Ufd1 exhibits the AAA-ATPase fold with two distinct ubiquitin interaction sites

Ufd1 mediates ubiquitin fusion degradation by association with Npl4 and Cdc48/p97. The Ufd1-ubiquitin interaction is essential for transfer of substrates to the proteasome. However, the mechanism and specificity of ubiquitin recognition by Ufd1 are poorly understood due to the lack of detailed structural information. Here, we present the solution structure of yeast Ufd1 N domain and show that it has two distinct binding sites for mono- and polyubiquitin. The structure exhibits striking similarities to the Cdc48/p97 N domain. It contains the double-psi beta barrel motif, which is thus identified as a ubiquitin binding domain. Significantly, Ufd1 shows higher affinity toward polyubiquitin than monoubiquitin, attributable to the utilization of separate binding sites with different affinities. Further studies revealed that the Ufd1-ubiquitin interaction involves hydrophobic contacts similar to those in well-characterized ubiquitin binding proteins. Our results provide a structural basis for a previously proposed synergistic binding of polyubiquitin by Cdc48/p97 and Ufd1.     

Structural basis for ubiquitin recognition by the Otu1 ovarian tumor domain protein

Ubiquitination of proteins modifies protein function by either altering their activities, promoting their degradation, or altering their subcellular localization. Deubiquitinating enzymes are proteases that reverse this ubiquitination. Previous studies demonstrate that proteins that contain an ovarian tumor (OTU) domain possess deubiquitinating activity. This domain of approximately 130 amino acids is weakly similar to the papain family of proteases and is highly conserved from yeast to mammals. Here we report structural and functional studies on the OTU domain-containing protein from yeast, Otu1. We show that Otu1 binds polyubiquitin chain analogs more tightly than monoubiquitin and preferentially hydrolyzes longer polyubiquitin chains with Lys(48) linkages, having little or no activity on Lys(63)- and Lys(29)-linked chains. We also show that Otu1 interacts with Cdc48, a regulator of the ER-associated degradation pathway. We also report the x-ray crystal structure of the OTU domain of Otu1 covalently complexed with ubiquitin and carry out structure-guided mutagenesis revealing a novel mode of ubiquitin recognition and a variation on the papain protease catalytic site configuration that appears to be conserved within the OTU family of ubiquitin hydrolases. Together, these studies provide new insights into ubiquitin binding and hydrolysis by yeast Otu1 and other OTU domain-containing proteins.     

The mitochondrial pathway and reactive oxygen species are critical contributors to interferon-α/β-mediated apoptosis in Ubp43-deficient hematopoietic cells

Stress associated proteins (SAPs) in plants contain A20-type zinc finger (A20_ZF) domains and are involved with abiotic stress response. A20-type zinc finger domains in animals reportedly recognize ubiquitin as a regulatory signal in cell. However, it remains unclear whether A20_ZF domains in plants perform similar roles. AtSAP5, a SAP from Arabidopsis thaliana, exhibits a unique sequence feature among 10 AtSAPs harboring A20_ZF domains. The highly conserved diaromatic patch is replaced by the dialipathic patch. Here we investigated whether AtSAP5 recognizes ubiquitin and the roles of the dialipathic patch in ubiquitin binding in vitro. GST pulldown assay reveals that AtSAP5 binds polyubiquitin rather than monoubiquitin. AtSAP5 shows preferences for linear and K63-linked polyubiquitin chains to K48-linked one. The A20_ZF domain of AtSAP5 is sufficient for linkage-specific polyubiquitin recognition. The dialipathic patch in AtSAP5 plays an important role in K48-linked polyubiquitin recognition. Taken together, our results suggest that AtSAP5 participates in polyubiquitin recognition in plants and that the dialipathic patch in AtSAP5 is critical in binding K48-linked polyubiquitn chains.     

E2/E3-mediated assembly of lysine 29-linked polyubiquitin chains.

