Cathepsin S Signals via PAR2 and Generates a Novel Tethered Ligand Receptor Agonist

Protease-activated receptor-2 is widely expressed in mammalian epithelial, immune and neural tissues. Cleavage of PAR2 by serine proteases leads to self-activation of the receptor by the tethered ligand SLIGRL. The contribution of other classes of proteases to PAR activation has not been studied in detail. Cathepsin S is a widely expressed cysteine protease that is upregulated in inflammatory conditions. It has been suggested that cathepsin S activates PAR2. However, cathepsin S activation of PAR2 has not been demonstrated directly nor has the potential mechanism of activation been identified. We show that cathepsin S cleaves near the N-terminus of PAR2 to expose a novel tethered ligand, KVDGTS. The hexapeptide KVDGTS generates downstream signaling events specific to PAR2 but is weaker than SLIGRL. Mutation of the cathepsin S cleavage site prevents receptor activation by the protease while KVDGTS retains activity. In conclusion, the range of actions previously ascribed to cysteine cathepsins in general, and cathepsin S in particular, should be expanded to include molecular signaling. Such signaling may link together observations that had been attributed previously to PAR2 or cathepsin S individually. These interactions may contribute to inflammation

Cathepsin S Signals via PAR2 and Generates a Novel Tethered Ligand Receptor Agonist

Abstract Protease-activated receptor-2 is widely expressed in mammalian epithelial, immune and neural tissues. Cleavage of PAR2 by serine proteases leads to self-activation of the receptor by the tethered ligand SLIGRL. The contribution of other classes of proteases to PAR activation has not been studied in detail. Cathepsin S is a widely expressed cysteine protease that is upregulated in inflammatory conditions. It has been suggested that cathepsin S activates PAR2. However, cathepsin S activation of PAR2 has not been demonstrated directly nor has the potential mechanism of activation been identified. We show that cathepsin S cleaves near the N-terminus of PAR2 to expose a novel tethered ligand, KVDGTS. The hexapeptide KVDGTS generates downstream signaling events specific to PAR2 but is weaker than SLIGRL. Mutation of the cathepsin S cleavage site prevents receptor activation by the protease while KVDGTS retains activity. In conclusion, the range of actions previously ascribed to cysteine cathepsins in general, and cathepsin S in particular, should be expanded to include molecular signaling. Such signaling may link together observations that had been attributed previously to PAR2 or cathepsin S individually. These interactions may contribute to inflammation

The CMS Outer Hadron Calorimeter

The CMS hadron calorimeter is a sampling calorimeter with brass absorber and plastic scintillator tiles with wavelength shifting fibres for carrying the light to the readout device. The barrel hadron calorimeter is complemented with a outer calorimeter to ensure high energy shower containment in CMS and thus working as a tail catcher. Fabrication, testing and calibrations of the outer hadron calorimeter are carried out keeping in mind its importance in the energy measurement of jets in view of linearity and resolution. It will provide a net improvement in missing \et measurements at LHC energies. The outer hadron calorimeter has a very good signal to background ratio even for a minimum ionising particle and can hence be used in coincidence with the Resistive Plate Chambers of the CMS detector for the muon trigger

Design, Performance, and Calibration of the CMS Hadron-Outer Calorimeter

The CMS hadron calorimeter is a sampling calorimeter with brass absorber and plastic scintillator tiles with wavelength shifting fibres for carrying the light to the readout device. The barrel hadron calorimeter is complemented with an outer calorimeter to ensure high energy shower containment in the calorimeter. Fabrication, testing and calibration of the outer hadron calorimeter are carried out keeping in mind its importance in the energy measurement of jets in view of linearity and resolution. It will provide a net improvement in missing \et measurements at LHC energies. The outer hadron calorimeter will also be used for the muon trigger in coincidence with other muon chambers in CMS

