The identification and functional characterization of ariadne-1 (ari-1), a novel and vital Drosophila gene required for the correct differentiation of most cell types in the adult organism is reported. A sequence-related gene, ari-2, and the corresponding mouse and human homologs of both genes, are described. All these sequences define a new protein family by the Acid-rich, RING finger, B-box, RING finger, coiled-coil (ARBRCC) motif string. In Drosophila, ari-1 is expressed throughout development in all tissues. The mutant phenotypes are most noticeable in cells that undergo a large and rapid membrane deposition, such as rewiring neurons during metamorphosis, large tubular muscles during adult myogenesis, and photoreceptors. Occasional survivors of null alleles exhibit reduced life span, motor impairments, and short and thin bristles. Single substitutions at key cysteines in each RING finger cause lethality with no survivors and a drastic reduction of rough endoplasmic reticulum that can be observed in the photoreceptors of mosaic eyes. In yeast two-hybrid assays, the protein ARI-1 interacts with a novel ubiquitin-conjugating enzyme, UbcD10, whose sequence is also reported in this study. The N-terminal RING-finger motif is necessary and sufficient to mediate this interaction. Mouse and fly homologs of both ARI proteins and the Ubc can substitute for each other in the yeast two-hybrid assay, indicating that ARI represents a conserved novel mechanism in development. In addition to ARI homologs, the RBR signature is also found in the Parkinson-disease-related protein Parkin, adjacent to an ubiquitin-like domain, suggesting that the study of this mechanism could be relevant for human pathology (Aguilera, 2000).
Ubiquitin-conjugating enzymes are a family of related proteins that participate in the ubiquitination of proteins. Studies on the crystal structures of Saccharomyces cerevisiae Ubc4 and Arabidopsis thaliana Ubc1 have indicated that the smallest enzymes (class I), which consist entirely of the conserved core domain, share a common tertiary fold. The three-dimensional structure of the S. cerevisiae class I enzyme encoded by the UBC7 gene is reported. The crystal structure has been solved using molecular replacement techniques and refined by simulated annealing to an R-factor of 0.183 at 2.93 Å resolution. Bond lengths and angles in the molecule have root-mean-square deviations from ideal values of 0.016 Å and 2.3 degrees, respectively. Ubc7 is an alpha/beta protein with four alpha-helices and a four-stranded antiparallel beta-sheet. With the exception of two regions where extra residues are present, the tertiary folding of Ubc7 is similar to those of the other two enzymes. The ubiquitin-accepting cysteine is located in a cleft between two loops. One of these loops is nonconserved, since this region of the Ubc7 molecule differs from the other two enzymes by having 13 extra residues. There is also a second single amino acid insertion that alters the orientation of the turn between the first two beta-strands. Analysis of the 13 ubiquitin-conjugating enzyme sequences in S. cerevisiae indicates that there may be two other regions where extra residues could be inserted into the common tertiary fold. Both of these other regions exhibit significant deviations in the superposition of the three structures and, like the two insertion regions in Ubc7, may represent hypervariable regions within a common tertiary fold. As ubiquitin-conjugating enzymes interact with different substrates or other accessory proteins in the ubiquitination pathway, these variable surface regions may confer distinct specificity to individual enzymes (Cook, 1997).
Substrate discrimination in the ubiquitin-proteasome system is believed to be dictated by specific combinations of ubiquitin-protein ligases (E3s) and ubiquitin-conjugating enzymes (E2s). Doa10/Ssm4 has been identified as a yeast E3 that is embedded in the endoplasmic reticulum (ER)/nuclear envelope yet can target the soluble transcription factor Matalpha2. Doa10 contains an unusual RING finger, which has ubiquitin-ligase activity in vitro and is essential in vivo for degradation of alpha2 via its Deg1 degradation signal. Doa10 functions with two E2s, Ubc6 and Ubc7, to ubiquitinate Deg1-bearing substrates, and it is also required for the degradation of at least one ER membrane protein. Interestingly, different short-lived ER proteins show distinct requirements for Doa10 and another ER-localized E3, Hrd1. Nevertheless, the two E3s overlap in function: A doa10Delta hrd1Delta mutant is far more sensitive to cadmium relative to either single mutant and displays strong constitutive induction of the unfolded protein response; this suggests a role for both E3s in eliminating aberrant ER proteins. The likely human ortholog of DOA10 is in the cri-du-chat syndrome critical region on chromosome 5p, suggesting that defective ubiquitin ligation might contribute to this common genetic disorder (Swanson, 2001).
