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1.
FASEB J ; 36(3): e22234, 2022 03.
Article in English | MEDLINE | ID: covidwho-1702985

ABSTRACT

The transmembrane protease angiotensin converting enzyme 2 (ACE2) is a protective regulator within the renin angiotensin system and additionally represents the cellular receptor for SARS-CoV. The release of soluble ACE2 (sACE2) from the cell surface is hence believed to be a crucial part of its (patho)physiological functions, as both, ACE2 protease activity and SARS-CoV binding ability, are transferred from the cell membrane to body fluids. Yet, the molecular sources of sACE2 are still not completely investigated. In this study, we show different sources and prerequisites for the release of sACE2 from the cell membrane. By using inhibitors as well as CRISPR/Cas9-derived cells, we demonstrated that, in addition to the metalloprotease ADAM17, also ADAM10 is an important novel shedding protease of ACE2. Moreover, we observed that ACE2 can also be released in extracellular vesicles. The degree of either ADAM10- or ADAM17-mediated ACE2 shedding is dependent on stimulatory conditions and on the expression level of the pro-inflammatory ADAM17 regulator iRhom2. Finally, by using structural analysis and in vitro verification, we determined for the first time that the susceptibility to ADAM10- and ADAM17-mediated shedding is mediated by the collectrin-like part of ACE2. Overall, our findings give novel insights into sACE2 release by several independent molecular mechanisms.


Subject(s)
ADAM10 Protein/metabolism , ADAM17 Protein/metabolism , Amyloid Precursor Protein Secretases/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Extracellular Vesicles/metabolism , Membrane Glycoproteins/metabolism , Membrane Proteins/metabolism , SARS Virus/metabolism , ADAM10 Protein/genetics , ADAM17 Protein/genetics , Amyloid Precursor Protein Secretases/genetics , Angiotensin-Converting Enzyme 2/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , Extracellular Vesicles/genetics , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Glycoproteins/genetics , Membrane Proteins/genetics , SARS Virus/genetics , SARS-CoV-2
2.
Nat Neurosci ; 24(11): 1522-1533, 2021 11.
Article in English | MEDLINE | ID: covidwho-1500484

ABSTRACT

Coronavirus disease 2019 (COVID-19) can damage cerebral small vessels and cause neurological symptoms. Here we describe structural changes in cerebral small vessels of patients with COVID-19 and elucidate potential mechanisms underlying the vascular pathology. In brains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals and animal models, we found an increased number of empty basement membrane tubes, so-called string vessels representing remnants of lost capillaries. We obtained evidence that brain endothelial cells are infected and that the main protease of SARS-CoV-2 (Mpro) cleaves NEMO, the essential modulator of nuclear factor-κB. By ablating NEMO, Mpro induces the death of human brain endothelial cells and the occurrence of string vessels in mice. Deletion of receptor-interacting protein kinase (RIPK) 3, a mediator of regulated cell death, blocks the vessel rarefaction and disruption of the blood-brain barrier due to NEMO ablation. Importantly, a pharmacological inhibitor of RIPK signaling prevented the Mpro-induced microvascular pathology. Our data suggest RIPK as a potential therapeutic target to treat the neuropathology of COVID-19.


Subject(s)
Blood-Brain Barrier/metabolism , Brain/metabolism , Coronavirus 3C Proteases/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Microvessels/metabolism , SARS-CoV-2/metabolism , Animals , Blood-Brain Barrier/pathology , Brain/pathology , Chlorocebus aethiops , Coronavirus 3C Proteases/genetics , Cricetinae , Female , Humans , Intracellular Signaling Peptides and Proteins/genetics , Male , Mesocricetus , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microvessels/pathology , SARS-CoV-2/genetics , Vero Cells
3.
Nat Neurosci ; 24(11): 1522-1533, 2021 11.
Article in English | MEDLINE | ID: covidwho-1483143

