In-Vitro Fluorescence Microscopy Studies Show Retention of Spike-Protein (SARS-Cov-2) on Cell Membrane in the Presence of Amodiaquin Dihydrochloride Dihydrate Drug

2021 ◽  
Author(s):  
Partha Pratim Mondal ◽  
Subhra Mandal
Keyword(s):  
Cell Membrane ◽  
Large Fraction ◽  
Degradation Process ◽  
Cell Receptor ◽  
Endocytic Pathway ◽  
Receptor Protein ◽  
Potential Candidate ◽  
S Protein ◽  
Cellular Environment

The ability of S-glycoprotein (S-protein) in SARS-Cov-2 to bind to the host cell receptor protein (angiotensinconverting enzyme 2 (ACE2)) leading to its entry in cellular system determines its contagious index and global spread. Three available drugs (Riboflavin, Amodiaquin dihydrochloride dihydrate (ADD) and Remidesivir) were investigated to understand the kinetics of S-protein and its entry inside a cellular environment. Optical microscopy and fluorescence-based assays on 293T cells (transfected with ACE2 plasmid) were used as the preamble for assessing the behaviour of S-protein in the presence of these drugs for the first 12 hours post S-protein - ACE2 binding. Preliminary results suggest relatively long retention of S-protein on the cell membrane in the presence of ADD drug. Evident from the %-overlap and colocalization of S-protein with endosome studies, a large fraction of S-protein entering the cell escape endosomal degradation process, suggesting S-protein takes non-endocytic mediated entry in the presence of ADD, whereas in the presence of Riboflavin, S-protein carry out normal endocytic pathway, comparable to control (no drug) group. Therefore, present study indicates ADD potentially affects S-protein’s entry mechanism (endocytic pathway) in addition to its reported target action mechanism. Hence, ADD substantially interfere with S-protein cellular entrance mechanism. However, further detailed studies at molecular scale will clarify our understanding of exact intermediate molecular processes. The present study (based on limited data) reveal ADD could be potential candidate to manage Covid-19 functions through yet unknown molecular mechanism.

Potential inhibitors for blocking the interaction of the coronavirus SARS-CoV-2 spike protein and its host cell receptor ACE2

2021 ◽  
Author(s):  
Changzhi Li ◽  
Hongjuan Zhou ◽  
Lingling Guo ◽  
Dehuan Xie ◽  
Huiping He ◽  
...  
Keyword(s):  
Cell Receptor ◽  
Economic Stability ◽  
Time Resolved ◽  
S Protein ◽  
Host Cell Receptor ◽  
Compound Libraries ◽  
Screening Platform ◽  
Potential Inhibitors ◽  
Validation Experiments

The outbreak of SARS-CoV-2 continues to pose a serious threat to human health and social and economic stability. In this study, we established an anti-coronavirus drug screening platform based on the Homogeneous Time Resolved Fluorescence (HTRF) technology and the interaction between the coronavirus S protein and its host receptor ACE2. This platform is a rapid, sensitive, specific, and high throughput system. With this platform, we screened two compound libraries of 2,864 molecules and identified three potential anti-coronavirus compounds: tannic acid (TA), TS-1276 (anthraquinone), and TS-984 (9-Methoxycanthin-6-one). Our in vitro validation experiments indicated that TS-984 strongly inhibits the interaction of the coronavirus S-protein and the human cell ACE2 receptor. This data suggests that TS-984 is a potent blocker of the interaction between the S-protein and ACE2, which might have the potential to be developed into an effective anti-coronavirus drug.

scholarly journals Mass Spectrometry and Structural Biology Techniques in the Studies on the Coronavirus-Receptor Interaction

Molecules ◽  
2020 ◽  
Vol 25 (18) ◽  
pp. 4133
Author(s):  
Danuta Witkowska
Keyword(s):  
Mass Spectrometry ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Three Dimensional ◽  
Cell Receptor ◽  
Receptor Protein ◽  
Receptor Interaction ◽  
S Protein ◽  
X Ray Crystallography ◽  
Huge Impact

Mass spectrometry and some other biophysical methods, have made substantial contributions to the studies on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human proteins interactions. The most interesting feature of SARS-CoV-2 seems to be the structure of its spike (S) protein and its interaction with the human cell receptor. Mass spectrometry of spike S protein revealed how the glycoforms are distributed across the S protein surface. X-ray crystallography and cryo-electron microscopy made huge impact on the studies on the S protein and ACE2 receptor protein interaction, by elucidating the three-dimensional structures of these proteins and their conformational changes. The findings of the most recent studies in the scope of SARS-CoV-2-Human protein-protein interactions are described here.

