The proteomic analysis of endogenous FAT10 substrates identifies p62/SQSTM1 as a substrate of FAT10ylation.
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 pro-inflammatory 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.
Biotechnology Institute Thurgau at the University of Constance, CH-8280 Kreuzlingen,  Switzerland.
Annette
A
Aichem
Birte
B
Kalveram
Valentina
V
Spinnenhirn
Kathrin
K
Kluge
Nicola
N
Catone
Terje
T
Johansen
Marcus
M
Groettrup
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D020816:Amino Acid Motifs; D001343:Autophagy / Q000187:drug effects; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D007371:Interferon-gamma / Q000494:pharmacology; D008239:Lysine / Q000378:metabolism; D013058:Mass Spectrometry; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D040901:Proteomics; D000071456:Sequestosome-1 Protein; D013379:Substrate Specificity / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014452:Ubiquitins / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D014409:Tumor Necrosis Factor-alpha; C109184:UBD protein, human; D014452:Ubiquitins; D007371:Interferon-gamma; D008239:Lysine