ABSTRACT

Coronavirus disease 2019 (COVID-19) can damage cerebral small vessels and cause neurological symptoms. Here we describe structural changes in cerebral small vessels of patients with COVID-19 and elucidate potential mechanisms underlying the vascular pathology. In brains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals and animal models, we found an increased number of empty basement membrane tubes, so-called string vessels representing remnants of lost capillaries. We obtained evidence that brain endothelial cells are infected and that the main protease of SARS-CoV-2 (Mpro) cleaves NEMO, the essential modulator of nuclear factor-κB. By ablating NEMO, Mpro induces the death of human brain endothelial cells and the occurrence of string vessels in mice. Deletion of receptor-interacting protein kinase (RIPK) 3, a mediator of regulated cell death, blocks the vessel rarefaction and disruption of the blood-brain barrier due to NEMO ablation. Importantly, a pharmacological inhibitor of RIPK signaling prevented the Mpro-induced microvascular pathology. Our data suggest RIPK as a potential therapeutic target to treat the neuropathology of COVID-19.


Subject(s)
Blood-Brain Barrier/metabolism , Brain/metabolism , Coronavirus 3C Proteases/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Microvessels/metabolism , SARS-CoV-2/metabolism , Animals , Blood-Brain Barrier/pathology , Brain/pathology , Chlorocebus aethiops , Coronavirus 3C Proteases/genetics , Cricetinae , Female , Humans , Intracellular Signaling Peptides and Proteins/genetics , Male , Mesocricetus , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microvessels/pathology , SARS-CoV-2/genetics , Vero Cells
4.
Blood ; 138(25): 2702-2713, 2021 12 23.
Article in English | MEDLINE | ID: covidwho-1365304

ABSTRACT

Multiple organ dysfunction is the most severe outcome of sepsis progression and is highly correlated with a worse prognosis. Excessive neutrophil extracellular traps (NETs) are critical players in the development of organ failure during sepsis. Therefore, interventions targeting NET release would likely effectively prevent NET-based organ injury associated with this disease. Herein, we demonstrate that the pore-forming protein gasdermin D (GSDMD) is active in neutrophils from septic humans and mice and plays a crucial role in NET release. Inhibition of GSDMD with disulfiram or genic deletion abrogated NET formation, reducing multiple organ dysfunction and sepsis lethality. Mechanistically, we demonstrate that during sepsis, activation of the caspase-11/GSDMD pathway controls NET release by neutrophils during sepsis. In summary, our findings uncover a novel therapeutic use for disulfiram and suggest that GSDMD is a therapeutic target to improve sepsis treatment.


Subject(s)
Extracellular Traps/genetics , Gene Deletion , Intracellular Signaling Peptides and Proteins/genetics , Multiple Organ Failure/genetics , Phosphate-Binding Proteins/genetics , Sepsis/genetics , Acetaldehyde Dehydrogenase Inhibitors/therapeutic use , Adoptive Transfer , Aged , Animals , Cells, Cultured , Disulfiram/therapeutic use , Female , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Male , Mice, Inbred C57BL , Middle Aged , Multiple Organ Failure/pathology , Multiple Organ Failure/therapy , Phosphate-Binding Proteins/antagonists & inhibitors , Sepsis/pathology , Sepsis/therapy
5.
Viruses ; 13(6)2021 06 09.
Article in English | MEDLINE | ID: covidwho-1282639

ABSTRACT

Mammalian cells have developed an elaborate network of immunoproteins that serve to identify and combat viral pathogens. Interferon-stimulated gene 15 (ISG15) is a 15.2 kDa tandem ubiquitin-like protein (UBL) that is used by specific E1-E2-E3 ubiquitin cascade enzymes to interfere with the activity of viral proteins. Recent biochemical studies have demonstrated how the E3 ligase HECT and RCC1-containing protein 5 (HERC5) regulates ISG15 signaling in response to hepatitis C (HCV), influenza-A (IAV), human immunodeficiency virus (HIV), SARS-CoV-2 and other viral infections. Taken together, the potent antiviral activity displayed by HERC5 and ISG15 make them promising drug targets for the development of novel antiviral therapeutics that can augment the host antiviral response. In this review, we examine the emerging role of ISG15 in antiviral immunity with a particular focus on how HERC5 orchestrates the specific and timely ISGylation of viral proteins in response to infection.


