A Ubiquitin Shuttle DC-UbP/UBTD2 Reconciles Protein Ubiquitination and Deubiquitination via Linking UbE1 and USP5 Enzymes

The ubiquitination levels of protein substrates in eukaryotic cells are delicately orchestrated by various protein cofactors and enzymes. Dendritic cell-derived ubiquitin (Ub)-like protein (DC-UbP), also named as Ub domain-containing protein 2 (UBTD2), is a potential Ub shuttle protein comprised of a Ub-like (UbL) domain and a Ub-binding domain (UBD), but its biological function remains largely unknown. We identified two Ub-related enzymes, the deubiquitinating enzyme USP5 and the Ub-activating enzyme UbE1, as interacting partners of DC-UbP from HEK 293T cells. Biochemical studies revealed that the tandem UBA domains of USP5 and the C-terminal Ub-fold domain (UFD) of UbE1 directly interacted with the C-terminal UbL domain of DC-UbP but on the distinct surfaces. Overexpression of DC-UbP in HEK 293T cells enhanced the association of these two enzymes and thus prompted cellular ubiquitination, whereas knockdown of the protein reduced the cellular ubiquitination level. Together, DC-UbP may integrate the functions of USP5 and UbE1 through interacting with them, and thus reconcile the cellular ubiquitination and deubiquitination processes.     

Binding surface mapping of intra- and interdomain interactions among hHR23B,ubiquitin, and polyubiquitin binding site 2 of S5a

hHR23B is the human homologue of the yeast protein RAD23 and is known to participate in DNA repair by stabilizing xeroderma pigmentosum group C protein. However, hHR23B and RAD23 also have many important functions related to general proteolysis. hHR23B consists of N-terminal ubiquitin-like (UbL), ubiquitin association 1 (UBA1), xeroderma pigmentosum group C binding, and UBA2 domains. The UBA domains interact with ubiquitin (Ub) and inhibit the assembly of polyubiquitin. On the other hand, the UbL domain interacts with the poly-Ub binding site 2 (PUbS2) domain of the S5a protein, which can carry polyubiquitinated substrates into the proteasome. We calculated the NMR structure of the UbL domain of hHR23B and determined binding surfaces of UbL and Ub to UBA1, UBA2, of hHR23B and PUbS2 of S5a by using chemical shift perturbation. Interestingly, the surfaces of UbL and Ub that bind to UBA1, UBA2, and PUbS2 are similar, consisting of five beta-strands and their connecting loops. This is the first report that an intramolecular interaction between UbL and UBA domains is possible, and this interaction could be important for the control of proteolysis by hHR23B. The binding specificities of UbL and Ub for PUbS1, PUbS2, and general ubiquitin-interacting motifs, which share the LALA motif, were evaluated. The UBA domains bind to the surface of Ub including Lys-48, which is required for multiubiquitin assembly, possibly explaining the observed inhibition of multiubiquitination by hHR23B. The UBA domains bind to UbL through electrostatic interactions supported by hydrophobic interactions and to Ub mainly through hydrophobic interactions supported by electrostatic interactions.     

Rad23 promotes the targeting of proteolytic substrates to the proteasome

Rad23 contains a ubiquitin-like domain (UbL(R23)) that interacts with catalytically active proteasomes and two ubiquitin (Ub)-associated (UBA) sequences that bind Ub. The UBA domains can bind Ub in vitro, although the significance of this interaction in vivo is poorly understood. Rad23 can interfere with the assembly of multi-Ub chains in vitro, and high-level expression caused stabilization of proteolytic substrates in vivo. We report here that Rad23 interacts with ubiquitinated cellular proteins through the synergistic action of its UBA domains. Rad23 plays an overlapping role with Rpn10, a proteasome-associated multi-Ub chain binding protein. Mutations in the UBA domains prevent efficient interaction with ubiquitinated proteins and result in poor suppression of the growth and proteolytic defects of a rad23 Delta rpn10 Delta mutant. High-level expression of Rad23 revealed, for the first time, an interaction between ubiquitinated proteins and the proteasome. This increase was not observed in rpn10 Delta mutants, suggesting that Rpn10 participates in the recognition of proteolytic substrates that are delivered by Rad23. Overexpression of UbL(R23) caused stabilization of a model substrate, indicating that an unregulated UbL(R23)-proteasome interaction can interfere with the efficient delivery of proteolytic substrates by Rad23. Because the suppression of a rad23 Delta rpn10 Delta mutant phenotype required both UbL(R23) and UBA domains, our findings support the hypothesis that Rad23 encodes a novel regulatory factor that translocates ubiquitinated substrates to the proteasome.     

