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Ubiquitin-Like Proteins
U Ubiquitin-Like Proteins L-S Lu, Texas Heart Institute at St. Luke’s Episcopal Hospital, Houston, TX, USA E T H Yeh, The University of Texas MD Anderson Cancer Center and Texas Heart Institute at St. Luke’s Episcopal Hospital, Houston, TX, USA ã 2013 Elsevier Inc. All rights reserved. This article is a revision of the previous edition article by Edward T.H. Yeh, volume 4, pp. 304–307, ã 2004, Elsevier Inc.
Glossary De-UBLylation Ubiquitin-like protein (UBL) proteasesmediated removal of conjugated UBL from substrate. E1, E2, E3 Components of UBL conjugation machinery, with nomenclature derived from ubiquitin conjugation pathway. E1 activates UBL; E2 transfers the activated UBL either directly to the substrate or to an E3, which facilitates covalent bond formation between the terminal diglycine motif and acceptor cysteine of the substrate.
Introduction Ubiquitin is a posttranslational modifier. The elaborate biochemical machinery for ubiquitin conjugation has its root in various metabolite/cofactor biosynthesis pathways in prokaryotes and is further diversified to attach ubiquitin-like proteins (UBLs) to their targets. Since the identification of interferoninduced 15-kDa protein (Isg15) as the first UBL in 1992, now there are 11 proteins (small ubiquitin-like modifier (SUMO)1, SUMO2, SUMO3, SUMO4, neural precursor cell expressed, developmentally down regulated 8 (NEDD8), Isg15, Atg8, Atg12, Fat10, Ufm1, and Urm1) in the eukaryotic UBL family. These proteins are defined not by sequence similarity but instead by the presence of a conserved b-grasp fold in their structures. A b-grasp fold is an ancient structure that can be found in Escherichia Coli cofactor synthesis proteins MoaD and ThiS. It consists of a stranded beta-sheet, an alpha-helix, and a C-terminally extruded tail with a di-glycine motif, which is crucial for covalent conjugation to substrates. A general scheme for UBL modification involves activation, conjugation, ligation, and deconjugation (see Figure 1). Some UBLs are capable of forming polymeric branching chains just like ubiquitin. Monomeric and polymeric chains of UBL add an extensive interacting surface to the target molecule and therefore are capable of regulating enzymatic activity, protein complex formation, subcellular distribution, and protein stability.
UBL-binding motif A protein motif that noncovalently interacts with conjugated UBL. Such interaction confers regulatory function and thus proteins with UBL-binding motif usually act as effectors of UBLylation pathway. UBLylation The process of protein covalent modification by a specific UBL. For example, SUMOylation and NEDDylation refers to small ubiquitin-like modifier (SUMO)-mediated and neural precursor cell expressed, developmentally down regulated 8 (NEDD8)-mediated covalent modification respectively.
Experimentally verified UBL targets are involved in diverse cellular processes that can be found in every facet of cell biology. With the multitude of UBL signals, a specialized set of protein domains co-evolves with UBLs to specifically recognize UBL-modified proteins and ensures the UBL codes are correctly interpreted. Proteins with such UBL-binding domains may bind to monomeric/polymeric UBLs with various affinities and, therefore, render UBL-mediated protein complex formation highly flexible in response to different biological cues. Intriguingly, UBLs not only exist as standalone proteins but are also found as structural domains in more complex polypeptides. They act like built-in UBL modification to mediate interaction with UBL-binding domains and generally are not subject to enzymatic processing and cleavage.
Activation, Conjugation, Ligation, and Deconjugation The dynamics of UBL attachment is regulated by four groups of enzymes: E1, the activating enzyme; E2, the conjugation enzyme; E3, the ligation enzyme; and UBL proteases (see Table 1). Most UBLs are synthesized as inactive precursors. They first have to be processed by specific UBL proteases or hydrolases to remove C-terminal tail and expose the di-glycine motif. E1 will adenylate the exposed glycine at the cost of adenosine triphosphate (ATP) hydrolysis. This is the only
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Protein/Enzyme Structure Function and Degradation | Ubiquitin-Like Proteins
UBL protease UBL GG
UBL
ATP E1
UBL
E1
Substrate UBL
E2
UBL
E3
UBL
UBL protease UBD-containing UBL protein Substrate
UBL Substrate
Substrate
Figure 1 A general scheme for protein UBLylation dynamics.
Table 1
An overview of the UBL conjugation/deconjugation machinery
UBL
E1
E2
E3
UBL proteases
SUMO1 SUMO2 SUMO3 NEDD8 ISG15 ATG12 ATG8 FAT10 UFM1 URM1
Aos1/Uba2
Ubc9
PIAS1, PIAS2a, PIAS2b, PIAS3, PIAS4, Pc2, RanBP2, MMS21, TOPORS
SENP1, SENP2, SENP3, SENP5, SENP6, SENP7
Uba3/Nae1 Uba7 Atg7 Atg7 Uba6 Uba5 Uba4
Ubc12, Ube2F UbcH8 Atg10 Atg3
Rbx1, Dcn1, Mdm2, Cbl Herc5, Efp
Den1, CSN, Usp21, ataxin-3, UCH-L3, UCH-L1 Ubp43
Atg12/Atg5
Atg4
Ufc1
Ufl1
UfSP1, UfSP2
step that consumes ATP in the UBL modification reaction. The UBLadenosine monophosphate (AMP) intermediate is then attacked by a cysteine in the E1 catalytic core to form a highenergy thioester bond with the UBL. Following that, E1UBL binds to an E2 through an ubiquitin-fold domain on E1 and transfers UBL to the catalytic cysteine of E2. While E2UBL in many cases is sufficient to relay UBL to the substrate, the ligation is always facilitated by an E3 which brings in the substrate, binds to E2UBL intermediate, and catalyzes the isopeptide bond formation between the carboxyl group of terminal glycine on UBL and the e–amino group of acceptor lysine on the substrate. Similar to ubiquitin E3, really interesting new gene (RING) domain or homologous to the E6-associated protein carboxyl terminus (HECT) domain in many UBL E3s are the major effectors for ligating UBL. The transfer of UBL between loaded E1, E2, and E3 is driven by the release of inorganic pyrophosphate and AMP. In its free status, E1 has low affinity for E2. The binding to E2 only takes place with E1UBL intermediate, which allosterically inhibits E2 binding to E3. On the other hand, free E3 has a high
affinity only for loaded E2 but not for free one. The differential affinity between free/UBL-loaded enzymes and its downstream cognates account for the unidirectional transfer of UBL between E1, E2, and E3. To deconjugate UBL from its substrate, a UBL protease will bind the UBL-modified substrate and catalytic cysteine of the protease will attack the isopeptide bond to cleave UBL from substrate and form a proteaseUBL intermediate. At the end, hydrolysis of the thioester bond will release UBL from the protease. Most UBL-modified substrates only account for a small portion of the steady-state pool, and it is generally accepted that UBL proteases are a crucial factor in determining steady-state level of UBL-modified substrate.
