Itch: a HECT-type E3 ligase regulating immunity, skin and cancer.

The HECT-type E3 ubiquitin ligase (E3) Itch is absent in the non-agouti-lethal 18H or Itchy mice, which develop a severe immunological disease, including lung and stomach inflammation and hyperplasia of lymphoid and hematopoietic cells. The involvement of Itch in multiple signaling pathways and pathological conditions is presently an area of extensive scientific interest. This review aims to bring together a growing body of work exploring Itch-regulated biological processes, and to highlight recent discoveries on the regulatory mechanisms modulating its catalytic activity and substrate recognition capability. Our contribution is also an endeavor to correlate Itch substrate specificity with the pathological defects manifested by the mutant Itchy mice.

Ubiquitin-activating enzyme, E1, is associated with maturation of autophagic vacuoles.

The ubiquitin-activating enzyme, E1, is required for initiating a multi-step pathway for the covalent linkage of ubiquitin to target proteins. A CHO cell line containing a mutant thermolabile E1, ts20, has been shown to be defective in stress-induced degradation of proteins at restrictive temperature (Gropper et al., 1991. J. Biol. Chem. 266:3602-3610). Parental E36 cells responded to restrictive temperature by stimulating lysosome-mediated protein degradation twofold. Such a response was not observed in ts20 cells. The absence of accelerated degradation in these cells at 39.5 degrees C was accompanied by an accumulation of autolysosomes. The fractional volume of these degradative autophagic vacuoles was at least sixfold greater than that observed for either E36 cells at 30.5 degrees or 39.5 degrees C, or ts20 cells at 30.5 degrees C. These vacuoles were acidic and contained both acid phosphatase and cathepsin L, but, unlike the autolysosomes observed in E36 cells, ubiquitin-conjugated proteins were conspicuously absent. Combined, our results suggest that in ts20 cells, which are unable to generate ubiquitin-protein conjugates due to heat inactivation of E1, the formation and maturation of autophagosomes into autolysosomes is normal, but the conversion of autolysosomes into residual bodies is disrupted.

Degradation of proteins with acetylated amino termini by the ubiquitin system.

A free NH2-terminal group has been previously shown to be an obligatory signal for recognition and subsequent degradation of proteins in a partially fractionated and reconstituted ubiquitin proteolytic system. Naturally occurring proteins with acetylated NH2-termini--most cellular proteins fall in this category--were not degraded by this system. Other studies have suggested that the identity of the NH2-terminal residue is important in determining the metabolic stability of a protein in vivo (N-end rule). Whole reticulocyte lysate and antibodies directed against the ubiquitin-activating enzyme (E1) have now been used to show that such acetylated proteins are degraded in a ubiquitin-dependent mode. Although fractionation of lysate does not affect its proteolytic activity toward substrates with free NH2-termini, it completely abolishes the activity toward the blocked substrates, indicating that an important component of the system was either removed or inactivated during fractionation. An NH2-terminal "unblocking" activity that removes the blocking group, thus exposing a free NH2-terminus for recognition according to the N-end rule, does not seem to participate in this pathway. Incubation of whole lysate with labeled histone H2A results in the formation of multiple ubiquitin conjugates. In contrast, the fractionated system is devoid of any significant conjugating activity. These results suggest that a novel conjugating enzyme (possibly a ubiquitin-protein ligase) may be responsible for the degradation of these acetylated proteins by recognizing structural features of the substrate that are downstream and distinct from the NH2-terminal residue.

How are substrates recognized by the ubiquitin-mediated proteolytic system?

A necessary step in the degradation of proteins by the ubiquitin system is recognition by the ubiquitin-protein ligases(s). Various structural features of the proteolytic substrate render it susceptible to conjugation with ubiquitin. The N-terminal residue plays a major role in this process, with distinct sites on the ligase(s) recognizing specific types of N-termini. Post-translational modification of some of these residues is required prior to their recognition. A free N terminus is not the only marker; proteins with either free or blocked N termini can be recognized via structural domains that are downstream and distinct from this residue.

E2A protein degradation by the ubiquitin-proteasome system is stage-dependent during muscle differentiation.

