Kim K, Lee EJ, Kang SS (2024) COVID-19 Spike Protein Enhances Cytosol Ca2+ Release from ER for its Infection through Stimulating the Association between ACE2 and STIM1. Ann Vaccines Immunization 8(1): 1024

STIM1 consists of a 685-amino acid type I transmembrane protein that was originally identified in the plasma membrane, where it is oriented with its N-terminal domain targeted to the extracellular space [1,2]. STIM1 was initially described as a protein that induces growth arrest and degeneration of several human tumor cell lines, and is involved in stromal-hematopoietic cell interactions [1-3]. When the intraluminal Ca2+ concentration is reduced, STIM1 [671RKKFPLKIFKKPLKK685] re-localizes within the ER membrane to punctae at ER-plasma membrane junctions, which facilitates its association with members of the Orai-1, E-cadherin, Transient Receptor Potential Cation (TRPC), and Transient Receptor Vanilloid Potential 4 (TRPV4) cation families [4-7]. All these proteins contain the acidic motifs (Asp-Asp) [4-7].

The Renin–Angiotensin System (RAS) is one of the most well investigated and clinically relevant homoeostatic systems in human physiology [8-10]. As a bioactive peptide of RAS, Ang-(1– 7) [Angiotensin-(1–7)] is involved in many biological processes such as neural plasticity, memory and anxiety, and possesses anti-angiogenesis, anti-proliferation, anti-fibrosis, anti- hypertrophy, anti-thrombosis, and vasodilatation, properties by a direct interaction with its special receptor ACE2 (Angiotensin Converting Enzyme 2) [11,12].

Even though many researchers focus on the binding between COVID 19 Spike (S) and ACE2 recently, we attend our curiosity to the C-terminal tail of ACE2 [13,14]. Previously, we demonstrated that ACE2 specifically associates with PSD95 via ACE2 last four amino acids [801QTSF805 (801Gln-Thr-Ser-Phe805)] which is known Type II PDZ motif (xFxF-COOH), in the line of elucidating ACE2 signaling functions [11]. In the line of this research, we also recognized that ACE2 in all vertebrates have a conserved typical STIM1 (Stromal Interaction Molecule 1 Precursor) binding motif [799DD800 (Asp-Asp)] at its C-terminus which is just upfront PDZ domain (801QTSF805), the modular protein-interaction domain for the important roles in receptor signaling and functions [15,16]. Base on the fact that STIM1 is able to gate TRPV4 and TRPC1 with the electrostatic interaction between two conserved, we hypothesized that the negatively-charged aspartates in ACE2 [799DD800] and the positively-charged lysine residues in STIM1 [671RKKFPLKIFKKPLKK685] located in the C-terminal polybasic region [17,18].

During COVID-19 infection, it has been well demonstrated the modulation of ACE2 as trimmer by association with Spike (S) protein [19-21]. However, because it is unclear yet the after event of S binding ACE2, we hypothesized that the protein binding between S1 and ACE2 triggers the cytoplasmic Ca2+ ion release for enhancing COVID 19 infection, to result in cell autophagy or apoptosis [22]. For this reason, after confirming the protein-protein interaction, we attempted to study the biological meaning of the protein-protein interaction between ACE2 and STIM1. Here, we at first present the experimental evidences that STIM1 is the novel ACE2 associated protein to help COVID 19 infection through enhancing cytoplasmic Ca2+ ion concentration, upon its S protein binding to the host cell membrane receptor ACE2. The released cytoplasmic Ca2+ ion seems to be the host cell final contribution for its membrane fusion between viral coat and eventually host plasma cell membrane, to be penetrated by COVID-19. Thus, our observations provide an important control point to prevent COVID-19 pandemic recurring and to cure it.

STIM1 (Stromal Interaction Molecule 1 Precursor) was identified as a novel ACE2-binding protein

After inspection of motifs and domains on ACE2, we find that ACE2 (805 aa) contains COVID-19 S1 protein binding domain (1- 19), peptidase M2 (374-402), collectrin (617-770), Zn2+ binding site (374,378,402; for HExxH motif), the trans-membrane regions, the STIM1 binding site (799DD800), and C-terminal basic domain motif tail (qTsF805) [8,9,17].