Polyubiquitin (Ub) chains linked through Lys-48-Gly-76 isopeptide bonds represent the principal signal by which substrates of the Ub-dependent protein degradation pathway are targeted to the 26 S proteasome, but the mechanism(s) whereby these chains are assembled on substrate proteins is poorly understood. Nor have assembly mechanisms or definitive functions been assigned to polyubiquitin chains linked through several other lysine residues of ubiquitin. We show that rabbit reticulocyte lysate harbors enzymatic components that catalyze the assembly of unanchored Lys-29-linked polyubiquitin chains. This reaction can be reconstituted using the ubiquitin-conjugating enzyme (E2) known as UbcH5A, a 120-kDa protein(s) that behaves as a ubiquitin-protein ligase (E3), and ubiquitin-activating enzyme (E1). The same partially purified E3 preparation also catalyzes the assembly of unanchored chains linked through Lys-48. Kinetic studies revealed a K(m) of approximately 9 microM for the acceptor ubiquitin in the synthesis of diubiquitin; this value is similar to the concentration of free ubiquitin in most cells. Similar kinetic behavior was observed for conjugation to Lys-48 versus Lys-29 and for conjugation to tetraubiquitin versus monoubiquitin. The properties of these enzymes suggest that there may be distinct pathways for ubiquitin-ubiquitin ligation versus substrate-ubiquitin ligation in vivo.     

Nonhydrolyzable diubiquitin analogues are inhibitors of ubiquitin conjugation and deconjugation

A series of nonhydrolyzable ubiquitin dimer analogues has been synthesized and evaluated as inhibitors of ubiquitin-dependent processes. Dimer analogues were synthesized by cross-linking ubiquitin containing a terminal cysteine (G76C) to ubiquitin containing cysteine at position 11 ((76-11)Ub(2)), 29 ((76-29)Ub(2)), 48 ((76-48)Ub(2)), or 63 ((76-63)Ub(2)). A head-to-head dimer of cysteine G76C ((76-76)Ub(2)) served as a control. These analogues are mimics of the different chain linkages observed in natural polyubiquitin chains. All analogues showed weak inhibition toward the catalytic domain of UCH-L3 and a UBP pseudogene. In the absence of ubiquitin, isopeptidase T was inhibited only by the dimer linked through residue 29. In the presence of 0.5 microM ubiquitin, isopeptidase T was inhibited by several of the dimer analogues, with the (76-29)Ub(2) dimer exhibiting a K(i) of 1.8 nM. However, USP14, the human homologue of yeast Ubp6, was not inhibited at the concentrations tested. Some analogues of ubiquitin dimer also acted as selective inhibitors of conjugation and deconjugation of ubiquitin catalyzed by reticulocyte fraction II. (76-76)Ub(2) and (76-11)Ub(2) did not inhibit the conjugation of ubiquitin, while (76-29)Ub(2), (76-48)Ub(2), and (76-63)Ub(2) were potent inhibitors of conjugation. This specificity is consistent with the known ability of cells to form K29-, K48-, and K63-linked polyubiquitin chains. While (76-11)Ub(2), (76-29)Ub(2), and (76-63)Ub(2) inhibited release of ubiquitin from a pool of total conjugates, (76-48)Ub(2) and (76-76)Ub(2) showed no significant inhibition. Isopeptidase T was shown to specifically disassemble two conjugates (assumed to be di- and triubiquitin with masses of 26 and 17 kDa) formed in the reticulocyte lysate system. This activity was inhibited differentially by all dimer analogues. The inhibitor selectivity for deconjugation of the 26 and 17 kDa conjugates was similar to that observed for isopeptidase T. The observations suggest that these two conjugated proteins of the reticulocyte lysate are specific substrates for isopeptidase T in lysates.     

Perturbation of the ubiquitin system causes leaf curling, vascular tissue alterations and necrotic lesions in a higher plant.

