scholarly journals Intracellular autoactivation of TMPRSS11A, an airway epithelial transmembrane serine protease

2020 ◽  
Vol 295 (36) ◽  
pp. 12686-12696 ◽  
Author(s):  
Ce Zhang ◽  
Yikai Zhang ◽  
Shengnan Zhang ◽  
Zhiting Wang ◽  
Shijin Sun ◽  
...  
Keyword(s):  
Cell Surface ◽  
Serine Proteases ◽  
Transmembrane Domain ◽  
Airway Epithelial Cells ◽  
Surface Proteins ◽  
Site Directed Mutagenesis ◽  
Soluble Form ◽  
Cell Organelle ◽  
Airway Epithelial ◽  
Zymogen Activation

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.

Glycosylation and annexin II cell surface translocation mediate airway epithelial wound repair

2007 ◽  
Vol 293 (2) ◽  
pp. L354-L363 ◽  
Author(s):  
Benjamin J. Patchell ◽  
Kimberly R. Wojcik ◽  
Ting-Lin Yang ◽  
Steven R. White ◽  
Delbert R. Dorscheid
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Wound Repair ◽  
Airway Epithelial Cells ◽  
Surface Proteins ◽  
Annexin Ii ◽  
Airway Epithelial ◽  
Mechanical Wounding ◽  
Epithelial Repair ◽  
Carbohydrate Ligand

Glycosylation of cell surface proteins can regulate multiple cellular functions. We hypothesized that glycosylation and expression of glycoproteins after epithelial injury is important in mediating repair. We report the use of an in vitro culture model of human airway epithelial cells (1HAEo−) to identify mediators of epithelial repair. We characterized carbohydrate moieties associated with repair by their interaction with the lectin from Cicer arietinum, chickpea agglutinin (CPA). Using CPA, we identified changes in cell surface glycosylation during wound repair. Following mechanical wounding of confluent monolayers of 1HAEo−cells, CPA staining increases on the cell surface of groups of cells in proximity to the wound edge. Blocking the CPA carbohydrate ligand inhibited wound repair highlighting the role of the CPA carbohydrate ligand in epithelial repair. Annexin II (AII), a calcium-dependent, membrane-associated protein, was identified as a protein associated with the CPA ligand. By membrane protein biotinylation and immunodetection, we have shown that following mechanical wounding, the presentation of AII on the cell surface increases coordinate with repair. Cell surface AII accumulates in proximity to the wound. Furthermore, translocation of AII to the cell surface is N-glycosylation dependent. We are the first to demonstrate that following injury, N-glycosylation events and AII presentation on the cell surface of airway epithelial cells are important mediators in repair.

scholarly journals Human Tryptase ε (PRSS22), a New Member of the Chromosome 16p13.3 Family of Human Serine Proteases Expressed in Airway Epithelial Cells

2001 ◽  
Vol 276 (52) ◽  
pp. 49169-49182 ◽  
Author(s):  
Guang W. Wong ◽  
Shinsuke Yasuda ◽  
Mallur S. Madhusudhan ◽  
Lixin Li ◽  
Yi Yang ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Serine Proteases ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Chromosome 16P13.3

scholarly journals Down-Regulation of Cell Surface Receptors Is Modulated by Polar Residues within the Transmembrane Domain

2000 ◽  
Vol 11 (8) ◽  
pp. 2643-2655 ◽  
Author(s):  
Lolita Zaliauskiene ◽  
Sunghyun Kang ◽  
Christie G. Brouillette ◽  
Jacob Lebowitz ◽  
Ramin B. Arani ◽  
...  
Keyword(s):  
Cell Surface ◽  
Transmembrane Domain ◽  
Gradient Centrifugation ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Surface Proteins ◽  
Down Regulation ◽  
Surface Receptors ◽  
Receptor Complexes ◽  
Polar Residues

