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1.
Cell Mol Life Sci ; 79(6): 316, 2022 May 27.
Article in English | MEDLINE | ID: covidwho-1866610

ABSTRACT

AXL, a TAM receptor tyrosine kinase (RTK), and its ligand growth arrest-specific 6 (GAS6) are implicated in cancer metastasis and drug resistance, and cellular entry of viruses. Given this, AXL is an attractive therapeutic target, and its inhibitors are being tested in cancer and COVID-19 clinical trials. Still, astonishingly little is known about intracellular mechanisms that control its function. Here, we characterized endocytosis of AXL, a process known to regulate intracellular functions of RTKs. Consistent with the notion that AXL is a primary receptor for GAS6, its depletion was sufficient to block GAS6 internalization. We discovered that upon receptor ligation, GAS6-AXL complexes were rapidly internalized via several endocytic pathways including both clathrin-mediated and clathrin-independent routes, among the latter the CLIC/GEEC pathway and macropinocytosis. The internalization of AXL was strictly dependent on its kinase activity. In comparison to other RTKs, AXL was endocytosed faster and the majority of the internalized receptor was not degraded but rather recycled via SNX1-positive endosomes. This trafficking pattern coincided with sustained AKT activation upon GAS6 stimulation. Specifically, reduced internalization of GAS6-AXL upon the CLIC/GEEC downregulation intensified, whereas impaired recycling due to depletion of SNX1 and SNX2 attenuated AKT signaling. Altogether, our data uncover the coupling between AXL endocytic trafficking and AKT signaling upon GAS6 stimulation. Moreover, our study provides a rationale for pharmacological inhibition of AXL in antiviral therapy as viruses utilize GAS6-AXL-triggered endocytosis to enter cells.


Subject(s)
Endocytosis , Intercellular Signaling Peptides and Proteins , Proto-Oncogene Proteins , Receptor Protein-Tyrosine Kinases , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/metabolism , COVID-19/therapy , Clathrin/metabolism , Clathrin/physiology , Endocytosis/drug effects , Endocytosis/genetics , Endocytosis/physiology , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/physiology , Neoplasms/metabolism , Neoplasms/therapy , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins/physiology , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-akt/physiology , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/physiology
2.
Bioengineered ; 12(2): 12461-12469, 2021 12.
Article in English | MEDLINE | ID: covidwho-1585255

ABSTRACT

Severe mortality due to the COVID-19 pandemic resulted from the lack of effective treatment. Although COVID-19 vaccines are available, their side effects have become a challenge for clinical use in patients with chronic diseases, especially cancer patients. In the current report, we applied network pharmacology and systematic bioinformatics to explore the use of biochanin A in patients with colorectal cancer (CRC) and COVID-19 infection. Using the network pharmacology approach, we identified two clusters of genes involved in immune response (IL1A, IL2, and IL6R) and cell proliferation (CCND1, PPARG, and EGFR) mediated by biochanin A in CRC/COVID-19 condition. The functional analysis of these two gene clusters further illustrated the effects of biochanin A on interleukin-6 production and cytokine-cytokine receptor interaction in CRC/COVID-19 pathology. In addition, pathway analysis demonstrated the control of PI3K-Akt and JAK-STAT signaling pathways by biochanin A in the treatment of CRC/COVID-19. The findings of this study provide a therapeutic option for combination therapy against COVID-19 infection in CRC patients.


Subject(s)
Anticarcinogenic Agents/therapeutic use , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Colorectal Neoplasms/drug therapy , Gene Expression Regulation, Neoplastic/drug effects , Genistein/therapeutic use , Phytoestrogens/therapeutic use , Atlases as Topic , COVID-19/immunology , COVID-19/pathology , COVID-19/virology , Colorectal Neoplasms/immunology , Colorectal Neoplasms/pathology , Colorectal Neoplasms/virology , Cyclin D1/genetics , Cyclin D1/immunology , ErbB Receptors/genetics , ErbB Receptors/immunology , Humans , Interleukin-1alpha/genetics , Interleukin-1alpha/immunology , Interleukin-2/genetics , Interleukin-2/immunology , Janus Kinases/genetics , Janus Kinases/immunology , Metabolic Networks and Pathways/drug effects , Metabolic Networks and Pathways/genetics , Molecular Targeted Therapy/methods , Multigene Family , PPAR gamma/genetics , PPAR gamma/immunology , Pharmacogenetics/methods , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/immunology , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/immunology , Receptors, Interleukin-6/genetics , Receptors, Interleukin-6/immunology , SARS-CoV-2/drug effects , SARS-CoV-2/growth & development , SARS-CoV-2/pathogenicity , STAT Transcription Factors/genetics , STAT Transcription Factors/immunology , Signal Transduction
3.
EBioMedicine ; 73: 103672, 2021 Nov.
Article in English | MEDLINE | ID: covidwho-1568646