The Deg1 degradation signal of the transcriptional repressor Matalpha2 confers compartment-specific turnover to a reporter protein. Rapid degradation of a Deg1-containing fusion protein is observed only when the reporter is efficiently imported into the nucleus. In contrast, a reporter that is constantly exported from the nucleus exhibits an extended half-life. Furthermore, nuclear import functions are crucial for both Deg1-induced degradation as well as for the turnover of the endogenous yeast transcription factor Matalpha2. The conjugation of ubiquitin to a Deg1-containing reporter protein is abrogated in mutants affected in nuclear import. Obviously, the Deg1 signal initiates rapid proteolysis within the nucleoplasm, whereas in the cytosol it mediates turnover via a slower pathway. In both pathways the ubiquitin-conjugating enzymes Ubc6p/Ubc7p play a pivotal role. These observations imply that both the cellular targeting of a substrate and the compartment-specific activity of components of the ubiquitin-proteasome system define the half-life of naturally short-lived proteins (Lenk, 2001).
The hect domain protein family was originally identified by sequence similarity of its members to the C-terminal region of E6-AP, an E3 ubiquitin-protein ligase. Since the C terminus of E6-AP mediates thioester complex formation with ubiquitin, a necessary intermediate step in E6-AP-dependent ubiquitination, it has been proposed that members of the hect domain family in general have E3 activity. The hect domain is approximately 350 amino acids in length, and the hect domain of E6-AP is necessary and sufficient for ubiquitin thioester adduct formation. Furthermore, the human genome encodes at least 20 different hect domain proteins, and in further support of the hypothesis that hect domain proteins represent a family of E3s, several of these have been shown to form thioester complexes with ubiquitin. In addition, some hect domain proteins interact preferentially with UbcH5 (Drosophila homolog: UbcD10), whereas others interact with UbcH7, indicating that human hect domain proteins can be grouped into at least two classes based on their E2 specificity. Since E3s are thought to play a major role in substrate recognition, the presence of a large family of E3s should contribute to ensure the specificity and selectivity of ubiquitin-dependent proteolytic pathways (Schwarz, 1998).
The structural basis by which ubiquitin (Ub)-conjugating enzymes (E2s) determine substrate specificity remains unclear. Rabbit reticulocyte E217K has been cloned because unlike the similarly sized class I E2s, E214K and UBC4, it is unable to support ubiquitin-protein ligase (E3)-dependent conjugation to endogenous proteins. RNA analysis reveals that this E2 is expressed in all tissues tested, with higher levels in the testis. Analysis of testis RNA from rats of different ages shows that E217K mRNA is induced from days 15 to 30. The predicted amino acid sequence indicates that E217K is a 19. 5-kDa class I E2 but differs from other class I enzymes in possessing an insertion of 13 amino acids distal to the active site cysteine. E217K shows 74% amino acid identity with Saccharomyces cerevisiae UBC7, and therefore, it has been renamed mammalian UBC7. Yeast UBC7 crystal structure indicates that this insertion forms a loop out of the otherwise conserved folding structure. Sequence analysis of E2s had previously suggested that this loop is a hypervariable region and may play a role in substrate specificity. Mutant UBC7 lacking the loop (ubc7Deltaloop) and a mutant E214k with an inserted loop (E214k+loop) have been created and their biochemical functions have been characterized. Ubc7Deltaloop has higher affinity for the E1-Ub thiol ester than native UBC7 and permits conjugation of Ub to selected proteins in the testis but does not permit the broad spectrum E3-dependent conjugation to endogenous reticulocyte proteins. Surprisingly, E214k+loop is unable to accept Ub from ubiquitin-activating enzyme (E1) but is able to accept NEDD8 from E1. E214k+loop is able to support conjugation of NEDD8 to endogenous reticulocyte proteins but with much lower efficiency than E214k. Thus, the loop can influence interactions of the E2 with charged E1 as well as with E3s or substrates, but the exact nature of these interactions depends on divergent sequences in the remaining conserved core domain (Lin, 1999).
