). Absence of the dimeric forms of α-syn,

which dissociate upon adding β-mercaptoethanol, also

proves that during protein production the variants

with substitution of the tyrosine 136 residue for a

cysteine residue were not produced.

Thus, to exclude possible influence of the elon-

gated forms of α-syn on its fibrillization and inter-

action with the coronavirus proteins, we conducted

experiments with the N  protein and RBD of the S  pro-

tein of SARS-CoV-2 using the wild-type α-syn prepa-

rations free of any impurities. It is known that un-

der fibrillization conditions, α-syn undergoes amyloid

transformation, which, in particular, is manifested by

the increase in the ThT fluorescence intensity due to

its interaction with beta-amyloid cross-beta struc-

tures. Lag period of fibrillization depends on the pro-

tein concentration and this is a single stage process.

Figure  3a shows the curve of the ThT fluorescence in-

tensity increase (red curve) during incubation of the

wild-type α-syn under fibrillization-stimulating condi-

tions (incubation at 37°C at neutral pH and constant

stirring). In accordance with our previously obtained

data, addition of the RBD of the S  protein significant-

ly slows down amyloid transformation of α-syn  [25].

However, fibrillization is not completely prevented,

and after 40-50  h of incubation, increase in the ThT

fluorescence intensity occurs (Fig.  3a, green curve).

Addition of the N  protein has the opposite effect on

α-syn fibrillization, significantly reducing duration of

the lag period. Moreover, in the presence of N-pro-

tein, fibrillization has a pronounced biphasic charac-

ter, since it accelerates sharply after 40  h of incuba-

tion (Fig.  3a, black curve).

Influence of the coronavirus proteins on the am-

yloid transformation of α-syn was also confirmed by

analyzing fluorescence spectra of ThT added to the

proteins at certain time points during their co-incu-

bation (Fig.  3, b-d). Such experiments are necessary

to exclude the influence of ThT on the amyloidization

process, since the fluorescent dye is added in signif-

icant excess at the very beginning of the experiment

during the continuous recording in the experiments

described above. Co-incubation of α-syn with the

N protein or the RBD of the S  protein under condi-

tions not promoting fibrillization does not lead to the

significant change in the fluorescence of the added

ThT (Fig.  3, b  and  c). However, incubation under con-

ditions facilitating α-syn fibrillization causes a signif-

icant increase in the fluorescence intensity of ThT,

especially after 48  h. Moreover, with addition of the

RBD of the S  protein, the amyloid transformation of

α-syn occurs less efficiently compared to the case of

N-protein addition (Fig.  3, d  and  e).

Structural features of the α-syn fibrils formed in

the presence of coronavirus proteins were studied

using a proteolysis resistance test. As follows from

the data presented in Fig.  S2 (Online Resource  1), the

α-syn fibrils are almost completely cleaved by pro-

teinase  K after 10  min of incubation (Fig.  S2, a  and  d,

Online Resource  1). The α-synfibrils formed in the

presence of the N  protein are more resistant to pro-

teolysis (Fig. S2, b and d, Online Resource 1), and the

N  protein itself (a band with a molecular mass of

approximately 45  kDa) is hydrolyzed almost instantly

(Fig.  S2b, Online Resource  1), same as α-syn that has

not been subjected to fibrillization conditions. Fibrils

formed in the presence of the RBD of the S-spike

protein turned out to be the most resistant to prote-

olysis (Fig.  S2, c  and  d, Online Resource  1). Moreover,

the S  protein RBD itself proved to be quite resistant

to proteinase  K (Fig.  S2,  b, band with a molecular

weight of approximately 35  kDa; Online Resource  1).

Study of the structure of α-syn fibrils using trans-

mission electron microscopy revealed that fibrils

are formed when coronavirus proteins were added

to the α-syn monomer under experimental condi-

tions (Fig.  4). However, in the presence of the S  pro-

tein RBD, structures with lower density are formed

(Fig.  4b), while addition of the N  protein resulted in

formation of well-contrasted fibrils (Fig.  4c).

