Dr. Oliver Schilling
Proteolysis in Tumorigenesis and Tumor-Stroma Interaction
Proteolysis is an irreversible post-translational modification that affects every protein. The large number of genetically encoded proteases in man (> 560) illustrates the biological importance of proteolysis. Proteolysis plays a crucial role in cellular signalling and pericellular homeostasis as generation of stable cleavage products from precursors leads to neo–proteins with altered, often antagonistic activities. Elevated proteolytic activity is a hallmark of many tumors. Many proteases promote tumor development. However, depending on the tumor context, some proteases, such as prostasin and neprilysin have tumor-suppressing roles.
The most crucial task in present proteolysis research is elucidation of protease substrate repertoires. Most cleavage events have remained elusive for most proteases. This is a serious deficit since proteases alter protein abundance through degradation and protein function by generating stable cleavage products. Recent key inventions in the area of “protease proteomics” now enable elucidation of protease substrate repertoires in vivo or in the cellular context. I have co–invented these techniques. Published in Nature Biotechnology and Nature Methods, these strategies enable a focused analysis of native and proteolytically generated protein N- or C-termini. Through combination with differential protease expression and stable isotope labeling, induced cleavage events are distinguished from background proteolysis and protease–specific substrate repertoires are determined. Protease substrate profiling is performed with loss– and gain–of–function systems in a panel of cancer cell lines together with determination of cellular parameters that mirror tumor aggressiveness, such as proliferation, invasion, and migration.
The combination of N– and C–terminal analysis is pivotal for the analysis of shedding by cell surface proteases irrespective of the transmembrane orientation of the substrate. Processing of selected substrates is studied in detail in order to understand how N– or C–terminal truncations and/or shedding alters key characteristics of a substrate. Examples include chemotactic properties of cytokines or activity status of enzymes. The aim is to understand how individual cleavage events contribute to an (anti)-tumor activity of a given protease. To this end we determine the ratio of native vs. processed substrate in wild–type control samples. In protease-depleted cells (silenced gene expression), we reconstruct this situation by dosable expression of the cleavage product and dosable repression of the intact substrate, followed by cellular characterization. By applying concepts and tools of synthetic biology we reconstruct individual cleavage events in the absence of the executing protease. This interplay of analysis and synthetic reconstruction is a unique opportunity to decipher proteolytic networks involved in cell-cell signaling.
10 selected publications
- Identification of Protease Specificity by Combining Proteome-Derived Peptide Libraries and Quantitative Proteomics.
Biniossek ML, Niemer M, Maksimchuk K, Mayer B, Fuchs J, Huesgen PF, McCafferty DG, Turk B, Fritz G, Mayer J, Haecker G, Mach L, Schilling O.
Mol Cell Proteomics. 2016 Jul;15(7):2515-24.
- Formalin-Fixed, Paraffin-Embedded Tissues (FFPE) as a Robust Source for the Profiling of Native and Protease-Generated Protein Amino Termini.
Lai ZW, Weisser J, Nilse L, Costa F, Keller E, Tholen M, Kizhakkedathu JN, Biniossek M, Bronsert P, Schilling O.
Mol Cell Proteomics. 2016 Jun;15(6):2203-13.
- Fibroblast activation protein-α, a stromal cell surface protease, shapes key features of cancer associated fibroblasts through proteome and degradome alterations.
Koczorowska MM, Tholen S, Bucher F, Lutz L, Kizhakkedathu JN, De Wever O, Wellner UF, Biniossek ML, Stahl A, Lassmann S, Schilling O.
Mol Oncol. 2016 Jan;10(1):40-58.
- The stromal cell-surface protease fibroblast activation protein-α localizes to lipid rafts and is recruited to invadopodia.
Knopf JD, Tholen S, Koczorowska MM, De Wever O, Biniossek ML, Schilling O.
Biochim Biophys Acta. 2015 Oct;1853(10 Pt A):2515-25
- miR-200c dampens cancer cell migration via regulation of protein kinase A subunits.
Sigloch FC, Burk UC, Biniossek ML, Brabletz T, Schilling O.
Oncotarget. 2015 Sep 15;6(27):23874-89.
- Secretome and degradome profiling shows that Kallikrein-related peptidases 4, 5, 6, and 7 induce TGFβ-1 signaling in ovarian cancer cells.
Shahinian H, Loessner D, Biniossek ML, Kizhakkedathu JN, Clements JA, Magdolen V, Schilling O.
Mol Oncol. 2014 Feb;8(1):68-82.
- Deletion of cysteine cathepsins B or L yields differential impacts on murine skin proteome and degradome.
Tholen S, Biniossek ML, Gansz M, Gomez-Auli A, Bengsch F, Noel A, Kizhakkedathu JN, Boerries M, Busch H, Reinheckel T, Schilling O.
Mol Cell Proteomics. 2013 Mar;12(3):611-25.
- Proteome-wide analysis of protein carboxy termini: C terminomics.
Schilling O, Barré O, Huesgen PF, Overall CM.
Nat Methods. 2010 Jul;7(7):508-11
- Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products.
Kleifeld O, Doucet A, auf dem Keller U, Prudova A, Schilling O, Kainthan RK, Starr AE, Foster LJ, Kizhakkedathu JN, Overall CM.
Nat Biotechnol. 2010 Mar;28(3):281-8.
- Proteome-derived, database-searchable peptide libraries for identifying protease cleavage sites.
Schilling O, Overall CM.
Nat Biotechnol. 2008 Jun;26(6):685-94.