How recycling receptors are segregated from down-regulated receptors in the endosome is unknown. In previous studies, we demonstrated that substitutions in the transferrin receptor (TR) transmembrane domain (TM) convert the protein from an efficiently recycling receptor to one that is rapidly down regulated. In this study, we demonstrate that the “signal” within the TM necessary and sufficient for down-regulation is Thr11Gln17Thr19 (numbering in TM). Transplantation of these polar residues into the wild-type TR promotes receptor down-regulation that can be demonstrated by changes in protein half-life and in receptor recycling. Surprisingly, this modification dramatically increases the TR internalization rate as well (∼79% increase). Sucrose gradient centrifugation and cross-linking studies reveal that propensity of the receptors to self-associate correlates with down-regulation. Interestingly, a number of cell surface proteins that contain TM polar residues are known to be efficiently down-regulated, whereas recycling receptors for low-density lipoprotein and transferrin conspicuously lack these residues. Our data, therefore, suggest a simple model in which specific residues within the TM sequences dramatically influence the fate of membrane proteins after endocytosis, providing an alternative signal for down-regulation of receptor complexes to the well-characterized cytoplasmic tail targeting signals.

scholarly journals Posttranslational Modifications Required for Cell Surface Localization and Function of the Fungal Adhesin Aga1p

2003 ◽  
Vol 2 (5) ◽  
pp. 1099-1114 ◽  
Author(s):  
Guohong Huang ◽  
Mingliang Zhang ◽  
Scott E. Erdman
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Posttranslational Modifications ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Cell Types ◽  
Fungal Species ◽  
Surface Proteins ◽  
Functional Requirements

ABSTRACT Adherence of fungal cells to host substrates and each other affects their access to nutrients, sexual conjugation, and survival in hosts. Adhesins are cell surface proteins that mediate these different cell adhesion interactions. In this study, we examine the in vivo functional requirements for specific posttranslational modifications to these proteins, including glycophosphatidylinositol (GPI) anchor addition and O-linked glycosylation. The processing of some fungal GPI anchors, creating links to cell wall β-1,6 glucans, is postulated to facilitate postsecretory traffic of proteins to cell wall domains conducive to their functions. By studying the yeast sexual adhesin subunit Aga1p, we found that deletion of its signal sequence for GPI addition eliminated its activity, while deletions of different internal domains had various effects on function. Substitution of the Aga1p GPI signal domain with those of other GPI-anchored proteins, a single transmembrane domain, or a cysteine capable of forming a disulfide all produced functional adhesins. A portion of the cellular pool of Aga1p was determined to be cell wall resident. Aga1p and the α-agglutinin Agα1p were shown to be under glycosylated in cells lacking the protein mannosyltransferase genes PMT1 and PMT2, with phenotypes manifested only in MATα cells for single mutants but in both cell types when both genes are absent. We conclude that posttranslational modifications to Aga1p are necessary for its biogenesis and activity. Our studies also suggest that in addition to GPI-glucan linkages, other cell surface anchorage mechanisms, such as transmembrane domains or disulfides, may be employed by fungal species to localize adhesins.

scholarly journals Activity and inhibition of prostasin and matriptase on apical and basolateral surfaces of human airway epithelial cells

2012 ◽  
Vol 303 (2) ◽  
pp. L97-L106 ◽  
Author(s):  
Shilpa Nimishakavi ◽  
Marina Besprozvannaya ◽  
Wilfred W. Raymond ◽  
Charles S. Craik ◽  
Dieter C. Gruenert ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Selective Inhibition ◽  
Airway Epithelial Cells ◽  
Bronchial Epithelial Cells ◽  
Apical Surface ◽  
Airway Epithelial ◽  
Wild Type ◽  
Bronchial Epithelial

Prostasin is a membrane-anchored protease expressed in airway epithelium, where it stimulates salt and water uptake by cleaving the epithelial Na+ channel (ENaC). Prostasin is activated by another transmembrane tryptic protease, matriptase. Because ENaC-mediated dehydration contributes to cystic fibrosis (CF), prostasin and matriptase are potential therapeutic targets, but their catalytic competence on airway epithelial surfaces has been unclear. Seeking tools for exploring sites and modulation of activity, we used recombinant prostasin and matriptase to identify substrate t-butyloxycarbonyl-l-Gln-Ala-Arg-4-nitroanilide (QAR-4NA), which allowed direct assay of proteases in living cells. Comparisons of bronchial epithelial cells (CFBE41o−) with and without functioning cystic fibrosis transmembrane conductance regulator (CFTR) revealed similar levels of apical and basolateral aprotinin-inhibitable activity. Although recombinant matriptase was more active than prostasin in hydrolyzing QAR-4NA, cell surface activity resisted matriptase-selective inhibition, suggesting that prostasin dominates. Surface biotinylation revealed similar expression of matriptase and prostasin in epithelial cells expressing wild-type vs. ΔF508-mutated CFTR. However, the ratio of mature to inactive proprostasin suggested surface enrichment of active enzyme. Although small amounts of matriptase and prostasin were shed spontaneously, prostasin anchored to the cell surface by glycosylphosphatidylinositol was the major contributor to observed QAR-4NA-hydrolyzing activity. For example, the apical surface of wild-type CFBE41o− epithelial cells express 22% of total, extractable, aprotinin-inhibitable, QAR-4NA-hydrolyzing activity and 16% of prostasin immunoreactivity. In conclusion, prostasin is present, mature and active on the apical surface of wild-type and CF bronchial epithelial cells, where it can be targeted for inhibition via the airway lumen.