The E6AP ubiquitin-protein ligase (E3) mediates the human papillomavirus-induced degradation of the p53 tumor suppressor in cervical cancer and is mutated in Angelman syndrome, a neurological disorder. The crystal structure of the catalytic hect domain of E6AP reveals a bi-lobal structure with a broad catalytic cleft at the junction of the two lobes. The cleft consists of conserved residues whose mutation interferes with ubiquitin-thioester bond formation and is the site of Angelman syndrome mutations. The crystal structure of the E6AP hect domain bound to the UbcH7 ubiquitin-conjugating enzyme (E2) reveals the determinants of E2-E3 specificity and provides insights into the transfer of ubiquitin from the E2 to the E3 (Huang, 1999).
c-Cbl plays a negative regulatory role in tyrosine kinase signaling by an as yet undefined mechanism. Using the yeast two-hybrid system and an in vitro binding assay, it has been demonstrated that the c-Cbl RING finger domain interacts with UbcH7, a ubiquitin-conjugating enzyme (E2). UbcH7 interacts with the wild-type c-Cbl RING finger domain but not with a RING finger domain that lacks the amino acids that are deleted in 70Z-Cbl, an oncogenic mutant of c-Cbl. The in vitro interaction is enhanced by sequences on both the N- and C-terminal sides of the RING finger. In vivo and in vitro experiments reveal that c-Cbl and UbcH7 synergistically promote the ligand-induced ubiquitination of the epidermal growth factor receptor (EGFR). In contrast, 70Z-Cbl markedly reduces the ligand-induced, UbcH7-mediated ubiquitination of the EGFR. MG132, a proteasome inhibitor, significantly prolongs the ligand-induced phosphorylation of both the EGFR and c-Cbl. Thus, c-Cbl plays an essential role in the ligand-induced ubiquitination of the EGFR by a mechanism that involves an interaction of the RING finger domain with UbcH7. This mechanism participates in the down-regulation of tyrosine kinase receptors and loss of this function, as occurs in the naturally occurring 70Z-Cbl isoform, probably contributes to oncogenic transformation (Yokouchi, 1999).
Ubiquitin-protein ligases (E3s) regulate diverse cellular processes by mediating protein ubiquitination. The c-Cbl proto-oncogene is a RING family E3 that recognizes activated receptor tyrosine kinases, promotes their ubiquitination by a ubiquitin-conjugating enzyme (E2) and terminates signaling. The crystal structure of c-Cbl bound to a cognate E2 (UbcH7) and a kinase peptide shows how the RING domain recruits the E2. A comparison with a HECT family E3-E2 complex indicates that a common E2 motif is recognized by the two E3 families. The structure reveals a rigid coupling between the peptide binding and the E2 binding domains and a conserved surface channel leading from the peptide to the E2 active site, suggesting that RING E3s may function as scaffolds that position the substrate and the E2 optimally for ubiquitin transfer (Zheng, 2000).
Ubiquitinylation of proteins appears to be mediated by the specific interplay between ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s). However, cognate E3s and/or substrate proteins have been identified for only a few E2s. To identify proteins that can interact with the human E2 UbcH7, a yeast two-hybrid screen was performed. Two proteins were identified and termed human homolog of Drosophila ariadne (HHARI) and UbcH7-associated protein (H7-AP1). Both proteins, which are widely expressed, are characterized by the presence of RING finger and in-between-RING-finger (IBR) domains. No other overt structural similarity was observed between the two proteins. In vitro binding studies reveal that an N-terminal RING finger motif (HHARI) and the IBR domain (HHARI and H7-AP1) are involved in the interaction of these proteins with UbcH7. Furthermore, binding of these two proteins to UbcH7 is specific insofar as both HHARI and H7-AP1 can bind to the closely related E2, UbcH8, but not to the unrelated E2s UbcH5 and UbcH1. Although it is not clear at present whether HHARI and H7-AP1 serve, for instance, as substrates for UbcH7 or represent proteins with E3 activity, these data suggest that a subset of RING finger/IBR proteins are functionally linked to the ubiquitin/proteasome pathway (Moynihan, 1999).
gp78, also known as the tumor autocrine motility factor receptor, is a transmembrane protein whose expression is correlated with tumor metastasis. gp78 is a RING finger-dependent ubiquitin protein ligase (E3) of the endoplasmic reticulum (ER). Consistent with this, gp78 specifically recruits MmUBC7, a ubiquitin-conjugating enzyme (E2) implicated in ER-associated degradation (ERAD), through a region distinct from the RING finger. gp78 can target itself for proteasomal degradation in a RING finger- and MmUBC7-dependent manner. Importantly, gp78 can also mediate degradation of CD3-delta, a well-characterized ERAD substrate. In contrast, gp78 lacking an intact RING finger or its multiple membrane-spanning domains stabilizes CD3-delta. gp78 has thus been found to be an example of a mammalian cellular E3 intrinsic to the ER, suggesting a potential link between ubiquitylation, ERAD, and metastasis (Fang, 2001).