The E2A proteins are basic helix-loop-helix transcription factors that regulate proliferation and differentiation in many cell types. In muscle cells, the E2A proteins form heterodimers with muscle regulatory factors such as MyoD, which then bind to DNA and regulate the transcription of target genes essential for muscle differentiation. We now demonstrate that E2A proteins are primarily localized in the nucleus in both C2C12 myoblasts and myotubes, and are degraded by the ubiquitin proteasome system evidenced by stabilization following treatment with the proteasome inhibitor, MG132. During the differentiation from myoblast to myotube, the cellular abundance of E2A proteins is relatively unaltered, despite significant changes (each approximately 5-fold) in the relative rates of protein synthesis and protein degradation via the ubiquitin-proteasome system. The rate of ubiquitin-proteasome-mediated E2A protein degradation depends on the myogenic differentiation state (t 1/2 approximately 2 h in proliferating myoblasts versus t 1/2 > 10 h in differentiated myotubes), and is also associated with cell cycle in non-muscle cells. Our findings reveal an important role for both translational and post-translational regulatory mechanisms in mediating the complex program of muscle differentiation determined by the E2A proteins.

Mutation of the E6-AP ubiquitin ligase reduces nuclear inclusion frequency while accelerating polyglutamine-induced pathology in SCA1 mice.

Mutant ataxin-1, the expanded polyglutamine protein causing spinocerebellar ataxia type 1 (SCA1), aggregates in ubiquitin-positive nuclear inclusions (NI) that alter proteasome distribution in affected SCA1 patient neurons. Here, we observed that ataxin-1 is degraded by the ubiquitin-proteasome pathway. While ataxin-1 [2Q] and mutant ataxin-1 [92Q] are polyubiquitinated equally well in vitro, the mutant form is three times more resistant to degradation. Inhibiting proteasomal degradation promotes ataxin-1 aggregation in transfected cells. And in mice, Purkinje cells that express mutant ataxin-1 but not a ubiquitin-protein ligase have significantly fewer NIs. Nonetheless, the Purkinje cell pathology is markedly worse than that of SCA1 mice. Taken together, NIs are not necessary to induce neurodegeneration, but impaired proteasomal degradation of mutant ataxin-1 may contribute to SCA1 pathogenesis.

Regulation of the Drosophila ubiquitin ligase DIAP1 is mediated via several distinct ubiquitin system pathways.

Inhibitors of apoptosis proteins (IAPs) suppress cell death by inactivating proapoptotic regulators, and therefore play important roles in controlling apoptosis in normal and malignant cells. Many IAPs are ubiquitin ligases, and their activity is mediated via ubiquitination and subsequent degradation of their targets. Here we corroborate a previous observation that DIAP1 (Drosophila IAP1) can be degraded via a two-step mechanism: (i) limited caspase-mediated cleavage and (ii) degradation of the released fragment via the ubiquitin N-end rule pathway. Yet, we demonstrate that this pathway is not the only one involved in DIAP1 degradation, and the intact protein can be degraded independent of prior caspase cleavage. Importantly, this mode of degradation does not require the RING-finger-mediated autoubiquitinating activity of DIAP1, believed to target many RING-finger E3s for self-destruction. Our preliminary data suggest that DIAP2 mediates DIAP1 degradation, suggesting a novel regulatory loop within the apoptotic pathway. Studying the role of the autoubiquitinating activity of DIAP1, we demonstrate that it does not involve formation of Lys48-based polyubiquitin chains, but probably chains linked via Lys63. Our preliminary data suggest that the autoubiquitination serves to attenuate the ligase activity of DIAP1 towards its exogenous substrates.

Degradation of the proto-oncogene product c-Fos by the ubiquitin proteolytic system in vivo and in vitro: identification and characterization of the conjugating enzymes.

The transcription factor c-Fos is a short-lived cellular protein. The levels of the protein fluctuate significantly and abruptly during changing pathophysiological conditions. Thus, it is clear that degradation of the protein plays an important role in its tightly regulated activity. We examined the involvement of the ubiquitin pathway in c-Fos breakdown. Using a mutant cell line, ts20, that harbors a thermolabile ubiquitin-activating enzyme, E1, we demonstrate that impaired function of the ubiquitin system stabilizes c-Fos in vivo. In vitro, we reconstituted a cell-free system and demonstrated that the protein is multiply ubiquitinated. The adducts serve as essential intermediates for degradation by the 26S proteasome. We show that both conjugation and degradation are significantly stimulated by c-Jun, with which c-Fos forms the active heterodimeric transcriptional activator AP-1. Analysis of the enzymatic cascade involved in the conjugation process reveals that the ubiquitin-carrier protein E2-F1 and its human homolog UbcH5, which target the tumor suppressor p53 for degradation, are also involved in c-Fos recognition. The E2 enzyme acts along with a novel species of ubiquitin-protein ligase, E3. This enzyme is distinct from other known E3s, including E3 alpha/UBR1, E3 beta, and E6-AP. We have purified the novel enzyme approximately 350-fold and demonstrated that it is a homodimer with an apparent molecular mass of approximately 280 kDa. It contains a sulfhydryl group that is essential for its activity, presumably for anchoring activated ubiquitin as an intermediate thioester prior to its transfer to the substrate. Taken together, our in vivo and in vitro studies strongly suggest that c-Fos is degraded in the cell by the ubiquitin-proteasome proteolytic pathway in a process that requires a novel recognition enzyme.

