K63-specific deubiquitination by two JAMM/MPN+ complexes: BRISC-associated Brcc36 and proteasomal Poh1.

An unusual deubiquitinating (DUB) activity exists in HeLa cell extracts that is highly specific for cleaving K63-linked but not K48-linked polyubiquitin chains. The activity is insensitive to both N-ethyl-maleimide and ubiquitin aldehyde, indicating that it lacks an active site cysteine residue, and gel filtration experiments show that it resides in a high molecular weight (approximately 600 kDa) complex. Using a biochemical approach, we found that the K63-specific DUB activity co-fractionated through seven chromatographic steps with three multisubunit complexes: the 19S (PA700) portion of the 26S proteasome, the COP9 signalosome (CSN) and a novel complex that includes the JAMM/MPN+ domain-containing protein Brcc36. When we analysed the individual complexes, we found that the activity was intrinsic to PA700 and the Brcc36 isopeptidase complex (BRISC), but that the CSN-associated activity was due entirely to an interaction with Brcc36. None of the complexes cleave K6, K11, K29, K48 or alpha-linked polyubiquitin, but they do cleave K63 linkages within mixed-linkage chains. Our results suggest that specificity for K63-linked polyubiquitin is a common property of the JAMM/MPN+ family of DUBs.

Mms2-Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific polyubiquitin chain formation.

Lys63-linked polyubiquitin chains participate in nonproteolytic signaling pathways, including regulation of DNA damage tolerance and NF-kappaB activation. E2 enzymes bound to ubiquitin E2 variants (UEV) are vital in these pathways, synthesizing Lys63-linked polyubiquitin chains, but how these complexes achieve specificity for a particular lysine linkage has been unclear. We have determined the crystal structure of an Mms2-Ubc13-ubiquitin (UEV-E2-Ub) covalent intermediate with donor ubiquitin linked to the active site residue of Ubc13. In the structure, the unexpected binding of a donor ubiquitin of one Mms2-Ubc13-Ub complex to the acceptor-binding site of Mms2-Ubc13 in an adjacent complex allows us to visualize at atomic resolution the molecular determinants of acceptor-ubiquitin binding. The structure reveals the key role of Mms2 in allowing selective insertion of Lys63 into the Ubc13 active site and suggests a molecular model for polyubiquitin chain elongation.

Synthesis of free and proliferating cell nuclear antigen-bound polyubiquitin chains by the RING E3 ubiquitin ligase Rad5.

In replicating yeast, lysine 63-linked polyubiquitin (polyUb) chains are extended from the ubiquitin moiety of monoubiquitinated proliferating cell nuclear antigen (monoUb-PCNA) by the E2-E3 complex of (Ubc13-Mms2)-Rad5. This promotes error-free bypass of DNA damage lesions. The unusual ability of Ubc13-Mms2 to synthesize unanchored Lys(63)-linked polyUb chains in vitro allowed us to resolve the individual roles that it and Rad5 play in the catalysis and specificity of PCNA polyubiquitination. We found that Rad5 stimulates the synthesis of free polyUb chains by Ubc13-Mms2 in part by enhancing the reactivity of the Ubc13 approximately Ub thiolester bond. Polyubiquitination of monoUb-PCNA was further enhanced by interactions between the N-terminal domain of Rad5 and PCNA. Thus, Rad5 acts both to align monoUb-PCNA with Ub-charged Ubc13 and to stimulate Ub transfer onto Lys(63) of a Ub acceptor. We also found that Rad5 interacts with PCNA independently of the number of monoubiquitinated subunits in the trimer and that it binds to both unmodified and monoUb-PCNA with similar affinities. These findings indicate that Rad5-mediated recognition of monoUb-PCNA in vivo is likely to depend upon interactions with additional factors at stalled replication forks.

Diverse polyubiquitin interaction properties of ubiquitin-associated domains.

