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Aichem A.,Biotechnology Institute Thurgau at the University of Constance | Kalveram B.,University of Konstanz | Spinnenhirn V.,University of Konstanz | Kluge K.,University of Konstanz | And 4 more authors.
Journal of Cell Science | Year: 2012

FAT10 is a ubiquitin-like modifier proposed to function in apoptosis induction, cell cycle control and NF-κB activation. Upon induction by pro-inflammatory cytokines, hundreds of endogenous substrates become covalently conjugated to FAT10 leading to their proteasomal degradation. Nevertheless, only three substrates have been identified so far to which FAT10 becomes covalently attached through a non-reducible isopeptide bond, and these are the FAT10-conjugating enzyme USE1 which auto-FAT10ylates itself in cis, the tumor suppressor p53 and the ubiquitin-activating enzyme UBE1 (UBA1). To identify additional FAT10 substrates and interaction partners, we used a new monoclonal FAT10-specific antibody to immunopurify endogenous FAT10 conjugates from interferon (IFN)γ- and tumor necrosis factor (TNF)α-stimulated cells for identification by mass spectrometry. In addition to two already known FAT10-interacting proteins, histone deacetylase 6 and UBA6, we identified 569 novel FAT10-interacting proteins involved in different functional pathways such as autophagy, cell cycle regulation, apoptosis and cancer. Thirty-one percent of all identified proteins were categorized as putative covalently linked substrates. One of the identified proteins, the autophagosomal receptor p62/SQSTM1, was further investigated. p62 becomes covalently mono-FAT10ylated at several lysines, and FAT10 colocalizes with p62 in p62 bodies. Strikingly, FAT10ylation of p62 leads to its proteasomal degradation, and prolonged induction of endogenous FAT10 expression by proinflammatory cytokines leads to a decrease of endogenous p62. The elucidation of the FAT10 degradome should enable a better understanding of why FAT10 has evolved as an additional transferable tag for proteasomal degradation. © 2012. Published by The Company of Biologists Ltd.

Moebius J.,University of Konstanz | Van Den Broek M.,University of Zürich | Groettrup M.,University of Konstanz | Groettrup M.,Biotechnology Institute Thurgau at the University of Constance | And 2 more authors.
European Journal of Immunology | Year: 2010

Immunoproteasomes containing the IFN-inducible subunits β1i (LMP2), β2i (MECL-1) and β5i (LMP7) alter proteasomal cleavage preference and optimize the generation of peptide ligands of MHC class I molecules. Here, we report on an unexpected new function of immunoproteasome subunits for the survival and expansion of CD4 + and CD8 + T cells during viral infection of mice. The effect of immunoproteasome subunit deficiency on T-cell survival upon adoptive transfer was most prominent for the lack of LMP7 followed by MECL-1 and LMP2. The survival of T cells in uninfected mice or the homeostatic expansion after transfer into RAG-2 -/- mice was not affected by the lack of the immunosubunits. Lymphocytic choriomeningitis virus (LCMV)-specific CD8 + T cells lacking LMP7 or MECL-1 started to divide after transfer into LCMV-infected mice but experienced a considerable cell loss within 2 days after transfer. We provide strong evidence that the loss of immunoproteasome-deficient T cells after transfer is not a consequence of graft rejection by the host, but instead is based on the requirement for immunoproteasomes for the survival of T cells in LCMV-infected mice. Therefore, the immunoproteasome may qualify as a potential new target for the suppression of undesired proinflammatory T-cell responses. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Basler M.,Biotechnology Institute Thurgau at the University of Constance | Basler M.,University of Konstanz | Groettrup M.,Biotechnology Institute Thurgau at the University of Constance | Groettrup M.,University of Konstanz
Methods in Molecular Biology | Year: 2013

The major histocompatibility complex (MHC) class I restricted pathway of antigen processing allows the presentation of intracellular antigens to cytotoxic T lymphocytes. The proteasome is the main protease in the cytoplasm and the nucleus, which is responsible for the generation of most peptide ligands of MHC-I molecules. Peptides produced by the proteasome can be further trimmed or destroyed by numerous cytosolic or endoplasmic reticulum (ER) lumenal proteases. Small molecule inhibitors are useful tools for probing the role of proteases in MHC class I antigen processing. Here, we describe different methods to test the impact of protease inhibitors in antigen presentation assays. © Springer Science+Business Media, LLC 2013.

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