The Ubiquitin-like Protein FAT10 Forms Covalent Conjugates and Induces Apoptosis

Abstract

FAT10 is a ubiquitin-like protein that is encoded in the major histocompatibility complex class I locus and is synergistically inducible with interferon-␥ and tumor necrosis factor ␣. The molecule consists of two ubiquitin-like domains in tandem arrangement and bears a conserved diglycine motif at its carboxyl terminus commonly used in ubiquitin-like proteins for isopeptide linkage to conjugated proteins. We investigated the function of FAT10 by expressing murine FAT10 in a hemagglutinin-tagged wild type form as well as a diglycine-deficient mutant form in mouse fibroblasts in a tetracycline-repressible manner. FAT10 expression did not affect major histocompatibility complex class I cell surface expression or antigen presentation. However, we found that wild type but not mutant FAT10 caused apoptosis within 24 h of induction in a caspase-dependent manner as indicated by annexin V cell surface staining and DNA fragmentation. Wild type FAT10, but not its diglycine mutant, was covalently conjugated to thus far unidentified proteins, indicating that specific FAT10 activating and conjugating enzymes must be operative in unstimulated fibroblasts. Because FAT10 expression causes apoptosis and is inducible with tumor necrosis factor ␣, it may be functionally involved in the programmed cell death mediated by this cytokine.The covalent posttranslational modification of proteins is a versatile principle of determining the half-life, intracellular localization, and activity of proteins. In addition to modification by small molecules like orthophosphate, acetic acid, lipids, or sugars, the attachment of protein tags via isopeptide linkage to lysine residues in target proteins has recently been recognized as a frequently used and very diverse theme in cell biology. The prototype of such a protein tag is ubiquitin, which, when assembled as a polyubiquitin chain onto substrate proteins, can target these proteins to the 26S proteasome for degradation (1). Recently, it has become clear that the manner in which ubiquitin is linked in polyubiquitin chains defines the fate of the modified protein. The specific targeting signal for the proteasome pathway is a polyubiquitin chain that uses the K48 residue of the proximal ubiquitin as a target for further rounds of ubiquitination. The formation of K63-linked ubiquitin polymers, in contrast, does not lead to degradation but seems to play a role in DNA repair (2) and endocytosis (3). The conjugation of receptors with a single ubiquitin moiety can also serve as a signal for endocytosis (4), whereas in the case of histone H2B, monoubiquitination appears to regulate nucleosome function and cell division (5).The specificity of substrate selection and the mode of ubiquitin conjugation are determined by an enzymatic cascade required for the activation and specific transfer of ubiquitin. It consists of ubiquitin-activating enzyme (E1) 1 , which uses the energy of ATP to form a thioester linkage between a cysteine residue of the E1 enzyme and the carboxyl terminus of ubiquitin consisting of two ...

The ubiquitin-like modifier FAT10 in cancer development

“…A recent publication showed, that a high induction of FAT10 expression is also achieved by a synergistic treatment of HepG2 cells with TNF-␣ in combination with IL-6 (Choi et al, 2014). Upregulation of FAT10 expression induced caspase-dependent apoptosis in HeLa cells, renal tubular epithelial cells as well as mouse fibroblasts (Liu et al, 1999;Raasi et al, 2001;Ross et al, 2006). However, the role of FAT10 in apoptosis is contradictory because other publications showed a pro-survival role of FAT10 (Canaan et al, 2006).…”

“…The Nand C-terminal ubiquitin-like domains are 29% and 36% identical to ubiquitin, respectively, arranged in a tandem array and separated by a short linker (Fan et al, 1996;Groettrup et al, 2008;Theng et al, 2014). 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).…”

“…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.…”

Cloning and Characterization of DULP, a Novel Ubiquitin-Like Molecule from Human Dendritic Cells

“…All cell lines were maintained in RPMI 1640 (Hyclone) or Dulbecco's modified Eagle's medium (Hyclone) supplemented with 10% (v/v) heat-inactivated fetal calf serum (Hyclone), antibiotics, 2 mM glutamine, and 50 M 2-ME in a 5% CO 2 …”

“…UDPs bear a sequence domain similar to ubiquitin, but they are not conjugated to proteins. UBLs can be covalently conjugated to proteins by specific enzymatic cascades similar to those for ubiquitin conjugation (2,3).…”