The hPLIC proteins may provide a link between the ubiquitination machinery and the proteasome

Although there is a binding site on the proteasome for the polyubiquitin chains attached to degradation substrates by the ubiquitination machinery, it is currently unclear whether in vivo the activities of the ubiquitination machinery and the proteasome are coupled. Here we show that two human homologs of the yeast ubiquitin-like Dsk2 protein, hPLIC-1 and hPLIC-2, physically associate with both proteasomes and ubiquitin ligases in large complexes. Overexpression of hPLIC proteins interferes with the in vivo degradation of two unrelated ubiquitin-dependent proteasome substrates, p53 and IkappaBalpha, but not a ubiquitin-independent substrate. Our findings raise the possibility that the hPLIC proteins, and possibly related ubiquitin-like family members, may functionally link the ubiquitination machinery to the proteasome to affect in vivo protein degradation.     

Structural studies of the interaction between ubiquitin family proteins and proteasome subunit S5a

The 26S proteasome is essential for the proteolysis of proteins that have been covalently modified by the attachment of polyubiquitinated chains. Although the 20S core particle performs the degradation, the 19S regulatory cap complex is responsible for recognition of polyubiquitinated substrates. We have focused on how the S5a component of the 19S complex interacts with different ubiquitin-like (ubl) modules, to advance our understanding of how polyubiquitinated proteins are targeted to the proteasome. To achieve this, we have determined the solution structure of the ubl domain of hPLIC-2 and obtained a structural model of hHR23a by using NMR spectroscopy and homology modeling. We have also compared the S5a binding properties of ubiquitin, SUMO-1, and the ubl domains of hPLIC-2 and hHR23a and have identified the residues on their respective S5a contact surfaces. We provide evidence that the S5a-binding surface on the ubl domain of hPLIC-2 is required for its interaction with the proteasome. This study provides structural insights into protein recognition by the proteasome, and illustrates how the protein surface of a commonly utilized fold has highly evolved for various biological roles.     

Defining the geometry of the two-component proteasome degron

The eukaryotic 26S proteasome controls cellular processes by degrading specific regulatory proteins. Most proteins are targeted for degradation by a signal or degron that consists of two parts: a proteasome-binding tag, typically covalently attached polyubiquitin chains, and an unstructured region that serves as the initiation region for proteasomal proteolysis. Here we have characterized how the arrangement of the two degron parts in a protein affects degradation. We found that a substrate is degraded efficiently only when its initiation region is of a certain minimal length and is appropriately separated in space from the proteasome-binding tag. Regions that are located too close or too far from the proteasome-binding tag cannot access the proteasome and induce degradation. These spacing requirements are different for a polyubiquitin chain and a ubiquitin-like domain. Thus, the arrangement and location of the proteasome initiation region affect a protein's fate and are important in selecting proteins for proteasome-mediated degradation.     

Ubiquitin receptor proteins hHR23a and hPLIC2 interact

Ubiquitin receptor proteins play an important role in delivering ubiquitylated protein substrates to the proteasome for degradation. HHR23a and hPLIC2 are two such ubiquitin receptors that contain ubiquitin-like (UBL) domains, which interact with the proteasome, and ubiquitin-associated (UBA) domains, which interact with ubiquitin. Depending on their abundance UBL/UBA family members can either promote or inhibit the degradation of other proteins, which suggests their participation in the delivery of substrates to the proteasome is highly regulated. In previous work, we determined UBL/UBA domain interactions to promote intramolecular interactions in hHR23a that are abrogated with the addition of either ubiquitin or the proteasome component S5a. In yeast, we determined the hHR23a ortholog (Rad23) to interact with another UBL/UBA family member (Ddi1) and to bind a common tetraubiquitin chain. Here, we use NMR spectroscopy to reveal that hHR23a interacts with hPLIC2 via UBL/UBA domain interactions and to map their binding surfaces. In addition, we demonstrate that these two proteins associate in mammalian cells. Intriguingly, inhibition of the proteasome mitigates hHR23a/hPLIC2 interaction.     

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.     