Subject(s)
Cytokines/genetics , Interferons/immunology , Intracellular Signaling Peptides and Proteins/genetics , Ubiquitins/genetics , Virus Diseases/immunology , Animals , COVID-19/immunology , Cytokines/immunology , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/immunology , Mice , SARS-CoV-2/immunology , Ubiquitins/immunology , Viral Proteins/genetics , Viral Proteins/metabolism
6.
Mol Med Rep ; 24(2)2021 Aug.
Article in English | MEDLINE | ID: covidwho-1271003

ABSTRACT

Coronavirus disease 2019 (COVID­19), caused by the severe acute respiratory syndrome coronavirus­2 (SARS­CoV­2), led to an outbreak of viral pneumonia in December 2019. The present study aimed to investigate the host inflammatory response signature­caused by SARS­CoV­2 in human corneal epithelial cells (HCECs). The expression level of angiotensin­converting enzyme 2 (ACE2) in the human cornea was determined via immunofluorescence. In vitro experiments were performed in HCECs stimulated with the SARS­CoV­2 spike protein. Moreover, the expression levels of ACE2, IL­8, TNF­α, IL­6, gasdermin D (GSDMD) and IL­1ß in HCECs were detected using reverse transcription­quantitative PCR and/or western blotting. It was identified that ACE2 was expressed in normal human corneal epithelium and HCECs cultured in vitro. Furthermore, the expression levels of IL­8, TNF­α and IL­6 in HCECs were decreased following SARS­CoV­2 spike protein stimulation, while the expression levels of GSDMD and IL­1ß were increased. In conclusion, the present results demonstrated that the SARS­CoV­2 spike protein suppressed the host inflammatory response and induced pyroptosis in HCECs. Therefore, blocking the ACE2 receptor in HCECs may reduce the infection rate of COVID­19.


Subject(s)
Epithelium, Corneal/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Adult , Aged , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Cells, Cultured , Cornea/cytology , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelial Cells/virology , Epithelium, Corneal/virology , Female , Humans , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Male , Middle Aged , Phosphate-Binding Proteins/genetics , Phosphate-Binding Proteins/metabolism , Pyroptosis , Spike Glycoprotein, Coronavirus/genetics , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , Up-Regulation
8.
Kidney Blood Press Res ; 46(1): 74-83, 2021.
Article in English | MEDLINE | ID: covidwho-978847

ABSTRACT

INTRODUCTION: Various viruses including a novel coronavirus (SARS-CoV-2) can infect the kidney. When viruses invade the glomeruli from the bloodstream, glomerular endothelial cells (GECs) initiate the innate immune reactions. We investigated the expression of interferon (IFN)-induced protein with tetratricopeptide repeats (IFIT) 1/2/3, antiviral molecules, in human GECs treated with a toll-like receptor (TLR) 3 agonist. Role of IFIT1/2/3 in the expression of C-X-C motif chemokine ligand 10 (CXCL10) was also examined. METHODS: Human GECs were cultured and stimulated with polyinosinic-polycytidylic acid (poly IC), a synthetic TLR3 agonist. Real-time qPCR, Western blotting, and ELISA were used to examine the expression of IFIT1/2/3, IFN-ß, and CXCL10. RNA interference against IFN-ß or IFIT1/2/3 was also performed. RESULTS: Expression of IFIT1/2/3 and CXCL10 was induced by poly IC in GECs. The inductions were inhibited by RNA interfering of IFN-ß. Knockdown of IFIT1/2/3 decreased the CXCL10 expression. Knockdown of IFIT3 decreased the expression of IFIT1 and IFIT2 proteins. CONCLUSION: IFIT1/2/3 and CXCL10 were induced by poly IC via IFN-ß in GECs. IFIT1/2/3 may increase the expression of CXCL10 which induces lymphocyte chemotaxis and may inhibit the replication of infected viruses. These molecules may play a role in GEC innate immune reactions in response to viruses.