It should be noted that the TEM data, at first

glance, contradict the results of the proteolysis re-

sistance test. It could be assumed that the greater

resistance to proteinase  K of the fibrils obtained in

the presence of the S  protein RBD is due to its di-

rect protective effect. As has been shown previously,

STROYLOVA et al.842

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

Fig.  3. Kinetics of α-syn fibril formation  (a) and ThT fluorescence spectra measured with addition of the N  protein and

RBD of the SARS-CoV-2 S  protein under conditions not promoting  (b,  c) and promoting  (d,  e) fibrillization. a)  Red kinetic

curve demonstrates kinetics of fibril formation of pure α-syn at concentration of 70  μM, black curve demonstrates kinetics

of α-syn (70  μM) fibrillization in the presence of 5  μM N  protein, and green curve demonstrates kinetics of α-syn (70  μM)

fibrillization in the presence of 5  μM  RBD. b,  c)  ThT fluorescence spectra in the samples containing 35  μM α-syn in the pres-

ence of 20  μM N-protein or 20  μM  RBD (B  and  C, respectively) under conditions not promoting fibrillization (PBS, pH  7.0,

25°C, and without stirring). d, e) ThT fluorescence spectra in the samples containing 35 μM α-syn with added N-protein

and RBD of S protein (D and E, respectively) under conditions promoting fibrillization (PBS, pH 7.0,37°C, and with stirring

at 600 rpm). Pink color in all graphs shows the protein mixtures at the initial time point; red color shows the mixtures

2 days after onset of α-syn fibrillization.

IMPACT OF SARS-CoV-2 PROTEINS ON ALPHA-SYNUCLEIN 843

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

Fig.  4. TEM of α-syn fibrils(a), mixed with S protein RBD(b), N-protein(c), under conditions not promoting fibrillization(d).

Fig.  5. Survival of SH-SY5Y cells: (a)  intact, (b)  with stable expression of WT α-syn, (c)  with stable expression of A53T α-syn

and addition of N  protein and RBD S  protein. Cells were incubated for 20  h with recombinant proteins, after which meta-

bolic activity was assessed (MTT assay). Treatment conditions: PBS (control, blue), N  protein (200  nM – blue; 800  nM – light

green), RBD (200  nM – pink; 800  nM – green), N  +  RBD mixture (200  nM each – red; 800  nM each – beige).

STROYLOVA et al.844

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

the S  protein RBD binds to α-syn  [25], which could

lead to shielding of the proteolytic cleavage sites thus

preventing proteolysis of the structures formed in the

presence of this coronavirus protein. The protective

effect is observed only for the S  protein RBD, which

itself is resistant to the action of proteases, unlike the

N  protein, which is cleaved under these conditions.

In addition to the previously obtained data on

the effect of the S  protein RBD and α-syn fibrils

formed in its presence on the SH-SY5Y cell line  [25],

the effect of N  protein added externally or synthe-

sized in the cells on their survival was studied.

Addition of N  protein to the SH-SY5Y cells did not

affect their survival (Fig.  5). It is logical to assume

that the effect could be manifested in the cells stably

expressing α-syn due to the increased amyloid forma-

tion. However, in this case, addition of N  protein did

not affect cell survival, regardless of the amount of

added proteins (Fig.  5, b  and  c).

Effect of N  protein on the formation of amyloid

structures directly in the cells was also tested in the

HEK293T cells with transient co-expression of the

N  protein and wild-type α-syn. Both proteins were

shown to be efficiently produced with both separate

and co-transient expression. Analysis of α-syn distri-

bution between the supernatant and pellet of the cell

extracts allows to reveal amyloid forms of this protein

(Fig.  S3, lane  3, Online Resource  1). No significant in-

crease in the α-syn content in the pellet was detected

upon additional transfection of the cells with the plas-

mid encoding Nprotein (Fig.  S3, lanes  3 and  7, Online

Resource 1).

