Defining how ubiquitin receptors hHR23a and S5a bind polyubiquitin

Ubiquitin receptors connect substrate ubiquitylation to proteasomal degradation. HHR23a binds proteasome subunit 5a (S5a) through a surface that also binds ubiquitin. We report that UIM2 of S5a binds preferentially to hHR23a over polyubiquitin, and we provide a model for the ternary complex that we expect represents one of the mechanisms used by the proteasome to capture ubiquitylated substrates. Furthermore, we demonstrate that hHR23a is surprisingly adept at sequestering the ubiquitin moieties of a polyubiquitin chain, and provide evidence that it and the ubiquitylated substrate are committed to each other after binding.     

Structural determinants for the binding of ubiquitin-like domains to the proteasome

HHR23A, a protein implicated in nucleotide excision repair, belongs to a class of proteins containing both a ubiquitin-like (Ubl) domain and one or more ubiquitin-associated (UBA) domains, suggesting a role in the ubiquitin-proteasome pathway as well. The Ubl domain binds with high affinity to the second ubiquitin-interacting motif (UIM) of the S5a subunit of the proteasome. Here we present the solution structures of the HHR23A Ubl domain, the second UIM of S5a (UIM-2), and the Ubl:S5a-UIM-2 complex. The HHR23A Ubl domain is structurally similar to ubiquitin. The S5a UIM forms an alpha-helix with an unexpected hairpin loop that contributes to the binding interface with Ubl. The molecular determinants of the Ubl-proteasome interaction are revealed by analysis of the structures, chemical shift mapping, mutant binding studies and sequence conservation.     

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.     

Binding of HIV-1 Vpr Protein to the Human Homolog of the Yeast DNA Repair Protein RAD23 (hHR23A) Requires Its Xeroderma Pigmentosum Complementation Group C Binding (XPCB) Domain as Well as the Ubiquitin-associated 2 (UBA2) Domain

The human homolog of the yeast DNA repair protein RAD23, hHR23A, has been found previously to interact with the human immunodeficiency virus, type 1 accessory protein Vpr. hHR23A is a modular protein containing an N-terminal ubiquitin-like (UBL) domain and two ubiquitin-associated domains (UBA1 and UBA2) separated by a xeroderma pigmentosum complementation group C binding (XPCB) domain. All domains are connected by flexible linkers. hHR23A binds ubiquitinated proteins and acts as a shuttling factor to the proteasome. Here, we show that hHR23A utilizes both the UBA2 and XPCB domains to form a stable complex with Vpr, linking Vpr directly to cellular DNA repair pathways and their probable exploitation by the virus. Detailed structural mapping of the Vpr contacts on hHR23A, by NMR, revealed substantial contact surfaces on the UBA2 and XPCB domains. In addition, Vpr binding disrupts an intramolecular UBL-UBA2 interaction. We also show that Lys-48-linked di-ubiquitin, when binding to UBA1, does not release the bound Vpr from the hHR23A-Vpr complex. Instead, a ternary hHR23A·Vpr·di-Ub^K48 complex is formed, indicating that Vpr does not necessarily abolish hHR23A-mediated shuttling to the proteasome.     

Interaction of hHR23 with S5a. The ubiquitin-like domain of hHR23 mediates interaction with S5a subunit of 26 S proteasome.

hHR23B is one of two human homologs of the Saccharomyces cerevisiae nucleotide excision repair (NER) gene product RAD23 and a component of a protein complex that specifically complements the NER defect of xeroderma pigmentosum group C (XP-C) cell extracts in vitro. Although a small proportion of hHR23B is tightly complexed with the XP-C responsible gene product, XPC protein, a vast majority exists as an XPC-free form, indicating that hHR23B has additional functions other than NER in vivo. Here we demonstrate that the human NER factor hHR23B as well as another human homolog of RAD23, hHR23A, interact specifically with S5a, a subunit of the human 26 S proteasome using the yeast two-hybrid system. Furthermore, hHR23 proteins were detected with S5a at the position where 26 S proteasome sediments in glycerol gradient centrifugation of HeLa S100 extracts. Intriguingly, hHR23B showed the inhibitory effect on the degradation of (125)I-lysozyme in the rabbit reticulocyte lysate. hHR23 proteins thus appear to associate with 26 S proteasome in vivo. From co-precipitation experiments using several series of deletion mutants, we defined the domains in hHR23B and S5a that mediate this interaction. From these results, we propose that part of hHR23 proteins are involved in the proteolytic pathway in cells.     

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.     