ABSTRACT

BACKGROUND: Phospho-Akt1 (pAkt1) undergoes prolyl hydroxylation at Pro125 and Pro313 by the prolyl hydroxylase-2 (PHD2) in a reaction decarboxylating α-ketoglutarate (αKG). We investigated whether the αKG supplementation could inhibit Akt-mediated activation of platelets and monocytes, in vitro as well as in vivo, by augmenting PHD2 activity. METHODS: We treated platelets or monocytes isolated from healthy individuals with αKG in presence of agonists in vitro and assessed the signalling molecules including pAkt1. We supplemented mice with dietary αKG and estimated the functional responses of platelets and monocytes ex vivo. Further, we investigated the impact of dietary αKG on inflammation and thrombosis in lungs of mice either treated with thrombosis-inducing agent carrageenan or infected with SARS-CoV-2. FINDINGS: Octyl αKG supplementation to platelets promoted PHD2 activity through elevated intracellular αKG to succinate ratio, and reduced aggregation in vitro by suppressing pAkt1(Thr308). Augmented PHD2 activity was confirmed by increased hydroxylated-proline and enhanced binding of PHD2 to pAkt in αKG-treated platelets. Contrastingly, inhibitors of PHD2 significantly increased pAkt1 in platelets. Octyl-αKG followed similar mechanism in monocytes to inhibit cytokine secretion in vitro. Our data also describe a suppressed pAkt1 and reduced activation of platelets and leukocytes ex vivo from mice supplemented with dietary αKG, unaccompanied by alteration in their number. Dietary αKG significantly reduced clot formation and leukocyte accumulation in various organs including lungs of mice treated with thrombosis-inducing agent carrageenan. Importantly, in SARS-CoV-2 infected hamsters, we observed a significant rescue effect of dietary αKG on inflamed lungs with significantly reduced leukocyte accumulation, clot formation and viral load alongside down-modulation of pAkt in the lung of the infected animals. INTERPRETATION: Our study suggests that dietary αKG supplementation prevents Akt-driven maladies such as thrombosis and inflammation and rescues pathology of COVID19-infected lungs. FUNDING: Study was funded by the Department of Biotechnology (DBT), Govt. of India (grants: BT/PR22881 and BT/PR22985); and the Science and Engineering Research Board, Govt. of India (CRG/000092).


Subject(s)
Ketoglutaric Acids/therapeutic use , Prolyl Hydroxylases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Thrombosis/prevention & control , Animals , Blood Platelets/cytology , Blood Platelets/drug effects , Blood Platelets/metabolism , COVID-19/pathology , COVID-19/prevention & control , COVID-19/veterinary , COVID-19/virology , Cricetinae , Dietary Supplements , Down-Regulation/drug effects , Humans , Ketoglutaric Acids/pharmacology , Lung/metabolism , Lung/pathology , Mesocricetus , Mice , Mice, Inbred BALB C , Monocytes/cytology , Monocytes/drug effects , Monocytes/metabolism , Phosphorylation , Platelet Aggregation/drug effects , Protein Isoforms/genetics , Protein Isoforms/metabolism , Proto-Oncogene Proteins c-akt/genetics , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Thrombosis/chemically induced , Thrombosis/pathology , Thrombosis/veterinary
4.
Int J Biol Sci ; 17(11): 2770-2771, 2021.
Article in English | MEDLINE | ID: covidwho-1341901

ABSTRACT

Commentary on 'Capivasertib restricts SARS-CoV-2 cellular entry: a potential clinical application for COVID-19' by Sun et al.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Phosphatidylinositol 3-Kinases/genetics , Proto-Oncogene Proteins c-akt/genetics
5.
Viruses ; 13(6)2021 06 03.
Article in English | MEDLINE | ID: covidwho-1259622