Subject(s)
Adaptor Proteins, Signal Transducing/biosynthesis , Apoptosis Regulatory Proteins/biosynthesis , Chemokine CXCL10/biosynthesis , Intracellular Signaling Peptides and Proteins/biosynthesis , Kidney Glomerulus/metabolism , RNA-Binding Proteins/biosynthesis , Toll-Like Receptor 3/agonists , Adaptor Proteins, Signal Transducing/genetics , Apoptosis Regulatory Proteins/genetics , Cells, Cultured , Chemokine CXCL10/genetics , Dose-Response Relationship, Drug , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Gene Expression , Humans , Intracellular Signaling Peptides and Proteins/genetics , Kidney Glomerulus/cytology , Kidney Glomerulus/drug effects , Poly I-C/pharmacology , RNA-Binding Proteins/genetics , Toll-Like Receptor 3/metabolism
9.
J Neurochem ; 156(6): 967-978, 2021 03.
Article in English | MEDLINE | ID: covidwho-780946

ABSTRACT

The lysosomal membrane protein Niemann-Pick type C1 (NPC1) and Niemann-Pick type C2 (NPC2) are main players of cholesterol control in the lysosome and it is known that the mutation on these proteins leads to the cholesterol trafficking-related neurodegenerative disease, which is called the NPC disease. The mutation R518W or R518Q on the NPC1 is one of the type of disease-related mutation that causes cholesterol transports to be cut in half, which results in the accumulation of cholesterol and lipids in the late endosomal/lysosomal compartment of the cell. Even though there has been significant progress with understanding the cholesterol transport by NPC1 in combination with NPC2, especially after the structural determination of the full-length NPC1 in 2016, many details such as the interaction of the full-length NPC1 with the NPC2, the molecular motions responsible for the cholesterol transport during and after this interaction, and the structure and the function relations of many mutations are still not well understood. In this study, we report the extensive molecular dynamics simulations in order to gain insight into the structure and the dynamics of NPC1 lumenal domain for the cholesterol transport and the disease behind the mutation (R518W). It was found that the mutation induces a structural shift of the N-terminal domain, toward the loop region in the middle lumenal domain, which is believed to play a central role in the interaction with NPC2 protein, so the interaction with the NPC2 protein might be less favorable compared to the wild NPC1. Also, the simulation indicates the possible re-orientation of the N-terminal domain with both the wild and the R518W-mutated NPC1 after receiving the cholesterol from the NPC2 that align to form an internal tunnel, which is a possible pose for further action in cholesterol trafficking. We believe the current study can provide a better understanding of the cholesterol transport by NPC1 especially the role of NTD of NPC1 in combination with NPC2 interactions.


Subject(s)
Cholesterol/metabolism , Niemann-Pick Disease, Type C/genetics , Biological Transport , Endosomes/metabolism , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Lipid Metabolism/genetics , Lysosomes/metabolism , Models, Molecular , Molecular Dynamics Simulation , Mutation/genetics , Niemann-Pick C1 Protein , Vesicular Transport Proteins/chemistry , Vesicular Transport Proteins/genetics , X-Ray Diffraction
10.
J Biol Chem ; 295(41): 14040-14052, 2020 10 09.
Article in English | MEDLINE | ID: covidwho-704089