HIV-1, ubiquitin and ubiquitin-like proteins: the dialectic interactions of a virus with a sophisticated network of post-translational modifications

The modification of intracellular proteins by ubiquitin (Ub) and ubiquitin-like (UbL) proteins is a central mechanism for regulating and fine-tuning all cellular processes. Indeed, these modifications are widely used to control the stability, activity and localisation of many key proteins and, therefore, they are instrumental in regulating cellular functions as diverse as protein degradation, cell signalling, vesicle trafficking and immune response. It is thus no surprise that pathogens in general, and viruses in particular, have developed multiple strategies to either counteract or exploit the complex mechanisms mediated by the Ub and UbL protein conjugation pathways. The aim of this review is to provide an overview on the intricate and conflicting relationships that intimately link HIV-1 and these sophisticated systems of post-translational modifications.     

Cloning and identification of a novel human ubiquitin-like protein,DC-UbP,from dendritic cells

Several ubiquitin-like proteins recently discovered have been confirmed to modify proteins akin to ubiquitinization for fine-regulation of intracellular proteins. In the present study, we report a novel ubiquitin-like protein from human dendritic cells (DC), named as dendritic cell-derived ubiquitin-like protein (DC-UbP). The full-length of DC-UbP cDNA is 565bp and encodes 106 amino acids. Ubiquitin domain (UBQ) in DC-UbP shares 28.6% identity and 55% similarity to ubiquitin, but does not possess the conserved C-terminus Gly-Gly of ubiquitin required for ubiquitinization. DC-UbP localized in cytoplasm, especially in mitochondrion, indicating that it may play a role in mitochondrial biology. DC-UbP mRNA was expressed in various tumor cells, but not in adult human normal tissues, suggesting that DC-UbP might be related to tumor genesis. In addition, DC-UbP mRNA expression decreased in the HL60 cells undergoing apoptosis after being stimulated with TRAIL and in the differentiated HL60 cells induced by ARTA. Taken together, DC-UbP might be downregulated during cellular differentiation and apoptosis.     

Solution structure of the N‐terminal domain of DC‐UbP/UBTD2 and its interaction with ubiquitin

DC‐UbP/UBTD2 is a ubiquitin (Ub) domain‐containing protein first identified from dendritic cells, and is implicated in ubiquitination pathway. The solution structure and backbone dynamics of the C‐terminal Ub‐like (UbL) domain were elucidated in our previous work. To further understand the biological function of DC‐UbP, we then solved the solution structure of the N‐terminal domain of DC‐UbP (DC‐UbP_N) and studied its Ub binding properties by NMR techniques. The results show that DC‐UbP_N holds a novel structural fold and acts as a Ub‐binding domain (UBD) but with low affinity. This implies that the DC‐UbP protein, composing of a combination of both UbL and UBD domains, might play an important role in regulating protein ubiquitination and delivery of ubiquitinated substrates in eukaryotic cells.     

Different roles for two ubiquitin-like domains of ISG15 in protein modification

ISG15 (interferon-stimulated gene 15) is a novel ubiquitin-like (UbL) modifier with two UbL domains in its architecture. We investigated different roles for the two UbL domains in protein modification by ISG15 (ISGylation) and the impact of Influenza B virus NS1 protein (NS1B) on regulation of the pathway. The results show that, although the C-terminal domain is sufficient to link ISG15 to UBE1L and UbcH8, the N-terminal domain is dispensable in the activation and transthiolation steps but required for efficient E3-mediated transfer of ISG15 from UbcH8 to its substrates. NS1B specifically binds to the N-terminal domain of ISG15 but does not affect ISG15 linkage via a thioester bond to its activating and conjugating enzymes. However, it does inhibit the formation of cellular ISG15 conjugates upon interferon treatment. We propose that the N-terminal UbL domain of ISG15 mainly functions in the ligation step and NS1B inhibits ISGylation by competing with E3 ligases for binding to the N-terminal domain.     