A ubiquitin variant with Lys48 changed to Arg acts in vitro as an inhibitor of ubiquitin dependent protein degradation. To assess the role of this proteolytic pathway in the life cycle of plants, we expressed the ubiquitin variant in Nicotiana tabacum. Expression of variant mono- or polyubiquitin leads to marked abnormalities in vascular tissue. In addition, overexpression of variant polyubiquitin induces discrete lesions on leaves. This indicates that perturbations of the ubiquitin system can induce a programmed necrotic response in plants.     

Solution structure of a CUE-ubiquitin complex reveals a conserved mode of ubiquitin binding

Monoubiquitination serves as a regulatory signal in a variety of cellular processes. Monoubiquitin signals are transmitted by binding to a small but rapidly expanding class of ubiquitin binding motifs. Several of these motifs, including the CUE domain, also promote intramolecular monoubiquitination. The solution structure of a CUE domain of the yeast Cue2 protein in complex with ubiquitin reveals intermolecular interactions involving conserved hydrophobic surfaces, including the Leu8-Ile44-Val70 patch on ubiquitin. The contact surface extends beyond this patch and encompasses Lys48, a site of polyubiquitin chain formation. This suggests an occlusion mechanism for inhibiting polyubiquitin chain formation during monoubiquitin signaling. The CUE domain shares a similar overall architecture with the UBA domain, which also contains a conserved hydrophobic patch. Comparative modeling suggests that the UBA domain interacts analogously with ubiquitin. The structure of the CUE-ubiquitin complex may thus serve as a paradigm for ubiquitin recognition and signaling by ubiquitin binding proteins.     

Activated inositol 1,4,5-trisphosphate receptors are modified by homogeneous Lys-48- and Lys-63-linked ubiquitin chains, but only Lys-48-linked chains are required for degradation

Inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) are large, ubiquitously expressed, endoplasmic reticulum membrane proteins that form tetrameric IP(3) and Ca(2+)-gated Ca(2+) channels. Endogenous IP(3)Rs provide very appealing tools for studying the ubiquitin-proteasome pathway in intact mammalian cells because, upon activation, they are rapidly ubiquitinated and degraded. Using mass spectrometry, we previously examined the ubiquitination of IP(3)R1 in αT3-1 pituitary gonadotrophs and found that IP(3)R1 ubiquitination is highly complex, with receptors being modified at multiple sites by monoubiquitin and polyubiquitin chains formed through both Lys-48 and Lys-63 linkages (Sliter, D. A., Kubota, K., Kirkpatrick, D. S., Alzayady, K. J., Gygi, S. P., and Wojcikiewicz, R. J. H. (2008) J. Biol. Chem. 283, 35319-35328). Here, we have extended these studies to determine whether IP(3)R2 and IP(3)R3 are similarly modified and if ubiquitination is cell type-dependent. Using mass spectrometry and linkage-specific ubiquitin antibodies, we found that all IP(3)R types are subject to ubiquitination at approximately the same locations and that, independent of cell type, IP(3)Rs are modified by monoubiquitin and Lys-48- and Lys-63-linked ubiquitin chains, although in differing proportions. Remarkably, the attached Lys-48- and Lys-63-linked ubiquitin chains are homogeneous and are segregated to separate IP(3)R subunits, and Lys-48-linked ubiquitin chains, but not Lys-63-linked chains, are required for IP(3)R degradation. Together, these data provide unique insight into the complexities of ubiquitination of an endogenous ubiquitin-proteasome pathway substrate in unperturbed mammalian cells. Importantly, although Lys-48-linked ubiquitin chains appear to trigger proteasomal degradation, the presence of Lys-63-linked ubiquitin chains suggests that ubiquitination of IP(3)Rs may have physiological consequences beyond signaling for degradation.     

Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology

Protein ubiquitination regulates many cellular processes, including protein degradation, signal transduction, DNA repair and cell division. In the classical model, a uniform polyubiquitin chain that is linked through Lys 48 is required for recognition and degradation by the 26S proteasome. Here, we used a reconstituted system and quantitative mass spectrometry to demonstrate that cyclin B1 is modified by ubiquitin chains of complex topology, rather than by homogeneous Lys 48-linked chains. The anaphase-promoting complex was found to attach monoubiquitin to multiple lysine residues on cyclin B1, followed by poly-ubiquitin chain extensions linked through multiple lysine residues of ubiquitin (Lys 63, Lys 11 and Lys 48). These heterogeneous ubiquitin chains were sufficient for binding to ubiquitin receptors, as well as for degradation by the 26S proteasome, even when they were synthesized with mutant ubiquitin that lacked Lys 48. Together, our observations expand the context of what can be considered to be a sufficient degradation signal and provide unique insights into the mechanisms of substrate ubiquitination.     

Reading the ubiquitin postal code

Polyubiquitin chains are assembled through the formation of an isopeptide bond between a lysine side-chain or terminal amino group of a proximal ubiquitin moiety and the carboxy-terminal of a distal ubiquitin moiety. Protein substrates tagged by polyubiquitin chains of different linkages undergo different fates. Many polyubiquitin chain types have been characterized so far, notably Lys11, Lys48, Lys63 and linear chains. These different types of chains are synthesized, disassembled and recognized by selective enzymes and receptors. Here I survey the structural basis for the selective binding of polyubiquitin chains of specific linkages, with an emphasis on recent advances in our understanding of polyubiquitin chain structure and functions. Recent work suggests linkage-type discrimination by members of the NF-κb signalling and DNA repair pathways and a specific role for Lys48-linked polyubiquitin chain recognition by proteasome-associated proteins.     

Complete cDNA sequence of a Dictyostelium ubiquitin with a carboxy-terminal tail and identification of the protein using an anti-peptide antibody

The complete sequence of a Dictyostelium discoideum cDNA is presented that codes for monoubiquitin extended at its C-terminus by a 52 amino acid tail. The sequence of both the ubiquitin portion and the tail is highly homologous to the one of Saccharomyces cerevisiae and to a partial mouse sequence. The highly basic tail sequence contains a putative metal and nucleic acid-binding motif. The gene encoding the 0.6 kb mRNA of the C-terminally extended ubiquitin is represented only once in the haploid genome. The 0.6 kb mRNA as well as its translation product, a 15 kDa protein, is expressed in exponentially growing cells and remains present for at least 5 h of development. Using antibodies against a synthetic peptide that corresponds to the C-terminal amino acid sequence, a 15 kDa protein containing the extension a synthetic peptide that corresponds to the C-terminal amino acid sequence, a 15 kDa protein containing the extension was also detected in yeast.     

Definitive evidence for Ufd2-catalyzed elongation of the ubiquitin chain through Lys48 linkage

Saccharomyces cerevisiae Ufd2 is a ubiquitin chain elongation factor in the ubiquitin fusion degradation (UFD) pathway and functions in stress tolerance. A recent study has suggested that the mammalian Ufd2 homologue UFD2a catalyzes formation of Lys27- and Lys33-linked polyubiquitin chains rather than the Lys48-linked chain, but the linkage type of the polyubiquitin chain formed by yeast Ufd2 remains unclear. To determine the property of Ufd2, we reconstituted the UFD pathway using purified enzymes from yeast. Direct determination of the ubiquitin chain linkage type in polyubiquitinated UFD substrates by MALDI-TOF mass spectrometry revealed that Ufd2 catalyzes elongation of the ubiquitin chain through Lys48 linkage.     

Purification and characterization of a novel protein that is required for degradation of N-alpha-acetylated proteins by the ubiquitin system.

