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2.
J Biol Chem ; 295(36): 12686-12696, 2020 09 04.
Article in English | MEDLINE | ID: covidwho-1387615

ABSTRACT

Type II transmembrane serine proteases (TTSPs) are a group of enzymes participating in diverse biological processes. Some members of the TTSP family are implicated in viral infection. TMPRSS11A is a TTSP expressed on the surface of airway epithelial cells, which has been shown to cleave and activate spike proteins of the severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome coronaviruses (CoVs). In this study, we examined the mechanism underlying the activation cleavage of TMPRSS11A that converts the one-chain zymogen to a two-chain enzyme. By expression in human embryonic kidney 293, esophageal EC9706, and lung epithelial A549 and 16HBE cells, Western blotting, and site-directed mutagenesis, we found that the activation cleavage of human TMPRSS11A was mediated by autocatalysis. Moreover, we found that TMPRSS11A activation cleavage occurred before the protein reached the cell surface, as indicated by studies with trypsin digestion to remove cell surface proteins, treatment with cell organelle-disturbing agents to block intracellular protein trafficking, and analysis of a soluble form of TMPRSS11A without the transmembrane domain. We also showed that TMPRSS11A was able to cleave the SARS-CoV-2 spike protein. These results reveal an intracellular autocleavage mechanism in TMPRSS11A zymogen activation, which differs from the extracellular zymogen activation reported in other TTSPs. These findings provide new insights into the diverse mechanisms in regulating TTSP activation.


Subject(s)
Epithelial Cells/metabolism , Membrane Proteins/metabolism , Proteolysis , Serine Proteases/metabolism , A549 Cells , Cells, Cultured , HEK293 Cells , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mutation , Protein Domains , Protein Transport , Respiratory Mucosa/cytology , Serine Proteases/chemistry , Serine Proteases/genetics , Spike Glycoprotein, Coronavirus/metabolism , Trypsin/metabolism
3.
Am J Physiol Lung Cell Mol Physiol ; 319(4): L670-L674, 2020 10 01.
Article in English | MEDLINE | ID: covidwho-798131

ABSTRACT

The severity of coronavirus disease 2019 (COVID-19) is linked to an increasing number of risk factors, including exogenous (environmental) stimuli such as air pollution, nicotine, and cigarette smoke. These three factors increase the expression of angiotensin I converting enzyme 2 (ACE2), a key receptor involved in the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the etiological agent of COVID-19-into respiratory tract epithelial cells. Patients with severe COVID-19 are managed with oxygen support, as are at-risk individuals with chronic lung disease. To date, no study has examined whether an increased fraction of inspired oxygen (FiO2) may affect the expression of SARS-CoV-2 entry receptors and co-receptors, including ACE2 and the transmembrane serine proteases TMPRSS1, TMPRSS2, and TMPRSS11D. To address this, steady-state mRNA levels for genes encoding these SARS-CoV-2 receptors were assessed in the lungs of mouse pups chronically exposed to elevated FiO2, and in the lungs of preterm-born human infants chronically managed with an elevated FiO2. These two scenarios served as models of chronic elevated FiO2 exposure. Additionally, SARS-CoV-2 receptor expression was assessed in primary human nasal, tracheal, esophageal, bronchial, and alveolar epithelial cells, as well as primary mouse alveolar type II cells exposed to elevated oxygen concentrations. While gene expression of ACE2 was unaffected, gene and protein expression of TMPRSS11D was consistently upregulated by exposure to an elevated FiO2. These data highlight the need for further studies that examine the relative contribution of the various viral co-receptors on the infection cycle, and point to oxygen supplementation as a potential risk factor for COVID-19.


Subject(s)
Coronavirus Infections/pathology , Membrane Proteins/metabolism , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/pathology , Respiratory Mucosa/metabolism , Serine Endopeptidases/metabolism , Serine Proteases/metabolism , Alveolar Epithelial Cells/metabolism , Angiotensin-Converting Enzyme 2 , Animals , Betacoronavirus , COVID-19 , Cells, Cultured , Female , Humans , Male , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Oxygen/administration & dosage , Oxygen/analysis , Pandemics , Receptors, Virus/metabolism , Risk Factors , SARS-CoV-2 , Serine Endopeptidases/genetics , Serine Proteases/genetics , Severity of Illness Index
4.
FEBS Open Bio ; 10(11): 2363-2374, 2020 11.
Article in English | MEDLINE | ID: covidwho-792010

