Proteom- und funktionelle Analyse der Glukokortikoid-induzierten Apoptose bei lymphoblastischen Leukämiezellen unter gleichzeitiger Gabe von 2-Desoxyglucose
Zusammenfassung der Projektergebnisse
Based on the reviewers’ recommendation, the project served as a technology core providing protein analyses to a KFO and associated projects. To that end, a multidimensional peptide separation platform for the differential quantitative analysis of subcellular proteomes (i.e., cytosolic, mitochondrial, and membrane-enriched) and phosphoproteomes using stable isotope labelling (iTRAQ) was implemented. Peptides were obtained by tryptic in-solution digestion and separated by either consecutive strong-cation exchange (SCX) and (acidic) reversed-phase chromatography (RP-HPLC) or a combination of pipette tip-based SCX-fractionation and two consecutive RP-HPLC dimensions at neutral and acidic pH, respectively. The latter, three-dimensional strategy yielded on average 50% more protein identifications by at least two unique peptides than the conventional two-dimensional approach. For phosphoproteome analysis, either TiO2-affinity or IMAC-based phosphopeptide enrichment were used prior to nanoLC/MS/MS on an AB Sciex TripleTOF 5600+ system. In addition, differential 2D-PAGE using fluorescence labelling (DIGE) of undigested cellular protein extracts was employed to detect differentially regulated proteins in S-methyl-5’-thioadenosine phosphorylase (MTAP) deficient and expressing melanoma cell lines. Following in-gel tryptic digestion, differential protein spots were identified by LC/MS/MS. Additionally, protein extracts were subjected to in-solution tryptic digestion and bottom-up shotgun proteomics using data-independent Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS) analysis. Regulation of most proteins could be confirmed by both approaches, but in some instances opposing directions of regulation were observed, which were attributed to posttranslational modifications, as these are typically not accounted for by SWATH-MS. For instance, the heterogeneous nuclear ribonucleoproteins HNRNPA2/B1 and HNRPK are known to be modified by methylation of arginine residues. Interestingly, methylosome protein 50 (MEP50), a regulatory factor of the methylosome complex, that is responsible for arginine methylation of proteins, was among the significantly regulated proteins (ratio +/-MTAP: 0.1245; p-value: 1.077E-06). Shotgun proteomics was also used to confirm paracrine factors secreted by activated hepatic stellate cells (HSC) that had been predicted to affect proliferation of hepatocellular carcinoma cells (HCC) based on a systems level analysis of observational transcriptome profiles of HCC cells cultivated in different HSC secretome-enriched media, combining reverse network engineering with causal effect estimation. Among the previously unknown cancer promoting stromal factors was Pregnancy-Associated Plasma Protein A (PAPPA), which contributed to activation of NFKB signaling and whose expression was associated with advanced stage HCC. The project also contributed to the identification of binding partners of the plasma-membrane-localized isoform of the voltage-dependent anion channel (VDAC). To that end, co-immunoprecipitation samples were first subjected to SDS-PAGE, followed by tryptic in-gel digestion of separated protein bands, whose chemical nature was determined by nano-LC-MS/MS-based peptide sequencing. Thereby, HSP-90 was identified as a potential interaction partner with high confidence. Finally, we employed a combination of differential protein and protein-interaction analysis in oral squamous cell carcinoma (OSCC) cell lines to show, that gamma-secretase is responsible for processing of P-cadherin, as inhibition of gamma-secretase resulted in a non-glycosylated full-length P-cadherin isoform of 100 kDa, thereby reducing cell adhesion in OSCC. Further, we could show, that c-Fos interacted in a collagen XVI-dependent manner with dyskerin, a small nucleolar ribonucleoprotein, whose upregulation correlated with proliferation of OSCC cells.
