FAT10, a Ubiquitin-Independent Signal for Proteasomal Degradation

Abstract

FAT10 is a small ubiquitin-like modifier that is encoded in the major histocompatibility complex and is synergistically inducible by tumor necrosis factor alpha and gamma interferon. It is composed of two ubiquitinlike domains and possesses a free C-terminal diglycine motif that is required for the formation of FAT10 conjugates. Here we show that unconjugated FAT10 and a FAT10 conjugate were rapidly degraded by the proteasome at a similar rate. Fusion of FAT10 to the N terminus of very long-lived proteins enhanced their degradation rate as potently as fusion with ubiquitin did. FAT10-green fluorescent protein fusion proteins were not cleaved but entirely degraded, suggesting that FAT10-specific deconjugating enzymes were not present in the analyzed cell lines. Interestingly, the prevention of ubiquitylation of FAT10 by mutation of all lysines or by expression in ubiquitylation-deficient cells did not affect FAT10 degradation. Thus, conjugation with FAT10 is an alternative and ubiquitin-independent targeting mechanism for degradation by the proteasome, which, in contrast to polyubiquitylation, is cytokine inducible and irreversible.The ubiquitin (Ub)-proteasome pathway is the main system for the targeted degradation of intracellular proteins (37). Depending on the metabolic and functional requirement of a cell, regulatory proteins like cell cycle regulators, transcription factors, or key enzymes can be specifically selected for degradation by the 26S proteasome. The basis for selectivity does not lie in the protease itself but rather in the selective covalent modification of target proteins with Lys48-linked polyubiquitin chains. Polyubiquitylation is achieved by an enzymatic cascade of a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). The specificity of substrate recognition is afforded by the numerous ubiquitin ligases which selectively bind a substrate as well as an E2 enzyme, thus facilitating the formation of isopeptide bonds between the ε-amino group of lysines in a substrate protein and the diglycine motif at the carboxy terminus of ubiquitin (11). Monomeric ubiquitin is processed from precursor proteins through ubiquitin-specific proteases which recognize the Cterminal diglycine motif along with the ubiquitin domain and which cleave after the diglycine motif irrespective of whether it is isopeptide linked or linked through a conventional peptide bond (38). Before degradation, the ubiquitin chains are removed from the substrate and disassembled into monomeric ubiquitin which can be reused. Ubiquitin levels are hence kept at a steady-state level, and the ubiquitin protein itself is long lived (9, 26).A growing number of proteins which contain domains with significant homology to ubiquitin have been discovered over the past 5 years. These ubiquitin-like proteins can be assigned either to the group of ubiquitin domain proteins which contain a ubiquitin homology domain but which do not become covalently linked to target proteins or to the group of ubiquitinli...

The ubiquitin-like modifier FAT10 in cancer development

“…The ULMs share a similar three-dimensional structure, the ubiquitin or ␤-grasp fold, as well as the C-terminal diglycine motif for activation and covalent attachment to substrate proteins by an E1-E3 enzymatic cascade (Hochstrasser, 2009). Of all these ULMs, FAT10 is the only modifier which acts as an autonomous transferable signal for degradation by the 26S proteasome and it was shown that this process can occur independently of ubiquitin (Hipp et al, 2005;Schmidtke et al, 2009). FAT10 is a protein of the immune system and it is strongly up-regulated by pro-inflammatory cytokines (Fan et al, 1996;Raasi et al, 1999) and during the maturation of antigen presenting cells (Buerger et al, 2015;Ebstein et al, 2009;Lukasiak et al, 2008).…”

“…In contrast to ubiquitin and other ubiquitin-like modifiers which are expressed as inactive precursors and which need to be activated by proteolytic processing at their C-terminus by specific proteases (Kerscher et al, 2006), FAT10 is already synthesized as a mature protein with a free diglycine motif at its C-terminus and can directly be activated and conjugated to substrate proteins (Raasi et al, 2001). A central function of FAT10 modification is to target proteins for degradation by the proteasome (Hipp et al, 2005;Raasi et al, 2001;Schmidtke et al, 2014). This process was shown to be independent of ubiquitylation but to be dependent on a noncovalent interaction partner of FAT10, namely NEDD8-ultimate buster 1 long (NUB1L) (Hipp et al, 2005;Schmidtke et al, 2009Schmidtke et al, , 2006.…”

“…A central function of FAT10 modification is to target proteins for degradation by the proteasome (Hipp et al, 2005;Raasi et al, 2001;Schmidtke et al, 2014). This process was shown to be independent of ubiquitylation but to be dependent on a noncovalent interaction partner of FAT10, namely NEDD8-ultimate buster 1 long (NUB1L) (Hipp et al, 2005;Schmidtke et al, 2009Schmidtke et al, , 2006. NUB1L, a likewise interferon-inducible protein (Kito et al, 2001), acts as a soluble receptor and brings FAT10 and FAT10ylated proteins to the proteasome where it binds to the 19S regulatory subunits RPN10 (S5a) and RPN1 (S2).…”

“…It was shown that FAT10 itself could also directly bind to the VWA domain of RPN10 in the absence of NUB1L but that the docking of NUB1L to the proteasome was necessary for the accelerated degradation of FAT10 (Rani et al, 2012;Schmidtke et al, 2009Schmidtke et al, , 2006. In contrast to ubiquitin, FAT10 has a very short half life of about 1 h and seems to be degraded by the proteasome along with its substrates Hipp et al, 2005). Since NUB1L negatively influences the stability of FAT10, the FAT10 degradation rate might be different in different cell lines or tissues expressing different amounts of NUB1L, as e.g.…”

USE1 is a bispecific conjugating enzyme for ubiquitin and FAT10, which FAT10ylates itself in cis

“…Th e biological function of FAT10 remains poorly understood, but it has been shown that overexpression of FAT10 leads to caspase-dependent apoptosis in a mouse fi broblast cell line 13 and renal tubular epithelial cells 15 . In addition, FAT10 has been shown to serve as a signal for proteasomal degradation independently of the ubiquitylation system 16,17 . NEDD8 ultimate buster long (NUB1L), a cytokineinducible linker protein that non-covalently interacts with FAT10 through its three C terminal ubiquitin-associated (UBA) domains and with the 26S proteasome through its N-terminal ubiquitin-like (UBL) domain, accelerates the degradation of FAT10 and FAT10-linked proteins 18,19 .…”