Degradation of FAT10 by the 26S proteasome is independent of ubiquitylation but relies on NUB1L

The ubiquitin-like modifier FAT10 targets proteins for degradation by the proteasome, a process accelerated by the UBL-UBA domain protein NEDD8 ultimate buster 1-long. Here, we show that FAT10-mediated degradation occurs independently of poly-ubiquitylation as purified 26S proteasome readily degraded FAT10-dihydrofolate reductase (DHFR) but not ubiquitin-DHFR in vitro. Interestingly, the 26S proteasome could only degrade FAT10-DHFR when NUB1L was present. Knock-down of NUB1L attenuated the degradation of FAT10-DHFR in intact cells suggesting that NUB1L determines the degradation rate of FAT10-linked proteins. In conclusion, our data establish FAT10 as a ubiquitin-independent but NUB1L-dependent targeting signal for proteasomal degradation.     

The UBA domains of NUB1L are required for binding but not for accelerated degradation of the ubiquitin-like modifier FAT10

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 ubiquitin-associated (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 ubiquitin-like proteins, but also as a facilitator of proteasomal degradation.     

Regulation of the NEDD8 conjugation system by a splicing variant,NUB1L

NEDD8 is a ubiquitin-like protein that controls vital biological events through its conjugation to target proteins. We previously identified a negative regulator of the NEDD8 conjugation system, NUB1, which works by recruiting NEDD8 and its conjugates to the proteasome for degradation. Recently, we found its splicing variant, NUB1L. It possesses an insertion of 14 amino acids that codes for a UBA domain. Structural study revealed that NUB1 has a NEDD8-binding site at the C terminus, whereas NUB1L has an additional site at the newly generated UBA domain. Interestingly, the sequence A(X4)L(X10)L(X3)L was conserved in these NEDD8-binding sites among human and other mammals. Mutational studies revealed that at least three Leu residues in the conserved sequence are required for binding with NEDD8. Functional study suggested that the NEDD8-binding ability at the C terminus of NUB1 and NUB1L is mainly involved in the down-regulation of NEDD8, but the NEDD8-binding ability at the UBA2 domain of NUB1L is minimally or not involved at all. The NEDD8-binding ability at the UBA2 domain might be required for an unknown function of NUB1L.     

NEDD8 Ultimate Buster 1 Long (NUB1L) Protein Suppresses Atypical Neddylation and Promotes the Proteasomal Degradation of Misfolded Proteins

Neddylation is a posttranslational modification that controls diverse biological processes by covalently conjugating the ubiquitin-like protein NEDD8 to specific targets. Neddylation is commonly mediated by NEDD8-specific enzymes (typical neddylation) and, sometimes, by ubiquitin enzymes (atypical neddylation). Although typical neddylation is known to regulate protein function in many ways, the regulatory mechanisms and biological consequence of atypical neddylation remain largely unexplored. Here we report that NEDD8 conjugates were accumulated in the diseased hearts from mouse models and human patients. Proteotoxic stresses induced typical and atypical neddylation in cardiomyocytes. Loss of NUB1L exaggerated atypical neddylation, whereas NUB1L overexpression repressed atypical neddylation through promoting the degradation of NEDD8. Activation of atypical neddylation accumulated a surrogate misfolded protein, GFPu. In contrast, suppression of atypical neddylation by NUB1L overexpression enhanced GFPu degradation. Moreover, NUB1L depletion accumulated a cardiomyopathy-linked misfolded protein, CryAB^R120G, whereas NUB1L overexpression promoted its degradation through suppressing neddylation of ubiquitinated proteins in cardiomyocytes. Consequently, NUB1L protected cells from proteotoxic stress-induced cell injury. In summary, these data indicate that NUB1L suppresses atypical neddylation and promotes the degradation of misfolded proteins by the proteasome. Our findings also suggest that induction of NUB1L could potentially become a novel therapeutic strategy for diseases with increased proteotoxic stress.     