ABSTRACT

Coronaviruses have caused several zoonotic infections in the past two decades, leading to significant morbidity and mortality globally. Balanced regulation of cell death and inflammatory immune responses is essential to promote protection against coronavirus infection; however, the underlying mechanisms that control these processes remain to be resolved. Here we demonstrate that infection with the murine coronavirus mouse hepatitis virus (MHV) activated the NLRP3 inflammasome and inflammatory cell death in the form of PANoptosis. Deleting NLRP3 inflammasome components or the downstream cell death executioner gasdermin D (GSDMD) led to an initial reduction in cell death followed by a robust increase in the incidence of caspase-8- and receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated inflammatory cell deathafter coronavirus infection. Additionally, loss of GSDMD promoted robust NLRP3 inflammasome activation. Moreover, the amounts of some cytokines released during coronavirus infection were significantly altered in the absence of GSDMD. Altogether, our findings show that inflammatory cell death, PANoptosis, is induced by coronavirus infection and that impaired NLRP3 inflammasome function or pyroptosis can lead to negative consequences for the host. These findings may have important implications for studies of coronavirus-induced disease.


Subject(s)
Caspase 8/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Pyroptosis , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Animals , Cells, Cultured , Coronavirus/physiology , Coronavirus Infections/metabolism , Coronavirus Infections/pathology , Coronavirus Infections/veterinary , Cytokines/metabolism , Inflammasomes/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Macrophages/cytology , Macrophages/metabolism , Macrophages/virology , Mice , Mice, Inbred C57BL , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Necroptosis , Phosphate-Binding Proteins/genetics , Phosphate-Binding Proteins/metabolism
11.
J Lipid Res ; 61(7): 972-982, 2020 07.
Article in English | MEDLINE | ID: covidwho-382050

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV)-2 has resulted in the death of more than 328,000 persons worldwide in the first 5 months of 2020. Herculean efforts to rapidly design and produce vaccines and other antiviral interventions are ongoing. However, newly evolving viral mutations, the prospect of only temporary immunity, and a long path to regulatory approval pose significant challenges and call for a common, readily available, and inexpensive treatment. Strategic drug repurposing combined with rapid testing of established molecular targets could provide a pause in disease progression. SARS-CoV-2 shares extensive structural and functional conservation with SARS-CoV-1, including engagement of the same host cell receptor (angiotensin-converting enzyme 2) localized in cholesterol-rich microdomains. These lipid-enveloped viruses encounter the endosomal/lysosomal host compartment in a critical step of infection and maturation. Niemann-Pick type C (NP-C) disease is a rare monogenic neurodegenerative disease caused by deficient efflux of lipids from the late endosome/lysosome (LE/L). The NP-C disease-causing gene (NPC1) has been strongly associated with viral infection, both as a filovirus receptor (e.g., Ebola) and through LE/L lipid trafficking. This suggests that NPC1 inhibitors or NP-C disease mimetics could serve as anti-SARS-CoV-2 agents. Fortunately, there are such clinically approved molecules that elicit antiviral activity in preclinical studies, without causing NP-C disease. Inhibition of NPC1 may impair viral SARS-CoV-2 infectivity via several lipid-dependent mechanisms, which disturb the microenvironment optimum for viral infectivity. We suggest that known mechanistic information on NPC1 could be utilized to identify existing and future drugs to treat COVID-19.


Subject(s)
Anticholesteremic Agents/therapeutic use , Antiviral Agents/therapeutic use , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Intracellular Signaling Peptides and Proteins/genetics , Niemann-Pick Disease, Type C/drug therapy , Pandemics , Pneumonia, Viral/drug therapy , Androstenes/therapeutic use , Angiotensin-Converting Enzyme 2 , Betacoronavirus/metabolism , Betacoronavirus/pathogenicity , COVID-19 , Cholesterol/metabolism , Coronavirus Infections/diagnosis , Coronavirus Infections/epidemiology , Drug Repositioning/methods , Humans , Hydroxychloroquine/therapeutic use , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/metabolism , Lysosomes/drug effects , Lysosomes/metabolism , Lysosomes/virology , Niemann-Pick C1 Protein , Niemann-Pick Disease, Type C/genetics , Niemann-Pick Disease, Type C/metabolism , Niemann-Pick Disease, Type C/pathology , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/diagnosis , Pneumonia, Viral/epidemiology , Protein Binding , Receptors, Virus/antagonists & inhibitors , Receptors, Virus/genetics , Receptors, Virus/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
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