Structural motifs involved in ubiquitin-mediated processing of the NF-kappaB precursor p105: roles of the glycine-rich region and a downstream ubiquitination domain.

The ubiquitin proteolytic system plays a major role in a variety of basic cellular processes. In the majority of these processes, the target proteins are completely degraded. In one exceptional case, generation of the p50 subunit of the transcriptional regulator NF-kappaB, the precursor protein p105 is processed in a limited manner: the N-terminal domain yields the p50 subunit, whereas the C-terminal domain is degraded. The identity of the mechanisms involved in this unique process have remained elusive. It has been shown that a Gly-rich region (GRR) at the C-terminal domain of p50 is an important processing signal. Here we show that the GRR does not interfere with conjugation of ubiquitin to p105 but probably does interfere with the processing of the ubiquitin-tagged precursor by the 26S proteasome. Structural analysis reveals that a short sequence containing a few Gly residues and a single essential Ala is sufficient to generate p50. Mechanistically, the presence of the GRR appears to stop further degradation of p50 and to stabilize the molecule. It appears that the localization of the GRR within p105 plays an important role in directing processing: transfer of the GRR within p105 or insertion of the GRR into homologous or heterologous proteins is not sufficient to promote processing in most cases, which is probably due to the requirement for an additional specific ubiquitination and/or recognition domain(s). Indeed, we have shown that amino acid residues 441 to 454 are important for processing. In particular, both Lys 441 and Lys 442 appear to serve as major ubiquitination targets, while residues 446 to 454 are independently important for processing and may serve as the ubiquitin ligase recognition motif.

Degradation of myogenic transcription factor MyoD by the ubiquitin pathway in vivo and in vitro: regulation by specific DNA binding.

MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle differentiation. Its activity is induced during the transition from proliferating, nondifferentiated myoblasts to resting, well-differentiated myotubes. Like many other transcriptional regulators, it is a short-lived protein; however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved in the process have remained obscure. It has recently been shown that many short-lived regulatory proteins are degraded by the ubiquitin system. Degradation of a protein by the ubiquitin system proceeds via two distinct and successive steps, conjugation of multiple molecules of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In intact cells, the degradation is inhibited by lactacystin, a specific inhibitor of the 26S proteasome. Inhibition is accompanied by accumulation of high-molecular-mass MyoD-ubiquitin conjugates. In a cell-free system, the proteolytic process requires both ATP and ubiquitin and, like the in vivo process, is preceded by formation of ubiquitin conjugates of the transcription factor. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds: conjugation and degradation of a MyoD mutant protein which lacks the DNA-binding domain are not inhibited. The inhibitory effect of the DNA requires the formation of a complex between the DNA and the MyoD protein. Id1, which inhibits the binding of MyoD complexes to DNA, abrogates the effect of DNA on stabilization of the protein.

c-Abl regulates p53 levels under normal and stress conditions by preventing its nuclear export and ubiquitination.

The p53 protein is subject to Mdm2-mediated degradation by the ubiquitin-proteasome pathway. This degradation requires interaction between p53 and Mdm2 and the subsequent ubiquitination and nuclear export of p53. Exposure of cells to DNA damage results in the stabilization of the p53 protein in the nucleus. However, the underlying mechanism of this effect is poorly defined. Here we demonstrate a key role for c-Abl in the nuclear accumulation of endogenous p53 in cells exposed to DNA damage. This effect of c-Abl is achieved by preventing the ubiquitination and nuclear export of p53 by Mdm2, or by human papillomavirus E6. c-Abl null cells fail to accumulate p53 efficiently following DNA damage. Reconstitution of these cells with physiological levels of c-Abl is sufficient to promote the normal response of p53 to DNA damage via nuclear retention. Our results help to explain how p53 is accumulated in the nucleus in response to DNA damage.

