IIB   20738
INSTITUTO DE INVESTIGACIONES BIOLOGICAS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Proteome Turnover analysis reveals substrates and physiological role of membrane proteases
Autor/es:
ROBERTO A PAGGI; ANSGAR POETSCH; CHRISTIAN TROETSCHEL; CARINA RAMALLO GUEVARA; MICAELA CERLETTI; ROSANA E DE CASTRO
Lugar:
Taipei
Reunión:
Congreso; 15th Human Proteome Organization World Congress; 2016
Institución organizadora:
Human Proteome Organization
Resumen:
Proteome Turnover analysis revealssubstrates and physiological role of membrane proteasesChristian Trötschel1; Micaela Cerletti1; Roberto A. Paggi1; Carina Ramallo-Guevara2; Rosana de Castro1; Ansgar Poetsch*,2 1IIB-CONICET-UNMdP Mar del Plata,Argentina; 2Ruhr University Bochum,Germany Introduction and Objectives Due to technical challenges, substrates andphysiological role of membrane proteases remain largely elusive. We reasonedthat a recently developed proteome turnover approach, employing metaboliclabeling for the dissection of protein synthesis and degradation, couldadequately address this challenge. Two prokaryotic/microbial membraneproteases, LonB from the archaeon Haloferax volcanii and FtsH from thebacterium Corynebacterium glutamicum were subjected to proteome turnoveranalysis in strains to discover substrates based on changing proteolysiskinetics.Methods For H. volcanii, a conditionallonB mutant producing suboptimal or physiological protease levels wereused. The mutant strain was first cultivated in Hv-Min medium with 14NH4Cl in absence of trp to mid-exponentialphase and then shifted to fresh medium containing 15N. Cultures were supplemented with 1 mM trpto induce LonB synthesis. For C. glutamicum, the WT and ftsH deletionmutant were grown on MME-SN medium containing 14NH4Cl as nitrogen source till the end ofthe exponential growth phase and then switched to 15N to monitor dynamic label incorporation.Different time points were compared via an internal standard grown on 13C-glucose. An LTQ-Orbitrap Elite wasused in DDA mode. Proteins were identified with Sequest embedded in ProteomeDiscoverer 1.4 and protein turnover, as well as statistical analysis, wasachieved with QuPE (Albaum et al., MCP 2012).Results and Discussion Of the 1100 quantified proteins in the H.volcanii membrane fraction, 204 proteins displayed significantly changeddegradation rates. Most noteworthy was phytoene synthase (PS), a key enzyme incarotenoid biosynthesis which showed a degradation pattern consistent with aLonB target. This makes sense considering that Lon deficiency causeshyperpigmentation. Interestingly, the concomitant increase of PS synthesissuggests a feedback regulation mechanism. Furthermore, several ABC transporters(among others) could be LonB substrates.Deletion of the ftsH gene did not result in a C. glutamicum growth phenotype inagreement with previous works (Luedke et al. BMC Microbiology 2007). Of the1200 quantified proteins in the membrane fraction, 211 differed in degradation.A striking decrease of degradation in the ftsH deletion strain wasobserved for the succinate dehydrogenase subunit A., though other proteins ofenergy metabolism were affected too.Conclusion This study demonstrates the great potentialof proteome turnover analysis for a sensitive and comprehensive analysis ofmembrane proteases and substrate discovery. Of note, the two opposingstrategies, i.e. increasing the protease amount in H. volcanii anddecreasing the protease amount in C. glutamicum, were found equallysuitable and successful to uncover substrates. We anticipate that proteometurnover analysis will substantially facilitate the discovery of substratecandidates not only of AAA-type, but also other substrate-degrading (membrane)proteases.Keywords AAAProtease, membrane proteolysis, proteome turnoverPresenting Type Selection Poster & would like to be considered as the Short Presentation(Accepted as Oral Presentation will also be requested to present as Poster)Category Selection 14.Proteome Dynamics: Turnover &Degradomics