Small Ubiquitin-Like Modifier SUMOs are the best characterized UBL. Since its discovery in 1996, hundreds of proteins have been experimentally identified to be modified by SUMO. SUMO genes are highly
Protein/Enzyme Structure Function and Degradation | Ubiquitin-Like Proteins
conserved among yeasts, worms, flies, and vertebrates. Their homologs are also found in plants and archae. There are four SUMO genes in the mammalian genome. SUMO1 is a nuclear protein with 101 amino acids and shares 18% sequence similarity to ubiquitin. SUMO2 and SUMO3, which are 97% identical before their C-terminal di-glycine motif, are also nuclear proteins and only share 50% sequence similarity with SUMO1. While SUMO1, SUMO2, and SUMO3 are ubiquitously expressed, SUMO4 is preferentially expressed in immune cells and pancreatic islets. Although genetic evidence suggests that SUMO4 M55V polymorphism confers susceptibility to type I diabetes, it remains controversial whether or not SUMO4 can be processed into mature UBL and conjugate target proteins. SUMO1, SUMO2, and SUMO3 attach to their targets as a monomer. In addition, SUMO2 and SUMO3 are found to form branching chains just like ubiquitin through Lys11 linkage. SUMO1 also forms polymeric chain via Lys7, Lys16, and Lys17 in vitro. However, there is no concrete evidence for polymeric SUMO1 chains in vivo. Instead, SUMO1 may conjugate to polymeric SUMO2/3 chain and act as a cap to stop its extension. The E1 for SUMO conjugation is a heterodimer composed of Aos1 and Uba2. Endogenous regulation of SUMO E1 is not yet understood. However, an avian adenovirus protein Gam1 targets Aos1 for ubiquitination and degradation through its SOCS domain via Cul2/5-Elongin B/C-Roc1 ubiquitin E3 ligase complex. As a consequence, Uba2 also degrades and the loss of functional E1 leads to impaired global SUMOylation. Ubc9 is the only E2 for SUMO. Sequence analysis of the first few SUMO substrates has revealed a common SUMO acceptor motif c-K-X-D/E, where c stands for aromatic amino acids and X stands for any amino acid. Over a decade, the rule is confirmed in many other molecules and is the only established acceptor consensus among UBLs. However, not all lysines in this motif are SUMO acceptors, and in many cases SUMO conjugates to a cryptic lysine that cannot be predicted based on protein-sequence analysis. The E3 for SUMO features the presence of RING-type plant homeo domain (PHD) finger. The largest collection of SUMO E3 is the protein inhibitors of activated STAT (signal transducers and activators of transcription) (PIAS) family members, in addition to Pc2, RanBP2, Mms21, Rhes, and Topors. Interestingly, the PHD finger of Trim25 can function as an intramolecular SUMO E3. Although Aos1/Uba2 and Ubc9 are sufficient for SUMOylation, SUMO E3s facilitate the reaction, result in functional compartmentaliization, and usually are the targets for regulating SUMOylation balance. A functional SUMO pathway is essential for life. Unbiased analysis of heat-shocked cells suggests that SUMO regulates transcriptional repression, chromatin regulation, DNA damage response, lipid metabolism, and cell-cycle progression. How does SUMO achieve such versatility? The first clue comes from the long list of SUMO targets. These proteins tend to cluster in functionally relevant categories, and therefore imply the crucial regulatory role of SUMO in those biological processes. In addition to its far-reaching footprints, molecular consequences of protein SUMOylation also diversify. SUMOylation may render stronger interaction with DNA and thus promote chromatin binding. SUMOylation may alter the components of the transcriptional complex and switch on/off transcription by
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differential recruitment of transcriptional cofactors. SUMOylation also interacts with other posttranslational modifications. For example, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IkBa) can be modified by SUMO at Lys21, which is also an ubiquitin acceptor. Lys21 SUMOylation is sufficient to inhibit tumor necrosis factor-alpha (TNFa)-induced ubiquitination and degradation of IkBa, and thus blocks subsequent nuclear factor kappa B (NFkB) activation. Another example comes from the SUMOylation of core histones H2B, H3, and H4 in yeast. SUMOylation of H2B and H4 reversely correlate to acetylation at galactokinase (GAL1) promoters during altered carbon supply. Meanwhile both ubc9 and Dsiz1Dsiz2 mutants, which cannot undergo normal SUMOylation due to the lack of either E2 or E3, display enhanced acetylation of gene-associated H3. On the contrary, SUMOylation can be attenuated by phosphorylation. In the case of Elk1, mitogen- activated protein (MAP) kinase-mediated phosphorylation of Ser383 blocks SUMOylation, which is required for its maximal transcriptional repression. In addition to covalent modification, SUMOs are also capable of noncovalent interaction with SUMO interaction motif (SIM). SIM, with a consensus of (V/I/)-(V/I/)-X-(V/I/L) or (V/I/)-X-(V/I/)-(V/I/L) and usually a flanking acidic residue, binds to the second b-strand and the first a-helix of SUMO. The terminal di-glycine of SUMO is not involved in SUMO– SIM interaction. Such interaction is one to two orders stronger than that between ubiquitin and ubiquitin-binding domains. SIM-containing protein binds and exerts its function depending on the SUMOylation status of its partner, and in this sense the SUMO–SIM pair provides a specific decoding mechanism of protein SUMOylation marks. A good example is RING finger protein 4 (RNF4), a SUMO-targeted ubiquitin ligase. It has four SIMs and one RING-type PHD finger which catalyzes ubiquitin conjugation. RNF4 only binds promyelocytic leukemia protein (PML), a crucial protein in the pathogenesis of acute promyelocytic leukemia, after arsenic-induced SUMOylation. As a consequence, PML is ubiquitylated by RNF4 on its poly-SUMO tags and degrades in a proteosome-dependent manner. This is believed to be one of the therapeutic mechanisms of arsenic in treating acute promyelocytic leukemia. Considering the large number of SUMO targets, it is surprising that six isopeptidase – sentrin/SUMO specific proteases (SENP) – in the mammalian genome are sufficient for keeping proper SUMO status of hundreds of proteins. SENPs are cysteine peptidase and all of them contain a C48 protease domain in the C-terminal. The six SENPs can be divided into three subfamilies according to the substrate specificity, sequence homology, and cellular localization. SENP1 and SENP2 are mammalian ortholog of the yeast Ulp1 and both readily deconjugate SUMO1 and SUMO2. SENP3 and SENP5 are nucleolar proteins with a preference for deconjugating SUMO2/3. SENP6 and SENP7 have a split C48 protease domain in the C-terminal. While the deSUMOylating activities of these two enzymes are controversial, they are capable of editing polymeric SUMO chains. Early embryonic lethality of mice deficient of either SENP1 or SENP2 suggests that these SENPs perform nonredundant functions. Accessibility of SENP to its substrate may dictate paralog-selective SUMOylation in cells. For example, SUMO1conjugated RanGAP1 forms a more stable complex with nucleoporin RanBP2 compared to its SUMO2 conjugates and this
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prevents binding of the yeast SENP Ulp1. As a result, RanGAP1 is preferentially SUMOylated by SUMO1 in vivo.