To study the 799DD800 motif function, we cloned EGFP ACE2 WT, (D781-805aa) mutant t ACE2 mutant (799AA800) (WT; Gene Bank no. BC127052), as shown in figure 1B. Alignment of ACE2 WT, 799AA800, and D781. Both 799AA800 and D781 were constructed using site-directed mutagenesis.

To confirm the protein-protein interaction between ACE2 and STIM1 in HEK293 cell, IP and IB were conduct as follow. Following Immunoprecipitation (IP) using an anti-ACE2 antibody, Immunoblot (IB) analysiswasperformed using an antibody against STIM1 (left). Reversely, STIM1 immunoprecipitated complexes were subjected to immunoblot analysis using an anti-ACE2 antibody (right). As shown figure 2A, co-immunoprecipitation of STIM1 with ACE2 (co-immunoprecipitation of ACE2 with STIM1, reciprocally) confirmed the formation of an ACE2– STIM1 complex. The corresponding human ACE2 (up panel) and STIM1(middle panel) are indicated with the arrow. The negative control for immunoprecipitation was an unrelated antibody. The control for western blot analysis was an antibody against actin (bottom panel). These results suggest that the protein-protein complexes between ACE2 and STIM1are present in HEK293 cell line. We observed same the protein-protein complexes between ACE2 and STIM1 in Hela cell and human liver tissue lysates (data not shown). Thus, we concluded that the protein-protein interaction between ACE2 and STIM1are common.

To define the binding motif of ACE2 for STIM1, we inspected the ACE2 amino acid sequences with the bioinformatics information of STIM1 [671RKKFPLKIFKKPLKK685] which is located in its C-terminal. With the information that STIM1 poly basic amino acid tail prefers to bind with the shot acidic motif [DD (Asp-Asp)] of its partner protein, we noticed the motif [799DD800] on ACE2 (Figure 1A).

Figure 1: A). Transmembrane topology of mouse ACE2 (805 aa) including its putative STIM1 binding acid motif (DD) and C-terminal basic domain motif tail. A). Scheme of motifs and domains of ACE2.Transmembrane topology of mouse ACE2 (805 aa). COVID19 S1 protein binding domain [1- 20], Peptidase M2 (374-402), Collectrin (617-770), Zn2+ binding site (374,378,402; HExxH) the trans-membrane regions, the STIM1 binding site (STIM1) 799DD800, and PDZ motif tail (qTsF805) Δ781 are indicated [12,13].

B). The position of 799AA800 or Δ781 mutant. ACE2 799AA800 or Δ781 mutant which was constructed using site-directed mutagenesis was compared with its wild type (Gene Bank no. BC127052).

C). STIM1 C-tail [671RKKFPLKIFKKPLKK685]. This sequences is known as the responsible motif interaction with the acidic motif (DD) of many proteins are shown. STIM1 has been reported to be the sensor of Ca2+ accumulation in the ER that activates store-operated Ca2+ entry, a mechanism for Ca2+ influx that is controlled by the filling state of the intracellular Ca2+ stores.

D). Comparison of several vertebrate ACE2 amino acids. All vertebrata contain the putative STIM1 binding acid motif [799DD800], except bat ACE2 (802ND803) and chicken (802EE803). Bat is known as a natural reservoir of COVID19. The motif difference may provide COVID19 infection resistance to bat or chicken (Figure 1D) [24,25].

STIM1 has been reported to be the sensor of Ca2+ accumulation in the ER that activates store-operated Ca2+ entry (SOCE), a mechanism for Ca2+ influx that is controlled by the filling state of the intracellular Ca2+ stores. STIM1 C-tail [671RKKFPLKIFKKPLKK685] sequences which was known as the responsible motif interaction with the acidic motif (DD) of many proteins was shown.

Comparison several vertebrate ACE2 amino acids. All vertebrata contain the putative STIM1 binding acid motif [799DD800], except chicken ACE2 (801EE802) bat ACE2 (801ND802). Bat is known as a natural reservoir of COVID-19 [8,9,16,19,23,24]. The motif difference may also provide COVID-19 infection resistance to bat or chicken (Figure 1D).