ABSTRACT

Comorbidities in COVID-19 patients often worsen clinical conditions and may represent death predictors. Here, the expression of five genes, known to encode coronavirus receptors/interactors (ACE2, TMPRSS2, CLEC4M, DPP4 and TMPRSS11D), was investigated in normal and cancer tissues, and their molecular relationships with clinical comorbidities were investigated. Using expression data from GENT2 databases, we evaluated gene expression in all anatomical districts from 32 normal tissues in 3902 individuals. Functional relationships with body districts were analyzed by chilibot. We performed DisGeNet, genemania and DAVID analyses to identify human diseases associated with these genes. Transcriptomic expression levels were then analyzed in 31 cancer types and healthy controls from approximately 43 000 individuals, using GEPIA2 and GENT2 databases. By performing receiver operating characteristic analysis, the area under the curve (AUC) was used to discriminate healthy from cancer patients. Coronavirus receptors were found to be expressed in several body districts. Moreover, the five genes were found to associate with acute respiratory syndrome, diabetes, cardiovascular diseases and cancer (i.e. the most frequent COVID-19 comorbidities). Their expression levels were found to be significantly altered in cancer types, including colon, kidney, liver, testis, thyroid and skin cancers (P < 0.0001); AUC > 0.80 suggests that TMPRSS2, CLEC4M and DPP4 are relevant markers of kidney, liver, and thyroid cancer, respectively. The five coronavirus receptors are related to all main COVID-19 comorbidities and three show significantly different expression in cancer versus control tissues. Further investigation into their role may help in monitoring other comorbidities, as well as for follow-up of patients who have recovered from SARS-CoV-2 infection.


Subject(s)
COVID-19/prevention & control , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , Neoplasms/genetics , SARS-CoV-2/isolation & purification , Angiotensin-Converting Enzyme 2/genetics , COVID-19/epidemiology , COVID-19/virology , Cell Adhesion Molecules/genetics , Comorbidity , Databases, Genetic , Dipeptidyl Peptidase 4/genetics , Epidemics , Female , Genetic Predisposition to Disease/genetics , Humans , Lectins, C-Type/genetics , Male , Membrane Proteins/genetics , Neoplasms/classification , Neoplasms/epidemiology , Receptors, Cell Surface/genetics , SARS-CoV-2/physiology , Serine Endopeptidases/genetics , Serine Proteases/genetics
5.
Life Sci ; 260: 118313, 2020 Nov 01.
Article in English | MEDLINE | ID: covidwho-725734

ABSTRACT

BACKGROUND: The prevalence and mortality of the outbreak of the COVID-19 pandemic show marked geographic variation. The presence of several subtypes of the coronavirus and the genetic differences in the populations could condition that variation. Thus, the objective of this study was to propose variants in genes that encode proteins related to the SARS-CoV-2 entry into the host cells as possible targets for genetic associations studies. METHODS: The allelic frequencies of the polymorphisms in the ACE2, TMPRSS2, TMPRSS11A, cathepsin L (CTSL), and elastase (ELANE) genes were obtained in four populations from the American, African, European, and Asian continents reported in the 1000 Genome Project. Moreover, we evaluated the potential biological effect of these variants using different web-based tools. RESULTS: In the coding sequences of these genes, we detected one probably-damaging polymorphism located in the TMPRSS2 gene (rs12329760) that produces a change of amino acid. Furthermore, forty-eight polymorphisms with possible functional consequences were detected in the non-coding sequences of the following genes: three in ACE2, seventeen in TMPRSS2, ten in TMPRSS11A, twelve in ELANE, and six in CTSL. These polymorphisms produce binding sites for transcription factors and microRNAs. The minor allele frequencies of these polymorphisms vary in each community; indeed, some of them are high in specific populations. CONCLUSION: In summary, using data of the 1000 Genome Project and web-based tools, we propose some polymorphisms, which, depending on the population, could be used for genetic association studies.


Subject(s)
Betacoronavirus , Cathepsin L/genetics , Coronavirus Infections/genetics , Leukocyte Elastase/genetics , Membrane Proteins/genetics , Peptidyl-Dipeptidase A/genetics , Pneumonia, Viral/genetics , Polymorphism, Genetic , Serine Endopeptidases/genetics , Serine Proteases/genetics , Angiotensin-Converting Enzyme 2 , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , COVID-19 , Coronavirus Infections/epidemiology , Coronavirus Infections/pathology , Coronavirus Infections/virology , Gene Frequency , Genetic Association Studies , Humans , Linkage Disequilibrium , Pandemics , Pneumonia, Viral/epidemiology , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , SARS-CoV-2
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