Role of UEV-1, an inactive variant of the E2 ubiquitin-conjugating enzymes, in in vitro differentiation and cell cycle behavior of HT-29-M6 intestinal mucosecretory cells.

By means of differential RNA display, we have isolated a cDNA corresponding to transcripts that are down-regulated upon differentiation of the goblet cell-like HT-29-M6 human colon carcinoma cell line. These transcripts encode proteins originally identified as CROC-1 on the basis of their capacity to activate transcription of c-fos. We show that these proteins are similar in sequence, and in predicted secondary and tertiary structure, to the ubiquitin-conjugating enzymes, also known as E2. Despite the similarities, these proteins lack a critical cysteine residue essential for the catalytic activity of E2 enzymes and, in vitro, they do not conjugate or transfer ubiquitin to protein substrates. These proteins constitute a distinct subfamily within the E2 protein family and are highly conserved in phylogeny from yeasts to mammals. Therefore, we have designated them UEV (ubiquitin-conjugating E2 enzyme variant) proteins, defined as proteins similar in sequence and structure to the E2 ubiquitin-conjugating enzymes but lacking their enzymatic activity (HW/GDB-approved gene symbol, UBE2V). At least two human genes code for UEV proteins, and one of them, located on chromosome 20q13.2, is expressed as at least four isoforms, generated by alternative splicing. All human cell types analyzed expressed at least one of these isoforms. Constitutive expression of exogenous human UEV in HT-29-M6 cells inhibited their capacity to differentiate upon confluence and caused both the entry of a larger proportion of cells in the division cycle and an accumulation in G2-M. This was accompanied with a profound inhibition of the mitotic kinase, cdk1. These results suggest that UEV proteins are involved in the control of differentiation and could exert their effects by altering cell cycle distribution.

Intracellular protein degradation: from a vague idea thru the lysosome and the ubiquitin-proteasome system and onto human diseases and drug targeting.

Between the 1950s and 1980s, scientists were focusing mostly on how the genetic code is transcribed to RNA and translated to proteins, but how proteins are degraded has remained a neglected research area. With the discovery of the lysosome by Christian de Duve, it was assumed that cellular proteins are degraded within this organelle. Yet, several independent lines of experimental evidence strongly suggested that intracellular proteolysis is largely nonlysosomal, but the mechanisms involved remained obscure. The discovery of the ubiquitin-proteasome system resolved the enigma. We now recognize that degradation of intracellular proteins is involved in regulation of a broad array of cellular processes, such as cell cycle and division, regulation of transcription factors, and assurance of the cellular quality control. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of human disease, such as malignancies and neurodegenerative disorders, which led subsequently to an increasing effort to develop mechanism-based drugs.

Adaptive preconditioning in neurological diseases - therapeutic insights from proteostatic perturbations.

In neurological disorders, both acute and chronic neural stress can disrupt cellular proteostasis, resulting in the generation of pathological protein. However in most cases, neurons adapt to these proteostatic perturbations by activating a range of cellular protective and repair responses, thus maintaining cell function. These interconnected adaptive mechanisms comprise a 'proteostasis network' and include the unfolded protein response, the ubiquitin proteasome system and autophagy. Interestingly, several recent studies have shown that these adaptive responses can be stimulated by preconditioning treatments, which confer resistance to a subsequent toxic challenge - the phenomenon known as hormesis. In this review we discuss the impact of adaptive stress responses stimulated in diverse human neuropathologies including Parkinson׳s disease, Wolfram syndrome, brain ischemia, and brain cancer. Further, we examine how these responses and the molecular pathways they recruit might be exploited for therapeutic gain. This article is part of a Special Issue entitled SI:ER stress.

Degradation of the E7 human papillomavirus oncoprotein by the ubiquitin-proteasome system: targeting via ubiquitination of the N-terminal residue.

The E7 oncoprotein of the high risk human papillomavirus type 16 (HPV-16), which is etiologically associated with uterine cervical cancer, is a potent immortalizing and transforming agent. It probably exerts its oncogenic functions by interacting and altering the normal activity of cell cycle control proteins such as p21WAF1, p27KIP1 and pRb, transcriptional activators such as TBP and AP-1, and metabolic regulators such as M2-pyruvate kinase (M2-PK). Here we show that E7 is a short-lived protein and its degradation both in vitro and in vivo is mediated by the ubiquitin-proteasome pathway. Interestingly, ubiquitin does not attach to any of the two internal Lysine residues of E7. Substitution of these residues with Arg does not affect the ability of the protein to be conjugated and degraded; in contrast, addition of a Myc tag to the N-terminal but not to the C-terminal residue, stabilizes the protein. Also, deletion of the first 11 amino acid residues stabilizes the protein in cells. Taken together, these findings strongly suggest that, like MyoD and the Epstein Barr Virus (EBV) transforming Latent Membrane Protein 1 (LMPI), the first ubiquitin moiety is attached linearly to the free N-terminal residue of E7. Additional ubiquitin moieties are then attached to an internal Lys residue of the previously conjugated molecule. The involvement of E7 in many diverse and apparently unrelated processes requires tight regulation of its function and cellular level, which is controlled in this case by ubiquitin-mediated proteolysis.