scholarly journals Rat Spleen Dendritic Cells Express Natural Killer Cell Receptor Protein 1 (NKR-P1) and Have Cytotoxic Activity to Select Targets via a Ca2+-dependent Mechanism

1997 ◽  
Vol 186 (3) ◽  
pp. 467-472 ◽  
Author(s):  
Régis Josien ◽  
Michèle Heslan ◽  
Jean-Paul Soulillou ◽  
Maria-Cristina Cuturi
Keyword(s):  
Dendritic Cells ◽  
Natural Killer Cell ◽  
Natural Killer ◽  
Fas Ligand ◽  
Killer Cell ◽  
Cell Receptor ◽  
Receptor Protein ◽  
Natural Killer Cell Receptor ◽  
Major Function

Dendritic cells (DC) are a subset of leukocytes whose major function is antigen presentation. We investigated the phenotype and function of enriched (95–98.5%) rat DC. We show that both spleen and thymus DC express the natural killer cell receptor protein 1 (NKR-P1) as a disulfide linked homodimer of 60 kD. Freshly isolated DC express a low level of NKR-P1, which is strongly upregulated after overnight culture. Spleen, but not thymus DC, were able to kill the NK-sensitive YAC-1 cell line in vitro, and since this killing was Ca2+ dependent, a Fas ligand–Fas interaction was probably not involved. Besides their potent antigen-presenting function, DC can thus be cytotoxic for some tumor targets.

scholarly journals CD45 and RPTPα display different protein tyrosine phosphatase activities in T lymphocytes

1997 ◽  
Vol 327 (3) ◽  
pp. 867-876 ◽  
Author(s):  
H. W. David NG ◽  
D. Mojgan JABALI ◽  
Arpita MAITI ◽  
Peter BORODCHAK ◽  
W. Kenneth HARDER ◽  
...  
Keyword(s):  
T Cells ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Cell Receptor ◽  
Artificial Substrates ◽  
Receptor Protein ◽  
Tyrosine Phosphatases ◽  
Recombinant Enzymes ◽  
Protein Tyrosine

To examine the substrate specificity and function of two receptor protein tyrosine phosphatases, CD45 and RPTPα, RPTPα was expressed in a CD45-, T-cell receptor (TCR)+, BW5147 T-lymphoma cell. High levels of expression of RPTPα did not fully restore either proximal or distal TCR-mediated signalling events. RPTPα was unable to reconstitute the phosphorylation of CD3ζ and did not increase the expression of the activation marker, CD69, on stimulation with TCR/CD3. RPTPα did not significantly alter the phosphorylation state or kinase activity of two CD45 substrates, p56lck or p59fyn, suggesting that RPTPα does not have the same specificity or function as CD45 in T-cells. Further comparison of the two phosphatases indicated that immunoprecipitated RPTPα was approx. one-seventh to one-tenth as active as CD45 when tested against artificial substrates. This difference in activity was also observed in vitro with purified recombinant enzymes at physiological pH. Additional analysis with Src family phosphopeptides and recombinant p56lck as substrates indicated that CD45 was consistently more active than RPTPα, having both higher Vmax and lower Km values. Thus CD45 is intrinsically a much more active phosphatase than RPTPα, which provides one reason why RPTPα cannot effectively dephosphorylate p56lck and substitute for CD45 in T-cells. This work establishes that these two related protein tyrosine phosphatases are not interchangeable in T-cells and that this is due, at least in part, to quantitative differences in phosphatase activity.

scholarly journals SARS-CoV-2 variants with mutations at the S1/S2 cleavage site are generated in vitro during propagation in TMPRSS2-deficient cells

2020 ◽  
Author(s):  
Michihito Sasaki ◽  
Kentaro Uemura ◽  
Akihiko Sato ◽  
Shinsuke Toba ◽  
Takao Sanaki ◽  
...  
Keyword(s):  
Cleavage Site ◽  
De Novo ◽  
Gene Mutations ◽  
Critical Factor ◽  
Cell Receptor ◽  
Cell Tropism ◽  
Protein Cleavage ◽  
S Protein ◽  
S Gene

