key: cord-1011933-j4880x3y
authors: Kalashnyk, Olena; Lykhmus, Olena; Izmailov, Mykhailo; Koval, Lyudmyla; Komisarenko, Serhiy; Skok, Maryna
title: SARS-Cov-2 spike protein fragment 674–685 protects mitochondria from releasing cytochrome c in response to apoptogenic influence
date: 2021-05-11
journal: Biochem Biophys Res Commun
DOI: 10.1016/j.bbrc.2021.05.018
sha: 82fc4df43a0df8f88cf052f68620819ba49957be
doc_id: 1011933
cord_uid: j4880x3y

In spite of numerous studies, many details of SARS-Cov-2 interaction with human cells are still poorly understood. The 674–685 fragment of SARS-Cov-2 spike protein is homologous to the fragment of α-cobratoxin underlying its interaction with α7 nicotinic acetylcholine receptors (nAChRs). The interaction of 674–685 peptide with α7 nAChR has been predicted in silico. In the present paper we confirm this prediction experimentally and investigate the effect of SARS-Cov-2 spike protein peptide on mitochondria, which express α7 nAChRs to regulate apoptosis-related events. We demonstrate that SARS-Cov-2 spike protein peptide 674–685 competes with the antibody against 179–190 fragment of α7 nAChR subunit for the binding to α7-expressing cells and mitochondria and prevents the release of cytochrome c from isolated mitochondria in response to 0.5 mM H(2)O(2) but does not protect intact U373 cells against apoptogenic effect of H(2)O(2.) Our data suggest that the α7 nAChR-binding portion of SARS-Cov-2 spike protein prevents mitochondria-driven apoptosis when the virus is uncoated inside the cell and, therefore, supports the infected cell viability before the virus replication cycle is complete.

has changed the life style of people all over the world by causing dozens of millions of illness cases and millions of deaths. During the last year multiple studies were performed to understand the peculiarities of SARS-Cov-2 infection causing the disease and to develop specific drugs and vaccines to cure it. However, many aspects of virus interaction with target cells and its consequences are still poorly understood. causes pathological impairment of many organs and tissues, the main clinical symptom being a severe acute respiratory syndrome (SARS). It is believed that the virus infects cells after interaction with angiotensin-converting enzyme-2 (ACE-2) receptor expressed, in particular, in respiratory epithelium [1] .

Unexpectedly, smoking, which strongly affects respiratory system, appeared not to be a risk factor for severe . Moreover, clinical observations have shown that smoking people were relatively rare among hospitalized patients [2] . These data attracted attention to a possible relation of SARS-Cov-2 virus to nicotinic acetylcholine receptors (nAChRs) underlying smoking addiction and nicotine effects. Structural studies have demonstrated that the fragment 674-685 of SARS-Cov-2 spike protein is homologous to the fragment [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] of α-cobratoxin underlying its interaction with α7 nAChR subtype [3] [4] [5] . Further in silico studies predicted a direct interaction of this SARS-Cov-2 spike protein fragment with the portion 179-190 of α7 nAChR [6] .

The α7 nAChRs are involved in regulating many physiological processes including inflammation [7] , memory and behavior [8] , immune response [9] and blood coagulation [10] .

Since all these processes seem to be impaired upon SARS-Cov-2 infection, the available data suggested an important role of SARS-Cov-2 -α7 nAChR interaction in and proposed α7-specific drugs as therapeutic tools to cure the infected patients [3] [4] 11] . In addition to the cell plasma membrane, the α7 nAChRs are expressed in the outer membrane of mitochondria and are involved in regulating the early events of mitochondria-driven apoptosis [12] [13] . In the present paper, we put an aim to study the interaction of SARS-Cov-2 spike J o u r n a l P r e -p r o o f protein peptide 674-685 with α7 nAChR and to elucidate if such interaction influences mitochondria. The data presented indicate for the first time that SARS-Cov-2 spike protein peptide can prevent the development of mitochondria-driven apoptosis by attenuating cytochrome c release from mitochondria.

All reagents and antibodies against VDAC1 and α-lamin 1B were purchased from Sigma-Aldrich (Saint Louis, USA). The peptides corresponding to SARS-Cov-2 spike protein fragment 674-685, α-cobratoxin fragment [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] and α3 nAChR subunit fragment [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] were synthesized by JPT Peptide Technologies GmbH (Berlin, Germany). Antibodies against α7(1-208) [14] , α7( [179] [180] [181] [182] [183] [184] [185] [186] [187] [188] [189] [190] ) and α4( [181] [182] [183] [184] [185] [186] [187] [188] [189] [190] [191] [192] [15] nAChR fragments and against cytochrome c [16] were obtained, validated and biotinylated previously in our lab. Mitochondria isolation kit, Bax-specific and IRE-1α-specific antibodies and Neutravidin-peroxidase conjugate were from Invitrogen and were purchased from ALT Ukraine Ltd (representative of Thermo Fisher Scientific in Ukraine). 