Differential interaction of plakoglobin and beta-catenin with the ubiquitin-proteasome system.

Beta-catenin and plakoglobin are closely related armadillo family proteins with shared and distinct properties; Both are associated with cadherins in actin-containing adherens junctions. Plakoglobin is also found in desmosomes where it anchors intermediate filaments to the desmosomal plaques. Beta-catenin, on the other hand, is a component of the Wnt signaling pathway, which is involved in embryonic morphogenesis and tumorigenesis. A key step in the regulation of this pathway involves modulation of beta-catenin stability. A multiprotein complex, regulated by Wnt, directs the phosphorylation of beta-catenin and its degradation by the ubiquitin-proteasome system. Plakoglobin can also associate with members of this complex, but inhibition of proteasomal degradation has little effect on its levels while dramatically increasing the levels of beta-catenin. Beta-TrCP, an F-box protein of the SCF E3 ubiquitin ligase complex, was recently shown to play a role in the turnover of beta-catenin. To elucidate the basis for the apparent differences in the turnover of beta-catenin and plakoglobin we compared the handling of these two proteins by the ubiquitin-proteasome system. We show here that a deletion mutant of beta-TrCP, lacking the F-box, can stabilize the endogenous beta-catenin leading to its nuclear translocation and induction of beta-catenin/LEF-1-directed transcription, without affecting the levels of plakoglobin. However, when plakoglobin was overexpressed, it readily associated with beta-TrCP, efficiently competed with beta-catenin for binding to beta-TrCP and became polyubiquitinated. Fractionation studies revealed that about 85% of plakoglobin in 293 cells, is Triton X-100-insoluble compared to 50% of beta-catenin. These results suggest that while both plakoglobin and beta-catenin can comparably interact with beta-TrCP and the ubiquitination system, the sequestration of plakoglobin by the membrane-cytoskeleton system renders it inaccessible to the proteolytic machinery and stabilizes it.

ATP-stimulated degradation of endogenous proteins in cell-free extracts of BHK 21/C13 fibroblasts. A key role for the proteinase, macropain, in the ubiquitin-dependent degradation of short-lived proteins.

Baby hamster kidney (BHK) 21/C13 cell proteins, labeled with [35S]methionine, [14C]leucine or [3H]leucine in intact cells, were degraded in soluble, cell-free extracts by an ATP-stimulated process. The stimulatory effect of ATP appeared to require ATP hydrolysis and was mediated to a large extent by ubiquitin. Although the cell extracts contained endogenous ubiquitin, supplementation with exogenous ubiquitin increased ATP-dependent proteolysis by up to 2-fold. Furthermore, antibodies against the E1 ubiquitin conjugating enzyme specifically inhibited both conjugation of [125I]ubiquitin to endogenous proteins and ATP/ubiquitin-dependent proteolysis. Addition of purified E1 to antibody-treated extracts restored conjugation and proteolysis. Proteins containing the amino acid analogues canavanine and azatryptophan were also degraded in vitro by an ATP/ubiquitin-dependent process but at a rate up to 2-fold faster than normal proteins. These results indicate that soluble, cell-free extracts of BHK cells can selectively degrade proteins whose rates of degradation are increased in intact cells. Treatment of cell-free extracts with antibodies against the high molecular weight proteinase, macropain, also greatly inhibited the ATP/ubiquitin-dependent degradation of endogenous proteins. Proteolysis was specifically restored when purified macropain L was added to the antibody-treated extracts. Treatment of cell extracts with both anti-macropain and anti-E1 antibodies reduced ATP/ubiquitin-dependent proteolysis to the same extent as treatment with either antibody alone. Furthermore, proteolysis could be restored to the double antibody treated extracts only after addition of both purified E1 and macropain. These results provide strong evidence for an important role for macropain in the ATP/ubiquitin-dependent degradation of endogenous proteins in BHK cell extracts.