Kluge, Kathrin


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Characterisation of the Interaction between FAT10 and its Substrate Protein p62

2014, Kluge, Kathrin

The ubiquitin-like modifier FAT10 has a C-terminal diglycine motif which is required for the conjugation to lysines (K) in its substrate proteins via isopeptide bonds. The biological function of FAT10, besides the proteasomal degradation of substrate proteins remains obscure. Sequestosome 1 (SQSTM1/p62) was found to be mono-FAT10ylated at several lysines and a non-covalent interaction between FAT10 and p62 was detectable too. The FAT10ylation of p62 leads to its proteasomal degradation. p62 can interact with a large number of proteins and changes its face by altering the binding partner(s). It is required for the formation of ubiquitylated protein aggregates and was found to act as a shuttling factor which links those aggregates to the autophagy machinery.

The aim of this study was to further characterise the covalent and non-covalent interaction between FAT10 and p62. Therefore, in vitro interaction studies with either recombinant proteins or proteins which were expressed in HEK293T cells were performed. By using a lysineless p62 mutant, the FAT10ylation was not always completely abolished. p62 deletion proteins were used in order to identify the covalent and non-covalent interaction domains. The deletion of the PB1, the NPI, the TRAF or the N-terminal PEST domain of p62 were found to impede the FAT10ylation. By the mutation of single lysines, it was shown that the lysines of p62 which become FAT10ylated seem to be redundant. For the non-covalent interaction with FAT10, the ZZ, the LIR, the CPI and the C-terminus of the C-terminal PEST domain of HA-p62 seem to be dispensable. By using non-phosphorylation, phospho-mimicking and non-oligomerisation p62 mutants it was shown that neither the phosphorylation status at seine 403, nor the oligomerisation capability of p62 seem to be prerequisites for the interaction with FAT10. An isolated PB1 domain of p62 did not interact with Flag-FAT10, neither covalently or non-covalently. According to the cycloheximide chase, proteasomal degradation rather than autophagosomal degradation is involved in the degradation of the FAT10-p62 conjugate. There was no interaction detectable between FAT10 and other autophagic adaptor proteins such as NBR1, NDP52 and OPTN.


The proteomic analysis of endogenous FAT10 substrates identifies p62/SQSTM1 as a substrate of FAT10ylation

2012-10-01, Aichem, Annette, Kalveram, Birte, Spinnenhirn, Valentina, Kluge, Kathrin, Catone, Nicola, Johansen, Terje, Gröttrup, Marcus

FAT10 is a ubiquitin-like modifier proposed to function in apoptosis induction, cell cycle control and NF-kB 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)cand tumor necrosis factor (TNF)alpha-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.