Thus, although both coronavirus proteins influ-

ence α-syn fibrillization in the in  vitro model system,

no significant effects of the RBD of the S  protein or

N  protein were detected in the experiments using dif-

ferent cell models. This may be due to existence of a

large number of other protein partners in the cells,

both for α-syn and for coronavirus proteins, or due

to insufficient sensitivity of the methods used to de-

tect the changes.

The most important observation is the divergent

effects of coronavirus proteins on amyloid transfor-

mation of α-syn: in particular, the N  protein acceler-

ates α-syn amyloidization, resulting in formation of

a larger number of well-contrasted fibrils, while the

RBD of the S  protein slows this process. Also of inter-

est is observation of the protective effect of the RBD

of the S  protein against proteolytic cleavage of α-syn

fibrils, which may be due to the shielding of proteoly-

sis-sensitive regions of the polypeptide chains, differ-

ent fibril densities, or their concentrations. This fact

demonstrates that clear interpretation of the effects

arising from the protein–protein interactions is not

always possible. Thus, inhibition of fibril formation

due to the presence of the RBD of the S  protein may

be offset by their greater stability due to formation of

the complexes with this protein. It is also crucial to

emphasize importance of using natural α-syn prepa-

rations, free of any impurities from other proteins

or different forms of α-syn, when conducting com-

parative analysis of the influence of various factors

on pathological transformation of this protein. Inour

opinion, conflicting information regarding the role of

various interventions on α-syn fibrillization may be

due to the presence of such impurities, which could

accelerate fibrillization, inhibit it, or interact with

the biomolecules being studied.

CONCLUSION

Diverse and multidirectional effects on the patho-

logical transformation of α-syn and other amyloi-

dogenic proteins, including effects of post-translation-

al modifications, small molecules, proteins, and other

factors, should be supplemented by investigation of

the interactions of α-syn with coronavirus proteins.

Further studies should explore how other viral

proteins affect amyloid formation, in order to better

understand possible roles of infectious diseases in

neurodegeneration.

Our findings indicate that both the N  protein and

the RBD of the SARS-CoV-2 S  protein could interact

with α-syn, and possibly with other amyloidogenic

proteins, which may be relevant for the development

of neurodegenerative disorders.

At the same time, our results do not support the

idea that vaccination, which leads to the presence of

RBD of the S protein in the body, directly triggers or

accelerates neurodegenerative diseases, at least based

on the experiments with isolated proteins. A more

definitive interpretation of this assumption requires

appropriate experiments with model animals and

clinical studies.

Main limitation of this study is that the experi-

ments were conducted with the isolated proteins and

cell lines, which does not allow us to unambiguous-

ly extrapolate the obtained information to similar

processes in living systems.

Abbreviations

α-syn alpha-synuclein

N protein nucleocapsid protein

RBD receptor-binding domain

S protein spike protein

SARS-CoV-2 severe acute respiratory syndrome

coronavirus  2

Supplementary information

The online version contains supplementary material

available at https://doi.org/10.1134/S0006297926600559.

IMPACT OF SARS-CoV-2 PROTEINS ON ALPHA-SYNUCLEIN 845

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

Acknowledgments

We are grateful to the Moscow State University De-

velopment Program (PNG 5.13) for providing access

to the UltrafleXtreme BrukerDaltonics MALDI-mass

spectrometer.

Contributions

Y.  Y.  Stroylova and V.  I.  Muronets designed and su-

pervised the study. A.  V.  Konstantinova, Y.  Y.  Stroylo-

va, and D.  V.  Pozdyshev performed the experiments.

A.  V.  Konstantinova, Y.  Y.  Stroylova, I.  A.  Katrukha,

R.  Yousefi, and V.  I.  Muronets discussed the results.

Y.  Y.  Stroylova and V.  I.  Muronets wrote the manu-

script. All authors contributed to editing the final text.

Funding

This work was supported by the joint Russian-

Iranian grant from the Russian Science Foundation

(no.  24-44-20003, https://rscf.ru/project/24-44-20003/)

and the Iran National Science Foundation (INSF,

no. 4023311).

Ethics approval and consent to participate

This work does not contain any studies involving hu-

man and animal subjects.