AbstractThe spike (S) protein of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) binds to a host cell receptor which facilitates viral entry. A polybasic motif detected at the cleavage site of the S protein has been shown to broaden the cell tropism and transmissibility of the virus. Here we examine the properties of SARS-CoV-2 variants with mutations at the S protein cleavage site that undergo inefficient proteolytic cleavage. Virus variants with S gene mutations generated smaller plaques and exhibited a more limited range of cell tropism compared to the wild-type strain. These alterations were shown to result from their inability to utilize the entry pathway involving direct fusion mediated by the host type II transmembrane serine protease, TMPRSS2. Notably, viruses with S gene mutations emerged rapidly and became the dominant SARS-CoV-2 variants in TMPRSS2-deficient cells including Vero cells. Our study demonstrated that the S protein polybasic cleavage motif is a critical factor underlying SARS-CoV-2 entry and cell tropism. As such, researchers should be alert to the possibility of de novo S gene mutations emerging in tissue-culture propagated virus strains.

scholarly journals Implications of Spike-glycoprotein processing at S1/S2 by Furin, at S2′ by Furin and/or TMPRSS2 and shedding of ACE2: cell-to-cell fusion, cell entry and infectivity of SARS-CoV-2

2021 ◽  
Author(s):  
Rachid Essalmani ◽  
Jaspreet Jain ◽  
Delia Susan-Resiga ◽  
Ursula Andreo ◽  
Alexandra Evagelidis ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Viral Entry ◽  
Hela Cells ◽  
Protein Trafficking ◽  
Cleavage Site ◽  
Cell Receptor ◽  
Hek293 Cells ◽  
Furin Cleavage ◽  
S Protein

The Spike (S)-protein of SARS-CoV-2 binds host-cell receptor ACE2 and requires proteolytic "priming" at PRRAR685↓ into S1 and S2 (cleavage at S1/S2), and "fusion-activation" at KPSKR815↓ (cleavage at S2′) for viral entry. Both cleavages occur at Furin-like motifs suggesting that proprotein convertases might promote virus entry. In vitro Furin cleaved peptides mimicking the S1/S2 cleavage site more efficiently than S2′, whereas TMPRSS2 cleaved at both sites. In HeLa cells endogenous Furin-like enzymes cleave mainly at S1/S2 during intracellular protein trafficking, as confirmed by mutagenesis. We also mapped the S2′ cleavage site by proteomics and further showed that S2′-processing by Furin, while limited, was strongly enhanced in the presence of ACE2. In contrast, the S2′ KRRKR815↓ mutant (μS2′) was considerably better cleaved by Furin, whereas individual/double KR815AA mutants are retained in the endoplasmic reticulum (ER). Pharmacological inhibitors of convertases (Boston Pharmaceuticals - BOS-inhibitors) effectively blocked endogenous S-protein processing in HeLa cells. However, under co-expression the S-protein was prematurely cleaved by TMPRSS2 into ER-retained, non-O-glycosylated S2 and S2′ products. Quantitative analysis of cell-to-cell fusion and Spike processing using Hela cells revealed the key importance of the Furin sites for syncytia formation and unveiled the enhanced fusogenic potential of the α- and δ-variants of the S-protein of SARS-CoV-2. Our fusion assay indicated that TMPRSS2 enhances S2′ formation, especially in the absence of Furin cleavage, as well as ACE2 shedding. Furthermore, we provide evidence using pseudoparticles that while entry by a "pH-dependent" endocytosis pathway in HEK293 cells did not require Furin processing at S1/S2, a "pH-independent" viral entry in lung-derived Calu-3 cells was sensitive to inhibitors of Furin and TMPRSS2. Consistently, in Calu-3 cells BOS-inhibitors or Camostat potently reduce infectious viral titer and cytopathic effects and this outcome was enhanced when both compounds were combined. Overall, our results show that Furin and TMPRSS2 play synergistic roles in generating fusion-competent S-protein, and promote viral entry, supporting the combination of Furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2.

scholarly journals SARS-CoV-2 variants with mutations at the S1/S2 cleavage site are generated in vitro during propagation in TMPRSS2-deficient cells

2021 ◽  
Vol 17 (1) ◽  
pp. e1009233 ◽  
Author(s):  
Michihito Sasaki ◽  
Kentaro Uemura ◽  
Akihiko Sato ◽  
Shinsuke Toba ◽  
Takao Sanaki ◽  
...  
Keyword(s):  
Cleavage Site ◽  
De Novo ◽  
Gene Mutations ◽  
Critical Factor ◽  
Cell Receptor ◽  
Cell Tropism ◽  
Protein Cleavage ◽  
S Protein ◽  
S Gene