We used female C57BL/6J mice, 2-5 months of age, 20-25 g of weight. Animals were kept in the animal facility of Palladin Institute of Biochemistry. They were housed in quiet, temperaturecontrolled rooms and were provided with water and food pellets ad libitum. Before removing the brain mice were sacrificed by cervical dislocation. All procedures conformed to the guidelines of Palladin Institute's IACUC. Before starting the experiments, the protocols were approved by the IACUC.

J o u r n a l P r e -p r o o f Mitochondria were isolated from mouse brain by differential ultracentrifugation according to standard published procedures [12] and from U373 cells according to instructions of the kit manufacturer. Their purity was characterized by ELISA using the antibodies against mitochondria-specific voltage-dependent anion channel (VDAC1, [17] ), nuclear-specific marker α-lamin B1 [18] and endoplasmic reticulum-specific marker IRE-1α [19] as described previously [20] .

To prepare detergent lysates, the pellets of cells or mitochondria were frozen at -20 Cytochrome c release assay J o u r n a l P r e -p r o o f derived peptide, α-cobratoxin peptide, α3 peptide or PNU282987 (10nM, 50 nM and 100 nM each) at room temperature, for 5 min, and immediately pelleted by centrifugation (10 min, 7,000

x g at 4°C). The mitochondria supernatants were collected and tested for the presence of cytochrome c by sandwich assay as described [16] . Briefly, the immunoplates were coated with protein A-purified rabbit cytochrome c-specific IgG (40 µg/ml) and were incubated with mitochondria supernatants (undiluted) for 2h at room temperature. The bound cytochrome c was revealed with biotinylated cytochrome c-specific IgG (20 µg/ml, 1h at room temperature) was assessed by MTT assay [21] .

All experimental schemes were repeated at least 3 times, all giving similar results. ELISAs have been performed in triplicates, MTT assay in 5 repeats. Ether raw or normalized mean values were used for statistical analysis using one-way ANOVA test and Origin 9.0 software. The data are presented as Mean±SD. p<0.05 was considered a significant difference.

To elucidate if SARS-Cov-2 peptide 674-685 (further mentioned as the SARS spike protein-derived peptide) binds to α7 nAChRs we used a competitive ELISA assay in which the peptide competed with the antibody elicited against (179-190) fragment of α7 subunit for the binding to the α7-containing cell preparations. The 27-37 fragment of α-cobratoxin (further referred as CTX peptide) was used as a positive control, while the peptide of α3 nAChR subunit possessing no structural homology with CTX-peptide or SARS spike protein-derived peptide ( Fig.1 ) was applied as a negative control.

As shown in Fig.2A , both CTX peptide and the SARS spike protein-derived peptide, but not α3 peptide inhibited the α7(179-190)-specific antibody binding to U373 cell detergent lysate.

In contrast, none of these peptides inhibited the binding of α4(181-192)-specific antibody, which recognizes corresponding peptide fragment of α4 nAChR subunit (Fig.2B ). This kind of experiment was repeated in several α7 nAChR-expressing human cells and cell lines (DAUDI, NHA-TS, platelets) all giving similar results (data not shown). The data obtained clearly indicated that the SARS spike protein-derived peptide competes with α7(179-190)-specific J o u r n a l P r e -p r o o f antibody for the binding to α7-containing cell preparations and, therefore, is able to interact with α7 nAChRs.

The α7 nAChRs expressed in mitochondria outer membrane regulate the opening/formation of mitochondrial channel responsible for the release of pro-apoptotic substances like cytochrome c: it was shown that α7-selective agonists or type 2 positive allosteric modulators attenuate cytochrome c release from mitochondria stimulated by Ca 2+ or H 2 O 2 [22] .To find out if SARS spike protein-derived peptide affects mitochondrial α7 nAChRs we tested its binding to mouse brain mitochondria followed by the assay of cytochrome c release.

As shown in Fig. 3A , both CTX peptide and SARS spike protein-derived peptide, but not α3 peptide competed with α7(179-190)-specific antibody for the binding to mouse brain mitochondria preparation. Either CTX peptide or SARS spike protein-derived peptide inhibited cyt c release from mitochondria similarly to α7-specific agonist PNU282987, while α3 peptide provided no effect (Fig.3B) . Therefore, binding of SARS spike protein-derived peptide to mitochondrial α7 nAChRs attenuated cytochrome c release stimulated by H 2 O 2 .

Cytochrome c is released from mitochondria through a specific channel formed in the outer membrane with the participation of voltage-dependent anion channel (VDAC1) and the proapoptotic Bcl-2 family protein Bax [23] [24] . Recently we reported that mitochondrial α7 nAChRs and attenuated cytochrome c release [25] . Here we asked if SARS spike protein-derived peptide affects mitochondria of U373 cells similarly to PNU282987. As shown in Fig.4A , either PNU282987 or SARS spike protein-derived peptide, but not α3 peptide, disrupted α7-Bax complexes formed in response to H 2 O 2 , restored α7-VDAC1 complexes destroyed by H 2 O 2 and prevented cytochrome c leakage from mitochondria of H 2 O 2 -treated U373 cells. However, none of the peptides prevented U373 cell death after incubation with H 2 O 2 for 24 hours (Fig. 4B ).

