The UBA Domains of NUB1L Are Required for Binding but Not for Accelerated Degradation of the Ubiquitin-like Modifier FAT10

Abstract

Proteins selected for degradation are labeled with multiple molecules of ubiquitin and are subsequently cleaved by the 26 S proteasome. A family of proteins containing at least one ubiquitinassociated (UBA) domain and one ubiquitin-like (UBL) domain have been shown to act as soluble ubiquitin receptors of the 26 S proteasome and introduce a new level of specificity into the degradation system. They bind ubiquitylated proteins via their UBA domains and the 26 S proteasome via their UBL domain and facilitate the contact between substrate and protease. NEDD8 ultimate buster-1 long (NUB1L) belongs to this class of proteins and contains one UBL and three UBA domains. We recently reported that NUB1L interacts with the ubiquitin-like modifier FAT10 and accelerates its degradation and that of its conjugates. Here we show that a deletion mutant of NUB1L lacking the UBL domain is still able to bind FAT10 but not the proteasome and no longer accelerates FAT10 degradation. A version of NUB1L lacking all three UBA domains, on the other hand, looses the ability to bind FAT10 but is still able to interact with the proteasome and accelerates the degradation of FAT10. The degradation of a FAT10 mutant containing only the C-terminal UBL domain is also still accelerated by NUB1L, even though the two proteins do not interact. In addition, we show that FAT10 and either one of its UBL domains alone can interact directly with the 26 S proteasome. We propose that NUB1L not only acts as a linker between the 26 S proteasome and ubiquitinlike proteins, but also as a facilitator of proteasomal degradation.Ubiquitin, a small protein of 76 amino acids is one of the most conserved proteins known and has been found in all eukaryotic cells studied. It is essential for a variety of cellular processes, including degradation, cell-cycle regulation, DNA repair, stress response, embryogenesis, apoptosis, signal transduction, and transmembrane and vesicular transport (1-6). Throughout the past years, a family of proteins containing structural motives related to ubiquitin has been described that can be grouped into the ubiquitin-like modifiers and the ubiquitin-domain proteins (7). The ubiquitin-like modifiers have substantial sequence or structural homology to ubiquitin and form covalent conjugates with their target proteins. However, unlike ubiquitin, which can form large polymeric conjugates, usually only monomeric modifications are observed. As is the case for ubiquitin, a C-terminal diglycine motif is essential for conjugation of most modifiers to their target proteins (8). Prominent members of this group include SUMO-1, which serves several functions including nuclear transport and budding (9, 10), NEDD8, which regulates SCF ubiquitin-ligases via cullin modification (11), ISG15, which plays a role in innate immunity and in the response to ␣-interferon (12, 13), and FAT10, which is inducible with ␥-interferon (IFN)-␥ 4 and tumor necrosis factor ␣ (TNF-␣) (14, 15), and has been shown to cause apoptosis upon ectopic expression (16).The ubiquitin-li...

The ubiquitin-like modifier FAT10 in cancer development

“…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).…”

“…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).…”

NUB1 promotes cytoplasmic localization of p53 through cooperation of the NEDD8 and ubiquitin pathways

“…NUB1 is an interferoninducible protein and possesses a UBL (ubiquitin-like) domain at the N-terminal region and two ubiquitinassociated domains at the C-terminus Kito et al, 2001). Interestingly, the NUB1 splice variant NUBL1, which contains an extra ubiquitinassociated domain was shown to interact with the FAT10 Ubl (Hipp et al, 2004;Schmidtke et al, 2006). Expression of NUB1 was shown to decrease the levels of free unconjugated NEDD8 and of NEDDylated proteins.…”

FAT10 and NUB1L bind to the VWA domain of Rpn10 and Rpn1 to enable proteasome-mediated proteolysis

“…2e); full-length HA-FAT10-GFP served as a positive control and HA-Ub-GFP as well as GFP alone did not show any binding to hRpn10 that served as negative controls. Thus, the N-terminal UBL domain of FAT10 binds to NUB1L 34 , whereas the C-terminal UBL domain binds to hRpn10.…”