Conflict of interest

The authors of this work declare that they have no

conflicts of interest.

REFERENCES

1. Ciechanover, A., and Kwon, Y. T. (2017) Protein quality control by molecular chaperones in neurodegeneration,

Front. Neurosci., 11, 185, https://doi.org/10.3389/fnins.2017.00185.

2. Holmqvist, S., Chutna, O., Bousset, L., Aldrin-Kirk, P., Li, W., Bjorklund, T., Wang, Z. Y., Roybon, L., Melki, R.,

and Li, J. Y. (2014) Direct evidence of Parkinson pathology spread from the gastrointestinal tract to the brain

in rats, Acta Neuropathol., 128, 805-820, https://doi.org/10.1007/s00401-014-1343-6.

3. Huang, M., Zhang, Y., and Liu, X. (2024) The mechanism of cuproptosis in Parkinson’s disease, Ageing Res. Rev.,

95, 102214, https://doi.org/10.1016/j.arr.2024.102214.

4. Hunter, R. L., Dragicevic, N., Seifert, K., Choi, D. Y., Liu, M., Kim, H. C., Cass, W. A., Sullivan, P. G., and Bing, G.

(2007) Inflammation induces mitochondrial dysfunction and dopaminergic neurodegeneration in the nigrostriatal

system, J. Neurochem., 100, 1375-1386, https://doi.org/10.1111/j.1471-4159.2006.04327.x.

5. Kim, S., Kwon, S. H., Kam, T. I., Panicker, N., Karuppagounder, S. S., Lee, S., Lee, J. H., Kim, W. R., Kook, M.,

Foss, C. A., Shen, C., Lee, H., Kulkarni, S., Pasricha, P. J., Lee, G., Pomper, M. G., Dawson, V. L., Dawson, T. M.,

and Ko, H. S. (2019) Transneuronal propagation of pathologic alpha-synuclein from the gut to the Brain models

Parkinson’s disease, Neuron, 103, 627-641.e7, https://doi.org/10.1016/j.neuron.2019.05.035.

6. Lee, S., and Kovacs, G. G. (2024) The irony of iron: the element with diverse influence on neurodegenerative

diseases, Int.J. Mol. Sci., 25, 4269, https://doi.org/10.3390/ijms25084269.

7. Muronets, V. I., Kudryavtseva, S. S., Kurochkina, L. P., Leisi, E. V., Stroylova, Y. Y., and Schmalhausen, E. V.

(2025) Factors affecting pathological amyloid protein transformation: from post-translational modifications to

chaperones, Biochemistry (Moscow), 90, S164-S192, https://doi.org/10.1134/S0006297924604003.

8. Elechi, K. W., Oyepeju Nkem, O., Timothy Chibueze, N., Elechi, U. S., and Franklin Chimaobi, K. (2025) Long-

term neurological consequences of COVID-19 in patients with pre-existing Alzheimer’s and Parkinson’s disease:

a comprehensive review, Neurosci. Insights, 20, 26331055251342755, https://doi.org/10.1177/26331055251342755.

9. Muronetz, V. I., Melnikova, A. K., Seferbekova, Z. N., Barinova, K. V., and Schmalhausen, E. V. (2017) Glycation,

glycolysis, and neurodegenerative diseases: is there any connection? Biochemistry (Moscow), 82, 874-886, https://

doi.org/10.1134/s0006297917080028.

10. Guerrero, E., Vasudevaraju, P., Hegde, M. L., Britton, G. B., and Rao, K. S. (2013) Recent advances in alpha-

synuclein functions, advanced glycation, and toxicity: implications for Parkinson’s disease, Mol. Neurobiol., 47,

525-536, https://doi.org/10.1007/s12035-012-8328-z.

11. Krey, L., Huber, M. K., Hoglinger, G. U., and Wegner, F. (2021) Can SARS-CoV-2 infection lead to neurodegener-

ation and Parkinson’s disease? Brain Sci., 11, 1654, https://doi.org/10.3390/brainsci11121654.