Structure of the ubiquitin-interacting motif of S5a bound to the ubiquitin-like domain of HR23B

Ubiquitination, a modification in which single or multiple ubiquitin molecules are attached to a protein, serves signaling functions that control several cellular processes. The ubiquitination signal is recognized by downstream effectors, many of which carry a ubiquitin-interacting motif (UIM). Such interactions can be modulated by regulators carrying a ubiquitin-like (UbL) domain, which binds UIM by mimicking ubiquitination. Of them, HR23B regulates the proteasomal targeting of ubiquitinated substrates, DNA repair factors, and other proteins. Here we report the structure of the UIM of the proteasome subunit S5a bound to the UbL domain of HR23B. The UbL domain presents one hydrophobic and two polar contact sites for interaction with UIM. The residues in these contact sites are well conserved in ubiquitin, but ubiquitin also presents a histidine at the interface. The pH-dependent protonation of this residue interferes with the access of ubiquitin to the UIM and the ubiquitin-associated domain (UBA), and its mutation to a smaller residue increases the affinity of ubiquitin for UIM.     

The effects of the polyglutamine repeat protein ataxin-1 on the UbL-UBA protein A1Up

The ataxin-1 interacting ubiquitin-like protein (A1Up) contains an amino-terminal ubiquitin-like (UbL) region, four stress-inducible, heat shock chaperonin-binding motifs (STI1), and an ubiquitin-associated domain (UBA) at the carboxyl terminus of A1Up. Although proteins that have both an UbL and UBA domain are thought to play a crucial role in proteasome-mediated activities, few are characterized, except for hHR23A/B. Similar to other UbL-containing proteins, the UbL of A1Up is essential for the interaction of A1Up with the S5a subunit of the 19S proteasome. Importantly, the interaction with the 19S proteasome was disrupted in the presence of the polyglutamine repeat protein, ataxin-1. The UbL domain of A1Up is ubiquitinated by both Lys(48)-linked and Lys(63)-linked chains. Intact A1Up is stable, suggesting that ubiquitination of A1Up is important for degradation-independent targeting of A1Up to the 19S proteasome. The UBA domain of A1Up binds polyubiquitin chains and has a role in the stability of A1Up and in the subcellular localization of A1Up. When the UBA domain was deleted, the localization of A1Up was entirely cytoplasmic, and it co-localized with the proteasome. Interestingly, the interaction between A1Up and mutant ataxin-1-(82Q) increased the half-life of A1Up, whereas nonpathogenic wild-type ataxin-1-(30Q) or ataxin-1-(82Q)-A776 did not.     

2',5'-oligoadenylate synthetase gene in chicken: gene structure, distribution of alleles and their expression

We have cloned the gene for chicken 2',5'-oligoadenylate synthetase (ChOAS) by the method of polymerase chain reaction with use of ChOAS cDNA sequence. The ChOAS gene is composed of five introns and six exons containing all of the sequence of the ChOAS cDNA from the start to the stop codon. The first five exons of ChOAS gene which encode the OAS catalytic domain have a similar structure to HuOAS1 gene including the exon-intron boundaries. However, the length of introns of ChOAS gene is only 1/7 of those of HuOAS1 gene. The sixth exon of the ChOAS gene encodes the ubiquitin-like (UbL) domain of two consecutive sequence (UbL1 and UbL2) homologous to ubiquitin. ChOAS encoded in a single copy gene has at least two alleles, OAS(*)A and OAS(*)B. The differences between these two alleles are in the sixth exon of the gene; a 96-nucleotide sequence in the UbL1 portion of OAS(*)A is deleted from OAS(*)B. No OAS(*)B gene was detected in nine lines of chickens tested other than Leghorns. Almost the same levels of ChOAS-A and -B proteins induced physiologically in erythrocytes were detected in infant chickens (2-week-old), but in grown-up chickens (6-month-old) the level of erythrocyte OAS-B was markedly reduced in most of B/B chickens. Thus, the UbL domain of ChOAS is responsible for the maintenance of the OAS level in the tissue.     