The ubiquitin-associated (UBA) domain occurs frequently in proteins involved in ubiquitin-dependent signaling pathways. Although polyubiquitin chain binding is considered to be a defining feature of the UBA domain family, the generality of this property has not been established. Here we have surveyed the polyubiquitin interaction properties of 30 UBA domains, including 16 of 17 occurrences in budding yeast. The UBA domains sort into four classes that include linkage-selective polyubiquitin binders and domains that bind different chains (and monoubiquitin) in a nondiscriminatory manner; one notable class ( approximately 30%) did not bind any ubiquitin ligand surveyed. The properties of a given UBA domain are conserved from yeast to mammals. Their functional relevance is further suggested by the ability of an ectopic UBA domain to alter the specificity of a deubiquitylating enzyme in a predictable manner. Conversely, non-UBA sequences can modulate the interaction properties of a UBA domain.

The ubiquitin-proteasome pathway in Parkinson's disease and other neurodegenerative diseases.

During the past decade, it has become apparent that a set of ostensibly unrelated neurodegenerative diseases, including Parkinson's disease and Huntington's disease, shares striking molecular and cell biology commonalities. Each of the diseases involves protein misfolding and aggregation, resulting in inclusion bodies and other aggregates within cells. These aggregates often contain ubiquitin, which is the signal for proteolysis by the 26S proteasome, and chaperone proteins that are involved in the refolding of misfolded proteins. The link between the ubiquitin-proteasome system and neurodegeneration has been strengthened by the identification of disease-causing mutations in genes coding for several ubiquitin-proteasome pathway proteins in Parkinson's disease. However, the exact molecular connections between these systems and pathogenesis remain uncertain and controversial. In this article, we summarize the state of current knowledge, focusing on important unresolved questions.

Crystal structure and solution NMR studies of Lys48-linked tetraubiquitin at neutral pH.

Ubiquitin modification of proteins is used as a signal in many cellular processes. Lysine side-chains can be modified by a single ubiquitin or by a polyubiquitin chain, which is defined by an isopeptide bond between the C terminus of one ubiquitin and a specific lysine in a neighboring ubiquitin. Polyubiquitin conformations that result from different lysine linkages presumably differentiate their roles and ability to bind specific targets and enzymes. However, conflicting results have been obtained regarding the precise conformation of Lys48-linked tetraubiquitin. We report the crystal structure of Lys48-linked tetraubiquitin at near-neutral pH. The two tetraubiquitin complexes in the asymmetric unit show the complete connectivity of the chain and the molecular details of the interactions. This tetraubiquitin conformation is consistent with our NMR data as well as with previous studies of diubiquitin and tetraubiquitin in solution at neutral pH. The structure provides a basis for understanding Lys48-linked polyubiquitin recognition under physiological conditions.

Ubiquitin: structures, functions, mechanisms.

Ubiquitin is the founding member of a family of structurally conserved proteins that regulate a host of processes in eukaryotic cells. Ubiquitin and its relatives carry out their functions through covalent attachment to other cellular proteins, thereby changing the stability, localization, or activity of the target protein. This article reviews the basic biochemistry of these protein conjugation reactions, focusing on ubiquitin itself and emphasizing recent insights into mechanism and specificity.

Controlled synthesis of polyubiquitin chains.

Many intracellular signaling processes depend on the modification of proteins with polymers of the conserved 76-residue protein ubiquitin. The ubiquitin units in such polyubiquitin chains are connected by isopeptide bonds between a specific lysine residue of one ubiquitin and the carboxyl group of G76 of the next ubiquitin. Chains linked through K48-G76 and K63-G76 bonds are the best characterized, signaling proteasome degradation and nonproteolytic outcomes, respectively. The molecular determinants of polyubiquitin chain recognition are under active investigation; both the chemical structure and the length of the chain can influence signaling outcomes. In this article, we describe the protein reagents necessary to produce K48- and K63-linked polyubiquitin chains and the use of these materials to produce milligram quantities of specific-length chains for biochemical and biophysical studies. The method involves reactions catalyzed by linkage-specific conjugating factors, in which proximally and distally blocked monoubiquitins (or chains) are joined to produce a particular chain product in high yield. Individual chains are then deblocked and joined in another round of reaction. Successive rounds of deblocking and synthesis give rise to a chain of the desired length.