The C-terminal acidic motif [799DD800] of ACE2 requires for interaction with STIM1

Protein-protein interaction between ACE2 and STIM1 in HEK 293 cells has been proven with IP-IB experiment. Following Immunoprecipitation (IP) using an anti-ACE2 antibody, Immunoblot (IB) analysis was performed using an antibody against STIM1 (up). Conversely, STIM1 immunoprecipitated complexes were subjected to immunoblot analysis using an anti-ACE2 antibody (middle). To monitor the total cell amount, the western blot of cell lysate with the actin Ab was performed (bottom). The negative control for immunoprecipitation was an unrelated antibody. Co-immunoprecipitation of STIM1 with ACE2 confirmed the formation of an ACE2–STIM1 complex in HEK293 cell (Figure 2A).

Figure 2: The C-terminal acidic motif [799DD800] of ACE2 is required for interaction with STIM1.

(A) Protein-protein interaction between ACE2 and STIM1 in HEK 293 cells. Following Immunoprecipitation (IP) using an anti-ACE2 antibody, Immunoblot (IB) analysis was performed using an antibody against STIM1 (up). Co STIM1 immunoprecipitated complexes were subjected to immunoblot analysis using an anti-ACE2 antibody (middle). To monitor the total cell amount, the western blot of cell lysate with the actin Ab was performed (bottom). The negative control for immunoprecipitation was an unrelated antibody. Co-immunoprecipitation of STIM1 with ACE2 confirmed the formation of an ACE2–STIM1 complex in HEK293 cell.

(B) Requirement of ACE2 the short acidic motif binding motif [799DD800] for the association with STIM1. To demonstrate the requirement of [799DD800] motif for the association with STIM1. we transfected EGFP-ACE2 WT, [799AA800] or Δ781 mutant to HEK293 cell and IP was performed with EGFP Ab and IB was done with STIM1 to demonstrate whether ACE2 [799DD800] is required for the binding to STIM1. Only ACE2 WT pull down STIM1, but ACE2 (799AA800 or Δ781) did not

We transfected EGFP-ACE2 WT, [799AA800] or D781 mutant to HEK293 cell and IP was performed with EGFP Ab and IB was done with STIM1 to demonstrate whether ACE2 [799DD800] is required for the binding to STIM1. Only ACE2 WT pull down STIM1, but ACE2 (799AA800 or D781) did not (Figure 2B). Blots are representative of three to five independent experiments. Thus, our results confirmed that ACE2 [799DD800] acidic motif is also required to form a protein complex with STIM1 in the HEK293 cell (Figure 2B).

Confocal microscopic images of cells transfected with EGFP-ACE2 WT or mutant (799AA800 ) in HEK cells

Cells were examined by direct immunofluorescence microscopytodetectACE2andSTIM1interactionanditssubcellular co-localization (Figure 3).

Figure 3: Confocal microscopic images of cells transfected with EGFP-ACE2 WT or mutant (799AA800 ) in HEK cells. Cells were examined by direct immunofluorescence microscopy. The figures show EGFP-ACE2 WT or mutant (799AA800) (green), STIM1[671RKKFPLKIFKKPLKK685] (red), and merged (yellow) confocal microscopic images.

(A) EGFP-ACE2 WT showed co-localization with STIM1 at the Golgi apparatus. PCC (Pearson Co-localization co-efficiency) are indicated below (0.84 +/- 0.05; N = 5).

(B) EGFP-ACE2 (799AA800) was not detected with Cherry STIM1 (right). PCC (Pearson Co-localization co-efficiency) are indicated below (0.14 +/- 0.03; N = 5). Consisting figure 2 results, the microscopy results also support that the C-terminal acidic motif [799DD800] of ACE2 is required for interaction with STIM1.

The figures show EGFP-ACE2 WT or mutant (799AA800) (green), STIM1[671RKKFPLKIFKKPLKK685] (red), and merged (yellow) confocal microscopic images. EGFP-ACE2 WT showed co-localization with STIM1 at the Golgi apparatus. PCC (Pearson Co-localization Co-efficiency) are indicated below (0.84 +/-0.05) (Figure 3A). However, EGFP-ACE2 (799AA800) was not detected with Cherry STIM1 (right). PCC (Pearson Co- localization co-efficiency) are indicated below (0.14 +/- 0.03) (Figure 3B).

Consisting with figure 2B results, the microscopy results also support that the C-terminal acidic motif [799DD800] of ACE2 is required for interaction with STIM1. This confocal microscopic analysis of the transfected EGFP-ACE2 WT, AA(green) also supported that ACE2 interacts with STIM1 and co localize together (Figure 3).