HHARI (human homologue of Drosophila ariadne) binds to the human ubiquitin-conjugating enzyme UbcH7 in vitro. HHARI interacts and co-localizes with UbcH7 in mammalian cells, particularly in the perinuclear region. A minimal interaction region of HHARI has been defined comprising residues 186-254. Individual amino acid residues essential for the interaction have been identified, and the distance between the RING1 finger and IBR domains has been shown to be critical to maintaining binding. The RING1 finger of HHARI cannot be substituted for by the highly homologous RING finger domains of either of the ubiquitin-protein ligase components c-CBL or Parkin, despite their similarity in structure and their independent capabilities to bind UbcH7. Furthermore, mutation of the RING1 finger domain of HHARI from a RING-HC to a RING-H2 type abolishes interaction with UbcH7. These studies demonstrate that very subtle changes to the domains that regulate recognition between highly conserved components of the ubiquitin pathway can dramatically affect their ability to interact (Ardley, 2001).
Autosomal recessive juvenile parkinsonism (AR-JP), one of the most common familial forms of Parkinson disease, is characterized by selective dopaminergic neural cell death and the absence of the Lewy body, a cytoplasmic inclusion body consisting of aggregates of abnormally accumulated proteins. PARK2, whose mutations cause AR-JP, has been cloned, but the function of the gene product, parkin, remains unknown. Parkin is reported here to be involved in protein degradation as a ubiquitin-protein ligase collaborating with the ubiquitin-conjugating enzyme UbcH7, and mutant parkins from AR-JP patients show loss of the ubiquitin-protein ligase activity. These findings indicate that accumulation of proteins that have yet to be identified causes a selective neural cell death without formation of Lewy bodies. These findings should enhance the exploration of the molecular mechanisms of neurodegeneration in Parkinson's disease as well as in other neurodegenerative diseases that are characterized by involvement of abnormal protein ubiquitination, including Alzheimer's disease, other tauopathies, CAG triplet repeat disorders and amyotrophic lateral sclerosis (Shimura, 2000).
Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive accumulation in selected neurons of protein inclusions containing alpha-synuclein and ubiquitin. Rare inherited forms of PD are caused by autosomal dominant mutations in alpha-synuclein or by autosomal recessive mutations in parkin, an E3 ubiquitin ligase. It is hypothesized that these two gene products interact functionally, namely, that parkin ubiquitinates alpha-synuclein normally and that this process is altered in autosomal recessive PD. A protein complex has been identified in normal human brain that includes parkin as the E3 ubiquitin ligase; UbcH7 is its associated E2 ubiquitin conjugating enzyme, and a new 22-kilodalton glycosylated form of alpha-synuclein (alphaSp22) is its substrate. In contrast to normal parkin, mutant parkin associated with autosomal recessive PD fails to bind alphaSp22. In an in vitro ubiquitination assay, alphaSp22 was modified by normal but not mutant parkin into a polyubiquitinated, high molecular weight species. Accordingly, alphaSp22 accumulated in a non-ubiquitinated form in parkin-deficient PD brains. It is concluded that alphaSp22 is a substrate for parkin's ubiquitin ligase activity in normal human brain and that loss of parkin function causes pathological alphaSp22 accumulation. These findings demonstrate a critical biochemical reaction between the two PD-linked gene products and suggest that this reaction underlies the accumulation of ubiquitinated alpha-synuclein in conventional PD (Shimura, 2001).
Parkin is a product of the Park2 gene, whose mutation causes autosomal recessive juvenile parkinsonism (AR-JP) characterized by selective dopaminergic neuronal death and absence of Lewy bodies. Parkin is directly linked to the ubiquitin (Ub)-proteasome pathway as a Ub-protein ligase (E3) collaborating with a Ub-conjugating enzyme (E2) UbcH7. The expression of mRNAs for parkin and UbcR7 (rat ortholog of human UbcH7) in the developing rat brain has been examined. Parkin mRNA increases in parallel with neuronal maturation, but is unevenly distributed in various brain regions after four postnatal days. The expression pattern of the UbcR7 mRNA is almost identical to that of the parkin mRNA in all cases examined. Both parkin and UbcR7 mRNAs are distributed in neurons but not glial cells. These findings indicate that parkin is expressed not only in the substantia nigra, but also uniformly in various brain regions in a development-dependent manner. Co-expression of UbcR7 with parkin suggests that UbcR7 may interact with parkin in vivo for ubiquitination of yet unidentified target protein(s) (Wang, 2001).
