HSPA5 Promotes Attachment and Internalization of Porcine Epidemic Diarrhea Virus through Interaction with the Spike Protein and the Endo-/Lysosomal Pathway.

Porcine epidemic diarrhea virus (PEDV) has caused huge economic losses to the global pig industry. The swine enteric coronavirus spike (S) protein recognizes various cell surface molecules to regulate viral infection. In this study, we identified 211 host membrane proteins related to the S1 protein by pulldown combined with liquid-chromatography tandem mass spectrometry (LC-MS/MS) analysis. Among these, heat shock protein family A member 5 (HSPA5) was identified through screening as having a specific interaction with the PEDV S protein, and positive regulation of PEDV infection was validated by knockdown and overexpression tests. Further studies verified the role of HSPA5 in viral attachment and internalization. In addition, we found that HSPA5 interacts with S proteins through its nucleotide-binding structural domain (NBD) and that polyclonal antibodies can block viral infection. In detail, HSPA5 was found to be involved in viral trafficking via the endo-/lysosomal pathway. Inhibition of HSPA5 activity during internalization would reduce the subcellular colocalization of PEDV with lysosomes in the endo-/lysosomal pathway. Together, these findings show that HSPA5 is a novel PEDV potential target for the creation of therapeutic drugs. IMPORTANCE PEDV infection causes severe piglet mortality and threatens the global pig industry. However, the complex invasion mechanism of PEDV makes its prevention and control difficult. Here, we determined that HSPA5 is a novel target for PEDV which interacts with its S protein and is involved in viral attachment and internalization, influencing its transport via the endo-/lysosomal pathway. Our work extends knowledge about the relationship between the PEDV S and host proteins and provides a new therapeutic target against PEDV infection.

New progresses on cell surface protein HSPA5/BiP/GRP78 in cancers and COVID-19.

Heat-shock-protein family A (Hsp70) member 5 (HSPA5), aliases GRP78 or BiP, is a protein encoded with 654 amino acids by the HSPA5 gene located on human chromosome 9q33.3. When the endoplasmic reticulum (ER) was stressed, HSPA5 translocated to the cell surface, the mitochondria, and the nucleus complexed with other proteins to execute its functions. On the cell surface, HSPA5/BiP/GRP78 can play diverse functional roles in cell viability, proliferation, apoptosis, attachments, and innate and adaptive immunity regulations, which lead to various diseases, including cancers and coronavirus disease 2019 (COVID-19). COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which caused the pandemic since the first outbreak in late December 2019. HSPA5, highly expressed in the malignant tumors, likely plays a critical role in SARS-CoV-2 invasion/attack in cancer patients via tumor tissues. In the current study, we review the newest research progresses on cell surface protein HSPA5 expressions, functions, and mechanisms for cancers and SARS-CoV-2 invasion. The therapeutic and prognostic significances and prospects in cancers and COVID-19 disease by targeting HSPA5 are also discussed. Targeting HSPA5 expression by natural products may imply the significance in clinical for both anti-COVID-19 and anti-cancers in the future.

Is There any Alternative Receptor for SARS-CoV-2?

Angiotensin-converting enzyme II (ACE2) in association with type II transmembrane serine protease (TMPRSS2) is considered the main receptor of SARS-CoV-2. However, considering the clinical complications of COVID-19 in different organs, there is no strong association between the abundance of ACE2/TMPRSS2 co-expression and clinical features of the disease and the severity of complications. Since SARS-CoV-2 affects certain organs that lack or have low expression of ACE2/TMPRSS2, it may be possible that the virus employs other receptors for colonization and entry. Based on recent studies, glucose-regulated protein 78 (GRP78) can be a potential alternative receptor for SARS-CoV-2 entry. In this letter, supporting evidence proposed GRP78 as an alternative receptor in SARS-CoV-2 infection.

HSPA5 Promotes the Proliferation, Metastasis and Regulates Ferroptosis of Bladder Cancer.

Heat shock protein family A (HSP70) member 5 (HSPA5) is aberrantly expressed in various tumors and closely associated with the progression and prognosis of cancer. Nevertheless, its role in bladder cancer (BCa) remains elusive. The results of our study demonstrated that HSPA5 was upregulated in BCa and correlated with patient prognosis. Cell lines with low expression level of HSPA5 were constructed to explore the role of this protein in BCa. HSPA5 knockdown promoted apoptosis and retarded the proliferation, migration and invasion of BCa cells by regulating the VEGFA/VEGFR2 signaling pathway. In addition, overexpression of VEGFA alleviated the negative effect of HSPA5 downregulation. Moreover, we found that HSPA5 could inhibit the process of ferroptosis through the P53/SLC7A11/GPX4 pathway. Hence, HSPA5 can facilitate the progression of BCa and may be used as a novel biomarker and latent therapeutic target in the clinic.

