The role of SPATA2 for inflammation and cell death
Cell Biology
Final Report Abstract
We have previously identified the protein SPATA2 as a member of the TNF Receptor 1- signaling complex (TNFR1-SC), and this project aimed at further understanding the role of SPATA2 for pro-inflammatory signaling. The original aims of the project were to characterize the role of SPATA2 for colonic inflammation, its regulation by caspase-8, its role for CYLD phosphorylation and whether SPATA2 has a role for the mid-gestation lethality of mice deficient for TAK-1 or caspase-8. In the course of mouse breeding experiments, we crossed Spata2-/- animals with Cyld-/- animals to explore possible consequences of the loss of SPATA2 in absence of CYLD. We observed that most double-knockout (DKO) offspring died right after birth, while the few surviving DKO animals exhibited reduced body weight and kinked tails. This was an unexpected phenotype, and we decided, in deviation of the original aims, to further explore the underlying molecular mechanism. Intestines of DKO mice showed signs of inflammation such as villus blunting, and DKO intestinal epithelial cells (IEC) exhibited increased pro-inflammatory cytokine expression as compared to IEC from Spata2-/- or Cyld-/- animals. Bone marrow derived macrophages (BMDM) from DKO mice displayed enhanced response to LPS and Pam3CSK4, and DKO mice had elevated numbers of marginal zone B cells. Mechanistically, upon TNFR1 stimulation, we observed increased M1-linked polyubiquitylation in DKO murine embryonic fibroblasts (MEF) as compared to Spata2-/- or Cyld-/- MEF. On the other hand, DKO MEF exhibited substantial protection from TNF/SMAC mimetic-induced apoptosis, while cell death was only partially prevented in Spata2-/- or Cyld-/- MEF. Therefore, we hypothesized that SPATA2 interferes with the activity of LUBAC, via competing for the binding of OTULIN, a deubiquitinase which had previously been shown to prevent the inhibitory autoubiquitylation of the linear ubiquitin chain assembly complex (LUBAC). Indeed, transfection experiments showed that increased SPATA2 levels reduced OTULIN binding to LUBAC and increased LUBAC autoubiquitylation. The interaction of SPATA2 and HOIP did not only depend on the PUB-interaction motif (PIM) in SPATA2, but also on a C-terminal SPATA2 zinc finger (ZnF) motif, which we found to mediate the homodimerization of SPATA2. Reconstitution of DKO MEF with SPATA2, but not with PIM- or ZnF-deficient SPATA2, reduced M1-ubiquitylation of RIPK1, and diminished NF-kB and JNK signaling. We could also show that the increased RIPK1 M1-ubiquitylation of DKO MEF upon TNFR1 stimulation depended on the presence of OTULIN, as it was not observed upon-CRISPRS/Cas9-mediated knockout of OTULIN in DKO MEF. In sum, our data provide a mechanism as to how SPATA2, independently of CYLD, restricts NF-kB and JNK signaling and pro-inflammatory gene expression.
Publications
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Keeping Cell Death in Check: Ubiquitylation-Dependent Control of TNFR1 and TLR Signaling. Frontiers in Cell and Developmental Biology, 7.
Griewahn, Laura; Köser, Aaron & Maurer, Ulrich
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SPATA2 and CYLD independently control pro-inflammatory signaling and B-cell homeostasis. Oral presentation, 18th TNF superfamily meeting, Les Diablerets, Switzerland
Maurer, U.
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SPATA2 and CYLD independently control proinflammatory signaling and B-cell homeostasis. Poster, 18th TNF superfamily meeting, Les Diablerets, Switzerland
Griewahn, L. & Maurer, U.
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SPATA2 and CYLD independently control proinflammatory signaling and B-cell homeostasis. Poster, Swiss Apoptosis and Autophagy Meeting, Bern, Switzerland
Griewahn, L. & Maurer, U.
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SPATA2 suppresses M1-linked ubiquitylation independently of CYLD. Oral presentation, EMBO Workshop ‘Dying in self-defense: Cell death signaling in animals and plants‘, Crete, Greece
Griewahn, L.
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SPATA2 restricts OTULIN-dependent LUBAC activity independently of CYLD. Cell Reports, 42(1), 111961.
Griewahn, Laura; Müller-Foxworth, Madeleine; Peintner, Lukas; Wissler, Manuela; Weiss, Martina; Brauns-Schubert, Prisca; Massoumi, Ramin; Borner, Christoph; Groß, Olaf; Yabal, Monica; Charvet, Céline & Maurer, Ulrich