Ca2+ live image comparison of ACE2 WT or (799AA800) mutant in HEK cells

The biological significance of the protein-protein inter2a+ction calcium concentration change [Ca2+] I of ACE2 WT or (799AA800) with / without S1 protein treatment (10nM) was measured with the absorption at 488 nm of argon-ion laser in HEK 293 cells (as an arbitrary % unit).

As shown in figure 4, S1 protein treatment on ACE2 WT enhanced the intracellular calcium concentration change [Ca2+] I, 3~5 times more than that of the no treated cell (Figure 4 upper graph).

Figure 4: Ca2+ live image Comparison of ACE2 WT or 799AA800mutant in HEK cells with S protein treatment. The effects on intracellular calcium concentration change [Ca2+] I of ACE2 WT or (799AA800) with/ without S1 protein treatment (10nM) was measured with the absorption at 488 nm of argon-ion laser in HEK 293 cells (as an arbitrary % unit). S1 protein treatment on ACE2 WT enhanced the intracellular calcium concentration change [Ca2+] I, 3 ~ 5 times more than that of the no treated cell (Figure 4 upper graph). ACE2 (799AA800) mutant showed no significant change with S protein treatment (10nM), compared with that of the non-treated cell (Figure 4 lower graph)

In contrast, ACE2 (799AA800) mutant showed no significant change with S protein treatment (10nM), compared with that of the no treated cell (Figure 4 lower graph). Gather together, we concluded that ACE2 acidic motif (799DD800) is required not only to interact with STIM1 (Figure 2) but also to release Ca2+ from ER into the cytoplasm for the COVID-19 infection (Figure 5).

Figure 5: Our working model for the regulation of ACE2 by STIM1 in the cytosol Ca2+ release after S protein binding with ACE2. ACE2 WT can activate STIM1 through association/dissociation from its C-terminal cytoplasmic domain (a short-term activation/inactivation loop on the left side). In normal state (A), ACE2 WT can bind STIM1 at basal level and STIM1 does not open to transport Ca2+ in the cytoplasm from ER. (A) ACE2 WT [799DD800] motif can bind STIM1 [671RKKFPLKIFKKPLKK685] and stimulate to form STIM1 channel complex which is able to transport Ca2+ in the cytoplasm from ER, resulting in Ca2+ release in the cytoplasm from ER. After S1 protein (processed from S by the protease: TMPRSS2) binds its host cell ACE2 receptor, ACE2 aggregation (such as trimmer) and stimulation causes STIM1 Ca2+ release from ER. The increased Ca2+ in the cytoplasm seems to promote the cell apoptosis (Table 1). (B) In contrast, ACE2 [799AA800] or Δ781 mutants cannot bind STIM1 [671RKKFPLKIFKKPLKK685] nor form STIM1 channel complex. Thus, the transport Ca2+ in the cytoplasm from ER is blockaded (Figure 4).

The effect of the interaction between ACE2 and STIM1 on apoptosis

In order to compare ACE2 WT biological activity with DAA, or D781, FACS analysis was performed with each cell line (WT, 799AA800, or D781) which was established from HEK293. The ACE2 protein was then immunoprecipitated with the anti-GFP antibody, and the ACE2 was analyzed by immunoblotting with an anti-ACE antibody. In contrast, the ACE2 AA or D781 deletion mutant was not affected by STIM1 [671RKKFPLKIFKKPLKK685] co- expression in comparison with that of mutant ACE2 expression alone, suggesting that ACE2 was inhibited by the interaction with STIM1. These data were consistent with the results from our confocal microscopy data that ACE2 partly co-localized with between ACE2 and STIM1, we attempted compared Ca live proteasomes in the cytoplasm, and the receptor was anchored in image of ACE2 WT, AA, in HEK cells. The effects on intracellular the plasma membrane by STIM1 co-expression, inhibiting ACE2 (799DD800) co-localization. The increased Ca2+ in the cytoplasm seems to promote the cell apoptosis (Table 1).

Table 1: The effect of the interaction between ACE2 and STIM1 on autophagy and apoptosis activity.

To compare ACE2 WT autophagy activity with ΔAA, or Δ781, FACS analysis was performed with each cell line (WT, 799AA800, or Δ781) which was established from HEK293 [22].

Therefore, we speculated that the protein interaction between ACE2 and STIM1 may effect on the host cell autophagy and apoptosis activity. However, because the increased Ca2+ effects on many biological activities in the cell, we cannot rule out that the change of host cell autophagy and apoptosis activity by ACE2 WT, 799AA800, or D781 is caused by other unknown facts (Table 1).