Cellular stress modulates severity of the inflammatory response in lungs via cell surface BiP.

Inflammation is a central pathogenic feature of the acute respiratory distress syndrome (ARDS) in COVID-19. Previous pathologies such as diabetes, autoimmune or cardiovascular diseases become risk factors for the severe hyperinflammatory syndrome. A common feature among these risk factors is the subclinical presence of cellular stress, a finding that has gained attention after the discovery that BiP (GRP78), a master regulator of stress, participates in the SARS-CoV-2 recognition. Here, we show that BiP serum levels are higher in COVID-19 patients who present certain risk factors. Moreover, early during the infection, BiP levels predict severe pneumonia, supporting the use of BiP as a prognosis biomarker. Using a mouse model of pulmonary inflammation, we observed increased levels of cell surface BiP (cs-BiP) in leukocytes during inflammation. This corresponds with a higher number of neutrophiles, which show naturally high levels of cs-BiP, whereas alveolar macrophages show a higher than usual exposure of BiP in their cell surface. The modulation of cellular stress with the use of a clinically approved drug, 4-PBA, resulted in the amelioration of the lung hyperinflammatory response, supporting the anti-stress therapy as a valid therapeutic strategy for patients developing ARDS. Finally, we identified stress-modulated proteins that shed light into the mechanism underlying the cellular stress-inflammation network in lungs.

Therapeutic potential of green tea catechin, (-)-epigallocatechin-3-O-gallate (EGCG) in SARS-CoV-2 infection: Major interactions with host/virus proteases.

Background: The current COVID-19 pandemic from the human pathogenic virus SARS-CoV-2 has resulted in a major health hazard globally. The morbidity and transmission modality of this disease are severe and uncontrollable. As no effective clinical drugs are available for treatment of COVID-19 infection till to date and only vaccination is used as prophylaxis and its efficacy is restricted due to emergent of new variants of SARS-CoV-2, there is an urgent need for effective drugs for its treatment. Purpose: The aim of this review was to provide a detailed analysis of anti-SARS-CoV-2 efficacy of (-)-epigallocatechin-3-O-gallate (EGCG), a major catechin constituent of green tea (Camellia sinensis (L.) Kuntze) beverage to highlight the scope of EGCG in clinical medicine as both prophylaxis and treatment of present COVID-19 infection. In addition, the factors related to poor oral bioavailabilty of EGCG was also analysed for a suggestion for future research in this direction. Study design: We collected the published articles related to anti-SARS-CoV-2 activity of EGCG against the original strain (Wuhan type) and its newly emerged variants of SARS-CoV-2 virus. Methods: A systematic search on the published literature was conducted in various databases including Google Scholar, PubMed, Science Direct and Scopus to collect the relevant literature. Results: The findings of this search demonstrate that EGCG shows potent antiviral activity against SARS-CoV-2 virus by preventing viral entry and replication in host cells in vitro models. The studies on the molecular mechanisms of EGCG in inhibition of SARS-CoV-2 infection in host cells reveal that EGCG blocks the entry of the virus particles by interaction with the receptor binding domain (RBD) of viral spike (S) protein to host cell surface receptor protease angiotensin-converting enzyme 2 (ACE2) as well as suppression of the expressions of host proteases, ACE2, TMPRSS2 and GRP78, required for viral entry, by Nrf2 activation in host cells. Moreover, EGCG inhibits the activities of SARS-CoV-2 main protease (Mpro), papain-like protease (PLpro), endoribonuclease Nsp15 in vitro models and of RNA-dependent RNA polymerase (RdRp) in molecular docking model for suppression of viral replication. In addition, EGCG significantly inhibits viral inflammatory cytokine production by stimulating Nrf2- dependent host immune response in virus-infected cells. EGCG significantly reduces the elevated levels of HMGB1, a biomarker of sepsis, lung fibrosis and thrombotic complications in viral infections. EGCG potentially inhibits the infection of original (Wuhan type) strain of SARS-CoV-2 and other newly emerged variants as well as the infections of SARS-CoV-2 virus spike-protein of WT and its mutants-mediated pseudotyped viruses . EGCG shows maximum inhibitory effect against SARS-CoV-2 infection when the host cells are pre-incubated with the drug prior to viral infection. A sorbitol/lecithin-based throat spray containing concentrated green tea extract rich in EGCG content significantly reduces SARS-CoV-2 infectivity in oral mucosa. Several factors including degradation in gastrointestinal environment, low absorption in small intestine and extensive metabolism of EGCG are responsible for its poor bioavailability in humans. Pharmacokinetic and metabolism studies of EGCG in humans reveal poor bioavailability of EGCG in human plasma and EGCG-4"-sulfate is its major metabolite. The concentration of EGCG-4"-sulfate in human plasma is almost equivalent to that of free EGCG (Cmax 177.9 vs 233.5 nmol/L). These findings suggest that inhibition of sulfation of EGCG is a crucial factor for improvement of its bioavailability. In vitro study on the mechanism of EGCG sulfonation indicates that sulfotransferases, SULT1A1 and SULT1A3 are responsible for sulfonation in human liver and small intestine, respectively. Some attempts including structural modifications, and nanoformulations of EGCG and addition of nutrients with EGCG have been made to improve the bioavailability of EGCG. Conclusions: The findings of this study suggest that EGCG has strong antiviral activity against SARS-CoV-2 infection independent of viral strains (Wuhan type (WT), other variants) by inhibition of viral entry and replication in host cells in vitro models. EGCG may be useful in reduction of this viral load in salivary glands of COVID-19 patients, if it is applied in mouth and throat wash formulations in optimal concentrations. EGCG could be a promising candidate in the development of effective vaccine for prevention of the infections of newly emergent strains of SARS-CoV-2 virus. EGCG might be useful also as a clinical medicine for treatment of COVID-19 patients if its bioavailability in human plasma is enhanced.