Neural Precursor Cell Expressed, Developmentally Downregulated 8 NEDD8, also known as related to ubiquitin 1, or Rub1, in budding yeast is the closet kin of ubiquitin in the UBL family (60% sequence identity). The 81-amino-acid protein was identified in 1992 with a substraction cloning approach from mouse-neural precursor cell complementary DNA (cDNA) library and its function as a UBL was first characterized in 1997 in our laboratory. It is ubiquitously expressed, evolutionary conserved, and is essential for cell viability. NEDD8 is a crucial regulator of ubiquitin E3 ligase complexes. Dysregulated NEDDylation has been observed in tumors and neurodegenerative diseases, implying its correlation with the onset and progression of human diseases. NEDD8 is synthesized as inactive precursor with a stretch of four glycines in the C-terminus. NEDD8 protease SENP8 (Den1) and hydrolase UchL3 are both capable of exposing the Gly76 for conjugation. The E1 for NEDD8, like the SUMO E1, is a heterodimer composed of Uba3 and Nae1. Despite the similarity, NEDD8 conjugating machinery is devoted for processing NEDD8 but not ubiquitin. Several mechanisms account for such specificity. First of all, Uba3/Nae1 is capable of discriminating NEDD8 from ubiquitin due to a strategically positioned basic residue in the UBL binding pocket of Uba3, which repels Arg72 in ubiquitin but not Ala72 in NEDD8. Second, the NEDD8 E2 Ubc12 has a unique N-terminal sequence that is required for Uba3/Nae1 binding, which prevents mischarging of ubiquitin-specific E2s to NEDD8 E1. In addition, Ubc12 has a smaller cysteine-binding pocket and a unique groove which accommodates N-terminal extrusion of NEDD8. These structural features allow Ubc12 to discriminate NEDD8 from ubiquitin. Recently, Ube2F is found to be the second NEDD8 E2. Ubc12 and Ube2F form a complex with different E3 (Rbx1 and Rbx2, respectively), neddylate distinct substrates, and result in selective ubiquitin E3 ligase complexes activation. Rbx1, a common component of Skp1-Cul1-F-box-protein (SCF) and Elongin B/C, Cullen-2, VHL-containing (ECV) ubiquitin E3 ligase complexes, is the first reported NEDD8 E3. It harbors a RING-finger domain which not only approximates ubiquitin E2 to the substrate but also promotes cullin neddylation in vitro. The second NEDD8 E3, Dcn1, contains a potentiating neddylation (PONY) domain that is required for neddylation activity in vitro and in vivo. Dcn1 may act as a scaffold to facilitate interaction between substrate and NEDD8-charged Ubc12. It is interesting that in vitro reconstitution of NEDDylation requires the presence of both Dcn1 and Rbx1, which indicates potential synergy between the two molecules. In addition to Dcn1 and Rbx1, ubiquitin E3 ligases Mdm2 and Cbl are also reported to promote NEDDylation in an E3-like fashion. All of the cullin family members are NEDD8 substrates. Cullins are structural scaffold for Cullin-RING ubiquitin ligase, and neddylation of cullins are essential to bring in E2 to promote the ubiquitination and degradation of their cognate
target cyclins, which are crucial regulators for cell cycle progression. Human Cul-1 is a major component of SCF ubiquitin E3 ligase that catalyzes the ubiquitination of IkBa, b-catenin, and p27Kip1; NEDDylation of Cul-1 is essential for the ubiquitin ligase activity. Molecular consequences of NEDDylation may involve changes in protein conformation. In the case of SCF, NEDDylation of Cul-1 exposes the RING finger of Rbx1 and therefore facilitates ubiquitin ligation to the proximal substrate. In another case, NEDDylation of cullins allosterically inhibits the binding of Cand1, which is an inhibitor of cullin E3 complex, and activates SCF ubiquitin ligase. NEDDylation can also recruit new binding partners. For example, epidermal growth factor receptor (EGFR) is a noncullin NEDD8 substrate. It can be neddylated by Ubc12-loaded Cbl at the cytoplasmic domain upon ligand binding, and then recruits Cbl precomplexed with ubiquitin E2 UbcH7 to EGFR. Subsequent ubiquitylation and degradation of EGFR ensures rapid receptor desensitization. Additional noncullin NEDD8 substrates include p53, p73, Mdm2, VHL, L11, and other ribosomal proteins. NEDD8 has been reported to form polymeric chains in vitro. The significance of poly-NEDD8 chain in vivo remains unknown. COP9 signalosome (CSN) is the best-characterized NEDD8 isopeptidase. CSN is a protein complex that interacts with Cul-1-containing SCF-type E3 ubiquitin ligases and promotes the cleavage of NEDD8 from neddylated Cul-1. The Jab1/Mpr/ Pad1 N-terminal (MPN) domain metalloenzyme motif in Csn5 subunit is responsible for the de-neddylation activity of CSN. Thus, CSN binds to SCF-type E3 ubiquitin ligases and probably regulates their activity through the de-neddylation of conjugated Cul-1. SENP8/Den1, a cysteine protease in the SENP family, is another NEDD8-specific isopeptidase. Unlike CSN, SENP8/Den1 is weak in de-neddylating cullins. SENP8/Den1 deletion results in accumulation of NEDD8 conjugates; removing one allele of SENP8/Den1 genetically counteracts the developmental lethality in NEDD8 hypomorphic mutants. Therefore, SENP8/Den1 may act as the major de-neddylase for noncullin NEDD8 substrates. In addition to CSN and Den1, NEDD8 conjugates can also be cleaved by ubiquitin–NEDD8 dual specific proteases such as Usp21, ataxin-3, PfUch54, Uch-L3, and Uch-L1. Regulation of NEDD8 pathway is more complicated than simply keeping balanced conjugation/deconjugation. Nub1 (NEDD8 ultimate buster 1), a 66-kDa NEDD8-interacting protein with a N-terminal ubiquitin-like domain, has been shown to direct NEDD8 and its conjugates to 19S subunit of proteosome and results in their degradation. NEDD8 pathway can also be manipulated pharmacologically. MLN4924 is a suicide inhibitor for NEDD8 E1 Nae1/Uba3 heterodimer and suppresses tumor cell growth. It is currently under phase II clinical trial for treating acute myelocytic leukemia and may become the first UBL-targeting compound for human cancer treatment.
Interferon-Induced 15-kDa Protein Isg15 was first identified as a 15-kDa protein induced by interferon treatment in 1979. The gene actually encodes a 17-kDa protein with two tandem ubiquitin-like domains and was found to form high molecular weight conjugates in 1992.