scholarly journals ACE2 Expression is elevated in Airway Epithelial Cells from aged and male donors but reduced in asthma

2020 ◽  
Author(s):  
Peter Wark ◽  
Prabuddha Pathinyake ◽  
Gerard Kaiko ◽  
Kristy Nichol ◽  
Ayesha Ali ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Serine Proteases ◽  
Airway Epithelial Cells ◽  
Lower Airway ◽  
Chronic Obstructive ◽  
Airway Epithelial ◽  
Obstructive Pulmonary Disease ◽  
Protein Levels ◽  
Asthma Patients

Rationale: COVID-19 is complicated by acute lung injury, and death in some individuals. It is caused by SARS-CoV-2 that requires the ACE2 receptor and serine proteases to enter airway epithelial cells (AECs). Objective: To determine what factors are associated with ACE2 expression particularly in patients with asthma and chronic obstructive pulmonary disease (COPD). Methods: We obtained upper and lower AECs from 145 people from two independent cohorts, aged 2-89, Newcastle (n=115), and from Perth (n= 30) Australia. The Newcastle cohort was enriched with people with asthma (n=37) and COPD (n=38). Gene expression for ACE2 and other genes potentially associated with SARS-CoV-2 cell entry were assessed by quantitative PCR, protein expression was confirmed with immunohistochemistry on endobronchial biopsies and cultured AECs. Results: Increased gene expression of ACE2 was associated with older age (p=0.02) and male sex (p=0.03), but not pack-years smoked. When we compared gene expression between adults with asthma, COPD and healthy controls, mean ACE2 expression was lower in asthma (p=0.01). Gene expression of furin, a protease that facilitates viral endocytosis, was also lower in asthma (p=0.02), while ADAM-17, a disintegrin that cleaves ACE2 from the surface was increased (p=0.02). ACE2 protein levels were lower in endobronchial biopsies from asthma patients. Conclusions: Increased ACE2 expression occurs in older people and males. Asthma patients have reduced expression. Altered ACE2 expression in the lower airway may be an important factor in virus tropism and may in part explain susceptibility factors and why asthma patients are not over-represented in those with COVID-19 complications.

IL-1β induces eotaxin gene transcription in A549 airway epithelial cells through NF-κB

2000 ◽  
Vol 279 (6) ◽  
pp. L1058-L1065 ◽  
Author(s):  
Sean Jedrzkiewicz ◽  
Hidetoshi Nakamura ◽  
Eric S. Silverman ◽  
Andrew D. Luster ◽  
Naresh Mansharamani ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Transcriptional Activation ◽  
Transcriptional Start Site ◽  
Nuclear Factor Κb ◽  
Airway Epithelial Cells ◽  
Site Directed Mutagenesis ◽  
Airway Epithelial ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
Necessary And Sufficient

Eotaxin is an asthma-related C-C chemokine that is produced in response to interleukin-1β (IL-1β). We detected an increase in newly transcribed eotaxin mRNA in IL-1β-stimulated airway epithelial cells. Transient transfection assays using promoter-reporter constructs identified a region as essential for IL-1β-induced increases in eotaxin transcription. Using site-directed mutagenesis, we found that a nuclear factor-κB (NF-κB) site located 46 bp upstream from the transcriptional start site was both necessary and sufficient for IL-1β induction of reporter construct activity. Electrophoretic mobility shift assay demonstrated that IL-1β-stimulated airway epithelial cells produced p50 and p65 protein that bound this site in a sequence-specific manner. The functional importance of the NF-κB site was demonstrated by coexpression experiments in which increasing doses of p65 expression vector were directly associated with reporter activity exclusively in constructs with an intact NF-κB site ( r 2 = 0.97, P = 0.002). Moreover, IL-1β-induced increases in eotaxin mRNA expression are inhibited by inhibitors of NF-κB. Our findings implicate NF-κB and its binding sequence in IL-1β-induced transcriptional activation of the eotaxin gene.