12. Motyl, J. A., Gromadzka, G., Czapski, G. A., and Adamczyk, A. (2024) SARS-CoV-2 infection and alpha-synu-

cleinopathies: potential links and underlying mechanisms, Int. J. Mol. Sci., 25, 12079, https://doi.org/10.3390/

ijms252212079.

13. Iravanpour, F., Farrokhi, M. R., Jafarinia, M., and Oliaee, R. T. (2024) The effect of SARS-CoV-2 on the devel-

opment of Parkinson’s disease: the role of alpha-synuclein, Human Cell, 37, 1-8, https://doi.org/10.1007/s13577-

023-00988-2.

14. Rahmati, M., Yon, D. K., Lee, S. W., Soysal, P., Koyanagi, A., Jacob, L., Li, Y., Park, J. M., Kim, Y. W., Shin, J. I.,

and Smith, L. (2023) New-onset neurodegenerative diseases as long-term sequelae of SARS-CoV-2 infection: a

systematic review and meta-analysis, J. Med. Virol., 95, e28909, https://doi.org/10.1002/jmv.28909.

STROYLOVA et al.846

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

15. Chana-Cuevas, P., Salles-Gandara, P., Rojas-Fernandez, A., Salinas-Rebolledo, C., and Milan-Sole, A. (2020) The

potential role of SARS-COV-2 in the pathogenesis of Parkinson’s disease, Front. Neurol., 11, 1044, https://

doi.org/10.3389/fneur.2020.01044.

16. Yang, L., Kim, T. W., Han, Y., Nair, M. S., Harschnitz, O., Zhu, J., Wang, P., Koo, S. Y., Lacko, L. A., Chandar, V.,

Bram, Y., Zhang, T., Zhang, W., He, F., Pan, C., Wu, J., Huang, Y., Evans, T., van der Valk, P., Titulaer, M. J.,

et al. (2024) SARS-CoV-2 infection causes dopaminergic neuron senescence, Cell Stem Cell, 31, 196-211.e6, https://

doi.org/10.1016/j.stem.2023.12.012.

17. Lee, B., Choi, H. N., Che, Y. H., Ko, M., Seong, H. M., Jo, M. G., Kim, S. H., Song, C., Yoon, S., Choi, J., Kim, J. H.,

Kim, M., Lee, M. Y., Park, S. W., Kim, H. J., Kim, S. J., Moon, D. S., Lee, S., Park, J. H., Yeo, S. G., et al. (2024)

SARS-CoV-2 infection exacerbates the cellular pathology of Parkinson’s disease in human dopaminergic neurons

and a mouse model, Cell Rep. Med., 5, 101570, https://doi.org/10.1016/j.xcrm.2024.101570.

18. Mysiris, D. S., Vavougios, G. D., Karamichali, E., Papoutsopoulou, S., Stavrou, V. T., Papayianni, E., Boutlas, S.,

Mavridis, T., Foka, P., Zarogiannis, S. G., Gourgoulianis, K., and Xiromerisiou, G. (2022) Post-COVID-19 parkin-

sonism and Parkinson’s disease pathogenesis: the exosomal cargo hypothesis, Int. J. Mol. Sci., 23, 9739, https://

doi.org/10.3390/ijms23179739.

19. Emmi, A., Rizzo, S., Barzon, L., Sandre, M., Carturan, E., Sinigaglia, A., Riccetti, S., Della Barbera, M., Boscolo-

Berto, R., Cocco, P., Macchi, V., Antonini, A., De Gaspari, M., Basso, C., De Caro, R., and Porzionato, A. (2023)

Detection of SARS-CoV-2 viral proteins and genomic sequences in human brainstem nuclei, NPJ Parkinson’s

Disease, 9, 25, https://doi.org/10.1038/s41531-023-00467-3.