 Our working model for the regulation of ACE2 by STIM1 in the cytosol Ca2+ release after S protein binding with ACE2

In normal state (A), ACE2 WT can bind STIM1 at basal level and STIM1 does not open to transport Ca2+ in the cytoplasm from ER. In activated state (Figure 5A), (in case of Spike (S1) protein association with ACE2), ACE2 forms protein oligomer (trimmer) complex, ACE2 WT [799DD800] motif can bind STIM1 [671RKKFPLKIFKKPLKK685] and stimulate to form STIM1 channel complex which transport Ca2+ in the cytoplasm from ER, resulting in Ca2+ release in the cytoplasm from ER. S protein is digested by TMPRSS2 (host cell protease) and cleaved to S1 (soluble) and S2 (remains in viral particle). After S1 protein binding to its host cell ACE2 receptor, ACE2 aggregates (as trimmer) and stimulates STIM1 to release Ca2+ from ER. Ultimately, the release Ca2+ in the cytoplasm seems to promote the cell apoptosis (Table 1) or autophagy.

ACE2 WT [799DD800] motif can bind STIM1 [671RKKFPLKIFKKPLKK685] (Figure 1) and stimulate to form STIM1 channel complex which transport Ca2+ in the cytoplasm from ER, resulting in Ca2+ release in the cytoplasm from ER. The increased Ca2+ in the cytoplasm seems to promote the cell apoptosis. However, ACE2 [799AA800] or D781 mutant (inactive state) is not able to bind STIM1 [671RKKFPLKIFKKPLKK685] nor form STIM1 channel complex, even though S1 binds to ACE2 [799AA800] or D781 mutant (Figure 4B). Thus, the transport Ca2+ in the cytoplasm from ER is blockaded (Figure 4).

In conclusion, our data indicate that STIM1 [671RKKFPLKIFKKPLKK685] can be activated by ACE2 WT (799DD800), to release Ca2+ ion from ER through both protein- protein interaction (Figure 5). ACE2 is a functional receptor of COVID 19 through the interaction between its S protein and ACE2, too [8,9,17,20,24,25]. The associated cellular protein STIM1 may also help COVID-19 S infection process through enhancing both Ca2+ concentration and cell apoptosis (Table 1). Further, because ACE2 is a functional receptor of COVID 19 through the interaction between COVID-19 S and ACE2, the characterization of its associated cellular protein may also be great helpful to elucidate in COVID-19 S infection process (Figure 5).

ACE2 is well characterized for the critical properties of the central nervous system, including neural plasticity, memory and anxiety [8,9,17]. Several case studies have shown that brief seizure episodes lead to a significant and transient increase in ACE2 mRNA expression in the hippocampus. Even though we identified that ACE2 interacts with STIM1 to Ca2+ release after S1 protein binds ACE2, it also required to identify ACE2 association proteins which modulate its signal transduction, in order to understand ACE2 signal transduction pathway further. The functions and signaling mechanisms of the ACE2 have been studied extensively. However, much less attention has been paid to the intracellular regulation of ACE2 protein which is COVID-19 receptor protein [8,9,17].

Comparing ACE2 amino acid sequences (Figure 1), we noticed that all vertebrata contain the putative STIM1 binding acid motif [799DD800], except chicken (801EE802) and bat ACE2 (801ND802) as a natural reservoir of COVID-19. Therefore, it seems to that STIM1 binding motif difference of ACE2 also allows the perseverance to COVID-19 together with its receptor binding motif [20,24,25]. However, it requires further research. In the present study, we at first demonstrate that ACE2-binding protein is STIM1 which has [671RKKFPLKIFKKPLKK685]. For this interaction, the acidic motif (799DD800) in ACE2 is required not only to interact with STIM1 but also to release Ca2+ from ER into the cytoplasm for the COVID-19 infection. Because the host cell membrane protein (eg.TMPRSS2) cuts at 655aa of COVID-19 S and generates S1 (1-655aa) and S2 (656-1273aa) [8,9,17,23], we used the purified S1 as ACE2 stimulator (Figure 4). Thus, the increased interaction between the acidic motif (799DD800) in ACE2 and STIM1 through COVID-19 S may inhibit the overall STIM1functions by the disruption of its normal protein-protein interaction or subcellular localization, resulting in COVID-19 virulence [11,15,23].

