Analysis of the Proteolytic Processing of ABCA3: Identification of Cleavage Site and Involved Proteases

Rationale ABCA3 is a lipid transporter in the limiting membrane of lamellar bodies in alveolar type II cells. Mutations in the ABCA3 gene cause respiratory distress syndrome in new-borns and childhood interstitial lung disease. ABCA3 is N-terminally cleaved by an as yet unknown protease, a process believed to regulate ABCA3 activity. Methods The exact site where ABCA3 is cleaved was localized using mass spectrometry (MS). Proteases involved in ABCA3 processing were identified using small molecule inhibitors and siRNA mediated gene knockdown. Results were verified by in vitro digestion of a synthetic peptide substrate mimicking ABCA3's cleavage region, followed by MS analysis. Results We found that cleavage of ABCA3 occurs after Lys174 which is located in the proteins' first luminal loop. Inhibition of cathepsin L and, to a lesser extent, cathepsin B resulted in attenuation of ABCA3 cleavage. Both enzymes showed activity against the ABCA3 peptide in vitro with cathepsin L being more active. Conclusion We show here that, like some other proteins of the lysosomal membrane, ABCA3 is a substrate of cathepsin L. Therefore, cathepsin L may represent a potential target to therapeutically influence ABCA3 activity in ABCA3-associated lung disease

Structure and Dynamics of Photosystem II Light-Harvesting Complex Revealed by High-Resolution FTICR Mass Spectrometric Proteome Analysis

Structure and dynamics of membrane-bound light-harvesting pigment protein complexes (LHCs), which collect and transmit light energy for photosynthesis and thereby play an essential role in the regulation of photosynthesis and photoprotection, were identified and characterized using high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). LHCs from photosystem II (LHCII) were isolated from the thylakoid membrane of Arabidopsis thaliana leaves after light stress treatment using sucrose density gradient centrifugation, and separated by gel-filtration into LHCII subcomplexes. Using reversed-phase high-performance liquid chromatography and two-dimensional (2D) gel electrophoresis, the LHCII proteins, Lhcb1 6 and fibrillins, were efficiently separated and identified by FTICR-MS. Some of the LHCII subcomplexes were shown to migrate from photosystem II to photosystem I as a result of short-term adaptation to changes in light intensity. In the mobile LHCII subcomplexes, decreased levels of fibrillins and a modified composition of LHCII protein isoforms were identified compared to the tightly bound LHCII subcomplexes. In addition, FTICR-MS analysis revealed several oxidative modifications of LHCII proteins. A number of protein spots in 2D gels were found to contain a mixture of proteins, illustrating the feasibility of high-resolution mass spectrometry to identify proteins that remain unseparated in 2D gels even upon extended pH gradients

Analysis of the Proteolytic Processing of ABCA3: Identification of Cleavage Site and Involved Proteases

Rationale ABCA3 is a lipid transporter in the limiting membrane of lamellar bodies in alveolar type II cells. Mutations in the ABCA3 gene cause respiratory distress syndrome in new-borns and childhood interstitial lung disease. ABCA3 is N-terminally cleaved by an as yet unknown protease, a process believed to regulate ABCA3 activity. Methods The exact site where ABCA3 is cleaved was localized using mass spectrometry (MS). Proteases involved in ABCA3 processing were identified using small molecule inhibitors and siRNA mediated gene knockdown. Results were verified by in vitro digestion of a synthetic peptide substrate mimicking ABCA3's cleavage region, followed by MS analysis. Results We found that cleavage of ABCA3 occurs after Lys174 which is located in the proteins' first luminal loop. Inhibition of cathepsin L and, to a lesser extent, cathepsin B resulted in attenuation of ABCA3 cleavage. Both enzymes showed activity against the ABCA3 peptide in vitro with cathepsin L being more active. Conclusion We show here that, like some other proteins of the lysosomal membrane, ABCA3 is a substrate of cathepsin L. Therefore, cathepsin L may represent a potential target to therapeutically influence ABCA3 activity in ABCA3-associated lung disease

Degradation and oxidation postmortem of myofibrillar proteins in porcine skeleton muscle revealed by high resolution mass spectrometric proteome analysis

Early postmortem changes of porcine muscle proteins including the rate and extent of pH decline, proteolysis and oxidation are key factors influencing the loss of water in meat, and proteolytic degradation may result in shrinking of muscle cells and drip loss. We report here the identification and structural characterisation of post-mortem degradation and oxidation of myofibrillar proteins using high resolution mass spectrometric proteomics. Soluble muscle proteins from M. Longissimus dorsi obtained 48 h postmortem at different drip loss were separated by two-dimensional gel electrophoresis (2D-PAGE), and degradation products were identified by Fourier-transform ion cyclotron resonance mass spectrometry. Oxidation products were detected by 2D-oxyblot analysis of 2,4-dinitrophenylhydrazine (DNPH)-treated proteins using an anti-DNP antibody, and selected spots were identified by liquid chromatography-tandem mass spectrometry (LC–MS/MS). Postmortem denaturation at low drip loss was found for four contractile proteins, myosin-light chain-1; myosin regulatory light chain; α-β-tropomyosin and α-actin. The combination of 2D-PAGE and FTICR-MS was found to be a powerful approach for identification of muscle protein degradation products, providing identification of several truncation forms of creatine kinase and troponin T. The comparison of 2D-oxyblot and silver-stained 2D-gels at low and high drip loss revealed approximately 70 oxidatively modified proteins from muscle cell lysate. Oxidative modifications, representing possible biomarker candidates, were identified at Lys-170 of creatine kinase (4-hydroxynonenal), Lys-326 of actin (amino-adipic semialdehyde), and at W-169 (kynurenine) of triosephosphate isomerase

