Comparative proteomics of respiratory exosomes in cystic fibrosis, primary ciliary dyskinesia and asthma.

Cystic fibrosis (CF) and primary ciliary dyskinesia (PCD) are pulmonary genetic disorders associated with inflammation and heterogeneous progression of the lung disease. We hypothesized that respiratory exosomes, nanovesicles circulating in the respiratory tract, may be involved in the progression of inflammation-related lung damage. We compared proteomic content of respiratory exosomes isolated from bronchoalveolar lavage fluid in CF and PCD to asthma (A), a condition also associated with inflammation but with less severe lung damage. BALF were obtained from 3 CF, 3 PCD and 6 A patients. Exosomes were isolated from BALF by ultracentrifugations and characterized using immunoelectron microscopy and western-blot. Exosomal protein analysis was performed by high-resolution mass spectrometry using label-free quantification. Exosome enrichment was validated by electron microscopy and immunodetection of CD9, CD63 and ALIX. Mass spectrometry analysis allowed the quantification of 665 proteins, of which 14 were statistically differential according to the disease. PCD and CF exosomes contained higher levels of antioxidant proteins (Superoxide-dismutase, Glutathione peroxidase-3, Peroxiredoxin-5) and proteins involved in leukocyte chemotaxis. All these proteins are known activators of the NF-KappaB pathway. Our results suggest that respiratory exosomes are involved in the pro-inflammatory propagation during the extension of CF or PCD lung diseases. SIGNIFICANCE: The mechanism of local propagation of lung disease in cystic fibrosis (CF) and primary ciliary dyskinesia (PCD) is not clearly understood. Differential Proteomic profiles of exosomes isolated from BAL from CF, PCD and asthmatic patients suggest that they carry pro-inflammatory proteins that may be involved in the progression of lung damage.

Molecular identification and functional characterization of the first Nα-acetyltransferase in plastids by global acetylome profiling.

Protein N(α) -terminal acetylation represents one of the most abundant protein modifications of higher eukaryotes. In humans, six N(α) -acetyltransferases (Nats) are responsible for the acetylation of approximately 80% of the cytosolic proteins. N-terminal protein acetylation has not been evidenced in organelles of metazoans, but in higher plants is a widespread modification not only in the cytosol but also in the chloroplast. In this study, we identify and characterize the first organellar-localized Nat in eukaryotes. A primary sequence-based search in Arabidopsis thaliana revealed seven putatively plastid-localized Nats of which AT2G39000 (AtNAA70) showed the highest conservation of the acetyl-CoA binding pocket. The chloroplastic localization of AtNAA70 was demonstrated by transient expression of AtNAA70:YFP in Arabidopsis mesophyll protoplasts. Homology modeling uncovered a significant conservation of tertiary structural elements between human HsNAA50 and AtNAA70. The in vivo acetylation activity of AtNAA70 was demonstrated on a number of distinct protein N(α) -termini with a newly established global acetylome profiling test after expression of AtNAA70 in E. coli. AtNAA70 predominately acetylated proteins starting with M, A, S and T, providing an explanation for most protein N-termini acetylation events found in chloroplasts. Like HsNAA50, AtNAA70 displays N(ε) -acetyltransferase activity on three internal lysine residues. All MS data have been deposited in the ProteomeXchange with identifier PXD001947 (http://proteomecentral.proteomexchange.org/dataset/PXD001947).