Interference of Chaga mushroom terpenoids with the attachment of SARS-CoV-2; in silico perspective.

Finding a potent inhibitor to the pandemic SARS-CoV-2 is indispensable nowadays. Currently, in-silico methods work as expeditious investigators to screen drugs for possible repurposing or design new ones. Targeting one of the possible SARS-CoV-2 attachment and entry receptors, Glucose-regulated protein 78 (GRP78), is an approach of major interest. Recently, GRP78 was reported as a recognized representative in recognition of the latest variants of SARS-CoV-2. In this work, molecular docking and molecular dynamics simulations were performed on the host cell receptor GRP78. With its many terpenoid compounds, Chaga mushroom was tested as a potential therapeutic against the SARS-CoV-2 receptor, GRP78. Results revealed low binding energies (high affinities) toward the GRP78 substrate-binding domain ß (SBDß) of Chaga mushroom terpenoids. Even the highly specific cyclic peptide Pep42, which selectively targeted GRP78 over cancer cells in vivo, showed lower binding affinity against GRP78 SBDß compared to the binding affinities of terpenoids. These are auspicious results that need to be tested experimentally. Intriguingly, terpenoids work as a double sword as they can be used to interfere with VUI 202,012/01, 501.V2, and B.1.1.248 variants of SARS-CoV-2 spike recognition.

A Fluorescence Polarization-Based High-Throughput Screen to Identify the First Small-Molecule Modulators of the Human Adenylyltransferase HYPE/FICD.

The covalent transfer of the AMP portion of ATP onto a target protein-termed adenylylation or AMPylation-by the human Fic protein HYPE/FICD has recently garnered attention as a key regulatory mechanism in endoplasmic reticulum homeostasis, neurodegeneration, and neurogenesis. As a central player in such critical cellular events, high-throughput screening (HTS) efforts targeting HYPE-mediated AMPylation warrant investigation. Herein, we present a dual HTS assay for the simultaneous identification of small-molecule activators and inhibitors of HYPE AMPylation. Employing the fluorescence polarization of an ATP analog fluorophore-Fl-ATP-we developed and optimized an efficient, robust assay that monitors HYPE autoAMPylation and is amenable to automated, high-throughput processing of diverse chemical libraries. Challenging our pilot screen with compounds from the LOPAC, Spectrum, MEGx, and NATx libraries yielded 0.3% and 1% hit rates for HYPE activators and inhibitors, respectively. Further, these hits were assessed for dose-dependency and validated via orthogonal biochemical AMPylation assays. We thus present a high-quality HTS assay suitable for tracking HYPE's enzymatic activity, and the resultant first small-molecule manipulators of HYPE-promoted autoAMPylation.

Evaluation and characterization of HSPA5 (GRP78) expression profiles in normal individuals and cancer patients with COVID-19.