Protein/Enzyme Structure Function and Degradation | Ubiquitin-Like Proteins
Unlike SUMO and NEDD8, Isg15 is only present in vertebrates and these homologs share limited sequence similarity. Isg15 protein is detected in lymphoid tissues, striated and smooth muscles, receptive uterine endometrium, epithelial cells, and neurons. In mammalians, Isg15 is an important regulator of antiviral defense. Despite the lack of a classical signal peptide and a membrane receptor, Isg15 is secreted and may act in an autocrine/paracrine fashion to modulate interferon production from CD3þ lymphocytes and subsequent NK cell proliferation and activation. While Isg15 knockout mice are viable and fertile, they are more vulnerable to viral infections. Interestingly, the antiviral property seems to be dependent on its UBL property rather than its cytokine property since reconstitution of nonconjugatable Isg15 cannot rescue the virus susceptibility. Uba7 is the only E1 dedicated to Isg15 activation. This protein is a monomeric E1. It has been reported that influenza virus protein NS1B specifically interacts with Uba7 and inhibits its E1 activity. The cognate E2 for Uba7 is UbcH8. There are only a few E3 that have been identified as Isg15 E3 including Herc5 and Efp. The fact that Isg15, Uba7, and UbcH8 are all upregulated after interferon treatment may explain enhanced protein ISGylation after type I interferon (IFN-a and IFN-b) stimulation. It is noteworthy that some cell lines (such as K562 and 293T cells) do not respond to interferons with detectable protein ISGylation. In the case of K562 cells, it is ascribed to the defective expression of Uba7. So far Ubp43 is the only one Isg15-specific protease that has been identified. An in vitro screen of ubiquitin-specific proteases has identified Usp13 as a dual-specificity protease that is capable of processing ubiquitin and Isg15. However, in vivo validation of this finding is still pending. Experimentally verified Isg15 targets include Sta1, Jak1, Erk1, PKR, Mx1, RIG-1, Ubc13, and influenza protein NS1A. ISGylation of these proteins do not lead to proteosomal degradation nor does it lead to altered subcellular localization. ISGylation of an ubiquitin E2 Ubc13 blocks its ability to form thioester bond with ubiquitin and therefore abolishes subsequent Lys63-linked polyubiquitination. Enhanced ISGylation of Jak1 and Stat1 in Ubp43-deficient bone marrow cells are associated with persistent Stat1 phosphorylation and enhanced expression of interferon-stimulated genes. As a result, bone marrow cells die soon after interferon treatment. However, whether ISGylation of Jak1/Stat1 accounts for the phenotype observed in Ubp43deficient mice was challenged by the observation that compound deletion of Isg15/Ubp43 does not rescue the Ubp43 deletion phenotypes.
Atg8 and Atg12 Autophagy is a tightly regulated catabolic process that turns over proteins and organelles with lysosomal degradation machinery in a eukaryote cell. There are approximately 30 proteins for the autophagy pathway in yeast. Among them are two UBLs, Atg8 and Atg12, and their conjugation system components. Both UBL pathways are required for efficient autophagy as deletion of any component leads to autophagy defect. Atg8 and Atg12 share little sequence similarity. However, structurally they both have ubiquitin-like folds. Both
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Atg12 and Atg8 conjugation systems are conserved in mammalian cells and they act in a similar manner. Atg12 is synthesized as mature UBL and does not require to be processed prior to E1 activation. The E1 for Atg12 is Atg7, which forms a thioester bond between its Cys507 and Gly186 of Atg12. Atg10 is the only E2 for Atg12 conjugation pathway. The transfer of Atg12 from Atg10 to its sole substrate Atg5 does not need an E3 and is constitutively active. Atg12–Atg5 conjugate localizes to the outer membrane of and promotes the expansion of autophagosome. Atg12 conjugation seems to be irreversible since there is no isopeptidase to cleave Atg12 from Atg5. Atg12-modified Atg5 interacts with coiled coil protein Atg16 and forms multimeric complex to direct Atg8-PE to tightly associate with the expanding autophagosome membrane. The other UBL, Atg8, modifies phosphatidylethanolamine (PE, a lipid moiety on autophagosome membrane). It is synthesized as a precursor as most UBLs do. The protease Atg4 cleaves and exposes Gly116 of Atg8 so that it can conjugate to Cys 507 of Atg7 (the same E1 and the same cysteine residue as used in Atg12 conjugation pathway) for activation. Atg7 is a noncanonical E1 because, unlike the ubiquitin and SUMO E1, it forms a homodimer and the resulting homodimeric adenlyation domains are likely to bind two UBL molecules simultaneously. This is more close to the prokaryotic E1 antecedent MoeB and ThiF. Atg7 also stands out among UBL E1s due to its capability to activate two unrelated UBL, Atg12 and Atg8, and specifically transfer these UBL to their cognate E2. The structural basis for such duality is not known. Atg8 is then transferred to the E2 Atg3 and then forms an amide bond between PE and Atg8. Atg12 interacts with Atg3 and such interaction allows Atg12–Atg5 complex to promote Atg8 modification of PE. Although Atg12–Atg5, in this sense, acts like an E3 but the complex lacks HECT or RING domains which is the hallmark of most UBL E3. Atg8–PE is the structural determinant of autophagosome membrane expansion, and quantitative fluorimetry analysis suggests a positive correlation between Atg8 abundance and the size of autophagosome. It is also capable of joining the ends of expanding lipid bilayer and finalizes autophagosome formation. On completion of autophagosome formation, Atg8-PE will be cleaved by the protease Atg4. Atg4-mediated recycling of Atg8 is crucial for efficient autophagy since reintroduction of processed Atg8 into DAtg4 mutant is capable of binding to PE, but cannot rescue hampered autophagosome formation upon nutrient depletion.
Fat10 Fat10, also known as diubiquitin or ubiquitin D, is originally identified as a UBL-encoding gene in the human leukocyte antigen F (HLA-F) locus. The molecule consists of two ubiquitin-like domains in tandem arrangement and bears a conserved di-glycine motif at its C-terminus. It is expressed in mature B cells, dendritic cells, B lymphoblasts, thymus, tonsil, pancreatic islets, mammary gland epithelia, and small intestine. Fat10 is capable of covalent conjugation as all UBLs do, but the conjugation machinery is poorly characterized. Fat10 is synthesized in mature form and forms thioester bond with Uba6, which is a monomeric E1 and also surprisingly activates ubiquitin. Uba6 does not load Fat10 onto its cognate ubiquitin E2
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including Ubc3, Ubc5, Ubc13, and E2-25K. The structural basis for such specificity is unknown. The other members of the Fat10 pathway, including E2, E3, protease, and the substrate are yet to be identified. Current knowledge on the function of Fat10 is limited. Like Isg15, Fat10 is an interferon-responsive gene and it can also be induced by tumor necrosis factor alpha. Strong upregulation of Fat10 has been observed in hepatocellular carcinoma, colorectal carcinoma, and HIV-infected renal tubular epithelial cells, which might be due to the proinflammatory microenvironment in these samples. Although Fat10 knockout mice are viable, fertile, and grossly normal, they are more vulnerable to endotoxin-induced sepsis. These evidences imply that Fat10 is involved in innate immunity. The best-characterized molecular property of Fat10 is its role in the control of protein quantity and quality. Linearly fused Fat10 serves as an ubiquitin-independent degron (the amino acid sequence that tags the protein for degradation) to promote protein degradation at a comparable kinetics to their ubiquitin-fused counterparts. Fat10-dependent degradation is not affected in cells defective in ubiquitin activation, or in cells expressing lysine-mutated Fat10 which prevents ubiquitin conjugation. Noncovalent interaction between Fat10 and NUB1L, which associates with the 26S proteosome, is required for Fat10-fused protein to be processed by proteosome
in vitro and greatly facilitates the degradation of Fat10 monomer and its conjugates in vivo. It is interesting to note that both Fat10 and NUB1L are interferon-responsive genes and overexpression of Fat10 but not its conjugation-defective mutant leads to apoptosis. Fat10 also promotes aggresome formation in case of proteosome failure through its interaction with histone deacetylase (HDAC6), which is involved in the transportation of ubiquitylated misfolded protein to aggresome for sequestration and removal. Other functions of Fat10 include its roles in regulating mitosis through its interaction with mitotic spindle assembly checkpoint protein (MAD2) and mediating NF-kB signal transduction.
See also: Protein/Enzyme Structure Function and Degradation: Ubiquitin System.
