N-end Rule Pathway Research Articles

Modulating huntingtin half-life alters polyglutamine-dependent aggregate formation and cell toxicity.

A common finding among the expanded polyglutamine disorders is intracellular protein aggregates. Although the precise role of these aggregates in the disease process is unclear, they are generally ubiquitinated, implicating the ubiquitin-proteasome pathway in neuronal degeneration. To investigate the mechanism of aggregate formation, we have developed a cell culture model to express huntingtin designed to have an altered degradation rate through the ubiquitin-dependent N-end rule pathway. We fused the first 171 amino acids of huntingtin, containing either a pathogenic or normal polyglutamine tract, to the enhanced green fluorescent protein (EGFP). The half-life of soluble huntingtin-EGFP was dependent on the degradation signal and the polyglutamine tract length. However, once huntingtin-EGFP with a pathogenic tract had aggregated, the protein was extremely stable. Huntingtin-EGFP with a pathogenic glutamine tract and a shorter half-life displayed a delayed onset of aggregate formation and was more toxic to transfected cells. These data suggest that rapid clearance through the ubiquitin-proteasome pathway slows aggregate formation, yet increases cellular toxicity. Polyglutamine-induced neurotoxicity may therefore be triggered by non-aggregated protein, and aggregate formation itself may be a cellular defense mechanism.

Female lethality and apoptosis of spermatocytes in mice lacking the UBR2 ubiquitin ligase of the N-end rule pathway.

Substrates of the ubiquitin-dependent N-end rule pathway include proteins with destabilizing N-terminal residues. UBR1(-/-) mice, which lacked the pathway's ubiquitin ligase E3alpha, were viable and retained the N-end rule pathway. The present work describes the identification and analysis of mouse UBR2, a homolog of UBR1. We demonstrate that the substrate-binding properties of UBR2 are highly similar to those of UBR1, identifying UBR2 as the second E3 of the mammalian N-end rule pathway. UBR2(-/-) mouse strains were constructed, and their viability was found to be dependent on both gender and genetic background. In the strain 129 (inbred) background, the UBR2(-/-) genotype was lethal to most embryos of either gender. In the 129/B6 (mixed) background, most UBR2(-/-) females died as embryos, whereas UBR2(-/-) males were viable but infertile, owing to the postnatal degeneration of the testes. The gross architecture of UBR2(-/-) testes was normal and spermatogonia were intact as well, but UBR2(-/-) spermatocytes were arrested between leptotene/zygotene and pachytene and died through apoptosis. A conspicuous defect of UBR2(-/-) spermatocytes was the absence of intact synaptonemal complexes. We conclude that the UBR2 ubiquitin ligase and, hence, the N-end rule pathway are required for male meiosis and spermatogenesis and for an essential aspect of female embryonic development.

PRT1 of Arabidopsis is a ubiquitin protein ligase of the plant N-end rule pathway with specificity for aromatic amino-terminal residues.

The gene PRT1 of Arabidopsis, encoding a 45-kD protein with two RING finger domains, is essential for the degradation of F-dihydrofolate reductase, a model substrate of the N-end rule pathway of protein degradation. We have determined the function of PRT1 by expression in yeast (Saccharomyces cerevisiae). PRT1 can act as a ubiquitin protein ligase in the heterologous host. The identified substrates of PRT1 have an aromatic residue at their amino-terminus, indicating that PRT1 mediates degradation of N-end rule substrates with aromatic termini but not of those with aliphatic or basic amino-termini. Expression of model substrates in mutant and wild-type plants confirmed this substrate specificity. A ligase activity exclusively devoted to aromatic amino-termini of the N-end rule pathway is apparently unique to plants. The results presented also imply that other known substrates of the plant N-end rule pathway are ubiquitylated by one or more different ubiquitin protein ligases.

The N-end rule and regulation of apoptosis.

The ubiquitin-dependent N-end rule pathway targets proteins for degradation through their destabilizing N-terminal residues. This pathway is known to control the import of peptides, chromosome stability and cardiovascular development. A new report identifies yet another function of the N-end rule pathway: the regulation of apoptosis through degradation of Drosophila melanogaster DIAP1.

Ubiquitin-protein ligases in muscle wasting: multiple parallel pathways?

Studies in a wide variety of animal models of muscle wasting have led to the concept that increased protein breakdown via the ubiquitin-proteasome pathway is responsible for the loss of muscle mass seen as muscle atrophy. The complexity of the ubiquitination apparatus has hampered our understanding of how this pathway is activated in atrophying muscles and which ubiquitin-conjugating enzymes in muscle are responsible. Recent experiments have shown that two newly identified ubiquitin-protein ligases (E3s), atrogin-1/MAFbx and MURF-1, are critical in the development of muscle atrophy. Other in-vitro studies also implicated E2(14k) and E3alpha, of the N-end rule pathway, as playing an important role in the process. It seems likely that multiple pathways of ubiquitin conjugation are activated in parallel in atrophying muscle, perhaps to target for degradation specific classes of muscle proteins. The emerging challenge will be to define the protein targets for, as well as inhibitors of, these E3s.

Gamma 2 subunit of G protein heterotrimer is an N-end rule ubiquitylation substrate.