Recently, in the course of our research of ACE2 tail motif (qTsF805) interaction with PSD95 PDZ III domain, we also recognized the COVID 19 S protein C-terminal acidic motif [1258DEDDSE1263] which seems to be essential for this interaction with  STIM1  [671RKKFPLKIFKKPLKK685]  motif  [23].  This information leaded us to pursue the biological significances that hide in the association between COVID 19 S (S2) protein acidic tail [1258DEDDSE1263] and STIM1 C-terminal basic amino acid domain [671RKKFPLKIFKKPLKK685] [11,12,16,23]. Thus, S1 protein binds its host cell ACE2, which causes ACE2 aggregation and stimulation, resulting in STIM1 Ca2+ release from ER (Figure 5).

COVID-19 may evolve to adapt the host physiology for maximizing its survival rate, as the nature anti-reaction force for the equilibrium. Thus, ACE2 seems to be naturally selected as the effective COVID-19 receptor during the virus evolution. After S protein accumulation in the host cell, it also contributes the dissociation of ACE2 from STIM1 with its [1258DEDDSE1263] motif, as a feedback inhibition/feedforward activation.

In the line of this assumption, we also speculated that the C-terminus of COVID-19 S protein [1258DEDDSE1263] motif which specifically interacts with the motif of STIM1 seems to be evolved for the viral maximum survival purpose through COVID-19 natural selection process. Because STIM1 is the most essential multifunctional host cell survival regulation point (such as Ca2+ ion regulation), the host target (disruption) motif of COVID-19 S protein interaction seems to be selected for its maximum survival effectiveness. However, after penetration into the host cell, whether COVID-19 S2 protein acidic tail [1258DEDDSE1263] interacts STIM1 C-terminal basic amino acid domain directly to blows up Ca2+ release from ER still remains to be characterized in a future [23]. Even though we used the purified S1 to stimulate ACE2 only (Figure 4), other unknown possibilities are not completely ruled out [22].

In summary, STIM1 was identified as a novel ACE2 (known as COVID-19 receptor) interacting molecule in this study. The C-terminus [799DD800] motif of ACE2 specifically interacts with the domain of STIM1 [671RKKFPLKIFKKPLKK685] (Figure 1). Therefore, our findings reveal a new mechanism for the regulation of ACE2 protein expression by STIM1. Thus, the elucidation of STIM1 mediated mechanism ACE2 regulation may help to clarify further the functions of the ACE2 in the central nervous system and to combat the dangerous COVID-19 epidemics [8,9,17,20,24,25]. In addition, ACE2 is a functional receptor of the SARS coronavirus 19 (COVID 19) through the interaction between COVID-19 S1 and ACE2, its associated cellular protein may also be involved in COVID-19 S infection process (Figure 5).

Preparation of plasmids

The pET30A vectors encoding individual domains (799DD800) of STIM1, pCherry-STIM1 constructs were kindly provided by Dr Randy Choi (Chungbuk national University, Cheongju, Korea.). The (799DD800) WT (wild-type) and its mutant (D781, 799AA800; 799DD800->799AA800) of human ACE2 were amplified by PCR from the pCGN-HA-ACE2 construct GeneCopoeia (USA) and subcloned into pEGFP-C1 expression vectors respectively [15,16]. In order to obtain the mutants, amino acid changes were introduced using mutated oligonucleotides for ACE2 799AA800 or 781 stop and wild- type ACE2 as a template.

Western blotting and antibodies

Samples were run on SDS/PAGE and then transferred on to a nitrocellulose membrane. The membrane was blocked in TBST [TBS (20 mM Tris/HCl, pH 7.5, and 500 mM NaCl) with 0.05% Tween 20] containing 5% (w/v) non-fat dried milk for 1 h at room temperature (23–25°C). Proteins of interest were probed with corresponding primary antibodies followed by HRP (Horseradish Peroxidase)-conjugated anti-rabbit or anti-mouse secondary antibodies. Immunoreactive bands were visualized by ECL detection reagents (Applygen Technologies) and analyzed with NIH Image. The anti-GST and anti-GFP antibodies were obtained from MBL. Anti-GST antibody was from Applygen Technologies. Anti-STIM1 antibody was obtained from Sigma– Aldrich. Anti-ACE2 antibody was from Novus Biologicals. HRP- conjugated anti-GST antibody was obtained from GE Healthcare. Anti-ACE2 was from Santa Cruz Biotechnology. Anti-GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase), anti-actin and HRP-conjugated secondary antibodies were obtained from ZSGB- BIO.

