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Projekt Druckansicht

Proteom- und funktionelle Analyse der Glukokortikoid-induzierten Apoptose bei lymphoblastischen Leukämiezellen unter gleichzeitiger Gabe von 2-Desoxyglucose

Fachliche Zuordnung Hämatologie, Onkologie
Förderung Förderung von 2011 bis 2015
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 190230491
 
Erstellungsjahr 2019

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)

 
 

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