Rap80 protein recruitment to DNA double-strand breaks requires binding to both small ubiquitin-like modifier (SUMO) and ubiquitin conjugates

Ubiquitin (Ub) modifications at sites of DNA double-strand breaks (DSBs) play critical roles in the assembly of signaling and repair proteins. The Ub-interacting motif (UIM) domain of Rap80, which is a component of the BRCA1-A complex, interacts with Ub Lys-63 linkage conjugates and mediates the recruitment of BRCA1 to DSBs. Small ubiquitin-like modifier (SUMO) conjugation also occurs at DSBs and promotes Ub-dependent recruitment of BRCA1, but its molecular basis is not clear. In this study, we identified that Rap80 possesses a SUMO-interacting motif (SIM), capable of binding specifically to SUMO2/3 conjugates, and forms a tandem SIM-UIM-UIM motif at its N terminus. The SIM-UIM-UIM motif binds to both Ub Lys-63 linkage and SUMO2 conjugates. Both the SIM and UIM domains are required for efficient recruitment of Rap80 to DSBs immediately after damage and confer cellular resistance to ionizing radiation. These findings propose a model in which SUMO and Ub modification is coordinated to recruit Rap80 and BRCA1 to DNA damage sites.     

A novel connexin43-interacting protein, CIP75, which belongs to the UbL-UBA protein family, regulates the turnover of connexin43

The degradation of connexin43 (Cx43) has been reported to involve both lysosomal and proteasomal degradation pathways; however, very little is known about the mechanisms regulating these Cx43 degradation pathways. Using yeast two-hybrid, glutathione S-transferase pull-down, and co-immunoprecipitation approaches, we have identified a novel Cx43-interacting protein of approximately 75 kDa, CIP75. Laser confocal microscopy showed that CIP75 is located primarily at the endoplasmic reticulum, as indicated by the calnexin marker, with Cx43 co-localization in this perinuclear region. CIP75 belongs to the UbL (ubiquitin-like)-UBA (ubiquitin-associated) domain-containing protein family with a N-terminal UbL domain and a C-terminal UBA domain. The UBA domain of CIP75 is the main element mediating the interaction with Cx43, whereas the CIP75-interacting region in Cx43 resides in the PY motif and multiphosphorylation sites located between Lys 264 and Asn 302. Interestingly, the UbL domain interacts with the S2/RPN1 and S5a/RPN10 protein subunits of the regulatory 19 S proteasome cap subunit of the 26 S proteasome complex. Overexpression experiments suggested that CIP75 is involved in the turnover of Cx43 as measured by a significant stimulation of Cx43 degradation and reduction in its half-life with the opposite effects on Cx43 degradation observed in small interference RNA knockdown experiments.     

Solution structure and dynamics of Ufm1, a ubiquitin-fold modifier 1

The ubiquitin-fold modifier 1 (Ufm1) is one of various ubiquitin-like modifiers and conjugates to target proteins in cells through Uba5 (E1) and Ufc1 (E2). The Ufm1-system is conserved in metazoa and plants, suggesting its potential roles in various multicellular organisms. Herein, we analyzed the solution structure and dynamics of human Ufm1 (hsUfm1) by nuclear magnetic resonance spectroscopy. Although the global fold of hsUfm1 is similar to those of ubiquitin (Ub) and NEDD8, the cluster of acidic residues conserved in Ub and NEDD8 does not exist on the Ufm1 surface. 15N spin relaxation data revealed that the amino acid residues of hsUfm1 exhibiting conformational fluctuations form a cluster at the C-terminal segment and its spatial proximity, which correspond to the versatile ligand-binding sites of Ub and other ubiquitin-like proteins (Ubls). We suggest that Ub and other Ubl-modifiers share a common feature of potential conformational multiplicity, which might be associated with the broad ligand specificities of these proteins.     