The degradation of subunits of the trimeric Sec61p complex, a key component of the protein translocation apparatus of the ER membrane, has been investigated. A mutant form of Sec6lp and one of the two associated proteins (Sss1p) are selectively degraded, while the third constituent of the complex (Sbh1p) is stable. These results demonstrate that the proteolysis of the multispanning membrane protein Sec61p is mediated by the ubiquitin-proteasome pathway, since it requires polyubiquitination, the presence of a membrane-bound (Ubc6) and a soluble (Ubc7) ubiquitin-conjugating enzyme and a functional proteasome. The process is proposed to be specific for unassembled Sec61p and Sss1p. Thus, these results suggest that one pathway of ER degradation of abnormal or unassembled membrane proteins is initiated at the cytoplasmic side of the ER (Biederer, 1996).
Ubiquitin conjugation during endoplasmic-reticulum-associated degradation (ERAD) depends on the activity of Ubc7. Ubc1 acts as a further ubiquitin-conjugating enzyme in this pathway. Absence of both enzymes results in marked stabilization of an ERAD substrate and induction of the unfolded-protein response (UPR). Furthermore, basic ERAD activity is sufficient to eliminate unfolded proteins under normal conditions. However, when stress is applied, the UPR is required to increase ERAD activity. This study thus demonstrates a regulatory loop between ERAD and the UPR, which is essential for normal growth of yeast cells (Friedlander, 2000).
Proteolysis by the ubiquitin-proteasome system is highly selective. Specificity is achieved by the cooperation of diverse ubiquitin-conjugating enzymes (Ubcs or E2s) with a variety of ubiquitin ligases (E3s) and other ancillary factors. These recognize degradation signals characteristic of their target proteins. Signals have been identified that direct the degradation of beta-galactosidase and Ura3p fusion proteins via a subsidiary pathway of the ubiquitin-proteasome system involving Ubc6p and Ubc7p. This pathway is essential for the degradation of misfolded and regulated proteins in the endoplasmic reticulum (ER) lumen and membrane: such proteins are transported to the cytoplasm via the Sec61p translocon. Mutant backgrounds which prevent retrograde transport of ER proteins (hrd1/der3Delta and sec61-2) do not inhibit the degradation of the beta-galactosidase and Ura3p fusions carrying Ubc6p/Ubc7p pathway signals. It is therefore concluded that the ubiquitination of these fusion proteins takes place on the cytosolic face of the ER without prior transfer to the ER lumen. The contributions of different sequence elements to a 16-amino-acid-residue Ubc6p-Ubc7p-specific signal were analyzed by mutation. A patch of bulky hydrophobic residues is an essential element. In addition, positively charged residues are essential. Unexpectedly, certain substitutions of bulky hydrophobic or positively charged residues with alanine create novel degradation signals, channeling the degradation of fusion proteins to an unidentified proteasomal pathway not involving Ubc6p and Ubc7p (Gilon, 2000).
In eukaryotes, endoplasmic reticulum-associated degradation (ERAD) functions in cellular quality control and regulation of normal ER-resident proteins. ERAD proceeds by the ubiquitin-proteasome pathway, in which the covalent attachment of ubiquitin to proteins targets them for proteasomal degradation. Ubiquitin-protein ligases (E3s) play a crucial role in this process by recognizing target proteins and initiating their ubiquitination. Hrd1p, which is identical to Der3p, is an E3 for ERAD. Hrd1p is required for the degradation and ubiquitination of several ERAD substrates and physically associates with relevant ubiquitin-conjugating enzymes (E2s). A soluble Hrd1 fusion protein shows E3 activity in vitro, catalysing the ubiquitination of itself and test proteins. In this capacity, Hrd1p has an apparent preference for misfolded proteins. Hrd1p functions as an E3 in vivo, using only Ubc7p or Ubc1p to specifically program the ubiquitination of ERAD substrates (Bays, 2001).
A new conditional-lethal mutation, ndc10-2, has been isolated in the NDC10/CBF2/CTF14 gene that encodes the 110-kD subunit of the Saccharomyces cerevisiae CBF3 kinetochore complex. At the restrictive temperature of 37 degrees, ndc10-2 cells are able to assemble anaphase spindles, but fail to segregate their DNA, consistent with a defect in kinetochore function. To identify other factors that play a role in kinetochore assembly or function, both dosage and second site suppressors of the ndc10-2 mutation have been isolated. These screens identified UBC6 as a dosage suppressor, and mutations in UBC6 and UBC7 as second-site suppressors of ndc10-2 heat sensitivity. Both UBC6 and UBC7 encode ubiquitin-conjugating enzymes that function in ubiquitin-mediated protein degradation. Furthermore, overexpression of a mutant ubiquitin suppresses the ndc10-2 mutation. These results implicate the ubiquitin system in the regulation of ndc10-2 function and suggest a role for the ubiquitin system in kinetochore function (Kopski, 1997).
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