Further Reading Ohsumi Y (2001) Molecular dissection of autophagy: Two ubiquitin-like systems. Nature Reviews Molecular Cell Biology 2: 211–216. Ritchie KJ and Zhang DE (2004) Isg15: The immunological kin of ubiquitin. Seminars in Cell and Developmental Biology 15: 237–246. Yeh ETH (2009) SUMOylation and De-SUMOylation: Wrestling with life’s processes. Journal of Biological Chemistry 284: 8223–8227.
Glossary De-UBLylation Ubiquitin-like protein (UBL) proteasesmediated removal of conjugated UBL from substrate. E1, E2, E3 Components of UBL conjugation machinery, with nomenclature derived from ubiquitin conjugation pathway. E1 activates UBL; E2 transfers the activated UBL either directly to the substrate or to an E3, which facilitates covalent bond formation between the terminal diglycine motif and acceptor cysteine of the substrate.
Introduction Ubiquitin is a posttranslational modifier. The elaborate biochemical machinery for ubiquitin conjugation has its root in various metabolite/cofactor biosynthesis pathways in prokaryotes and is further diversified to attach ubiquitin-like proteins (UBLs) to their targets. Since the identification of interferoninduced 15-kDa protein (Isg15) as the first UBL in 1992, now there are 11 proteins (small ubiquitin-like modifier (SUMO)1, SUMO2, SUMO3, SUMO4, neural precursor cell expressed, developmentally down regulated 8 (NEDD8), Isg15, Atg8, Atg12, Fat10, Ufm1, and Urm1) in the eukaryotic UBL family. These proteins are defined not by sequence similarity but instead by the presence of a conserved b-grasp fold in their structures. A b-grasp fold is an ancient structure that can be found in Escherichia Coli cofactor synthesis proteins MoaD and ThiS. It consists of a stranded beta-sheet, an alpha-helix, and a C-terminally extruded tail with a di-glycine motif, which is crucial for covalent conjugation to substrates. A general scheme for UBL modification involves activation, conjugation, ligation, and deconjugation (see Figure 1). Some UBLs are capable of forming polymeric branching chains just like ubiquitin. Monomeric and polymeric chains of UBL add an extensive interacting surface to the target molecule and therefore are capable of regulating enzymatic activity, protein complex formation, subcellular distribution, and protein stability.
UBL-binding motif A protein motif that noncovalently interacts with conjugated UBL. Such interaction confers regulatory function and thus proteins with UBL-binding motif usually act as effectors of UBLylation pathway. UBLylation The process of protein covalent modification by a specific UBL. For example, SUMOylation and NEDDylation refers to small ubiquitin-like modifier (SUMO)-mediated and neural precursor cell expressed, developmentally down regulated 8 (NEDD8)-mediated covalent modification respectively.
Experimentally verified UBL targets are involved in diverse cellular processes that can be found in every facet of cell biology. With the multitude of UBL signals, a specialized set of protein domains co-evolves with UBLs to specifically recognize UBL-modified proteins and ensures the UBL codes are correctly interpreted. Proteins with such UBL-binding domains may bind to monomeric/polymeric UBLs with various affinities and, therefore, render UBL-mediated protein complex formation highly flexible in response to different biological cues. Intriguingly, UBLs not only exist as standalone proteins but are also found as structural domains in more complex polypeptides. They act like built-in UBL modification to mediate interaction with UBL-binding domains and generally are not subject to enzymatic processing and cleavage.
Activation, Conjugation, Ligation, and Deconjugation The dynamics of UBL attachment is regulated by four groups of enzymes: E1, the activating enzyme; E2, the conjugation enzyme; E3, the ligation enzyme; and UBL proteases (see Table 1). Most UBLs are synthesized as inactive precursors. They first have to be processed by specific UBL proteases or hydrolases to remove C-terminal tail and expose the di-glycine motif. E1 will adenylate the exposed glycine at the cost of adenosine triphosphate (ATP) hydrolysis. This is the only
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Protein/Enzyme Structure Function and Degradation | Ubiquitin-Like Proteins
UBL protease UBL GG
UBL
ATP E1
UBL
E1
Substrate UBL
E2
UBL
E3
UBL
UBL protease UBD-containing UBL protein Substrate
UBL Substrate
Substrate
Figure 1 A general scheme for protein UBLylation dynamics.
Table 1
An overview of the UBL conjugation/deconjugation machinery
UBL
E1
E2
E3
UBL proteases
SUMO1 SUMO2 SUMO3 NEDD8 ISG15 ATG12 ATG8 FAT10 UFM1 URM1
Aos1/Uba2
Ubc9
PIAS1, PIAS2a, PIAS2b, PIAS3, PIAS4, Pc2, RanBP2, MMS21, TOPORS
SENP1, SENP2, SENP3, SENP5, SENP6, SENP7
Uba3/Nae1 Uba7 Atg7 Atg7 Uba6 Uba5 Uba4
Ubc12, Ube2F UbcH8 Atg10 Atg3
Rbx1, Dcn1, Mdm2, Cbl Herc5, Efp
Den1, CSN, Usp21, ataxin-3, UCH-L3, UCH-L1 Ubp43
Atg12/Atg5
Atg4
Ufc1
Ufl1
UfSP1, UfSP2
step that consumes ATP in the UBL modification reaction. The UBLadenosine monophosphate (AMP) intermediate is then attacked by a cysteine in the E1 catalytic core to form a highenergy thioester bond with the UBL. Following that, E1UBL binds to an E2 through an ubiquitin-fold domain on E1 and transfers UBL to the catalytic cysteine of E2. While E2UBL in many cases is sufficient to relay UBL to the substrate, the ligation is always facilitated by an E3 which brings in the substrate, binds to E2UBL intermediate, and catalyzes the isopeptide bond formation between the carboxyl group of terminal glycine on UBL and the e–amino group of acceptor lysine on the substrate. Similar to ubiquitin E3, really interesting new gene (RING) domain or homologous to the E6-associated protein carboxyl terminus (HECT) domain in many UBL E3s are the major effectors for ligating UBL. The transfer of UBL between loaded E1, E2, and E3 is driven by the release of inorganic pyrophosphate and AMP. In its free status, E1 has low affinity for E2. The binding to E2 only takes place with E1UBL intermediate, which allosterically inhibits E2 binding to E3. On the other hand, free E3 has a high
affinity only for loaded E2 but not for free one. The differential affinity between free/UBL-loaded enzymes and its downstream cognates account for the unidirectional transfer of UBL between E1, E2, and E3. To deconjugate UBL from its substrate, a UBL protease will bind the UBL-modified substrate and catalytic cysteine of the protease will attack the isopeptide bond to cleave UBL from substrate and form a proteaseUBL intermediate. At the end, hydrolysis of the thioester bond will release UBL from the protease. Most UBL-modified substrates only account for a small portion of the steady-state pool, and it is generally accepted that UBL proteases are a crucial factor in determining steady-state level of UBL-modified substrate.