Crystal structure of ubiquitin-like small archaeal modifier protein 1 (SAMP1) from Haloferax volcanii

The ubiquitin-like (Ubl) system has been shown to be ubiquitous in all three kingdoms of life following the very recent characterization of ubiquitin-like small archaeal modifier proteins (SAMP1 and 2) from Haloferax volcanii. The ubiquitin (Ub) and Ubl molecules in eukaryotes have been studied extensively and their cellular functions are well established. Biochemical and structural data pertaining to prokaryotic Ubl protein (Pup) continue to be reported. In contrast to eukaryotes and prokaryotes, no structural information on the archaeal Ubl molecule is available. Here we determined the crystal structure of SAMP1 at 1.55 Å resolution and generated a model of SAMP2. These were then compared with other Ubl molecules from eukaryotes as well as prokaryotes. The structure of SAMP1 shows a β-grasp fold of Ub, suggesting that the archaeal Ubl molecule is more closely related to eukaryotic Ub and Ubls than to its prokaryotic counterpart. The current structure identifies the location of critical elements such a single lysine residue (Lys4), C-terminal di-glycine motif, hydrophobic patches near leucine 60, and uniquely inserted α-helical segments (α1 and α3) in SAMP1. Based on the structure of SAMP1, several Ub-like features of SAMPs such as poly-SAMPylation and non-covalent interactions have been proposed, which should provide the basis for further investigations concerning the molecular function of archaeal Ubls and the large super-family of β-grasp fold proteins in the archaeal kingdom.     

Rad23 stabilizes Rad4 from degradation by the Ub/proteasome pathway

Rad23 protein interacts with the nucleotide excision-repair (NER) factor Rad4, and the dimer can bind damaged DNA. Rad23 also binds ubiquitinated proteins and promotes their degradation by the proteasome. Rad23/proteasome interaction is required for efficient NER, although the specific role of the Ub/proteasome system in DNA repair is unclear. We report that the availability of Rad4 contributes significantly to the cellular tolerance to UV light. Mutations in the proteasome, and in genes encoding the ubiquitin-conjugating enzymes Ubc4 and Ubc5, stabilized Rad4 and increased tolerance to UV light. A short amino acid sequence, previously identified in human Rad23, mediates the interaction between Rad23 and Rad4. We determined that this motif was required for stabilizing Rad4, and could function independently of the intact protein. A ubiquitin-like (UbL) domain in Rad23 binds the proteasome, and is required for conferring full resistance to DNA damage. However, Rad23/proteasome interaction appears unrelated to Rad23-mediated stabilization of Rad4. Specifically, simultaneous expression of a Rad23 mutant that could not bind the proteasome, with a mutant that could not interact with Rad4, fully suppressed the UV sensitivity of rad23Δ, demonstrating that Rad23 performs two independent, but concurrent roles in NER.     

Taking it step by step: mechanistic insights from structural studies of ubiquitin/ubiquitin-like protein modification pathways

Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins regulate a diverse array of cellular pathways through post-translational attachment to protein substrates. Ub/Ubl-mediated signaling is initiated through E1, E2, and E3-mediated conjugation, transduced by proteins that recognize Ub/Ubl-modified substrates, and terminated by proteases which remove the Ub/Ubl from the substrate. Recent structural studies have elucidated mechanisms pertinent to Ub/Ubl conjugation, recognition, and deconjugation, highlighting essential steps during Ub/Ubl modification that illustrate common and divergent mechanistic themes within this important process.     