Interaction of NUB1 with the proteasome subunit S5a

NUB1 interacts with a ubiquitin-like protein NEDD8 to target the NEDD8 monomer and neddylated proteins to the proteasome for degradation. Therefore, NUB1 is thought to be a potent downregulator of NEDD8 conjugation system. Since NUB1 possesses a UBL domain, which was previously shown to be an S5a-interacting motif in RAD23/HHR23, we initially hypothesized that NUB1 interacts with the S5a subunit of the proteasome through its UBL domain. To examine this, we performed an in vitro GST pull-down assay and a yeast two-hybrid assay. Unexpectedly, our studies revealed that NUB1 directly interacts with the S5a subunit through its C-terminal region between amino acid residues 536 and 584, not through its UBL domain. Although the UBL domain was not an S5a-interacting motif in NUB1, our further studies revealed that the UBL domain is required for the function of NUB1.     

NUB1-mediated targeting of the ubiquitin precursor UbC1 for its C-terminal hydrolysis.

NEDD8 is a ubiquitin-like protein that controls vital biological events through its conjugation to target proteins. Previously, we identified a negative regulator of the NEDD8 conjugation system, NEDD8 ultimate buster-1 (NUB1), that recruits NEDD8 and its conjugates to the proteasome for degradation. Recently, we performed yeast two-hybrid screening with NUB1 as bait and isolated a ubiquitin precursor UbC1 that is composed of nine tandem repeats of a ubiquitin unit through alpha-peptide bonds. Interestingly, NUB1 interacted with UbC1 through its UBA domain. Further study revealed that the UBA domain interacted with alpha-peptide bond-linked polyubiquitin, but not with isopeptide bond-linked polyubiquitin, indicating that the UBA domain of NUB1 is a specific acceptor for the linear ubiquitin precursor. A functional study revealed that an unidentified protein that was immunoprecipitated with NUB1 served as a ubiquitin C-terminal hydrolase for UbC1. Thus, NUB1 seems to form a protein complex with the unidentified ubiquitin C-terminal hydrolase and recruit UbC1 to this complex. This might allow the ubiquitin C-terminal hydrolase to hydrolyze UbC1, in order to generate ubiquitin monomers. Northern blot analysis showed that the mRNAs of both NUB1 and UbC1 were enriched in the testis. Furthermore, in situ hybridization showed that both mRNAs were strongly expressed in seminiferous tubules of the testis. These results may imply that the UbC1 hydrolysis mediated by NUB1 is involved in cellular functions in the seminiferous tubules such as spermatogenesis.     

NEDD8 Ultimate Buster-1 Long (NUB1L) Protein Promotes Transfer of NEDD8 to Proteasome for Degradation through the P97UFD1/NPL4 Complex

The NEDD8 protein and neddylation levels in cells are modulated by NUB1L or NUB1 through proteasomal degradation, but the underlying molecular mechanism is not well understood. Here, we report that NUB1L down-regulated the protein levels of NEDD8 and neddylation through specifically recognizing NEDD8 and P97/VCP. NUB1L directly interacted with NEDD8, but not with ubiquitin, on the key residue Asn-51 of NEDD8 and with P97/VCP on its positively charged VCP binding motif. In coordination with the P97-UFD1-NPL4 complex (P97^UFD1/NPL4), NUB1L promotes transfer of NEDD8 to proteasome for degradation. This mechanism is also exemplified by the canonical neddylation of cullin 1 for SCF (SKP1-cullin1-F-box) ubiquitin E3 ligases that is exquisitely regulated by the turnover of NEDD8.     