The data presented here demonstrate that SARS spike protein-derived peptide 674-685 is able to bind α7 nAChRs found in either α7-expressing cells or mitochondria and to prevent the formation of mitochondrial channel necessary for release of pro-apoptotic signals like cytochrome c. However, when added to intact cells, the SARS spike protein-derived peptide does not protect against apoptogenic effect of H 2 O 2 .

The ability of SARS spike protein-derived peptide and CTX peptide to bind α7 nAChRs is probably due to the presence of positively charged cluster of amino acid residues: RGKR in CTX peptide and RRAR in SARS spike protein-derived peptide, which mimic the quaternary nitrogen of choline (but not acetylcholine, because the peptides did not bind to α4-containing nAChRs, which require acetylcholine for activation). Then, the SARS-Cov-2 spike protein containing this motif can influence functioning of α7 nAChRs upon infection. A fragment containing CTX-like motif (RGKR) is also present in the glycoprotein of rabies virus and was suggested to be the reason for behavioral alterations caused by rabies infection [26] . The α7 nAChRs are expressed in many organs and tissues including central nervous system, respiratory epithelium, vascular endothelium, skin and blood cells. Interaction of SARS-Cov-2 with α7 nAChRs could facilitate virus intervention and explain multiple pathological effects observed upon . Here we show for the first time that SARS spike protein-derived peptide can influence α7 nAChRs expressed in mitochondria to inhibit apoptosis-inducing events.

It is known that coronaviruses, including SARS-Cov, encode both pro-apoptotic and antiapoptotic proteins and can either initiate or delay the progression of apoptosis [27] [28] . In parallel, viruses affect intracellular mechanisms to prevent the infected cell death before their replication cycle is complete [29] [30] . According to established model, after penetrating into the cell cytoplasm, the virus particle is uncoated to enable its RNA translation by the host cell's J o u r n a l P r e -p r o o f ribosomes [31] . Correspondingly, the coating proteins are degraded inside the cell and their fragments can influence intracellular structures including mitochondria. Our data suggest that the α7 nAChR-binding portion in SARS-Cov-2 spike protein is one of molecular tools to attenuate apoptosis and support the infected cell viability in the course of SARS-Cov-2 replication. This mechanism is efficient only after the virus is inside the cell and does not work if the peptide affects cell surface nAChRs.

The data presented provide the first experimental evidence for SARS-Cov-2 spike proteinderived peptide 674-685 interaction with α7 nAChRs in cells and mitochondria and demonstrate that such interaction attenuates cytochrome c release from isolated mitochondria of the brain cells. These data suggest a molecular mechanism by which SARS-Cov-2 virus supports the infected cell viability until its replication cycle is complete.

Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

[31] T.S. Fung, D.X. Liu, Human coronavirus: host-pathogen interaction, Annu. Rev. Microbiol. 73(2019) 529-557. 

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Apoptosis in animal models of virus-induced disease, Nat. Rev. peptide concentrations corresponded to the molar ratios of 1:1, 5:1 and 10:1 related to α7(179-190)-specific antibody concentration (4 nM) assuming the molecular weights of SARS-Cov-2 Sprotein-derived peptide 674-685 being 1.478 kDa

Cytochrome c (Cyto c) release from isolated mitochondria was induced by 0.5 mM H 2 O 2 in the presence or absence of SARS spike protein-derived, CTX, α3 peptides or PNU282987 (10, 50 or 100 nM each) and was measured by Sandwich ELISA as described in Methods. Each data point in A and B corresponds to means ±SD of triplicate measurements

The effects of SARS spike protein-derived, CTX, α3 peptides or PNU282987 on α7-containing complexes and cytochrome c content in mitochondria isolated from U373 cells (A) and on viability of U373 cells treated with H 2 O 2 (B). A: U373 cells were incubated with 1mM H 2 O 2 during 1h and mitochondria isolated with mitochondria isolation kit were either treated or not with PNU282987 (30 nM)

The mitochondria were lysed and analyzed for the levels of α7-Bax, α7-VDAC1 complexes and cytochrome c by Sandwich ELISA. The immunoplates coated with α7(1-208)-specific antibody (20 µg/ml) were incubated with mitochondria detergent lysates (100 µg/ml) and the bound Bax, or VDAC1 were revealed with biotinylated Bax

µg/ml) antibodies. For cytochrome c determination, the plates were coated with polyclonal cytochrome c-specific antibody (40 µg/ml) and incubated with mitochondria lysates followed by biotinylated cytochrome c-specific antibody (20µg/ml) as described in Methods. B: The U373 cells viability was measured by MTT assay after 24 h incubation with (A)