Regulation of the internalization and intracellular transport of Tumor Necrosis-Factor Receptor 1 (TNF-R1) and its impact on signal transduction and biological outcomes of TNF
Final Report Abstract
In our project, we presented a versatile toolkit for the targeted isolation of various sorts of membrane enclosed cellular organelles from cytokine activated cells(receptosomers) using selective labelling with tagged ligands and/or antibodies combined with magnetic nanoparticles and a novel free-flow magnetic chamber. With this approach, spacial and temporal intracellular signaling events eminating from trafficking organalles can be analyzed. By investigating the regulation of TNF-R1 regulation, we identified the constitutive palmitoylation of TNF-R1. Mutation analysis of the putative palmitoylation sites in U937 and HeLa80 cells revealed that TNF-R1 is triple palmitoylated at the cysteines C223, C248, C304. Mutation of one putative palmitoylation site C248 alreadychanged different signaling outcomes of TNF-R1, regarding cell death induction and NF-κB activation by interfering with its subcellular localization and thus, reducing the receptor surface expression. In addition, we could identify the protein thioesterase (PTE) APT2 which catalyzes the depalmitoylation of TNF-R1 upon TNF stimulation. The inhibition of APT2 altered the biological outcome of the TNF-R1 signal transduction, regarding cell death induction, NF-κB activation, receptor internalization and subcellular translocation of TNFR-1. Activated TNF-R1 was trapped at the cell surface by APT2 inhibition which resulted in an increase of apoptosis caused by the neutral sphingomyelinase (nSMase) mediated production of ceramide. Also, additional palmitoylated proteins in TNF signaling were identified: It could be shown that the protein acyltransferase (PAT) DHHC5 is involved in the intracellular maturation, transport and apoptosis induction of TNF-receptosomes after receptor internalization. Our investigations on down-stream signaling events mediatedb from internalized TNF receptosomes led to the identification of the chaperon HSP90β as target for TNF-induced cathepsin D cleavage within the lysosomal death pathway of TNF-R1. Co-incubation of cells with either TNF or TRAIL in combination with the HSP90β inhibitor KUNB105 but not HSP90α selective inhibition promotes apoptosis induction. In addition, we identified the pro survival transcription factor hypoxia induced factor 1 alpha (HIF1α) as a novel HSDP90β substrate and validate its ligand:inhibitor triggered degradation and ist link to apoptosis induction. The selective inhibition of HSP90 isoforms together with death ligand stimulation may provide novel strategies for therapy of inflammatory diseases or cancer, in future.
Publications
- Differences and similarities in TRAIL- and TNF-mediated necroptotic signaling in cancer cells. Mol. Cell Biol. 2016 Aug 15
Sosna, J., Philipp, S., Plengea, J., Arenz, C., Pinkert, T., Kalthoff, H., Trauzold, A., Schütze, S., Adam, D.
(See online at https://doi.org/10.1128/MCB.00941-15) - TNF induced cleavage of HSP90 by Cathepsin D potentiates apoptotic cell death. Oncotarget, 2016. Nov. 15:7(46): 75774-75789
Fritsch, J., Fickers, R., Klawitter, J., Särchen, V., Zingler, P., Adam, D., Janssen, O., Krause, E., Schütze, S
(See online at https://doi.org/10.18632/oncotarget.12411) - Biphasic activation of acid sphingomyelinase by CD95 Ligand stimulation. Oncotarget, 2017, Mar 21;8(12):20067-20085
Stephan, M., Edelmann, B., Janssen, O., Schütze, S. Fritsch, J.
(See online at https://doi.org/10.18632/oncotarget.15379) - Role of ubiqutination and proteolysis in the regulation of pro- and anti-apoptotic TNF-R1 signaling. BBA – Mol. Cell Res. 2017 Jul 29. pii: S0167-4889(17)30203-3
Fritsch, J., Zingler, P., Särchen, V., Heck, A.-L., Schütze, S
(See online at https://doi.org/10.1016/j.bbamcr.2017.07.017) - A toolbox for the immunomagnetic purifiction of signaling organelles. Traffic. 2019 Mar;20(3):246-258
Fritsch J, Tchikov V, Hennig L, Lucius R, Schütze S
(See online at https://doi.org/10.1111/tra.12631) - Palmitoylation is required for TNF-R1 signaling. Cell Commun Signal. 2019 Aug 5;17(1):90
Zingler P, Särchen V, Glatter T, Caning L, Saggau C, Kathayat RS, Dickinson BC, Adam D, Schneider-Brachert W, Schütze S, Fritsch J
(See online at https://doi.org/10.1186/s12964-019-0405-8)