Genomic-based-breeding tools for tropical maize improvement

Maize has traditionally been the main staple diet in the Southern Asia and Sub-Saharan Africa and widely grown by millions of resource poor small scale farmers. Approximately, 35.4 million hectares are sown to tropical maize, constituting around 59% of the developing worlds. Tropical maize encounters tremendous challenges besides poor agro-climatic situations with average yields recorded <3 tones/hectare that is far less than the average of developed countries. On the contrary to poor yields, the demand for maize as food, feed, and fuel is continuously increasing in these regions. Heterosis breeding introduced in early 90 s improved maize yields significantly, but genetic gains is still a mirage, particularly for crop growing under marginal environments. Application of molecular markers has accelerated the pace of maize breeding to some extent. The availability of array of sequencing and genotyping technologies offers unrivalled service to improve precision in maize-breeding programs through modern approaches such as genomic selection, genome-wide association studies, bulk segregant analysis-based sequencing approaches, etc. Superior alleles underlying complex traits can easily be identified and introgressed efficiently using these sequence-based approaches. Integration of genomic tools and techniques with advanced genetic resources such as nested association mapping and backcross nested association mapping could certainly address the genetic issues in maize improvement programs in developing countries. Huge diversity in tropical maize and its inherent capacity for doubled haploid technology offers advantage to apply the next generation genomic tools for accelerating production in marginal environments of tropical and subtropical world. Precision in phenotyping is the key for success of any molecular-breeding approach. This article reviews genomic technologies and their application to improve agronomic traits in tropical maize breeding has been reviewed in detail

Cathepsin S fails to activate mutant PAR2 but KVDGTS retains activity on mutant PAR2.

<p>a) Calcium imaging was performed in HeLa cells transfected with native or mutant PAR2 receptors following treatment with cathepsin S. Cathepsin S (2 µM) was able to stimulate native PAR2 and PAR2M1 but not PAR2M2 or PAR2M3. b) In contrast, KVDGTS activated PAR2 and all three substitution mutants. Cathepsin S (2 µM), KVDGTS (100 µM).</p

KVDGTS alters PAR2 response to cathepsin S.

<p>a) HeLa cells transfected with PAR2 cDNA were treated with KVDGTS and subsequently with cathepsin S after a 2 minute interval (dotted line). Pre-treatment with KVDGTS attenuates the response to cathepsin S. HeLa cells treated with cathepsin S failed to respond subsequently to KVDGTS (100 µM) (solid line). b) KVDGTS (100 µM) elicted calcium responses in NHEKs and the subsequent response to cathepsin S (2 µM) was attenuated. Treatment with cathepsin S abolishes the response to the subsequent addtion of KVDGTS. The second agent was delivered at the 300 second timepoint. KVDGTS (100 µM), cathepsin S (2 µM).</p

LC/MS/MS data from cathepsin S digestion of the human PAR2 N-terminal peptide G²⁸TNRSSKGRSLIGKVDGTSHVTGKGVT.

<p>MS/MS analysis of cathepsin S cleavage of synthetic N-terminal PAR2. Column 1 lists the amino acid in the P1 postion. Column 2 lists the identified cleaved peptide. Column 3 lists the number of times the cleaved peptide occurred in the scan. Details of the method are provided in the Experimental Procedures section.</p

PAR2 N-terminal hexapeptides are capable of activating the receptor.

<p>a) Single-cell calcium imaging in HeLa cells transfected with PAR2 cDNA demonstrated a similar response to KVDGTS (thick solid line) and SLIGRL (thick dotted line). A weaker response was observed in response to IGKVDG (thin solid line). HeLa cells transfected with salmon sperm DNA and stimulated with KVDGTS did not respond (horizontal dotted line). b) Cathepsin S (solid line) and KVDGTS (100 µM) (dotted line) elicit similar calcium responses in NHEKs. KVDGTS (100 µM) and SLIGRL (10 µM), IGKVDG (100 µM) and cathepsin S (2 µM).</p