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible RNA virus that is the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Patients with severe COVID-19 may develop acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) and require mechanical ventilation. Key features of SARS-CoV-2 induced pulmonary complications include an overexpression of pro-inflammatory chemokines and cytokines that contribute to a 'cytokine storm.' In the current study an inflammatory state in Calu-3 human lung epithelial cells was characterized in which significantly elevated transcripts of the immunostimulatory chemokines CXCL9, CXCL10, and CXCL11 were present. Additionally, an increase in gene expression of the cytokines IL-6, TNFα, and IFN-γ was observed. The transcription of CXCL9, CXCL10, IL-6, and IFN-γ was also induced in the lungs of human transgenic angiotensin converting enzyme 2 (ACE2) mice infected with SARS-CoV-2. To elucidate cell signaling pathways responsible for chemokine upregulation in SARS-CoV-2 infected cells, small molecule inhibitors targeting key signaling kinases were used. The induction of CXCL9, CXCL10, and CXCL11 gene expression in response to SARS-CoV-2 infection was markedly reduced by treatment with the AKT inhibitor GSK690693. Samples from COVID-19 positive individuals also displayed marked increases in CXCL9, CXCL10, and CXCL11 transcripts as well as transcripts in the AKT pathway. The current study elucidates potential pathway specific targets for reducing the induction of chemokines that may be contributing to SARS-CoV-2 pathogenesis via hyperinflammation.


Subject(s)
COVID-19/immunology , Chemokine CXCL10/genetics , Chemokine CXCL11/genetics , Chemokine CXCL9/genetics , Proto-Oncogene Proteins c-akt/metabolism , Up-Regulation , Angiotensin-Converting Enzyme 2/genetics , Animals , Cell Line , Chemokine CXCL10/immunology , Chemokine CXCL11/immunology , Chemokine CXCL9/immunology , Cytokine Release Syndrome/genetics , Cytokine Release Syndrome/immunology , Epithelial Cells/immunology , Epithelial Cells/virology , Female , Humans , Inflammation , Lung/cytology , Mice , Mice, Transgenic , Proto-Oncogene Proteins c-akt/genetics , Signal Transduction/genetics , Signal Transduction/immunology
6.
Cells ; 9(9)2020 09 16.
Article in English | MEDLINE | ID: covidwho-1148288

ABSTRACT

Vaccine design traditionally focuses on inducing adaptive immune responses against a sole target pathogen. Considering that many microbes evade innate immune mechanisms to initiate infection, and in light of the discovery of epigenetically mediated innate immune training, the paradigm of vaccine design has the potential to change. The Bacillus Calmette-Guérin (BCG) vaccine induces some level of protection against Mycobacterium tuberculosis (Mtb) while stimulating trained immunity that correlates with lower mortality and increased protection against unrelated pathogens. This review will explore BCG-induced trained immunity, including the required pathways to establish this phenotype. Additionally, potential methods to improve or expand BCG trained immunity effects through alternative vaccine delivery and formulation methods will be discussed. Finally, advances in new anti-Mtb vaccines, other antimicrobial uses for BCG, and "innate memory-based vaccines" will be examined.


Subject(s)
Adaptive Immunity/drug effects , BCG Vaccine/administration & dosage , COVID-19/prevention & control , Epigenesis, Genetic/drug effects , Myeloid Cells/drug effects , SARS-CoV-2/pathogenicity , Tuberculosis, Pulmonary/prevention & control , Acetylmuramyl-Alanyl-Isoglutamine/immunology , Acetylmuramyl-Alanyl-Isoglutamine/metabolism , COVID-19/immunology , COVID-19/virology , Cross Protection , Epigenesis, Genetic/immunology , Histones/genetics , Histones/immunology , Humans , Mycobacterium tuberculosis , Myeloid Cells/immunology , Myeloid Cells/pathology , Nod2 Signaling Adaptor Protein/genetics , Nod2 Signaling Adaptor Protein/immunology , Pathogen-Associated Molecular Pattern Molecules/immunology , Pathogen-Associated Molecular Pattern Molecules/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/immunology , Signal Transduction , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/immunology , Tuberculosis, Pulmonary/immunology , Tuberculosis, Pulmonary/microbiology
7.
J Cell Physiol ; 236(9): 6597-6606, 2021 09.
Article in English | MEDLINE | ID: covidwho-1098899

