A., Mecham R. cleft and close structural resemblance. These studies allowed us to synthesize for the first time novel sensitive fluorescence resonance energy transfer substrates for individual mouse NSPs. Our findings and the newly identified substrates should better our understanding about the role of NSPs in the pathogenesis of cigarette-associated chronic obstructive pulmonary disease as well as other neutrophils-associated inflammatory Rabbit polyclonal to HSD17B13 diseases. Introduction Neutrophil serine proteases (NSPs),3 neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (Pr3), are mainly stored in neutrophil primary granules in readily active forms. NSPs are structurally related and share the conserved charge-relay triad, His57CAsp102CSer195, where Ser is the active residue (chymotrypsinogen numbering) (1). NSPs contribute to neutrophil oxygen-independent system-mediated protection of the host against invading pathogens (2). Indeed, NSPs 6-Benzylaminopurine serve a physiological role for killing of microbes (3). Activated neutrophils are also known to release NSPs in the setting of inflammation. = 15) had C57/BL6 genetic background and were 8C10 weeks aged. Mice were maintained in the animal barrier facility with a 12-h light/dark cycle and provided with water and food (maximum, 1 m). The final concentration of all three enzymes was in the nanomolar range. The sites in the FRET substrates cleaved by mouse and human proteases were identified by mass spectrometry and/or N-terminal sequencing. Fluorogenic substrates (5C8 m final) were incubated with human or mouse Pr3, NE, and CG (1C10 nm) in reaction buffer at 37 C. The reaction was stopped by adding 4 volumes of absolute ethanol and incubating for 15 min on ice. Precipitated protein was removed by centrifugation at 13,000 for 10 min. The supernatant made up of the hydrolysis products was dried under vacuum and dissolved in 200 l of 0.01% trifluoroacetic acid (v/v). Hydrolysis fragments were purified by reversed-phase chromatography on a C18 column (2.1 mm 30 mm) eluted at 0.3 ml/min with a linear (0C60%, v/v) gradient of acetonitrile in 0.01% trifluoroacetic acid for 20 min. Eluted peaks were monitored at three wavelengths (220, 320, and 360 nm) simultaneously to directly identify EDDnp-containing peptides prior to sequencing. Modeling of Murine Neutrophil Serine Proteases Because human and mouse NSPs share 70% sequence identity, we used various automated comparative modeling tools to generate models of murine NSPs based on the three-dimensional structure templates of HNE, hPr3, and hCG, respectively. These include Swiss-Model, (PS)2, and ModWeb. The sequences of mNE (UniProtKB entry name ELNE_MOUSE), mPr3 (UniProtKB entry name PRTN3_MOUSE), and mCG (UniProtKB entry name CATG_MOUSE) were used as inputs for the automatic modeling mode. All the models were then evaluated using ANAOLEA to analyze the non-local environment of each heavy atom in the protein structure and EVAL123D, which allows evaluation of three-dimensional protein structures by different methods (Verify3D, Eval23D, ProsaII, EvTree, and SFE) 6-Benzylaminopurine in a single run. Based on this analysis, we found that Swiss-Model provides the best models for murine NSPs. All subsequent studies were carried based on these models. Electrostatic Potential Calculations The electrostatic potentials of mNE, mPr3, and mCG were calculated using the finite difference Poisson Boltzmann method as implemented in DELPHI software (INSIGHT II suite, Accelrys Inc.). The ionic strength was set to 0.15 m, and the dielectric constant to 4 for the protein interior and 80 for the solvent. Calculations were performed at pH 7.4 at a heat of 300 K. Formal charges, rather than all charges, were assigned as follows: arginine, lysine, N terminus, 6-Benzylaminopurine +1; glutamate, aspartate, C terminus, ?1; and histidine, neutral. Contours of electrostatic potentials were displayed at 5 kTe?1 using INSIGHT II. The locations of the various subsites in the active site of each protease were inferred from the positions of the side chains of the corresponding residues of the OMT3 inhibitor complexed with HNE (PDB 6-Benzylaminopurine entry code 1PPF). The solvent-accessible molecular surface was calculated and colored.