Interplay between poxviruses and the cellular ubiquitin/ubiquitin-like pathways

Post-translational polypeptide tagging by conjugation with ubiquitin and ubiquitin-like (Ub/Ubl) molecules is a potent way to alter protein functions and/or sort specific protein targets to the proteasome for degradation. Many poxviruses interfere with the host Ub/Ubl system by encoding viral proteins that can usurp this pathway. Some of these include viral proteins of the membrane-associated RING-CH (MARCH) domain, p28/Really Interesting New Gene (RING) finger, ankyrin-repeat/F-box and Broad-complex, Tramtrack and Bric-a-Brac (BTB)/Kelch subgroups of the E3 Ub ligase superfamily. Here we describe and discuss the various strategies used by poxviruses to target and subvert the host cell Ub/Ubl systems.     

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.     

Budding yeast Dsk2 protein forms a homodimer via its C-terminal UBA domain

Budding yeast Dsk2 is a family of UbL-UBA proteins that can interact with both polyubiquitin and the proteasome, and is thereby thought to function as a shuttle protein in the ubiquitin-proteasome pathway. Here we show that Dsk2 can homodimerize via its C-terminal UBA domain in the absence of ubiquitin. Dsk2 mutants defective in the UBA domain do not dimerize and do not bind polyubiquitin. The expression of Dsk2 UBA mutants fails to restore the growth defect caused by DSK2 disruption although that of wild-type Dsk2 can restore the defect. These results suggest that Dsk2 homodimerization via the UBA domain plays a role in regulating polyubiquitin binding in the ubiquitin-proteasome pathway.     

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.     

Mechanistic Study of Uba5 Enzyme and the Ufm1 Conjugation Pathway

E1 enzymes activate ubiquitin or ubiquitin-like proteins (Ubl) via an adenylate intermediate and initiate the enzymatic cascade of Ubl conjugation to target proteins or lipids. Ubiquitin-fold modifier 1 (Ufm1) is activated by the E1 enzyme Uba5, and this pathway is proposed to play an important role in the endoplasmic reticulum (ER) stress response. However, the mechanisms of Ufm1 activation by Uba5 and subsequent transfer to the conjugating enzyme (E2), Ufc1, have not been studied in detail. In this work, we found that Uba5 activated Ufm1 via a two-step mechanism and formed a binary covalent complex of Uba5∼Ufm1 thioester. This feature contrasts with the three-step mechanism and ternary complex formation in ubiquitin-activating enzyme Uba1. Uba5 displayed random ordered binding with Ufm1 and ATP, and its ATP-pyrophosphate (PP_i) exchange activity was inhibited by both AMP and PP_i. Ufm1 activation and Uba5∼Ufm1 thioester formation were stimulated in the presence of Ufc1. Furthermore, binding of ATP to Uba5∼Ufm1 thioester was required for efficient transfer of Ufm1 from Uba5 to Ufc1 via transthiolation. Consistent with the two-step activation mechanism, the mechanism-based pan-E1 inhibitor, adenosine 5′-sulfamate (ADS), reacted with the Uba5∼Ufm1 thioester and formed a covalent, tight-binding Ufm1-ADS adduct in the active site of Uba5, which prevented further substrate binding or catalysis. ADS was also shown to inhibit the Uba5 conjugation pathway in the HCT116 cells through formation of the Ufm1-ADS adduct. This suggests that further development of more selective Uba5 inhibitors could be useful in interrogating the roles of the Uba5 pathway in cells.     

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.     

A conditional yeast E1 mutant blocks the ubiquitin-proteasome pathway and reveals a role for ubiquitin conjugates in targeting Rad23 to the proteasome

E1 ubiquitin activating enzyme catalyzes the initial step in all ubiquitin-dependent processes. We report the isolation of uba1-204, a temperature-sensitive allele of the essential Saccharomyces cerevisiae E1 gene, UBA1. Uba1-204 cells exhibit dramatic inhibition of the ubiquitin-proteasome system, resulting in rapid depletion of cellular ubiquitin conjugates and stabilization of multiple substrates. We have employed the tight phenotype of this mutant to investigate the role ubiquitin conjugates play in the dynamic interaction of the UbL/UBA adaptor proteins Rad23 and Dsk2 with the proteasome. Although proteasomes purified from mutant cells are intact and proteolytically active, they are depleted of ubiquitin conjugates, Rad23, and Dsk2. Binding of Rad23 to these proteasomes in vitro is enhanced by addition of either free or substrate-linked ubiquitin chains. Moreover, association of Rad23 with proteasomes in mutant and wild-type cells is improved upon stabilizing ubiquitin conjugates with proteasome inhibitor. We propose that recognition of polyubiquitin chains by Rad23 promotes its shuttling to the proteasome in vivo.     