Proteasome subunit Rpn1 binds ubiquitin-like protein domains

The yeast protein Rad23 belongs to a diverse family of proteins that contain an amino-terminal ubiquitin-like (UBL) domain. This domain mediates the binding of Rad23 to proteasomes, which in turn promotes DNA repair and modulates protein degradation, possibly by delivering ubiquitinylated cargo to proteasomes. Here we show that Rad23 binds proteasomes by directly interacting with the base subcomplex of the regulatory particle of the proteasome. A component of the base, Rpn1, specifically recognizes the UBL domain of Rad23 through its leucine-rich-repeat-like (LRR-like) domain. A second UBL protein, Dsk2, competes with Rad23 for proteasome binding, which suggests that the LRR-like domain of Rpn1 may participate in the recognition of several ligands of the proteasome. We propose that the LRR domain of Rpn1 may be positioned in the base to allow the cargo proteins carried by Rad23 to be presented to the proteasomal ATPases for unfolding. We also report that, contrary to expectation, the base subunit Rpn10 does not mediate the binding of UBL proteins to the proteasome in yeast, although it can apparently contribute to the binding of ubiquitin chains by intact proteasomes.     

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.     

Interaction with a ubiquitin-like protein enhances the ubiquitination and degradation of hepatitis C virus RNA-dependent RNA polymerase

To identify potential cellular regulators of hepatitis C virus (HCV) RNA-dependent RNA polymerase (NS5B), we searched for cellular proteins interacting with NS5B protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a ubiquitin-like protein, hPLIC1 (for human homolog 1 of protein linking intergrin-associated protein and cytoskeleton), which is expressed in the liver (M. F. Kleijnen, A. H. Shih, P. Zhou, S. Kumar, R. E. Soccio, N. L. Kedersha, G. Gill, and P. M. Howley, Mol. Cell 6: 409-419, 2000). In vitro binding assays and in vivo coimmunoprecipitation studies confirmed the interaction between hPLIC1 and NS5B, which occurred through the ubiquitin-associated domain at the C terminus of the hPLIC1 protein. As hPLICs have been shown to physically associate with two E3 ubiquitin protein ligases as well as proteasomes (Kleijnen et al., Mol. Cell 6: 409-419, 2000), we investigated whether the stability and posttranslational modification of NS5B were affected by hPLIC1. A pulse-chase labeling experiment revealed that overexpression of hPLIC1, but not the mutant lacking the NS5B-binding domain, significantly shortened the half-life of NS5B and enhanced the polyubiquitination of NS5B. Furthermore, in Huh7 cells that express an HCV subgenomic replicon, the amounts of both NS5B and the replicon RNA were reduced by overexpression of hPLIC1. Thus, hPLIC1 may be a regulator of HCV RNA replication through interaction with NS5B.     

VWA domain of S5a restricts the ability to bind ubiquitin and Ubl to the 26S proteasome

The 26S proteasome recognizes a vast number of ubiquitin-dependent degradation signals linked to various substrates. This recognition is mediated mainly by the stoichiometric proteasomal resident ubiquitin receptors S5a and Rpn13, which harbor ubiquitin-binding domains. Regulatory steps in substrate binding, processing, and subsequent downstream proteolytic events by these receptors are poorly understood. Here we demonstrate that mammalian S5a is present in proteasome-bound and free states. S5a is required for efficient proteasomal degradation of polyubiquitinated substrates and the recruitment of ubiquitin-like (Ubl) harboring proteins; however, S5a-mediated ubiquitin and Ubl binding occurs only on the proteasome itself. We identify the VWA domain of S5a as a domain that limits ubiquitin and Ubl binding to occur only upon proteasomal association. Multiubiquitination events within the VWA domain can further regulate S5a association. Our results provide a molecular explanation to how ubiquitin and Ubl binding to S5a is restricted to the 26S proteasome.     

Integral UBL domain proteins: a family of proteasome interacting proteins

The family of ubiquitin-like (UBL) domain proteins (UDPs) comprises a conserved group of proteins involved in a multitude of different cellular activities. However, recent studies on UBL-domain proteins indicate that these proteins appear to share a common property in their ability to interact with 26S proteasomes. The 26S proteasome is a multisubunit protease which is responsible for the majority of intracellular proteolysis in eukaryotic cells. Before degradation commences most proteins are first marked for destruction by being coupled to a chain of ubiquitin molecules. Some UBL-domain proteins catalyse the formation of ubiquitin-protein conjugates, whereas others appear to target ubiquitinated proteins for degradation and interact with chaperones. Hence, by binding to the 26S proteasome the UBL-domain proteins seem to tailor and direct the basic proteolytic functions of the particle to accommodate various cellular substrates.     