Chemical and genetic strategies for manipulating polyubiquitin chain structure.

Ubiquitin can be conjugated to lysine residues of other ubiquitin molecules to form polymers called polyubiquitin chains. Ubiquitin has seven lysine residues, creating the potential for seven distinct types of chains, at least five of which have been observed in vitro or in vivo. A subset of these chains mediates substrate targeting to proteasomes, whereas other types of chains have been implicated in nonproteolytic signaling pathways. In this chapter, we outline chemical and genetic strategies that can be used to deduce (or control) the structures of polyubiquitin chains in vitro and in living cells.

Polyubiquitin chains: polymeric protein signals.

The 76-residue protein ubiquitin exists within eukaryotic cells both as a monomer and in the form of isopeptide-linked polymers called polyubiquitin chains. In two well-described cases, structurally distinct polyubiquitin chains represent functionally distinct intracellular signals. Recently, additional polymeric structures have been detected in vivo and in vitro, and several large families of proteins with polyubiquitin chain-binding activity have been discovered. Although the molecular mechanisms governing specificity in chain synthesis and recognition are still incompletely understood, the scope of signaling by polyubiquitin chains is likely to be broader than originally envisioned.

Binding of polyubiquitin chains to ubiquitin-associated (UBA) domains of HHR23A.

Ubiquitin-associated (UBA) domains are small protein domains that occur in the context of larger proteins and are likely to function as inter- and intramolecular communication elements in ubiquitin/polyubiquitin signaling. Although monoubiquitin/UBA complexes are well characterized, much less is known about UBA/polyubiquitin complexes, even though polyubiquitin chains are believed to be biologically relevant ligands of many UBA domain proteins. Here, we report the results of a quantitative study of the interaction of K48-linked polyubiquitin chains with UBA domains of the DNA repair/proteolysis protein HHR23A, using surface plasmon resonance and other approaches. We present evidence that the UBL domain of HHR23A negatively regulates polyubiquitin/UBA interactions and identify leucine 8 of ubiquitin as an important determinant of chain recognition. A striking relationship between binding affinity and chain length suggests that maximum affinity is associated with a conformational feature that is fully formed in chains of n = 4-6 and can be recognized by a single UBA domain of HHR23A. Our findings provide new insights into polyubiquitin chain recognition and set the stage for future structural investigations of UBA/polyubiquitin complexes.

Ubiquitin chain synthesis.

Several important signaling processes depend on the tagging of cellular proteins with "polyubiquitin chains"-ubiquitin polymers whose building blocks are connected by isopeptide bonds between G76 of one ubiquitin and a specific lysine residue of the next one. Here we describe procedures for the synthesis of polyubiquitin chains of defined lengths that are linked through the K48 or K63 side chains. The method involves a series of enzymatic reactions in which proximally and distally blocked monoubiquitins (or chains) are conjugated to produce a particular chain in high yield. Individual chains are then deblocked and joined in another round of reaction. Successive rounds of deblocking and synthesis can give rise to a chain of any desired length.

Dynamics of Ubiquitin Pools in Developing Sea Urchin Embryos: (ubiquitin/embryogenesis/proteolysis).

The sea urchin embryo is a closed metabolic system in which embryogenesis is accompanied by significant protein degradation. We report results which are consistent with a function for the ubiquitinmediated proteolytic pathway in selective protein degradation during embryogenesis in this system. Quantitative solid- and solution-phase immunochemical assays, employing anti-ubiquitin antibodies, showed that unfertilized eggs of Strongylocentrotus purpuratus have a high content of unconjugated ubiquitin (ca. 8 × 108 molecules), and also contain abundant conjugates involving ubiquitin and maternal proteins. The absolute content of ubiquitin in the conjugated form increases about 13-fold between fertilization and the pluteus larva stage; 90% or more of embryonic ubiquitin molecules are conjugated to embryonic proteins in hatched blastulae and later-stage embryos. Qualitatively similar results were obtained with embryos of Lytechinus variegatus. The results of pulse-labeling and immunoprecipitation experiments indicate that synthesis of ubiquitin in S. purpuratus is developmentally regulated, with an overall increase in synthetic rate of 12-fold between fertilization and hatching. Regulation is likely to occur at the level of translation, since others have shown that levels of ubiquitin-encoding mRNA remain virtually constant in echinoid embryos during this developmental interval. The sea urchin embryo should be a useful system for characterizing the role of ubiquitination in embryogenesis.