Small Ubiquitin-Like Modifier SUMOs are the best characterized UBL. Since its discovery in 1996, hundreds of proteins have been experimentally identified to be modified by SUMO. SUMO genes are highly
Protein/Enzyme Structure Function and Degradation | Ubiquitin-Like Proteins
conserved among yeasts, worms, flies, and vertebrates. Their homologs are also found in plants and archae. There are four SUMO genes in the mammalian genome. SUMO1 is a nuclear protein with 101 amino acids and shares 18% sequence similarity to ubiquitin. SUMO2 and SUMO3, which are 97% identical before their C-terminal di-glycine motif, are also nuclear proteins and only share 50% sequence similarity with SUMO1. While SUMO1, SUMO2, and SUMO3 are ubiquitously expressed, SUMO4 is preferentially expressed in immune cells and pancreatic islets. Although genetic evidence suggests that SUMO4 M55V polymorphism confers susceptibility to type I diabetes, it remains controversial whether or not SUMO4 can be processed into mature UBL and conjugate target proteins. SUMO1, SUMO2, and SUMO3 attach to their targets as a monomer. In addition, SUMO2 and SUMO3 are found to form branching chains just like ubiquitin through Lys11 linkage. SUMO1 also forms polymeric chain via Lys7, Lys16, and Lys17 in vitro. However, there is no concrete evidence for polymeric SUMO1 chains in vivo. Instead, SUMO1 may conjugate to polymeric SUMO2/3 chain and act as a cap to stop its extension. The E1 for SUMO conjugation is a heterodimer composed of Aos1 and Uba2. Endogenous regulation of SUMO E1 is not yet understood. However, an avian adenovirus protein Gam1 targets Aos1 for ubiquitination and degradation through its SOCS domain via Cul2/5-Elongin B/C-Roc1 ubiquitin E3 ligase complex. As a consequence, Uba2 also degrades and the loss of functional E1 leads to impaired global SUMOylation. Ubc9 is the only E2 for SUMO. Sequence analysis of the first few SUMO substrates has revealed a common SUMO acceptor motif c-K-X-D/E, where c stands for aromatic amino acids and X stands for any amino acid. Over a decade, the rule is confirmed in many other molecules and is the only established acceptor consensus among UBLs. However, not all lysines in this motif are SUMO acceptors, and in many cases SUMO conjugates to a cryptic lysine that cannot be predicted based on protein-sequence analysis. The E3 for SUMO features the presence of RING-type plant homeo domain (PHD) finger. The largest collection of SUMO E3 is the protein inhibitors of activated STAT (signal transducers and activators of transcription) (PIAS) family members, in addition to Pc2, RanBP2, Mms21, Rhes, and Topors. Interestingly, the PHD finger of Trim25 can function as an intramolecular SUMO E3. Although Aos1/Uba2 and Ubc9 are sufficient for SUMOylation, SUMO E3s facilitate the reaction, result in functional compartmentaliization, and usually are the targets for regulating SUMOylation balance. A functional SUMO pathway is essential for life. Unbiased analysis of heat-shocked cells suggests that SUMO regulates transcriptional repression, chromatin regulation, DNA damage response, lipid metabolism, and cell-cycle progression. How does SUMO achieve such versatility? The first clue comes from the long list of SUMO targets. These proteins tend to cluster in functionally relevant categories, and therefore imply the crucial regulatory role of SUMO in those biological processes. In addition to its far-reaching footprints, molecular consequences of protein SUMOylation also diversify. SUMOylation may render stronger interaction with DNA and thus promote chromatin binding. SUMOylation may alter the components of the transcriptional complex and switch on/off transcription by
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differential recruitment of transcriptional cofactors. SUMOylation also interacts with other posttranslational modifications. For example, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IkBa) can be modified by SUMO at Lys21, which is also an ubiquitin acceptor. Lys21 SUMOylation is sufficient to inhibit tumor necrosis factor-alpha (TNFa)-induced ubiquitination and degradation of IkBa, and thus blocks subsequent nuclear factor kappa B (NFkB) activation. Another example comes from the SUMOylation of core histones H2B, H3, and H4 in yeast. SUMOylation of H2B and H4 reversely correlate to acetylation at galactokinase (GAL1) promoters during altered carbon supply. Meanwhile both ubc9 and Dsiz1Dsiz2 mutants, which cannot undergo normal SUMOylation due to the lack of either E2 or E3, display enhanced acetylation of gene-associated H3. On the contrary, SUMOylation can be attenuated by phosphorylation. In the case of Elk1, mitogen- activated protein (MAP) kinase-mediated phosphorylation of Ser383 blocks SUMOylation, which is required for its maximal transcriptional repression. In addition to covalent modification, SUMOs are also capable of noncovalent interaction with SUMO interaction motif (SIM). SIM, with a consensus of (V/I/)-(V/I/)-X-(V/I/L) or (V/I/)-X-(V/I/)-(V/I/L) and usually a flanking acidic residue, binds to the second b-strand and the first a-helix of SUMO. The terminal di-glycine of SUMO is not involved in SUMO– SIM interaction. Such interaction is one to two orders stronger than that between ubiquitin and ubiquitin-binding domains. SIM-containing protein binds and exerts its function depending on the SUMOylation status of its partner, and in this sense the SUMO–SIM pair provides a specific decoding mechanism of protein SUMOylation marks. A good example is RING finger protein 4 (RNF4), a SUMO-targeted ubiquitin ligase. It has four SIMs and one RING-type PHD finger which catalyzes ubiquitin conjugation. RNF4 only binds promyelocytic leukemia protein (PML), a crucial protein in the pathogenesis of acute promyelocytic leukemia, after arsenic-induced SUMOylation. As a consequence, PML is ubiquitylated by RNF4 on its poly-SUMO tags and degrades in a proteosome-dependent manner. This is believed to be one of the therapeutic mechanisms of arsenic in treating acute promyelocytic leukemia. Considering the large number of SUMO targets, it is surprising that six isopeptidase – sentrin/SUMO specific proteases (SENP) – in the mammalian genome are sufficient for keeping proper SUMO status of hundreds of proteins. SENPs are cysteine peptidase and all of them contain a C48 protease domain in the C-terminal. The six SENPs can be divided into three subfamilies according to the substrate specificity, sequence homology, and cellular localization. SENP1 and SENP2 are mammalian ortholog of the yeast Ulp1 and both readily deconjugate SUMO1 and SUMO2. SENP3 and SENP5 are nucleolar proteins with a preference for deconjugating SUMO2/3. SENP6 and SENP7 have a split C48 protease domain in the C-terminal. While the deSUMOylating activities of these two enzymes are controversial, they are capable of editing polymeric SUMO chains. Early embryonic lethality of mice deficient of either SENP1 or SENP2 suggests that these SENPs perform nonredundant functions. Accessibility of SENP to its substrate may dictate paralog-selective SUMOylation in cells. For example, SUMO1conjugated RanGAP1 forms a more stable complex with nucleoporin RanBP2 compared to its SUMO2 conjugates and this
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prevents binding of the yeast SENP Ulp1. As a result, RanGAP1 is preferentially SUMOylated by SUMO1 in vivo.