Anion exchange chromatography of reticulocyte lysates revealed that the ubiquitin cell-free system can be resolved into two essential fractions: unadsorbed material (Fraction I) that contains ubiquitin and a high salt eluate (Fraction II) that contains the conjugating enzymes and the conjugate-degrading protease. Many proteins with exposed NH2 termini are degraded in a ubiquitin-supplemented Fraction II. However, this partially purified and reconstituted system does not degrade N-alpha-acetylated proteins. These proteins are degraded in whole lysates in a ubiquitin-dependent manner (Mayer, A. Siegel, N. R., Schwartz, A. L., and Ciechanover, A. (1989) Science 244, 1480-1483). It appears that a protein factor which is specifically required for the degradation of N-alpha-acetylated proteins is removed or inactivated during the fractionation of the lysate. Here we report the purification and characterization of a novel protein that is required along with the protease for the degradation of ubiquitin conjugates of histone H2A, an N-alpha-acetylated protein. The protein is not required for the degradation of ubiquitin conjugates of proteins with free NH2 termini. The protein, which is found in crude Fraction I, was purified approximately 200-fold by (NH4)2SO4 precipitation, Sephadex G-100 gel-filtration chromatography, Mono Q anion exchange chromatography, and an additional Sephadex G-100 gel filtration chromatography step. The protein is removed from Fraction I during the purification of ubiquitin and has not been previously recognized since the majority of the protein substrates evaluated in the cell-free system have free NH2 termini. The protein has an apparent molecular mass of approximately 92 kDa. It is a homodimer that is composed of two identical 46-kDa subunits. Initial analysis of the mechanism of action of this protein revealed that it must interact with the conjugates in order to allow proteolysis to occur. We designated the protein Factor H (Factor Hedva).     

Methylated ubiquitin inhibits cyclin degradation in clam embryo extracts.

A derivative of ubiquitin in which amino groups were blocked by reductive methylation was used to study the possible role of the ubiquitin pathway in the cell cycle-programmed degradation of cyclin. It was shown previously that methylated ubiquitin can be efficiently ligated to protein substrates, but cannot form polyubiquitin chains. In the well-characterized ubiquitin-dependent proteolytic system from reticulocytes, it was found that rates of protein breakdown obtained with methylated ubiquitin are generally slower than those with ubiquitin; and thus, this derivative can be used, in some cases, as an inhibitor of ubiquitin-dependent protein degradation. The addition of methylated ubiquitin to a cell-free system from fertilized clam oocytes inhibited the degradation of both cyclins A and B. That this was due to specific interference with ubiquitin function was indicated by the observation that the supplementation of excess ubiquitin completely overcame the inhibitory action of methylated ubiquitin on cyclin degradation. These findings suggest that polyubiquitin chain formation is required for cyclin degradation.     

Mass spectrometric and mutational analyses reveal Lys-6-linked polyubiquitin chains catalyzed by BRCA1-BARD1 ubiquitin ligase

The breast and ovarian cancer suppressor BRCA1 acquires significant ubiquitin ligase activity when bound to BARD1 as a RING heterodimer. Although the activity may well be important for the role of BRCA1 as a tumor suppressor, the biochemical consequence of the activity is not yet known. Here we report that BRCA1-BARD1 catalyzes Lys-6-linked polyubiquitin chain formation. K6R mutation of ubiquitin dramatically reduces the polyubiquitin products mediated by BRCA1-BARD1 in vitro. BRCA1-BARD1 preferentially utilizes ubiquitin with a single Lys residue at Lys-6 or Lys-29 to mediate autoubiquitination of BRCA1 in vivo. Furthermore, mass spectrometry analysis identified the Lys-6-linked branched ubiquitin fragment from the polyubiquitin chain produced by BRCA1-BARD1 using wild type ubiquitin. The BRCA1-BARD1-mediated Lys-6-linked polyubiquitin chains are deubiquitinated by 26 S proteasome in vitro, whereas autoubiquitinated CUL1 through Lys-48-linked polyubiquitin chains is degraded. Proteasome inhibitors do not alter the steady state level of the autoubiquitinated BRCA1 in vivo. Hence, the results indicate that BRCA1-BARD1 mediates novel polyubiquitin chains that may be distinctly edited by 26 S proteasome from conventional Lys-48-linked polyubiquitin chains.     

