Matrix metalloproteinase-14 mediates a phenotypic shift in the airways to increase mucin production.

RATIONALE: Induced mainly by cigarette smoking, chronic obstructive pulmonary disease (COPD) is a global public health problem characterized by progressive difficulty in breathing and increased mucin production. Previously, we reported that acrolein levels found in COPD sputum could activate matrix metalloproteinase-9 (MMP9). OBJECTIVES: To determine whether acrolein increases expression and activity of MMP14, a critical membrane-bound endopeptidase that can initial a MMP-activation cascade. METHODS: MMP14 activity and adduct formation were measured following direct acrolein treatment. MMP14 expression and activity was measured in human airway epithelial cells. MMP14 immunohistochemistry was performed with COPD tissue, and in acrolein- or tobacco-exposed mice. MEASUREMENTS AND MAIN RESULTS: In a cell-free system, acrolein, in concentrations equal to those found in COPD sputum, directly adducted cysteine 319 in the MMP14 hemopexin-like domain and activated MMP14. In cells, acrolein increased MMP14 activity, which was inhibited by a proprotein convertase inhibitor, hexa-d-arginine. In the airway epithelium of COPD subjects, immunoreactive MMP14 protein increased. In mouse lung, acrolein or tobacco smoke increased lung MMP14 activity and protein. In cells, acrolein-induced MMP14 transcripts were inhibited by an epidermal growth factor receptor (EGFR) neutralizing antibody, EGFR kinase inhibitor, metalloproteinase inhibitor, or mitogen-activated protein kinase (MAPK) 3/2 or MAPK8 inhibitors, but not a MAPK14 inhibitor. Decreasing the MMP14 protein and activity in vitro by small interfering (si)RNA to MMP14 diminished the acrolein-induced MUC5AC transcripts. In acrolein-exposed mice or transgenic mice with lung-specific transforming growth factor-alpha (an EGFR ligand) expression, lung MMP14 and MUC5AC levels increased and these effects were inhibited by a EGFR inhibitor, erlotinib. CONCLUSIONS: Taken together, these findings implicate acrolein-induced MMP14 expression and activity in mucin production in COPD.

Characterizing the reproducibility of a protein profiling method for the analysis of mouse bronchoalveolar lavage fluid.

The detection of biomarkers in biological fluids has been advanced by the introduction of mass spectrometry screening methods such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), which enables the detection of the presence and the molecular mass of proteins in unfractionated mixtures. The generation of reproducible mass spectra over the course of an experiment is vital in obtaining data in which differences in protein profiles between diseased and healthy states can be assessed correctly. We have developed a protocol to automate the collection of protein profiling data from a large number of samples using MALDI-TOFMS, and we used these samples to characterize the technical reproducibility of the method. This protocol has been used for the analysis of proteins found in bronchoalveolar lavage fluid samples from mice with the ultimate goal of enabling the discovery of differential expression patterns predictive of the development of chronic obstructive pulmonary disease. Samples were purified using magnetic bead-based technology and analyzed on an AnchorChip target plate. Our results demonstrate that the number of peaks detected reproducibly decreases significantly as sample size increases, which motivates the need for technical replicates to be explicitly included in the analysis of MALDI-TOF-based protein profiling studies.

Association of the 17-kDa extrinsic protein with photosystem II in higher plants.

The structural association of the spinach 17-kDa extrinsic protein of photosystem II with other extrinsic and membrane-bound components of the photosystem was investigated by labeling the 17-kDa extrinsic protein with the amino-group-specific reagent N-hydroxysuccinimidobiotin both on intact photosystem II membranes or as a free protein in solution. After isolation of the biotinylated molecules, the modified 17-kDa proteins were allowed to rebind to photosystem II membranes which were depleted of the 17-kDa component. Differential binding of the protein biotinylated in solution compared to unmodified 17-kDa protein or 17-kDa protein modified on PS II membranes was observed. This indicated possible steric or ionic interference because of biotinylated lysyl residues present on the protein modified in solution. Biotinylated sites on the different modified 17-kDa proteins were identified by trypsin and Staphylococcus V8 protease digestion, followed by affinity chromatography enrichment of the biotinylated peptides and analysis of the peptide fragment mixture by nanospray liquid chromatography-tandem mass spectrometry. Four lysyl residues that were modified when the protein was biotinylated in solution were not biotinylated when the protein was modified on the PS II membrane (90K, 96K, 101K, and 102K). These residues appear to identify a protein domain involved in the interaction of the 17-kDa protein with the other components of the photosystem.