The E3 Ubiquitin Ligase Parkin Is Recruited to the 26 S Proteasome via the Proteasomal Ubiquitin Receptor Rpn13

Mutations in the Park2 gene, encoding the RING-HECT hybrid E3 ubiquitin ligase parkin, are responsible for a common familial form of Parkinson disease. By mono- and polyubiquitinating target proteins, parkin regulates various cellular processes, including degradation of proteins within the 26 S proteasome, a large multimeric degradation machine. In our attempt to further elucidate the function of parkin, we have identified the proteasomal ubiquitin receptor Rpn13/ADRM1 as a parkin-interacting protein. We show that the N-terminal ubiquitin-like (Ubl) domain of parkin binds directly to the pleckstrin-like receptor for ubiquitin (Pru) domain within Rpn13. Using mutational analysis and NMR, we find that Pru binding involves the hydrophobic patch surrounding Ile-44 in the parkin Ubl, a region that is highly conserved between ubiquitin and Ubl domains. However, compared with ubiquitin, the parkin Ubl exhibits greater than 10-fold higher affinity for the Pru domain. Moreover, knockdown of Rpn13 in cells increases parkin levels and abrogates parkin recruitment to the 26 S proteasome, establishing Rpn13 as the major proteasomal receptor for parkin. In contrast, silencing Rpn13 did not impair parkin recruitment to mitochondria or parkin-mediated mitophagy upon carbonyl cyanide m-chlorophenyl hydrazone-induced mitochondrial depolarization. However, it did delay the clearance of mitochondrial proteins (TIM23, TIM44, and TOM20) and enhance parkin autoubiquitination. Taken together, these findings implicate Rpn13 in linking parkin to the 26 S proteasome and regulating the clearance of mitochondrial proteins during mitophagy.     

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.     

Structure of the XPC binding domain of hHR23A reveals hydrophobic patches for protein interaction

Rad23 proteins are involved both in the ubiquitin-proteasome pathway and in nucleotide excision repair (NER), but the relationship between these two pathways is not yet understood. The two human homologs of Rad23, hHR23A and B, are functionally redundant in NER and interact with xeroderma pigmentosum complementation group C (XPC) protein. The XPC-hHR23 complex is responsible for the specific recognition of damaged DNA, which is an early step in NER. The interaction of the XPC binding domain (XPCB) of hHR23A/B with XPC protein has been shown to be important for its optimal function in NER. We have determined the solution structure of XPCB of hHR23A. The domain consists of five amphipathic helices and reveals hydrophobic patches on the otherwise highly hydrophilic domain surface. The patches are predicted to be involved in interaction with XPC. The XPCB domain has limited sequence homology with any proteins outside of the Rad23 family except for sacsin, a protein involved in spastic ataxia of Charlevoix-Saguenay, which contains a domain with 35% sequence identity.     

Ubiquitin family proteins and their relationship to the proteasome: a structural perspective

Many biological processes rely on targeted protein degradation, the dysregulation of which contributes to the pathogenesis of various diseases. Ubiquitin plays a well-established role in this process, in which the covalent attachment of polyubiquitin chains to protein substrates culminates in their degradation via the proteasome. The three-dimensional structural topology of ubiquitin is highly conserved as a domain found in a variety of proteins of diverse biological function. Some of these so-called "ubiquitin family proteins" have recently been shown to bind components of the 26S proteasome via their ubiquitin-like domains, thus implicating proteasome activity in pathways other than protein degradation. In this chapter, we provide a structural perspective of how the ubiquitin family of proteins interacts with the proteasome.     

E2-binding surface on Uba3 β-grasp domain undergoes a conformational transition

The covalent attachment of ubiquitin (Ub) and ubiquitin‐like (Ubl) proteins to various eukaryotic targets plays critical roles in regulating numerous cellular processes. E1‐activating enzymes are critical, because they catalyze activation of their cognate Ub/Ubl protein and are responsible for its transfer to the correct E2‐conjugating enzyme(s). The activating enzyme for neural‐precursor‐cell‐expressed developmentally downregulated 8 (NEDD8) is a heterodimer composed of APPBP1 and Uba3 subunits. The carboxyl terminal ubiquitin‐like β‐grasp domain of human Uba3 (Uba3‐βGD) has been suggested as a key E2‐binding site defining E2 specificity. In crystal structures of free E1 and the NEDD8‐E1 complex, the E2‐binding surface on the domain was missing from the electron density. However, when complexed with various E2s, this missing segment adopts a kinked α‐helix. Here, we demonstrate that Uba3‐βGD is an independently folded domain in solution and that residues involved in E2 binding are absent from the NMR spectrum, indicating that the E2‐binding surface on Uba3‐βGD interconverts between multiple conformations, analogous to a similar conformational transition observed in the E2‐binding surface of SUMO E1. These results suggest that access to multiple conformational substates is an important feature of the E1–E2 interaction. Proteins 2012;. © 2012 Wiley Periodicals, Inc.