Lung alveolar proteomics of bronchoalveolar lavage from a pulmonary alveolar proteinosis patient using high-resolution FTICR mass spectrometry

High-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometry was developed and applied to the proteome analysis of bronchoalveolar lavage fluid (BALF) from a patient with pulmonary alveolar proteinosis. With use of 1-D and 2-D gel electrophoresis, surfactant protein A (SP-A) and other surfactant-related lung alveolar proteins were efficiently separated and identified by matrix-assisted laser desorption/ionization FTICR mass spectrometry . Low molecular mass BALF proteins were separated using a gradient 2-D gel. An efficient extraction/precipitation system was developed and used for the enrichment of surfactant proteins. The result of the BALF proteome analysis show the presence of several isoforms of SP-A, in which an N-non-glycosylierte form and several proline hydroxylations were identified. Furthermore, a number of protein spots were found to contain a mixture of proteins unresolved by 2-D gel electrophoresis, illustrating the feasibility of high-resolution mass spectrometry to provide identifications of proteins that remain unseparated in 2-D gels even upon extended pH gradients

Widespread bacterial lysine degradation proceeding via glutarate and L-2-hydroxyglutarate

Lysine degradation has remained elusive in many organisms including Escherichia coli. Here we report catabolism of lysine to succinate in E. coli involving glutarate and L-2-hydroxyglutarate as intermediates. We show that CsiD acts as an α-ketoglutarate-dependent dioxygenase catalysing hydroxylation of glutarate to L-2-hydroxyglutarate. CsiD is found widespread in bacteria. We present crystal structures of CsiD in complex with glutarate, succinate, and the inhibitor N-oxalyl-glycine, demonstrating strong discrimination between the structurally related ligands. We show that L-2-hydroxyglutarate is converted to α-ketoglutarate by LhgO acting as a membrane-bound, ubiquinone-linked dehydrogenase. Lysine enters the pathway via 5-aminovalerate by the promiscuous enzymes GabT and GabD. We demonstrate that repression of the pathway by CsiR is relieved upon glutarate binding. In conclusion, lysine degradation provides an important link in central metabolism. Our results imply the gut microbiome as a potential source of glutarate and L-2-hydroxyglutarate associated with human diseases such as cancer and organic acidurias.publishe

Analysis of ABCA3 cleavage region.

<p><b>A</b>: ABCA3 containing gel bands were excised, digested with trypsin and analysed. <b>B</b>: Partial sequences of unprocessed (190 kDa) and processed (170 kDa) ABCA3 forms identified by LC-MS/MS. Identified sequence parts of ABCA3 precursor protein including HA-TAG with spacer (shown in blue) are underlined. Sequences identified only in unprocessed form and absent in processed form are shown in red. Cleavage region (162–174) is highlighted in yellow. <b>C</b>: Peptide intensity ratios from MS ion chromatograms (three measurements), leading to the predicted cleavage region.</p

Partial sequence (162–174) is present in unprocessed ABCA3 and missing in processed protein form.

<p><b>A</b>: Intensity profiles of an <i>m/z</i> range corresponding to the monoisotopic peak of the doubly-charged ion at <i>m/z</i> 819.885 (<i>m/z</i> window ±5 ppm) for 190 kDa and 170 kDa gel bands acquired in Orbitrap. <b>B</b>: Identification of the ABCA3 tryptic peptide comprising amino acid residues 162–174 of the precursor protein and containing methionine sulfoxide at position 164. MS/MS spectra after CID fragmentation measured in the LTQ ion trap are shown. Major <i>b</i> and <i>y</i> ions are labelled on the spectra and all fragment ions obtained are marked on the identified sequence. The ion at <i>m/z</i> 811 corresponds to the loss of water from the parent ion.</p

Cathepsin L cleaves ABCA3 peptide preferentially after Lys¹⁷⁴.

<p>Shown are signal intensities of C-terminal cathepsins L and B cleavage products of ABCA3 peptide (151–194) within the sequence </p><p>¹⁶²NYMWTQTGSFFLK¹⁷⁴</p> analyzed by LC-MS/MS. Extracted MS ion chromatograms of the accurate m/z values ±5 ppm related to the corresponding peptide ions (peak areas) from three experiments were used for the diagram.<p></p

Effect of knockdown of single proteases on ABCA3 processing.

<p>Expression of lysosomal cathepsins was silenced using siRNA mediated knockdown and ABCA3 cleavage was assessed by Western blotting. Experiments were performed thrice, every time in triplicates. Representative Immunoblots are shown.</p