The inherited blindness protein AIPL1 regulates the ubiquitin-like FAT10 pathway

Mutations in AIPL1 cause the inherited blindness Leber congenital amaurosis (LCA). AIPL1 has previously been shown to interact with NUB1, which facilitates the proteasomal degradation of proteins modified with the ubiquitin-like protein FAT10. Here we report that AIPL1 binds non-covalently to free FAT10 and FAT10ylated proteins and can form a ternary complex with FAT10 and NUB1. In addition, AIPL1 antagonised the NUB1-mediated degradation of the model FAT10 conjugate, FAT10-DHFR, and pathogenic mutations of AIPL1 were defective in inhibiting this degradation. While all AIPL1 mutants tested still bound FAT10-DHFR, there was a close correlation between the ability of the mutants to interact with NUB1 and their ability to prevent NUB1-mediated degradation. Interestingly, AIPL1 also co-immunoprecipitated the E1 activating enzyme for FAT10, UBA6, suggesting AIPL1 may have a role in directly regulating the FAT10 conjugation machinery. These studies are the first to implicate FAT10 in retinal cell biology and LCA pathogenesis, and reveal a new role of AIPL1 in regulating the FAT10 pathway.     

NEDD化修饰系统及其在肿瘤发展中的作用

目前已经鉴定出17种类泛素蛋白（ubiquitin like proteins,UBLs）,这些蛋白与底物的结合方式与泛素相似．根据进化特征,可将UBLs分为9类,分别为：NEDD8、SUMO、ISG15、FUB1、FAT10、Atg8、Atg12、Urm1和UFM1．NEDD8是目前研究最多的UBLs之一,与泛素的氨基酸序列具有高度相似性．NEDD化修饰是一种动态的可逆蛋白质翻译后修饰方式,可以将NEDD8共价结合到靶蛋白之上,也可以将NEDD8从靶蛋白上去除．NEDD化修饰对蛋白功能具有重要的调节作用,如改变蛋白质的空间构象、阻碍底物与其它蛋白质的相互作用和招募与NEDD8相互作用的蛋白等．最新研究表明,NEDD化与肿瘤的发生发展密切相关,但具体的机制还不清楚．本文将就NEDD化修饰在肿瘤发展过程中的作用机制做一综述．     

FAT10, a ubiquitin-independent signal for proteasomal degradation

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 ubiquitin-like 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.     

FAT10 protein binds to polyglutamine proteins and modulates their solubility

Expansion of polyglutamine (pQ) chain by expanded CAG repeat causes dominantly inherited neurodegeneration such as Huntington disease, dentatorubral-pallidoluysian atrophy (DRPLA), and numbers of other spinocerebellar ataxias. Expanded pQ disrupts the stability of the pQ-harboring protein and increases its susceptibility to aggregation. Aggregated pQ protein is recognized by the ubiquitin proteasome system, and the enzyme ubiquitin ligase covalently attaches ubiquitin, which serves as a degradation signal by the proteasome. However, accumulation of the aggregated proteins in the diseased brain suggests insufficient degradation machinery. Ubiquitin has several functionally related proteins that are similarly attached to target proteins through its C terminus glycine residue. They are called ubiquitin-like molecules, and some of them are similarly related to the protein degradation pathway. One of the ubiquitin-like molecules, FAT10, is known to accelerate protein degradation through a ubiquitin-independent manner, but its role in pQ aggregate degradation is completely unknown. Thus we investigated its role in a Huntington disease cellular model and found that FAT10 molecules were covalently attached to huntingtin through their C terminus glycine. FAT10 binds preferably to huntingtin with a short pQ chain and completely aggregated huntingtin was FAT10-negative. In addition, ataxin-1,3 and DRPLA proteins were both positive for FAT10, and aggregation enhancement was observed upon FAT10 knockdown. These findings were similar to those for huntingtin. Our new finding will provide a new role for FAT10 in the pathogenesis of polyglutamine diseases.     