scholarly journals Hydrogen Sulfide Inhibits TMPRSS2 in Human Airway Epithelial Cells: Implications for SARS-CoV-2 Infection

Biomedicines ◽  
2021 ◽  
Vol 9 (9) ◽  
pp. 1273
Author(s):  
Giulia Pozzi ◽  
Elena Masselli ◽  
Giuliana Gobbi ◽  
Prisco Mirandola ◽  
Luis Taborda-Barata ◽  
...  
Keyword(s):  
Hydrogen Sulfide ◽  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Surface Proteins ◽  
Lower Airway ◽  
Target Cells ◽  
Airway Epithelial ◽  
Respiratory Epithelial Cells ◽  
Respiratory Epithelial

The COVID-19 pandemic has now affected around 190 million people worldwide, accounting for more than 4 million confirmed deaths. Besides ongoing global vaccination, finding protective and therapeutic strategies is an urgent clinical need. SARS-CoV-2 mostly infects the host organism via the respiratory system, requiring angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) to enter target cells. Therefore, these surface proteins are considered potential druggable targets. Hydrogen sulfide (H2S) is a gasotransmitter produced by several cell types and is also part of natural compounds, such as sulfurous waters that are often inhaled as low-intensity therapy and prevention in different respiratory conditions. H2S is a potent biological mediator, with anti-oxidant, anti-inflammatory, and, as more recently shown, also anti-viral activities. Considering that respiratory epithelial cells can be directly exposed to H2S by inhalation, here we tested the in vitro effects of H2S-donors on TMPRSS2 and ACE2 expression in human upper and lower airway epithelial cells. We showed that H2S significantly reduces the expression of TMPRSS2 without modifying ACE2 expression both in respiratory cell lines and primary human upper and lower airway epithelial cells. Results suggest that inhalational exposure of respiratory epithelial cells to natural H2S sources may hinder SARS-CoV-2 entry into airway epithelial cells and, consequently, potentially prevent the virus from spreading into the lower respiratory tract and the lung.

scholarly journals TMPRSS2 structure-phylogeny repositions Avoralstat for SARS-CoV-2 prophylaxis in mice

2021 ◽  
Author(s):  
Young Joo Sun ◽  
Gabriel Velez ◽  
Dylan Parsons ◽  
Kun Li ◽  
Miguel Ortiz ◽  
...  
Keyword(s):  
Viral Entry ◽  
Serine Proteases ◽  
Tertiary Structure ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Cell Infection ◽  
Effective Inhibition ◽  
Transmembrane Serine Protease

Drugs targeting host proteins can act prophylactically to reduce viral burden early in disease and limit morbidity, even with antivirals and vaccination. Transmembrane serine protease 2 (TMPRSS2) is a human protease required for SARS-CoV-2 viral entry and may represent such a target. We hypothesized drugs selected from proteins related by their tertiary structure, rather than their primary structure, were likely to interact with TMPRSS2. We created a structure-based phylogenetic computational tool 3DPhyloFold to systematically identify structurally similar serine proteases with known therapeutic inhibitors and demonstrated effective inhibition of SARS-CoV-2 infection in vitro and in vivo. Several candidate compounds, Avoralstat, PCI-27483, Antipain, and Soybean-Trypsin-Inhibitor, inhibited TMPRSS2 in biochemical and cell infection assays. Avoralstat, a clinically tested Kallikrein-related B1 inhibitor, inhibited SARS-CoV-2 entry and replication in human airway epithelial cells. In an in vivo proof of principle, Avoralstat significantly reduced lung tissue titers and mitigated weight-loss when administered prophylactically to SARS-CoV-2 susceptible mice indicating its potential to be repositioned for COVID-19 prophylaxis in humans.

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