A single MIU motif of MINDY‐1 recognizes K48‐linked polyubiquitin chains

The eight different types of ubiquitin (Ub) chains that can be formed play important roles in diverse cellular processes. Linkage‐selective recognition of Ub chains by Ub‐binding domain (UBD)‐containing proteins is central to coupling different Ub signals to specific cellular responses. The motif interacting with ubiquitin (MIU) is a small UBD that has been characterized for its binding to monoUb. The recently discovered deubiquitinase MINDY‐1/FAM63A contains a tandem MIU repeat (tMIU) that is highly selective at binding to K48‐linked polyUb. We here identify that this linkage‐selective binding is mediated by a single MIU motif (MIU2) in MINDY‐1. The crystal structure of MIU2 in complex with K48‐linked polyubiquitin chains reveals that MIU2 on its own binds to all three Ub moieties in an open conformation that can only be accommodated by K48‐linked triUb. The weak Ub binder MIU1 increases overall affinity of the tMIU for polyUb chains without affecting its linkage selectivity. Our analyses reveal new concepts for linkage selectivity and polyUb recognition by UBDs.     

Chemistry-based functional proteomics: mechanism-based activity-profiling tools for ubiquitin and ubiquitin-like specific proteases

Determining the biological function of newly discovered gene products requires the development of novel functional approaches. To facilitate this task, recent developments in proteomics include small molecular probes that target proteolytic enzyme families including serine, threonine, and cysteine proteases. For the families of ubiquitin (Ub) and ubiquitin-like (UBL)-specific proteases, such tools were lacking until recently. Here, we review the advances made in the development of protein-based active site-directed probes that target proteases specific for ubiquitin and ubiquitin-like proteins. Such probes were applied successfully to discover and characterize novel Ub/UBL-specific proteases. Ub/UBL processing and deconjugation are performed by a diverse set of proteases belonging to several different enzyme families, including members of the ovarian tumor domain (OTU) protease family. A further definition of this family of enzymes will benefit from a directed chemical proteomics approach. Some of the Ub/UBL-specific proteases react with multiple Ub/UBLs and members of the same protease family can recognize multiple Ub/UBLs, underscoring the need for tools that appropriately address enzyme specificity.     

A family of ubiquitin-like proteins binds the ATPase domain of Hsp70-like Stch

We have isolated two human ubiquitin-like (UbL) proteins that bind to a short peptide within the ATPase domain of the Hsp70-like Stch protein. Chap1 is a duplicated homologue of the yeast Dsk2 gene that is required for transit through the G2/M phase of the cell cycle and expression of the human full-length cDNA restored viability and suppressed the G2/M arrest phenotype of dsk2Delta rad23Delta Saccharomyces cerevisiae mutants. Chap2 is a homologue for Xenopus scythe which is an essential component of reaper-induced apoptosis in egg extracts. While the N-terminal UbL domains were not essential for Stch binding, Chap1/Dsk2 contains a Sti1-like repeat sequence that is required for binding to Stch and is also conserved in the Hsp70 binding proteins, Hip and p60/Sti1/Hop. These findings extend the association between Hsp70 members and genes encoding UbL sequences and suggest a broader role for the Hsp70-like ATPase family in regulating cell cycle and cell death events.     

Rad23 ubiquitin-associated domains (UBA) inhibit 26 S proteasome-catalyzed proteolysis by sequestering lysine 48-linked polyubiquitin chains

Most substrates of the 26 S proteasome are recognized only following conjugation to a Lys48-linked polyubiquitin chain. Rad23 is one member of a family of proteins that possesses an N-terminal ubiquitin-like domain (UbL) and a C-terminal ubiquitin-associated domain(s) (UBA). Recent studies have shown that UbLs interact with 26 S proteasomes, whereas UBAs bind polyubiquitin chains. These biochemical properties suggest that UbL-UBA proteins may shuttle polyubiquitinated substrates to proteasomes. Here we show that contrary to prediction from this model, the effect of human Rad23A on the degradation of polyubiquitinated substrates catalyzed by purified proteasomes is exclusively inhibitory. Strong inhibition is dependent on the presence of both UBAs, independent of the UbL, and can be explained by competition between the UBA domains and the proteasome for binding to substrate-linked polyubiquitin chains. The UBA domains bind Lys48-linked polyubiquitin chains in strong preference to Lys63 or Lys29-linked chains, leading to selective inhibition of the assembly and disassembly of Lys48-linked chains. These results place constraints on the mechanism(s) by which UbL-UBA proteins promote proteasome-catalyzed proteolysis and reveal new properties of UBA domains.