NMR reveals a different mode of binding of the Stam2 VHS domain to ubiquitin and diubiquitin

The VHS domain of the Stam2 protein is a ubiquitin binding domain involved in the recognition of ubiquitinated proteins committed to lysosomal degradation. Among all VHS domains, the VHS domain of Stam proteins is the strongest binder to monoubiqiuitin and exhibits preferences for K63-linked chains. In the present paper, we report the solution NMR structure of the Stam2-VHS domain in complex with monoubiquitin by means of chemical shift perturbations, spin relaxation, and paramagnetic relaxation enhancements. We also characterize the interaction of Stam2-VHS with K48- and K63-linked diubiquitin chains and report the first evidence that VHS binds differently to these two chains. Our data reveal that VHS enters the hydrophobic pocket of K48-linked diubiquitin and binds the two ubiquitin subunits with different affinities. In contrast, VHS interacts with K63-linked diubiquitin in a mode similar to its interaction with monoubiquitin. We also suggest possible structural models for both K48- and K63-linked diubiquitin in interaction with VHS. Our results, which demonstrate a different mode of binding of VHS for K48- and K63-linked diubiquitin, may explain the preference of VHS for K63- over K48-linked diubiquitin chains and monoubiquitin.     

Structural basis of ubiquitin recognition by the ubiquitin-associated (UBA) domain of the ubiquitin ligase EDD

EDD (or HYD) is an E3 ubiquitin ligase in the family of HECT (homologous to E6-AP C terminus) ligases. EDD contains an N-terminal ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin-mediated processes. Here, we use isothermal titration calorimetry (ITC), NMR titrations, and pull-down assays to show that the EDD UBA domain binds ubiquitin. The 1.85 A crystal structure of the complex with ubiquitin reveals the structural basis of ubiquitin recognition by UBA helices alpha1 and alpha3. The structure shows a larger number of intermolecular hydrogen bonds than observed in previous UBA/ubiquitin complexes. Two of these involve ordered water molecules. The functional importance of residues at the UBA/ubiquitin interface was confirmed using site-directed mutagenesis. Surface plasmon resonance (SPR) measurements show that the EDD UBA domain does not have a strong preference for polyubiquitin chains over monoubiquitin. This suggests that EDD binds to monoubiquitinated proteins, which is consistent with its involvement in DNA damage repair pathways.     

Essential factors determining codon usage in ubiquitin genes

Summary Ubiquitin is ubiquitous in all eukaryotes and its amino acid sequence shows extreme conservation. Ubiquitin genes comprise direct repeats of the ubiquitin coding unit with no spacers. The nucleotide sequences coding for 13 ubiquitin genes from 11 species reported so far have been compiled and analyzed. The G+C content of codon third base reveals a positive linear correlation with the genome G+C content of the corresponding species. The slope strongly suggests that the overall G+C content of codons of polyubiquitin genes clearly reflects the genome G+C content by AT/GC substitutions at the codon third position. The G+C content of ubiquitin codon third base also shows a positive linear correlation with the overall G+C content of coding regions of compiled genes, indicating the codon choices among synonymous codons reflect the average codon usage pattern of corresponding species. On the other hand, the monoubiquitin gene, which is different from the polyubiquitin gene in gene organization, gene expression, and function of the encoding protein, shows a different codon usage pattern compared with that of the polyubiquitin gene. From comparisons of the levels of synonymous substitutions among ubiquitin repeats and the homology of the amino acid sequence of the tail of monomeric ubiquitin genes, we propose that the molecular evolution of ubiquitin genes occurred as follows: Plural primitive ubiquitin sequences were dispersed on genome in ancestral eukaryotes. Some of them situated in a particular environment fused with the tail sequence to produce monomeric ubiquitin genes that were maintained across species. After divergence of species, polyubiquitin genes were formed by duplication of the other primitive ubiquitin sequences on different chromosomes. Differences in the environments in which ubiquitin genes are embedded reflect the differences in codon choice and in gene expression pattern between poly- and monomeric ubiquitin genes.