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 that causes coronavirus disease 2019 (COVID-19) binds to the angiotensin-converting enzyme 2 (ACE2) to gain cellular entry. Akt inhibitor triciribine (TCBN) has demonstrated promising results in promoting recovery from advanced-stage acute lung injury in preclinical studies. In the current study, we tested the direct effect of TCBN on ACE2 expression in human bronchial (H441) and lung alveolar (A549) epithelial cells. Treatment with TCBN resulted in the downregulation of both messenger RNA and protein levels of ACE2 in A549 cells. Since HMGB1 plays a vital role in the inflammatory response in COVID-19, and because hyperglycemia has been linked to increased COVID-19 infections, we determined if HMGB1 and hyperglycemia have any effect on ACE2 expression in lung epithelial cells and whether TCBN has any effect on reversing HMGB1- and hyperglycemia-induced ACE2 expression. We observed increased ACE2 expression with both HMGB1 and hyperglycemia treatment in A549 as well as H441 cells, which were blunted by TCBN treatment. Our findings from this study, combined with our previous reports on the potential benefits of TCBN in the treatment of acute lung injury, generate reasonable optimism on the potential utility of TCBN in the therapeutic management of patients with COVID-19.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/drug therapy , HMGB1 Protein/genetics , Proto-Oncogene Proteins c-akt/genetics , A549 Cells , Bronchi/metabolism , Bronchi/pathology , Bronchi/virology , COVID-19/genetics , COVID-19/pathology , Epithelial Cells/drug effects , Epithelial Cells/virology , Gene Expression Regulation/drug effects , Humans , Lung/drug effects , Lung/pathology , RNA, Viral/genetics , Ribonucleosides/administration & dosage , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity
8.
Stem Cell Res Ther ; 11(1): 508, 2020 11 27.
Article in English | MEDLINE | ID: covidwho-948402

ABSTRACT

BACKGROUND: Mesenchymal stem cells (MSCs), including adipose-derived mesenchymal stem cells (ADSCs), have been shown to attenuate organ damage in acute respiratory distress syndrome (ARDS) and sepsis; however, the underlying mechanisms are not fully understood. In this study, we aimed to explore the potential roles and molecular mechanisms of action of ADSCs in histone-induced endothelial damage. METHODS: Male C57BL/6 N mice were intravenously injected with ADSCs, followed by histones or a vehicle. The mice in each group were assessed for survival, pulmonary vascular permeability, and histological changes. A co-culture model with primary human umbilical vein endothelial cells (HUVECs) exposed to histones was used to clarify the paracrine effect of ADSCs. Overexpression and inhibition of miR-126 ADSCs were also examined as causative factors for endothelial protection. RESULTS: The administration of ADSCs markedly improved survival, inhibited histone-mediated lung hemorrhage and edema, and attenuated vascular hyper-permeability in mice. ADSCs were engrafted in the injured lung and attenuated histone-induced endothelial cell apoptosis. ADSCs showed endothelial protection (via a paracrine effect) and Akt phosphorylation in the histone-exposed HUVECs. Notably, increased Akt phosphorylation by ADSCs was mostly mediated by exosomes in histone-induced cytotoxicity and lung damage. Moreover, the expression of miR-126 was increased in exosomes from histone-exposed ADSCs. Remarkably, the inhibition of miR-126 in ADSCs failed to increase Akt phosphorylation in histone-exposed HUVECs. CONCLUSION: ADSC-derived exosomes may exert protective effects on endothelial cells via activation of the PI3K/Akt pathway.


Subject(s)
Acute Lung Injury , Exosomes , Mesenchymal Stem Cells , Adipose Tissue/metabolism , Animals , Exosomes/metabolism , Histones , Male , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism
9.
Chem Biol Interact ; 332: 109299, 2020 Dec 01.
Article in English | MEDLINE | ID: covidwho-885217

ABSTRACT

COVID-2019 pandemic is affecting people worldwide in the absence of an effective treatment strategy. Several suggestive therapeutic options through drug repurposing are recommended, but a complete consensus is not reached. A combination of Hydroxychloroquine (HCQ) and Azithromycin (AZM) has been widely tried and discussed but its administration has also led to potential adversities in patients. Studies are suggesting that most prominent adverse event with HCQ and AZM combination is QT interval prolongation. We studied interaction of HCQ with AZM and subsequent effect of this drug combination on QT interval prolongation. We performed system biological investigation of HCQ and AZM targets and screened important targets and pathways possibly involved in QT interval prolongation. The best core hub protein drug targets involved in QT interval prolongation were identified as HSP90AA1 exclusively associated with HCQ, while AKT1 exclusively associated with AZM on the basis of node degree value. It was found that PI3K/Akt, VEGF, ERBB2 pathways must be given consideration for understanding the role of HCQ and AZM in QT interval prolongation. Conclusion: Computational methods have certain limitations based on source database coverage and prediction algorithms and therefore this data needs experimental correlation to draw final conclusion, but current findings screen targets for QT interval prolongation associated with HCQ and AZM. These proteins and pathways may provide ways to reduce this major risk associated with this combination.