Projektbezogene Publikationen (Auswahl)
- (2012) Early changes in the liver-soluble proteome from mice fed a nonalcoholic steatohepatitis inducing diet. Proteomics 12:1437-1451
Thomas, A., A. P. Stevens, M. S. Klein, C. Hellerbrand, K. Dettmer, W. Gronwald, P. J. Oefner, and J. Reinders
(Siehe online unter https://doi.org/10.1002/pmic.201100628) - (2012) Poplar extrafloral nectar is protected against plant and human pathogenic fungus. Mol Plant 5:1157-1159
Escalante-Perez, M., M. Jaborsky, J. Reinders, O. Kurzai, R. Hedrich, and P. Ache
(Siehe online unter https://doi.org/10.1093/mp/sss072) - (2012) Raf kinases mediate the phosphorylation of eukaryotic translation elongation factor 1A and regulate its stability in eukaryotic cells. Cell Death Dis 3:e276
Sanges, C., C. Scheuermann, R. P. Zahedi, A. Sickmann, A. Lamberti, N. Migliaccio, A. Baljuls, M. Marra, S. Zappavigna, J. Reinders, U. Rapp, A. Abbruzzese, M. Caraglia, and P. Arcari
(Siehe online unter https://doi.org/10.1038/cddis.2012.16) - (2013) Changes in the hepatic mitochondrial and membrane proteome in mice fed a non-alcoholic steatohepatitis inducing diet. J Proteomics 80C:107-122
Thomas, A., M. S. Klein, A. P. Stevens, J. Reinders, C. Hellerbrand, K. Dettmer, W. Gronwald, P. J. Oefner, and J. Reinders
(Siehe online unter https://doi.org/10.1016/j.jprot.2012.12.027) - (2013) N-cadherin promoter polymorphisms and risk of osteoarthritis. Faseb J, Oct 22
Ruedel, A., K. Stark, S. Kaufmann, R. Bauer, J. Reinders, J. Rovensky, S. Blazickova, P. J. Oefner, and A. K. Bosserhoff
(Siehe online unter https://doi.org/10.1096/fj.13-238295) - (2013) Presenilin 1/γsecretase modulates P-cadherin processing and influences cell adhesion in oral squamous cell carcinoma cell lines. Carcinogenesis 34(11):2622-26288
Bauer, K., M. Gosau, J. Reinders, P. Oefner, T. E. Reichert, and R. Bauer
(Siehe online unter https://doi.org/10.1093/carcin/bgt211) - (2013) Strong reduction of AGO2 expression in melanoma and cellular consequences. Br J Cancer 109:3116-3124
Voller, D., J. Reinders, G. Meister, and A. K. Bosserhoff
(Siehe online unter https://doi.org/10.1038/bjc.2013.646) - (2014) Collagen XVI induces expression of MMP9 via modulation of AP-1 transcription factors and facilitates invasion of oral squamous cell carcinoma. PLoS One 9(1): e86777
Bedal, K. B., S. Grässel, P. J. Oefner, J. Reinders, T. E. Reichert, and R. Bauer
(Siehe online unter https://doi.org/10.1371/journal.pone.0086777) - (2014) Mistargeting of peroxisomal EHHADH and inherited renal Fanconi's syndrome. N Engl J Med 370:129-138
Klootwijk, E. D., M. Reichold, A. Helip-Wooley, A. Tolaymat, C. Broeker, S. L. Robinette, J. Reinders, D. Peindl, K. Renner, K. Eberhart, N. Assmann, P. J. Oefner, K. Dettmer, C. Sterner, J. Schroeder, N. Zorger, R. Witzgall, S. W. Reinhold, H. C. Stanescu, D. Bockenhauer, G. Jaureguiberry, H. Courtneidge, A. M. Hall, A. D. Wijeyesekera, E. Holmes, J. K. Nicholson, K. O'Brien, I. Bernardini, D. M. Krasnewich, M. Arcos-Burgos, Y. Izumi, H. Nonoguchi, Y. Jia, J. K. Reddy, M. Ilyas, R. J. Unwin, W. A. Gahl, R. Warth, and R. Kleta
(Siehe online unter https://doi.org/10.1056/NEJMoa1307581)