20. Matschke, J., Lutgehetmann, M., Hagel, C., Sperhake, J. P., Schroder, A. S., Edler, C., Mushumba, H., Fitzek, A.,

Allweiss, L., Dandri, M., Dottermusch, M., Heinemann, A., Pfefferle, S., Schwabenland, M., Sumner Magruder, D.,

Bonn, S., Prinz,M., Gerloff, C., Puschel, K., Krasemann, S., et al. (2020) Neuropathology of patients with COVID-19

in Germany: a post-mortem case series, Lancet Neurol., 19, 919-929, https://doi.org/10.1016/S1474-4422(20)30308-2.

21. Semerdzhiev, S. A., Fakhree, M. A. A., Segers-Nolten, I., Blum, C., and Claessens, M. M. A. E. (2022) Interactions

between SARS-CoV-2 N-protein and α-synuclein accelerate amyloid formation, ACS Chem. Neurosci., 13, 143-150,

https://doi.org/10.1021/acschemneuro.1c00666.

22. Wu, Z., Zhang, X., Huang, Z., and Ma, K. (2022) SARS-CoV-2 proteins interact with alpha synuclein and induce

lewy body-like pathology in vitro, Int. J. Mol. Sci., 23, 3394, https://doi.org/10.3390/ijms23063394.

23. Idrees, D., and Kumar, V. (2021) SARS-CoV-2 spike protein interactions with amyloidogenic proteins: potential

clues to neurodegeneration, Biochem. Biophys. Res. Commun., 554, 94-98, https://doi.org/10.1016/j.bbrc.2021.03.100.

24. Nystrom, S., and Hammarstrom, P. (2022) Amyloidogenesis of SARS-CoV-2 spike protein, J.Am. Chem. Soc., 144,

8945-8950, https://doi.org/10.1021/jacs.2c03925.

25. Stroylova, Y., Konstantinova, A., Stroylov, V., Katrukha, I., Rozov, F., and Muronetz, V. (2023) Does the SARS-CoV-2

spike receptor-binding domain hamper the amyloid transformation of alpha-synuclein after all? Biomedicines,

11, 498, https://doi.org/10.3390/biomedicines11020498.

26. Tavassoly, O., Safavi, F., and Tavassoly, I. (2020) Seeding brain protein aggregation by SARS-CoV-2 as a possi-

ble long-term complication of COVID-19 infection, ACS Chem. Neurosci., 11, 3704-3706, https://doi.org/10.1021/

acschemneuro.0c00676.

27. Semerdzhiev, S. A., Segers-Nolten, I., van der Schoot, P., Blum, C., and Claessens, M. (2023) SARS-CoV-2 N-protein

induces the formation of composite alpha-synuclein/N-protein fibrils that transform into a strain of alpha-synu-

clein fibrils, Nanoscale, 15, 18337-18346, https://doi.org/10.1039/d3nr03556e.

28. Barinova, K. V., Kuravsky, M. L., Arutyunyan, A. M., Serebryakova, M.V., Schmalhausen, E. V., and Muronetz, V. I.

(2017) Dimerization of Tyr136Cys alpha-synuclein prevents amyloid transformation of wild type alpha-synuclein,

Int. J. Biol. Macromol., 96, 35-43, https://doi.org/10.1016/j.ijbiomac.2016.12.011.

29. Kolesova, Y. S., Stroylova, Y. Y., Maleeva, E. E., Moysenovich, A. M., Pozdyshev, D. V., Muronetz, V. I., and

Andreev, Y. A. (2023) Modulation of TRPV1 and TRPA1 channels function by sea anemones’ peptides enhanc-

es the viability of SH-SY5Y cell model of Parkinson’s disease, Int. J. Mol. Sci., 25, 368, https://doi.org/10.3390/

ijms25010368.

30. Lu, K. V., Rohde, M. F., Thomason, A. R., Kenney, W. C., and Lu, H. S. (1995) Mistranslation of a TGA termination

codon as tryptophan in recombinant platelet-derived growth factor expressed in Escherichia coli, Biochem. J.,

309, 411-417, https://doi.org/10.1042/bj3090411.

Publisher’s Note. Pleiades Publishing remains neutral with regard to jurisdictional claims in published

maps and institutional affiliations. AI tools may have been used in the translation or editing of this article.