Proteasomes and protein conjugation across domains of life

Like other energy-dependent proteases, proteasomes, which are found across the three domains of life, are self-compartmentalized and important in the early steps of proteolysis. Proteasomes degrade improperly synthesized, damaged or misfolded proteins and hydrolyse regulatory proteins that must be specifically removed or cleaved for cell signalling. In eukaryotes, proteins are typically targeted for proteasome-mediated destruction through polyubiquitylation, although ubiquitin-independent pathways also exist. Interestingly, actinobacteria and archaea also covalently attach small proteins (prokaryotic ubiquitin-like protein (Pup) and small archaeal modifier proteins (Samps), respectively) to certain proteins, and this may serve to target the modified proteins for degradation by proteasomes.     

Modeling and analysis of molecularinteraction between Smurf1-WW2 domain and various isoforms of LIM mineralization protein

LIM Mineralization Protein-1 (LMP-1) has been cloned and shown to be osteoinductive. Our efforts to understand the mode of action of LMP-1 led to the determination that LMP-1 interacts with Smad Ubiquitin Regulatory Factor-1 (Smurf1). Smurf1 targets osteogenic Smads, Smad1/5, for ubiquitin-mediated proteasomal degradation. Smurf1 interaction with LMP-1 or Smads is based on the presence of unique WW-domain interacting motif in these target molecules. By performing site-directed mutagenesis and binding studies in vitro on purified recombinant proteins, we identified a specific motif within the osteogenic region of several LMP isoforms that is necessary for Smurf1 interaction. Similarly, we have identified that the WW2 domain of Smurf1 is necessary for target protein interaction. Here, we present a homology-based modeling of the Smurf1 WW2 domain and its interacting motif of LMP-1. We performed computational docking of the interacting domains in Smurf1 and LMPs to identify the key amino acid residues involved in their binding regions. In support of the computational predictions, we also present biochemical evidence supporting the hypothesis that the physical interaction of Smurf1 and osteoinductive forms of LMP may prevent Smurf1 from targeting osteogenic Smads by ubiquitin-mediated proteasomal degradation.     

The structure of the APPBP1-UBA3-NEDD8-ATP complex reveals the basis for selective ubiquitin-like protein activation by an E1

E1 enzymes initiate ubiquitin-like protein (ubl) transfer cascades by catalyzing adenylation of the ubl's C terminus. An E1's selectivity for its cognate ubl is essential because the E1 subsequently coordinates the ubl with its correct downstream pathway. We report here the structure of the 120 kDa quaternary complex between human APPBP1-UBA3, a heterodimeric E1, its ubl NEDD8, and ATP. The E1 selectively recruits NEDD8 through a bipartite interface, involving a domain common to all ubl activating enzymes including bacterial ancestors, and also eukaryotic E1-specific sequences. By modeling ubiquitin into the NEDD8 binding site and performing mutational analysis, we identify a single conserved arginine in APPBP1-UBA3 that acts as a selectivity gate, preventing misactivation of ubiquitin by NEDD8's E1. NEDD8 residues that interact with E1 correspond to residues in ubiquitin important for binding the proteasome and other ubiquitin-interacting proteins, suggesting that the conjugation and recognition machineries have coevolved for each specific ubl.     

The Yeast E4 Ubiquitin Ligase Ufd2 Interacts with the Ubiquitin-like Domains of Rad23 and Dsk2 via a Novel and Distinct Ubiquitin-like Binding Domain

Proteins containing ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains interact with various binding partners and function as hubs during ubiquitin-mediated protein degradation. A common interaction of the budding yeast UBL-UBA proteins Rad23 and Dsk2 with the E4 ubiquitin ligase Ufd2 has been described in endoplasmic reticulum-associated degradation among other pathways. The UBL domains of Rad23 and Dsk2 play a prominent role in this process by interacting with Ufd2 and different subunits of the 26 S proteasome. Here, we report crystal structures of Ufd2 in complex with the UBL domains of Rad23 and Dsk2. The N-terminal UBL-interacting region of Ufd2 exhibits a unique sequence pattern, which is distinct from any known ubiquitin- or UBL-binding domain identified so far. Residue-specific differences exist in the interactions of these UBL domains with Ufd2, which are coupled to subtle differences in their binding affinities. The molecular details of their differential interactions point to a role for adaptive evolution in shaping these interfaces.     