The spike (S) protein of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) binds to a host cell receptor which facilitates viral entry. A polybasic motif detected at the cleavage site of the S protein has been shown to broaden the cell tropism and transmissibility of the virus. Here we examine the properties of SARS-CoV-2 variants with mutations at the S protein cleavage site that undergo inefficient proteolytic cleavage. Virus variants with S gene mutations generated smaller plaques and exhibited a more limited range of cell tropism compared to the wild-type strain. These alterations were shown to result from their inability to utilize the entry pathway involving direct fusion mediated by the host type II transmembrane serine protease, TMPRSS2. Notably, viruses with S gene mutations emerged rapidly and became the dominant SARS-CoV-2 variants in TMPRSS2-deficient cells including Vero cells. Our study demonstrated that the S protein polybasic cleavage motif is a critical factor underlying SARS-CoV-2 entry and cell tropism. As such, researchers should be alert to the possibility of de novo S gene mutations emerging in tissue-culture propagated virus strains.

scholarly journals Anti-SARS-CoV-2 Activity of Rhamnan Sulfate from Monostroma nitidum

Marine Drugs ◽  
2021 ◽  
Vol 19 (12) ◽  
pp. 685
Author(s):  
Yuefan Song ◽  
Peng He ◽  
Andre L. Rodrigues ◽  
Payel Datta ◽  
Ritesh Tandon ◽  
...  
Keyword(s):  
Structural Characteristics ◽  
Monosaccharide Composition ◽  
Cell Receptor ◽  
Host Cells ◽  
Health Concern ◽  
Wild Type ◽  
Binding Studies ◽  
S Protein ◽  
Monostroma Nitidum

The COVID-19 pandemic is a major human health concern. The pathogen responsible for COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), invades its host through the interaction of its spike (S) protein with a host cell receptor, angiotensin-converting enzyme 2 (ACE2). In addition to ACE2, heparan sulfate (HS) on the surface of host cells also plays a significant role as a co-receptor. Our previous studies demonstrated that sulfated glycans, such as heparin and fucoidans, show anti-COVID-19 activities. In the current study, rhamnan sulfate (RS), a polysaccharide with a rhamnose backbone from a green seaweed, Monostroma nitidum, was evaluated for binding to the S-protein from SARS-CoV-2 and inhibition of viral infectivity in vitro. The structural characteristics of RS were investigated by determining its monosaccharide composition and performing two-dimensional nuclear magnetic resonance. RS inhibition of the interaction of heparin, a highly sulfated HS, with the SARS-CoV-2 spike protein (from wild type and different mutant variants) was studied using surface plasmon resonance (SPR). In competitive binding studies, the IC50 of RS against the S-protein receptor binding domain (RBD) binding to immobilized heparin was 1.6 ng/mL, which is much lower than the IC50 for heparin (~750 ng/mL). RS showed stronger inhibition than heparin on the S-protein RBD or pseudoviral particles binding to immobilized heparin. Finally, in an in vitro cell-based assay, RS showed strong antiviral activities against wild type SARS-CoV-2 and the delta variant.

Light Chain LC and TAT-EGFP-HCS of Botulinum Toxin Expression and Biological Function in vitro and in vivo

2019 ◽  
Vol 16 (3) ◽  
pp. 175-180
Author(s):  
Fengjin Hao ◽  
Yueqin Feng ◽  
Yifu Guan
Keyword(s):  
Biological Activity ◽  
Botulinum Toxin ◽  
Cell Membrane ◽  
Western Blot ◽  
Biological Function ◽  
C6 Cells ◽  
Green Fluorescence ◽  
Bv2 Cells

Objective: To verify whether the botulinum toxin heavy chain HCS has specific neuronal targeting function and to confirm whether TAT-EGFP-LC has hydrolyzable SNAP-25 and has transmembrane biological activity. Methods: We constructed the pET-28a-TAT-EGFP-HCS/LC plasmid. After the plasmid is expressed and purified, we co-cultured it with nerve cells or tumors. In addition, we used Western-Blot to identify whether protein LC and TAT-EGFP-LC can digest the protein SNAP-25. Results: Fluorescence imaging showed that PC12, BV2, C6 and HeLa cells all showed green fluorescence, and TAT-EGFP-HCS had the strongest fluorescence. Moreover, TAT-EGFP-LC can hydrolyze intracellular SNAP-25 in PC12 cells, C6 cells, BV2 cells and HeLa, whereas LC alone cannot. In addition, the in vivo protein TAT-EGFP-HCS can penetrate the blood-brain barrier and enter mouse brain tissue. Conclusion: TAT-EGFP-HSC expressed in vitro has neural guidance function and can carry large proteins across the cell membrane without influencing the biological activity.

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