Subject(s)
Azithromycin/therapeutic use , COVID-19/drug therapy , Hydroxychloroquine/therapeutic use , Long QT Syndrome/etiology , Azithromycin/adverse effects , COVID-19/complications , COVID-19/epidemiology , Drug Combinations , HSP90 Heat-Shock Proteins/genetics , HSP90 Heat-Shock Proteins/metabolism , Humans , Hydroxychloroquine/adverse effects , Pandemics , Protein Interaction Maps , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , SARS-CoV-2/drug effects , Signal Transduction/drug effects
10.
Virus Res ; 280: 197901, 2020 04 15.
Article in English | MEDLINE | ID: covidwho-833197

ABSTRACT

Transmissible gastroenteritis virus (TGEV) primarily replicates in intestinal epithelial cells and causes severe damage to host cells, resulting in diarrhea. Surface NHE3 serves as the key regulatory site controlling electroneutral Na+ absorption. In this study, our results showed that the surface NHE3 content was significantly reduced following TGEV infection, whereas the total level of protein expression was not significantly changed, and NHE3 activity gradually decreased with prolonged infection time. We then inhibited SGLT1 expression by lentiviral interference and drug inhibition, respectively. Inhibition studies showed that the level of phosphorylation of the downstream key proteins, MAPKAPK-2 and EZRIN, in the SGLT1-mediated p38MAPK/AKt2 signaling pathway was significantly increased. The surface NHE3 expression was also significantly increased, and NHE3 activity was also significantly enhanced. These results demonstrate that a TGEV infection can inhibit NHE3 translocation and attenuates sodium-hydrogen exchange activity via the SGLT1-mediated p38MAPK/AKt2 signaling pathway, affecting cellular electrolyte absorption leading to diarrhea.


Subject(s)
Enterocytes/virology , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Sodium-Glucose Transporter 1/genetics , Sodium-Hydrogen Exchanger 3/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Cell Line , Proto-Oncogene Proteins c-akt/genetics , Sodium-Glucose Transporter 1/metabolism , Sodium-Hydrogen Exchanger 3/genetics , Swine , Transmissible gastroenteritis virus , p38 Mitogen-Activated Protein Kinases/genetics
11.
Emerg Microbes Infect ; 9(1): 1748-1760, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-658315

ABSTRACT

How severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections engage cellular host pathways and innate immunity in infected cells remains largely elusive. We performed an integrative proteo-transcriptomics analysis in SARS-CoV-2 infected Huh7 cells to map the cellular response to the invading virus over time. We identified four pathways, ErbB, HIF-1, mTOR and TNF signaling, among others that were markedly modulated during the course of the SARS-CoV-2 infection in vitro. Western blot validation of the downstream effector molecules of these pathways revealed a dose-dependent activation of Akt, mTOR, S6K1 and 4E-BP1 at 24 hours post infection (hpi). However, we found a significant inhibition of HIF-1α through 24hpi and 48hpi of the infection, suggesting a crosstalk between the SARS-CoV-2 and the Akt/mTOR/HIF-1 signaling pathways. Inhibition of the mTOR signaling pathway using Akt inhibitor MK-2206 showed a significant reduction in virus production. Further investigations are required to better understand the molecular sequelae in order to guide potential therapy in the management of severe coronavirus disease 2019 (COVID-19) patients.


Subject(s)
Betacoronavirus/pathogenicity , Coronavirus Infections/virology , Gene Expression Profiling/methods , Pneumonia, Viral/virology , Proteomics/methods , Signal Transduction , COVID-19 , Cell Line , Chromatography, Liquid , Coronavirus Infections/genetics , Coronavirus Infections/metabolism , Gene Expression Regulation , High-Throughput Nucleotide Sequencing , Humans , Hypoxia-Inducible Factor 1/genetics , Hypoxia-Inducible Factor 1/metabolism , Pandemics , Pneumonia, Viral/genetics , Pneumonia, Viral/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , SARS-CoV-2 , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Tandem Mass Spectrometry
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