Evidence for distinct functions for human DNA repair factors hHR23A and hHR23B

Rad23 proteins bind ubiquitinated substrates and the proteasome, consistent with an important role in protein degradation. Although human Rad23 proteins (hHR23A and hHR23B) have redundant roles in DNA repair, we determined they formed distinct interactions with proteasomes and multiubiquitinated proteins, but similar binding to Ataxin-3. Threonine-79 contributed to the weak proteasome-binding property of hHR23A, and its conversion to proline (T79P), which is the residue present in hHR23B, increased proteasome interaction. We also determined that hHR23A and hHR23B could be co-purified with unique proteolytic and stress-responsive factors from human breast cancer tissues, indicating that they have unique functions in vivo.     

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.     

Stalled proteasomes are directly relieved by P97 recruitment

The 26 S proteasome is the eukaryotic protease responsible for the degradation of most cellular proteins. As such it accommodates the ability to function under diverse conditions that the cell may encounter. This function is supported by various adaptors that modulate various aspects in protein degradation, these include regulation of substrate delivery, deubiquitination, unfolding, and 20 S gate dilation. Here we show a new functional complex between the P97 and the proteasome that is assembled in response to proteasomal impairment. This entails P97 binding to the 26 S proteasome via the 19 S particle thereby forming an additional hexameric ATPase ring to relieve repression. P97-bound proteasomes showed selective binding toward the Npl4-ufd1 P97 co-factors, indicating a unique cellular role for P97 binding to proteasomes. P97-bound proteasomes display enhanced activity, showing a relief in proteolysis impairment. Our findings place P97 directly in non-ERAD proteasomal functions and establish a new checkpoint in UPS impairment. The ability to modulate proteasome activity and properly respond to protein misfolding, is of great importance in cellular regulation.     

RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation

In acute promyelocytic leukaemia (APL), the promyelocytic leukaemia (PML) protein is fused to the retinoic acid receptor alpha (RAR). This disease can be treated effectively with arsenic, which induces PML modification by small ubiquitin-like modifiers (SUMO) and proteasomal degradation. Here we demonstrate that the RING-domain-containing ubiquitin E3 ligase, RNF4 (also known as SNURF), targets poly-SUMO-modified proteins for degradation mediated by ubiquitin. RNF4 depletion or proteasome inhibition led to accumulation of mixed, polyubiquitinated, poly-SUMO chains. PML protein accumulated in RNF4-depleted cells and was ubiquitinated by RNF4 in a SUMO-dependent fashion in vitro. In the absence of RNF4, arsenic failed to induce degradation of PML and SUMO-modified PML accumulated in the nucleus. These results demonstrate that poly-SUMO chains can act as discrete signals from mono-SUMOylation, in this case targeting a poly-SUMOylated substrate for ubiquitin-mediated proteolysis.     

Why do cellular proteins linked to K63-polyubiquitin chains not associate with proteasomes?

Although cellular proteins conjugated to K48‐linked Ub chains are targeted to proteasomes, proteins conjugated to K63‐ubiquitin chains are directed to lysosomes. However, pure 26S proteasomes bind and degrade K48‐ and K63‐ubiquitinated substrates similarly. Therefore, we investigated why K63‐ubiquitinated proteins are not degraded by proteasomes. We show that mammalian cells contain soluble factors that selectively bind to K63 chains and inhibit or prevent their association with proteasomes. Using ubiquitinated proteins as affinity ligands, we found that the main cellular proteins that associate selectively with K63 chains and block their binding to proteasomes are ESCRT0 (Endosomal Sorting Complex Required for Transport) and its components, STAM and Hrs. In vivo, knockdown of ESCRT0 confirmed that it is required to block binding of K63‐ubiquitinated molecules to the proteasome. In addition, the Rad23 proteins, especially hHR23B, were found to bind specifically to K48‐ubiquitinated proteins and to stimulate proteasome binding. The specificities of these proteins for K48‐ or K63‐ubiquitin chains determine whether a ubiquitinated protein is targeted for proteasomal degradation or delivered instead to the endosomal‐lysosomal pathway.