Solution structure of the ubiquitin-like domain of human DC-UbP from dendritic cells

The previously identified dendritic cell-derived ubiquitin-like protein (DC-UbP) was implicated in cellular differentiation and apoptosis. Sequence alignment suggested that it contains a ubiquitin-like (UbL) domain in the C terminus. Here, we present the solution NMR structure and backbone dynamics of the UbL domain of DC-UbP. The overall structure of the domain is very similar to that of Ub despite low similarity (<30%) in amino-acid sequence. One distinct feature of the domain structure is its highly positively charged surface that is different from the corresponding surfaces of the well-known UbL modifiers, Ub, NEDD8, and SUMO-1. The key amino-acid residues responsible for guiding polyubiquitinated proteins to proteasome degradation in Ub are not conserved in the UbL domain. This implies that the UbL domain of DC-UbP may have its own specific interaction partners with other yet unknown cellular functions related to the Ub pathway.     

But1 and But2 proteins bind to Uba3, a catalytic subunit of E1 for neddylation, in fission yeast

NEDD8/Rub1 is the most homologous protein to ubiquitin among the ubiquitin-like proteins, and it is covalently linked to target proteins via the C-terminal glycine residue in a manner analogous to ubiquitylation. However, the mechanism(s) involved in the regulation of the NEDD8 ligation pathway remains elusive. Using the two-hybrid system, we isolated novel genes from the Schizosaccharomyces pombe cDNA library whose products bind to Uba3, which is a catalytic protein for E1-like activity of the NEDD8 pathway. We designated these genes but1(+) and but2(+) (for proteins that bind to Uba three). But1 is a nuclear protein and its overexpression caused cell elongation, which is a common phenotype of the NEDD8 pathway defective mutant in S. pombe. Furthermore, overexpression of but1(+) in ned8-temperature sensitive mutant had a deleterious effect even under permissive temperatures. Our results suggest that But1 may have an inhibitory role in the NEDD8 pathway.     

Changes in the ratio of free NEDD8 to ubiquitin triggers NEDDylation by ubiquitin enzymes

Ubiquitin and UBL (ubiquitin-like) modifiers are small proteins that covalently modify other proteins to alter their properties or behaviours. Ubiquitin modification (ubiquitylation) targets many substrates, often leading to their proteasomal degradation. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is the UBL most closely related to ubiquitin, and its best-studied role is the activation of CRLs (cullin-RING ubiquitin ligases) by its conjugation to a conserved C-terminal lysine residue on cullin proteins. The attachment of UBLs requires three UBL-specific enzymes, termed E1, E2 and E3, which are usually well insulated from parallel UBL pathways. In the present study, we report a new mode of NEDD8 conjugation (NEDDylation) whereby the UBL NEDD8 is linked to proteins by ubiquitin enzymes in vivo. We found that this atypical NEDDylation is independent of classical NEDD8 enzymes, conserved from yeast to mammals, and triggered by an increase in the NEDD8 to ubiquitin ratio. In cells, NEDD8 overexpression leads to this type of NEDDylation by increasing the concentration of NEDD8, whereas proteasome inhibition has the same effect by depleting free ubiquitin. We show that bortezomib, a proteasome inhibitor used in cancer therapy, triggers atypical NEDDylation in tissue culture, which suggests that a similar process may occur in patients receiving this treatment.     

Inhibition of a NEDD8 Cascade Restores Restriction of HIV by APOBEC3G

Cellular restriction factors help to defend humans against human immunodeficiency virus (HIV). HIV accessory proteins hijack at least three different Cullin-RING ubiquitin ligases, which must be activated by the small ubiquitin-like protein NEDD8, in order to counteract host cellular restriction factors. We found that conjugation of NEDD8 to Cullin-5 by the NEDD8-conjugating enzyme UBE2F is required for HIV Vif-mediated degradation of the host restriction factor APOBEC3G (A3G). Pharmacological inhibition of the NEDD8 E1 by MLN4924 or knockdown of either UBE2F or its RING-protein binding partner RBX2 bypasses the effect of Vif, restoring the restriction of HIV by A3G. NMR mapping and mutational analyses define specificity determinants of the UBE2F NEDD8 cascade. These studies demonstrate that disrupting host NEDD8 cascades presents a novel antiretroviral therapeutic approach enhancing the ability of the immune system to combat HIV.