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Proteolytic cleavages in the extracellular domain of receptor tyrosine kinases by membrane-associated serine proteases.

ABSTRACT: The epithelial extracellular membrane-associated serine proteases matriptase, hepsin, and prostasin are proteolytic modifying enzymes of the extracellular domain (ECD) of the epidermal growth factor receptor (EGFR). Matriptase also cleaves the ECD of the vascular endothelial growth factor receptor 2 (VEGFR2) and the angiopoietin receptor Tie2. In this study we tested the hypothesis that these serine proteases may cleave the ECD of additional receptor tyrosine kinases (RTKs). We co-expressed the proteases in an epithelial cell line with Her2, Her3, Her4, insulin receptor (INSR), insulin-like growth factor I receptor (IGF-1R), the platelet-derived growth factor receptors (PDGFRs) ? and ?, or nerve growth factor receptor A (TrkA). Western blot analysis was performed to detect the carboxyl-terminal fragments (CTFs) of the RTKs. Matriptase and hepsin were found to cleave the ECD of all RTKs tested, while TMPRSS6/matriptase-2 cleaves the ECD of Her4, INSR, and PDGFR ? and ?. Prostasin was able to cleave the ECD of Her3 and PDGFR?. Matriptase cleaves phosphorylated Her2 at Arg558 and Arg599 and the Arg599 cleavage produces a CTF not recognized by the monoclonal antibody trastuzumab/Herceptin. Her2 cleavages by matriptase can be inhibited by the hepatocyte growth factor activator inhibitor 1 (HAI-1) in the MDA-MB-231 human breast cancer cells. Matriptase silencing in the Her2, matriptase, and HAI-1 triple-positive SKBR3 human breast cancer cells enhanced Her2 protein down-regulation induced by a sustained exposure to phorbol 12-myristate 13-acetate (PMA), which down-regulated matriptase protein. The novel Her2 cleavage and expression regulation mechanisms mediated by matriptase may have potential impacts in Her2-targeting therapies.

SUBMITTER: Chen LM

PROVIDER: S-EPMC5593577 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Proteolytic cleavages in the extracellular domain of receptor tyrosine kinases by membrane-associated serine proteases.

Chen Li-Mei LM Chai Karl X KX

Oncotarget 20170410 34

The epithelial extracellular membrane-associated serine proteases matriptase, hepsin, and prostasin are proteolytic modifying enzymes of the extracellular domain (ECD) of the epidermal growth factor receptor (EGFR). Matriptase also cleaves the ECD of the vascular endothelial growth factor receptor 2 (VEGFR2) and the angiopoietin receptor Tie2. In this study we tested the hypothesis that these serine proteases may cleave the ECD of additional receptor tyrosine kinases (RTKs). We co-expressed the ...[more]

PMID: 28915606

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Project description:Streptococcus pneumoniae has evolved versatile strategies to colonize the nasopharynx of humans. Colonization is facilitated by direct interactions with host cell receptors or via binding to components of the extracellular matrix. In addition, pneumococci hijack host-derived extracellular proteases such as the serine protease plasmin(ogen) for ECM and mucus degradation as well as colonization. S. pneumoniae expresses strain-dependent up to four serine proteases. In this study, we assessed the role of secreted or cell-bound serine proteases HtrA, PrtA, SFP, and CbpG, in adherence assays and in a mouse colonization model. We hypothesized that the redundancy of serine proteases compensates for the deficiency of a single enzyme. Therefore, double and triple mutants were generated in serotype 19F strain EF3030 and serotype 4 strain TIGR4. Strain EF3030 produces only three serine proteases and lacks the SFP encoding gene. In adherence studies using Detroit-562 epithelial cells, we demonstrated that both TIGR4Δcps and 19F mutants without serine proteases or expressing only CbpG, HtrA, or PrtA have a reduced ability to adhere to Detroit-562 cells. Consistent with these results, we show that the mutants of strain 19F, which preferentially colonizes mice, abrogate nasopharyngeal colonization in CD-1 mice after intranasal infection. The bacterial load in the nasopharynx was monitored for 14 days. Importantly, mutants showed significantly lower bacterial numbers in the nasopharynx two days after infection. Similarly, we detected a significantly reduced pneumococcal colonization on days 3, 7, and 14 post-inoculations. To assess the impact of pneumococcal serine proteases on acute infection, we infected mice intranasally with bioluminescent and invasive TIGR4 or isogenic triple mutants expressing only CbpG, HtrA, PrtA, or SFP. We imaged the acute lung infection in real-time and determined the survival of the mice. The TIGR4lux mutant expressing only PrtA showed a significant attenuation and was less virulent in the acute pneumonia model. In conclusion, our results showed that pneumococcal serine proteases contributed significantly to pneumococcal colonization but played only a minor role in pneumonia and invasive diseases. Because colonization is a prerequisite for invasive diseases and transmission, these enzymes could be promising candidates for the development of antimicrobials to reduce pneumococcal transmission.

Dataset Information

Proteolytic cleavages in the extracellular domain of receptor tyrosine kinases by membrane-associated serine proteases.

Publications

Proteolytic cleavages in the extracellular domain of receptor tyrosine kinases by membrane-associated serine proteases.

Shared Molecules

Only show the datasets with similarity scores above: 0.5

Threshold

0.5

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