Neural Precursor Cell Expressed, Developmentally Downregulated 8 NEDD8, also known as related to ubiquitin 1, or Rub1, in budding yeast is the closet kin of ubiquitin in the UBL family (60% sequence identity). The 81-amino-acid protein was identified in 1992 with a substraction cloning approach from mouse-neural precursor cell complementary DNA (cDNA) library and its function as a UBL was first characterized in 1997 in our laboratory. It is ubiquitously expressed, evolutionary conserved, and is essential for cell viability. NEDD8 is a crucial regulator of ubiquitin E3 ligase complexes. Dysregulated NEDDylation has been observed in tumors and neurodegenerative diseases, implying its correlation with the onset and progression of human diseases. NEDD8 is synthesized as inactive precursor with a stretch of four glycines in the C-terminus. NEDD8 protease SENP8 (Den1) and hydrolase UchL3 are both capable of exposing the Gly76 for conjugation. The E1 for NEDD8, like the SUMO E1, is a heterodimer composed of Uba3 and Nae1. Despite the similarity, NEDD8 conjugating machinery is devoted for processing NEDD8 but not ubiquitin. Several mechanisms account for such specificity. First of all, Uba3/Nae1 is capable of discriminating NEDD8 from ubiquitin due to a strategically positioned basic residue in the UBL binding pocket of Uba3, which repels Arg72 in ubiquitin but not Ala72 in NEDD8. Second, the NEDD8 E2 Ubc12 has a unique N-terminal sequence that is required for Uba3/Nae1 binding, which prevents mischarging of ubiquitin-specific E2s to NEDD8 E1. In addition, Ubc12 has a smaller cysteine-binding pocket and a unique groove which accommodates N-terminal extrusion of NEDD8. These structural features allow Ubc12 to discriminate NEDD8 from ubiquitin. Recently, Ube2F is found to be the second NEDD8 E2. Ubc12 and Ube2F form a complex with different E3 (Rbx1 and Rbx2, respectively), neddylate distinct substrates, and result in selective ubiquitin E3 ligase complexes activation. Rbx1, a common component of Skp1-Cul1-F-box-protein (SCF) and Elongin B/C, Cullen-2, VHL-containing (ECV) ubiquitin E3 ligase complexes, is the first reported NEDD8 E3. It harbors a RING-finger domain which not only approximates ubiquitin E2 to the substrate but also promotes cullin neddylation in vitro. The second NEDD8 E3, Dcn1, contains a potentiating neddylation (PONY) domain that is required for neddylation activity in vitro and in vivo. Dcn1 may act as a scaffold to facilitate interaction between substrate and NEDD8-charged Ubc12. It is interesting that in vitro reconstitution of NEDDylation requires the presence of both Dcn1 and Rbx1, which indicates potential synergy between the two molecules. In addition to Dcn1 and Rbx1, ubiquitin E3 ligases Mdm2 and Cbl are also reported to promote NEDDylation in an E3-like fashion. All of the cullin family members are NEDD8 substrates. Cullins are structural scaffold for Cullin-RING ubiquitin ligase, and neddylation of cullins are essential to bring in E2 to promote the ubiquitination and degradation of their cognate
target cyclins, which are crucial regulators for cell cycle progression. Human Cul-1 is a major component of SCF ubiquitin E3 ligase that catalyzes the ubiquitination of IkBa, b-catenin, and p27Kip1; NEDDylation of Cul-1 is essential for the ubiquitin ligase activity. Molecular consequences of NEDDylation may involve changes in protein conformation. In the case of SCF, NEDDylation of Cul-1 exposes the RING finger of Rbx1 and therefore facilitates ubiquitin ligation to the proximal substrate. In another case, NEDDylation of cullins allosterically inhibits the binding of Cand1, which is an inhibitor of cullin E3 complex, and activates SCF ubiquitin ligase. NEDDylation can also recruit new binding partners. For example, epidermal growth factor receptor (EGFR) is a noncullin NEDD8 substrate. It can be neddylated by Ubc12-loaded Cbl at the cytoplasmic domain upon ligand binding, and then recruits Cbl precomplexed with ubiquitin E2 UbcH7 to EGFR. Subsequent ubiquitylation and degradation of EGFR ensures rapid receptor desensitization. Additional noncullin NEDD8 substrates include p53, p73, Mdm2, VHL, L11, and other ribosomal proteins. NEDD8 has been reported to form polymeric chains in vitro. The significance of poly-NEDD8 chain in vivo remains unknown. COP9 signalosome (CSN) is the best-characterized NEDD8 isopeptidase. CSN is a protein complex that interacts with Cul-1-containing SCF-type E3 ubiquitin ligases and promotes the cleavage of NEDD8 from neddylated Cul-1. The Jab1/Mpr/ Pad1 N-terminal (MPN) domain metalloenzyme motif in Csn5 subunit is responsible for the de-neddylation activity of CSN. Thus, CSN binds to SCF-type E3 ubiquitin ligases and probably regulates their activity through the de-neddylation of conjugated Cul-1. SENP8/Den1, a cysteine protease in the SENP family, is another NEDD8-specific isopeptidase. Unlike CSN, SENP8/Den1 is weak in de-neddylating cullins. SENP8/Den1 deletion results in accumulation of NEDD8 conjugates; removing one allele of SENP8/Den1 genetically counteracts the developmental lethality in NEDD8 hypomorphic mutants. Therefore, SENP8/Den1 may act as the major de-neddylase for noncullin NEDD8 substrates. In addition to CSN and Den1, NEDD8 conjugates can also be cleaved by ubiquitin–NEDD8 dual specific proteases such as Usp21, ataxin-3, PfUch54, Uch-L3, and Uch-L1. Regulation of NEDD8 pathway is more complicated than simply keeping balanced conjugation/deconjugation. Nub1 (NEDD8 ultimate buster 1), a 66-kDa NEDD8-interacting protein with a N-terminal ubiquitin-like domain, has been shown to direct NEDD8 and its conjugates to 19S subunit of proteosome and results in their degradation. NEDD8 pathway can also be manipulated pharmacologically. MLN4924 is a suicide inhibitor for NEDD8 E1 Nae1/Uba3 heterodimer and suppresses tumor cell growth. It is currently under phase II clinical trial for treating acute myelocytic leukemia and may become the first UBL-targeting compound for human cancer treatment.
Interferon-Induced 15-kDa Protein Isg15 was first identified as a 15-kDa protein induced by interferon treatment in 1979. The gene actually encodes a 17-kDa protein with two tandem ubiquitin-like domains and was found to form high molecular weight conjugates in 1992.
Protein/Enzyme Structure Function and Degradation | Ubiquitin-Like Proteins
Unlike SUMO and NEDD8, Isg15 is only present in vertebrates and these homologs share limited sequence similarity. Isg15 protein is detected in lymphoid tissues, striated and smooth muscles, receptive uterine endometrium, epithelial cells, and neurons. In mammalians, Isg15 is an important regulator of antiviral defense. Despite the lack of a classical signal peptide and a membrane receptor, Isg15 is secreted and may act in an autocrine/paracrine fashion to modulate interferon production from CD3þ lymphocytes and subsequent NK cell proliferation and activation. While Isg15 knockout mice are viable and fertile, they are more vulnerable to viral infections. Interestingly, the antiviral property seems to be dependent on its UBL property rather than its cytokine property since reconstitution of nonconjugatable Isg15 cannot rescue the virus susceptibility. Uba7 is the only E1 dedicated to Isg15 activation. This protein is a monomeric E1. It has been reported that influenza virus protein NS1B specifically interacts with Uba7 and inhibits its E1 activity. The cognate E2 for Uba7 is UbcH8. There are only a few E3 that have been identified as Isg15 E3 including Herc5 and Efp. The fact that Isg15, Uba7, and UbcH8 are all upregulated after interferon treatment may explain enhanced protein ISGylation after type I interferon (IFN-a and IFN-b) stimulation. It is noteworthy that some cell lines (such as K562 and 293T cells) do not respond to interferons with detectable protein ISGylation. In the case of K562 cells, it is ascribed to the defective expression of Uba7. So far Ubp43 is the only one Isg15-specific protease that has been identified. An in vitro screen of ubiquitin-specific proteases has identified Usp13 as a dual-specificity protease that is capable of processing ubiquitin and Isg15. However, in vivo validation of this finding is still pending. Experimentally verified Isg15 targets include Sta1, Jak1, Erk1, PKR, Mx1, RIG-1, Ubc13, and influenza protein NS1A. ISGylation of these proteins do not lead to proteosomal degradation nor does it lead to altered subcellular localization. ISGylation of an ubiquitin E2 Ubc13 blocks its ability to form thioester bond with ubiquitin and therefore abolishes subsequent Lys63-linked polyubiquitination. Enhanced ISGylation of Jak1 and Stat1 in Ubp43-deficient bone marrow cells are associated with persistent Stat1 phosphorylation and enhanced expression of interferon-stimulated genes. As a result, bone marrow cells die soon after interferon treatment. However, whether ISGylation of Jak1/Stat1 accounts for the phenotype observed in Ubp43deficient mice was challenged by the observation that compound deletion of Isg15/Ubp43 does not rescue the Ubp43 deletion phenotypes.