Cells and transfection

HEK293 (Human Embryonic Kidney), NIH 3T3 cells were maintained in DMEM (Dulbecco’s Modified Eagle’s Medium) plus 10% (v/v) FBS and 1% penicillin/streptoCherryin (Sigma–Aldrich). The cells were cultured on polylysine-coated glass coverslips in DMEM supplemented with 10% (v/v) FBS, 10% (v/v) horse serum and 1% penicillin/streptoCherryin. Cell transfection was performed with Lipofectamine™ 2000 (Invitrogen). For cell stable transfection, constructs of ACE2 (WT, AA, 801) were transfected into HEK293 or 3T3 cells respectively with Lipofectamine™ 2000, and selected with the growth medium containing 1200 μg/ml neoCherryin (Amresco).

Co-immunoprecipitation

Co-immunoprecipitation was performed as described previously. Briefly, transfected cells were harvested or rabbit brain was homogenized in ice-cold lysis buffer. Supernatants were incubated with anti-GFP or anti-STIM1 antibody (for transfected cells) pre-bound to Protein A/G–agarose beads (Calbiochem). The immunoprecipitated proteins were then analyzed by Western blotting.

Immunofluorscence Co-Localization

Immunofluorescence co-localization was performed as described previously. Briefly, HEK293 cells were transfected with ACE2 (799DD800) and/or STIM1 constructs. After fixation and permeabilization, cells were stained with anti-STIM1 antibody and anti-ACE2 antibody (Abcam) followed by rhodamine- conjugated anti-(mouse IgG) antibody (ZSGB-BIO), Alexa Fluor® 647-conjugated anti-(rabbit IgG) antibody (Invitrogen) and Hoechst 33258 (5 μg/ml). The cellular distribution of these proteins was then visualized under a confocal microscope (Leica Microsystems, LAS AF-TCS SP5).

Fluorescence Measurements of [Ca2+]i

We measured [Ca2 +]i using a fluorescent Ca2+ indicator Fluo4- acetoxymethyl ester (Fluo4-AM), as previously described (Lee EJ, et al. ). In brief, cells growing on coverslips were incubated for 40 min in DMSO solution containing 1 μM Fluo4-AM at 24°C in darkness, and then washed and incubated for 15 min to hydrolyze internalized Fluo4-AM, with/ without the purified S1 (purchased from Abcam). We measured [Ca2 +]i in single cells that emitted fluorescence, using confocal microscopy (LSM710 Zeiss, Germany) at wavelengths of 495 nm (excitation), and 519 nm (emission). The absorption (as an arbitrary unit) at 488 nm with an argon-ion laser was measured as a relative intracellular Ca2 + ion concentration [Ca2 +]i. All experiments were carried out at 24°C. After stimulation with mild heat (from 24 to 42 °C within 45 s for 2 min), [Ca2 +]i was measured in single cells at a 24 °C solution [15,16].

FACS

FACS was performed with our previous method in elsewhere [15,16]. COVID-19 Spike, its AA mutant and EGFP vector, were transfected into cells and the rates of apoptosis measured with an Annexin V-PE apoptosis detection kit I (BD Biosciences, USA). Transfected cells were washed twice in cold PBS and resuspended in binding buffer [0.01 M Hepes/NaOH (pH 7.4) 0.14 M NaCl, 2.5 mM CaCl2]. 1 × 105 cells in 100 ml were transferred to 5ml culture tubes and 5 ml of Annexin V-PE and 5 ml of 7-Amino-actinomycin D were added. The cells were vortexed and incubated for 15 min at 25°C in the dark. 400 ml of binding buffer was added to each tube. Within 1 h FACS was performed on a Coulter Epics Elite equipped with a gated amplifier and upgraded to give enhanced system performance in the Core Facility of Chungbuk National University.

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Sang Sun Kang designed, directed and carried out the most experiments and data analysis with Lee and Kim. All authors have contributed to this works and have approved the final paper.

This work was supported by the Korea Natural Science Foundation, Chungbuk Natural Science Foundation [grant numbers 7131003 and 5112007].

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