Evidence for bidentate substrate binding as the basis for the K48 linkage specificity of otubain 1.

Otubain 1 belongs to the ovarian tumor (OTU) domain class of cysteine protease deubiquitinating enzymes. We show here that human otubain 1 (hOtu1) is highly linkage-specific, cleaving Lys48 (K48)-linked polyubiquitin but not K63-, K29-, K6-, or K11-linked polyubiquitin, or linear alpha-linked polyubiquitin. Cleavage is not limited to either end of a polyubiquitin chain, and both free and substrate-linked polyubiquitin are disassembled. Intriguingly, cleavage of K48-diubiquitin by hOtu1 can be inhibited by diubiquitins of various linkage types, as well as by monoubiquitin. NMR studies and activity assays suggest that both the proximal and distal units of K48-diubiquitin bind to hOtu1. Reaction of Cys23 with ubiquitin-vinylsulfone identified a ubiquitin binding site that is distinct from the active site, which includes Cys91. Occupancy of the active site is needed to enable tight binding to the second site. We propose that distinct binding sites for the ubiquitins on either side of the scissile bond allow hOtu1 to discriminate among different isopeptide linkages in polyubiquitin substrates. Bidentate binding may be a general strategy used to achieve linkage-specific deubiquitination.

Molecular determinants of polyubiquitin linkage selection by an HECT ubiquitin ligase.

Ubiquitin (Ub)-protein ligases (E3s) frequently modify their substrates with multiple Ub molecules in the form of a polyubiquitin (poly-Ub) chain. Although structurally distinct poly-Ub chains (linked through different Ub lysine (Lys) residues) can confer different fates on target proteins, little is known about how E3s select the Lys residue to be used in chain synthesis. Here, we used a combination of mutagenesis, biochemistry, and mass spectrometry to map determinants of linkage choice in chain assembly catalyzed by KIAA10, an HECT (Homologous to E6AP C-Terminus) domain E3 that synthesizes K29- and K48-linked chains. Focusing on the Ub molecule that contributes the Lys residue for chain formation, we found that specific surface residues adjacent to K48 and K29 are critical for the usage of the respective Lys residues in chain synthesis. This direct mechanism of linkage choice bears similarities to the mechanism of substrate site selection in sumoylation catalyzed by Ubc9, but is distinct from the mechanism of chain linkage selection used by the Mms2/Ubc13 (Ub E2 variant (UEV)/E2) complex.

E2-25K mediates US11-triggered retro-translocation of MHC class I heavy chains in a permeabilized cell system.

In cells expressing human cytomegalovirus US11 protein, newly synthesized MHC class I heavy chains (HCs) are rapidly dislocated from the endoplasmic reticulum (ER) and degraded in the cytosol, a process that is similar to ER-associated degradation (ERAD), the pathway used for degradation of misfolded ER proteins. US11-triggered movement of HCs into the cytosol requires polyubiquitination, but it is unknown which ubiquitin-conjugating and ubiquitin-ligase enzymes are involved. To identify the ubiquitin-conjugating enzyme (E2) required for dislocation, we used a permeabilized cell system, in which endogenous cytosol can be replaced by cow liver cytosol. By fractionating the cytosol, we show that E2-25K can serve as the sole E2 required for dislocation of HCs in vitro. Purified recombinant E2-25K, together with components that convert this E2 to the active E2-ubiquitin thiolester form, can substitute for crude cytosol. E2-25K cannot be replaced by the conjugating enzymes HsUbc7/Ube2G2 or Ube2G1, even though HsUbc7/Ube2G2 and its yeast homolog Ubc7p are known to participate in ERAD. The activity of E2-25K, as measured by ubiquitin dimer formation, is strikingly enhanced when added to permeabilized cells, likely by membrane-bound ubiquitin protein ligases. To identify these ligases, we tested RING domains of various ligases for their activation of E2-25K in vitro. We found that RING domains of gp78/AMFR, a ligase previously implicated in ERAD, and MARCHVII/axotrophin, a ligase of unknown function, greatly enhanced the activity of E2-25K. We conclude that in permeabilized, US11-expressing cells polyubiquitination of the HC substrate can be catalyzed by E2-25K, perhaps in cooperation with the ligase MARCHVII/axotrophin.