Atg8 and Atg12 Autophagy is a tightly regulated catabolic process that turns over proteins and organelles with lysosomal degradation machinery in a eukaryote cell. There are approximately 30 proteins for the autophagy pathway in yeast. Among them are two UBLs, Atg8 and Atg12, and their conjugation system components. Both UBL pathways are required for efficient autophagy as deletion of any component leads to autophagy defect. Atg8 and Atg12 share little sequence similarity. However, structurally they both have ubiquitin-like folds. Both
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Atg12 and Atg8 conjugation systems are conserved in mammalian cells and they act in a similar manner. Atg12 is synthesized as mature UBL and does not require to be processed prior to E1 activation. The E1 for Atg12 is Atg7, which forms a thioester bond between its Cys507 and Gly186 of Atg12. Atg10 is the only E2 for Atg12 conjugation pathway. The transfer of Atg12 from Atg10 to its sole substrate Atg5 does not need an E3 and is constitutively active. Atg12–Atg5 conjugate localizes to the outer membrane of and promotes the expansion of autophagosome. Atg12 conjugation seems to be irreversible since there is no isopeptidase to cleave Atg12 from Atg5. Atg12-modified Atg5 interacts with coiled coil protein Atg16 and forms multimeric complex to direct Atg8-PE to tightly associate with the expanding autophagosome membrane. The other UBL, Atg8, modifies phosphatidylethanolamine (PE, a lipid moiety on autophagosome membrane). It is synthesized as a precursor as most UBLs do. The protease Atg4 cleaves and exposes Gly116 of Atg8 so that it can conjugate to Cys 507 of Atg7 (the same E1 and the same cysteine residue as used in Atg12 conjugation pathway) for activation. Atg7 is a noncanonical E1 because, unlike the ubiquitin and SUMO E1, it forms a homodimer and the resulting homodimeric adenlyation domains are likely to bind two UBL molecules simultaneously. This is more close to the prokaryotic E1 antecedent MoeB and ThiF. Atg7 also stands out among UBL E1s due to its capability to activate two unrelated UBL, Atg12 and Atg8, and specifically transfer these UBL to their cognate E2. The structural basis for such duality is not known. Atg8 is then transferred to the E2 Atg3 and then forms an amide bond between PE and Atg8. Atg12 interacts with Atg3 and such interaction allows Atg12–Atg5 complex to promote Atg8 modification of PE. Although Atg12–Atg5, in this sense, acts like an E3 but the complex lacks HECT or RING domains which is the hallmark of most UBL E3. Atg8–PE is the structural determinant of autophagosome membrane expansion, and quantitative fluorimetry analysis suggests a positive correlation between Atg8 abundance and the size of autophagosome. It is also capable of joining the ends of expanding lipid bilayer and finalizes autophagosome formation. On completion of autophagosome formation, Atg8-PE will be cleaved by the protease Atg4. Atg4-mediated recycling of Atg8 is crucial for efficient autophagy since reintroduction of processed Atg8 into DAtg4 mutant is capable of binding to PE, but cannot rescue hampered autophagosome formation upon nutrient depletion.
Fat10 Fat10, also known as diubiquitin or ubiquitin D, is originally identified as a UBL-encoding gene in the human leukocyte antigen F (HLA-F) locus. The molecule consists of two ubiquitin-like domains in tandem arrangement and bears a conserved di-glycine motif at its C-terminus. It is expressed in mature B cells, dendritic cells, B lymphoblasts, thymus, tonsil, pancreatic islets, mammary gland epithelia, and small intestine. Fat10 is capable of covalent conjugation as all UBLs do, but the conjugation machinery is poorly characterized. Fat10 is synthesized in mature form and forms thioester bond with Uba6, which is a monomeric E1 and also surprisingly activates ubiquitin. Uba6 does not load Fat10 onto its cognate ubiquitin E2
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including Ubc3, Ubc5, Ubc13, and E2-25K. The structural basis for such specificity is unknown. The other members of the Fat10 pathway, including E2, E3, protease, and the substrate are yet to be identified. Current knowledge on the function of Fat10 is limited. Like Isg15, Fat10 is an interferon-responsive gene and it can also be induced by tumor necrosis factor alpha. Strong upregulation of Fat10 has been observed in hepatocellular carcinoma, colorectal carcinoma, and HIV-infected renal tubular epithelial cells, which might be due to the proinflammatory microenvironment in these samples. Although Fat10 knockout mice are viable, fertile, and grossly normal, they are more vulnerable to endotoxin-induced sepsis. These evidences imply that Fat10 is involved in innate immunity. The best-characterized molecular property of Fat10 is its role in the control of protein quantity and quality. Linearly fused Fat10 serves as an ubiquitin-independent degron (the amino acid sequence that tags the protein for degradation) to promote protein degradation at a comparable kinetics to their ubiquitin-fused counterparts. Fat10-dependent degradation is not affected in cells defective in ubiquitin activation, or in cells expressing lysine-mutated Fat10 which prevents ubiquitin conjugation. Noncovalent interaction between Fat10 and NUB1L, which associates with the 26S proteosome, is required for Fat10-fused protein to be processed by proteosome
in vitro and greatly facilitates the degradation of Fat10 monomer and its conjugates in vivo. It is interesting to note that both Fat10 and NUB1L are interferon-responsive genes and overexpression of Fat10 but not its conjugation-defective mutant leads to apoptosis. Fat10 also promotes aggresome formation in case of proteosome failure through its interaction with histone deacetylase (HDAC6), which is involved in the transportation of ubiquitylated misfolded protein to aggresome for sequestration and removal. Other functions of Fat10 include its roles in regulating mitosis through its interaction with mitotic spindle assembly checkpoint protein (MAD2) and mediating NF-kB signal transduction.
See also: Protein/Enzyme Structure Function and Degradation: Ubiquitin System.
Further Reading Ohsumi Y (2001) Molecular dissection of autophagy: Two ubiquitin-like systems. Nature Reviews Molecular Cell Biology 2: 211–216. Ritchie KJ and Zhang DE (2004) Isg15: The immunological kin of ubiquitin. Seminars in Cell and Developmental Biology 15: 237–246. Yeh ETH (2009) SUMOylation and De-SUMOylation: Wrestling with life’s processes. Journal of Biological Chemistry 284: 8223–8227.