HSPA5 (BiP, GRP78) has been reported as a potential host-cell receptor for SARS-Cov-2, but its expression profiles on different tissues including tumors, its susceptibility to SARS-Cov-2 virus and severity of its adverse effects on malignant patients are unclear. In the current study, HSPA5 has been found to be expressed ubiquitously in normal tissues and significantly increased in 14 of 31 types of cancer tissues. In lung cancer, mRNA levels of HSPA5 were 253-fold increase than that of ACE2. Meanwhile, in both malignant tumors and matched normal samples across almost all cancer types, mRNA levels of HSPA5 were much higher than those of ACE2. Higher expression of HSPA5 significantly decreased patient overall survival (OS) in 7 types of cancers. Moreover, systematic analyses found that 7.15% of 5,068 COVID-19 cases have malignant cancer coincidental situations, and the rate of severe events of COVID-19 patients with cancers present a higher trend than that for all COVID-19 patients, showing a significant difference (33.33% vs 16.09%, p<0.01). Collectively, these data imply that the tissues with high HSPA5 expression, not low ACE2 expression, are susceptible to be invaded by SARS-CoV-2. Taken together, this study not only indicates the clinical significance of HSPA5 in COVID-19 disease and cancers, but also provides potential clues for further medical treatments and managements of COVID-19 patients.

SARS-CoV-2 Spike-Heat Shock Protein A5 (GRP78) Recognition may be Related to the Immersed Human Coronaviruses.

The human coronavirus (HCoV), SARS-CoV-2, caused more than 34 M confirmed infections from which more than 1 M deaths are reported until now (the WHO situation report-154). The current pandemic causes severe socio-economic burden. Due to the importance of understanding of the mode of recognition and viral entry, spike protein shed drug designers as the first look protein target with the first released solved structure on 26 February 2020 (PDB ID: 6VSB). It is proposed that the recognition site for GRP78 is found in SARS-CoV-2 and the immersed human coronaviruses but experimental validation is still required.

Interaction of HSPA5 (Grp78, BIP) with negatively charged phospholipid membranes via oligomerization involving the N-terminal end domain.

Heat shock proteins (HSPs) are ubiquitous polypeptides expressed in all living organisms that participate in several basic cellular processes, including protein folding, from which their denomination as molecular chaperones originated. There are several HSPs, including HSPA5, also known as 78-kDa glucose-regulated protein (GRP78) or binding immunoglobulin protein (BIP) that is an ER resident involved in the folding of polypeptides during their translocation into this compartment prior to the transition to the Golgi network. HSPA5 is detected on the surface of cells or secreted into the extracellular environment. Surface HSPA5 has been proposed to have various roles, such as receptor-mediated signal transduction, a co-receptor for soluble ligands, as well as a participant in tumor survival, proliferation, and resistance. Recently, surface HSPA5 has been reported to be a potential receptor of some viruses, including the novel SARS-CoV-2. In spite of these observations, the association of HSPA5 within the plasma membrane is still unclear. To gain information about this process, we studied the interaction of HSPA5 with liposomes made of different phospholipids. We found that HSPA5 has a high affinity for negatively charged phospholipids, such as palmitoyl-oleoyl phosphoserine (POPS) and cardiolipin (CL). The N-terminal and C-terminal domains of HSPA5 were independently capable of interacting with negatively charged phospholipids, but to a lesser extent than the full-length protein, suggesting that both domains are required for the maximum insertion into membranes. Interestingly, we found that the interaction of HSPA5 with negatively charged liposomes promotes an oligomerization process via intermolecular disulfide bonds in which the N-terminus end of the protein plays a critical role.

Natural products may interfere with SARS-CoV-2 attachment to the host cell.

SARS-CoV-2 has been emerged in December 2019 in China, causing deadly (5% mortality) pandemic pneumonia, termed COVID-19. More than one host-cell receptor is reported to be recognized by the viral spike protein, among them is the cell-surface Heat Shock Protein A5 (HSPA5), also termed GRP78 or BiP. Upon viral infection, HSPA5 is upregulated, then translocating to the cell membrane where it is subjected to be recognized by the SARS-CoV-2 spike. In this study, some natural product compounds are tested against the HSPA5 substrate-binding domain ß (SBDß), which reported to be the recognition site for the SARS-CoV-2 spike. Molecular docking and molecular dynamics simulations are used to test some natural compounds binding to HSPA5 SBDß. The results show high to a moderate binding affinity for the phytoestrogens (Diadiazin, Genistein, Formontein, and Biochanin A), chlorogenic acid, linolenic acid, palmitic acid, caffeic acid, caffeic acid phenethyl ester, hydroxytyrosol, cis-p-Coumaric acid, cinnamaldehyde, thymoquinone, and some physiological hormones such as estrogens, progesterone, testosterone, and cholesterol to the HSPA5 SBDß. Based on its binding affinities, the phytoestrogens and estrogens are the best in binding HSPA5, hence may interfere with SARS-CoV-2 attachment to the stressed cells. These compounds can be successful as anti-COVID-19 agents for people with a high risk of cell stress like elders, cancer patients, and front-line medical staff.Communicated by Ramaswamy H. Sarma.