An arsenite-inducible 19S regulatory particle-associated protein adapts proteasomes to proteotoxicity.

Protein misfolding caused by exposure to arsenite is associated with transcriptional activation of the AIRAP gene. We report here that AIRAP is an arsenite-inducible subunit of the proteasome's 19S cap that binds near PSMD2 at the 19S base. Compared to the wild-type, knockout mouse cells or C. elegans lacking AIRAP accumulate more polyubiquitylated proteins and exhibit higher levels of stress when exposed to arsenite, and proteasomes isolated from arsenite-treated AIRAP knockout cells are relatively impaired in substrate degradation in vitro. AIRAP's association with the 19S cap reverses the stabilizing affect of ATP on the 26S proteasome during particle purification, and AIRAP-containing proteasomes, though constituted of 19S and 20S subunits, acquire features of hybrid proteasomes with both 19S and 11S regulatory caps. These features include enhanced cleavage of peptide substrates and suggest that AIRAP adapts the cell's core protein degradation machinery to counteract proteotoxicity induced by an environmental toxin.

Different HECT domain ubiquitin ligases employ distinct mechanisms of polyubiquitin chain synthesis.

Individual ubiquitin (Ub)-protein ligases (E3s) cooperate with specific Ub-conjugating enzymes (E2s) to modify cognate substrates with polyubiquitin chains. E3s belonging to the Really Interesting New Gene (RING) and Homologous to E6-Associated Protein (E6AP) C-Terminus (HECT) domain families utilize distinct molecular mechanisms. In particular, HECT E3s, but not RING E3s, form a thiol ester with Ub before transferring Ub to the substrate lysine. Here we report that different HECT domain E3s can employ distinct mechanisms of polyubiquitin chain synthesis. We show that E6AP builds up a K48-linked chain on its HECT cysteine residue, while KIAA10 builds up K48- and K29-linked chains as free entities. A small region near the N-terminus of the conserved HECT domain helps to bring about this functional distinction. Thus, a given HECT domain can specify both the linkage of a polyubiquitin chain and the mechanism of its assembly.

Ubiquitin binding site of the ubiquitin E2 variant (UEV) protein Mms2 is required for DNA damage tolerance in the yeast RAD6 pathway.

Different ubiquitin modifications to proliferating cell nuclear antigen (PCNA) signal distinct modes of lesion bypass in the RAD6 pathway of DNA damage tolerance. The modification of PCNA with monoubiquitin signals an error-prone bypass, whereas the extension of this modification into a Lys-63-linked polyubiquitin chain promotes error-free bypass. Chain formation is catalyzed by the Mms2/Ubc13 conjugating enzyme variant/conjugating enzyme (UEV.E2) complex together with the Rad5 ubiquitin ligase. In vitro studies of this UEV.E2 complex have identified a ubiquitin binding site that is mainly localized on Mms2. However, the role of this site in DNA damage tolerance and the molecular features of the ubiquitin/Mms2 interaction are poorly understood. Here we identify two molecular determinants, the side chains of Mms2-Ile-57 and ubiquitin-Ile-44, that are required for chain assembly in vitro and error-free lesion bypass in vivo. Mutating either of these side chains to alanine elicits a severe 10-20-fold inhibition of chain synthesis that is caused by compromised binding of the acceptor ubiquitin to Mms2. These results suggest that the ubiquitin binding site of Mms2 is necessary for error-free lesion bypass in the RAD6 pathway and provide new insights into ubiquitin recognition by UEV proteins.