Heterotrimeric G proteins transduce signals from activated transmembrane G protein-coupled receptors to appropriate downstream effectors within cells. Signaling specificity is achieved in part by the specific alpha, beta, and gamma subunits that compose a given heterotrimer. Additional structural and functional diversity in these subunits is generated at the level of posttranslational modification, offering alternate regulatory mechanisms for G protein signaling. Presented here is the identification of a variant of the gamma(2) subunit of G protein heterotrimer purified from bovine brain and the demonstration that this RDTASIA gamma(2) variant, containing unique amino acid sequence at its N terminus, is a substrate for ubiquitylation and degradation via the N-end rule pathway. Although N-end-dependent degradation has been shown to have important functions in peptide import, chromosome segregation, angiogenesis, and cardiovascular development, the identification of cellular substrates in mammalian systems has remained elusive. The isolation of RDTASIA gamma(2) from a native tissue represents identification of a mammalian N-end rule substrate from a physiological source, and elucidates a mechanism for the targeting of G protein gamma subunits for ubiquitylation and degradation.

Degradation of DIAP1 by the N-end rule pathway is essential for regulating apoptosis.

Some members of the inhibitor of apoptosis (IAP) protein family block apoptosis by binding to and neutralizing active caspases. We recently demonstrated that a physical association between IAP and caspases alone is insufficient to regulate caspases in vivo and that an additional level of control is provided by IAP-mediated ubiquitination of both itself and the associated caspases. Here we show that Drosophila IAP 1 (DIAP1) is degraded by the 'N-end rule' pathway and that this process is indispensable for regulating apoptosis. Caspase-mediated cleavage of DIAP1 at position 20 converts the more stable pro-N-degron of DIAP1 into the highly unstable, Asn-bearing, DIAP1 N-degron of the N-end rule degradation pathway. Thus, DIAP1 represents the first known metazoan substrate of the N-end rule pathway that is targeted for degradation through its amino-terminal Asn residue. We demonstrate that the N-end rule pathway is required for regulation of apoptosis induced by Reaper and Hid expression in the Drosophila melanogaster eye. Our data suggest that DIAP1 instability, mediated through caspase activity and subsequent exposure of the N-end rule pathway, is essential for suppression of apoptosis. We suggest that DIAP1 safeguards cell viability through the coordinated mutual destruction of itself and associated active caspases.

Pairs of dipeptides synergistically activate the binding of substrate by ubiquitin ligase through dissociation of its autoinhibitory domain.

Protein degradation by the ubiquitin (Ub) system controls the concentrations of many regulatory proteins. The degradation signals (degrons) of these proteins are recognized by the system's Ub ligases (complexes of E2 and E3 enzymes). Two substrate-binding sites of UBR1, the E3 of the N-end rule pathway in the yeast Saccharomyces cerevisiae, recognize basic (type 1) and bulky hydrophobic (type 2) N-terminal residues of proteins or short peptides. A third substrate-binding site of UBR1 targets CUP9, a transcriptional repressor of the peptide transporter PTR2, through an internal (non-N-terminal) degron of CUP9. Previous work demonstrated that dipeptides with destabilizing N-terminal residues allosterically activate UBR1, leading to accelerated in vivo degradation of CUP9 and the induction of PTR2 expression. Through this positive feedback, S. cerevisiae can sense the presence of extracellular peptides and react by accelerating their uptake. Here, we show that dipeptides with destabilizing N-terminal residues cause dissociation of the C-terminal autoinhibitory domain of UBR1 from its N-terminal region that contains all three substrate-binding sites. This dissociation, which allows the interaction between UBR1 and CUP9, is strongly increased only if both type 1- and type 2-binding sites of UBR1 are occupied by dipeptides. An aspect of autoinhibition characteristic of yeast UBR1 also was observed with mammalian (mouse) UBR1. The discovery of autoinhibition in Ub ligases of the UBR family indicates that this regulatory mechanism may also control the activity of other Ub ligases.

Ubiquitin-conjugating enzyme E214k/HR6B is dispensable for increased protein catabolism in muscle of fasted mice.

Activated skeletal muscle proteolysis in catabolic states has been linked to an upregulation of the ATP-ubiquitin-dependent proteolytic system. Previous studies suggested that the N-end rule pathway is primarily responsible for the bulk of skeletal muscle proteolysis. The activity of this pathway is dependent on the 14-kDa ubiquitin-conjugating enzyme E2(14k) (HR6B) and the ubiquitin protein ligase Ubr1. To address the requirement of E2(14k) in muscle proteolysis, we examined muscle protein metabolism in wild-type (WT) mice and mice lacking the E2(14k) gene (KO) in fed and fasted (48 h) states. Baseline body weight, muscle mass, and protein content were similar, and these parameters decreased similarly upon fasting in the two genotypes. There were also no effects of genotype on the rate of proteolysis in soleus muscle. The fasting-induced increase in the amount of ubiquitinated proteins was the same in WT and KO mice. The absence of any significant effect of loss of E2(14k) function was not due to a compensatory induction of the closely related isoform HR6A. Total intracellular concentration of E2(14k) and HR6A in the WT mice was 290 +/- 40 nM, but the level in the KO mice (reflecting the level of HR6A) was 110 +/- 9 nM. This value is about threefold the apparent Michaelis-Menten constant (K(m)) of E2(14k) (approximately 40 nM) for stimulating conjugation in muscle extracts. Because the HR6A isoform has a K(m) of 16 nM for stimulating conjugation, the HR6A levels in the muscles of KO mice appear sufficient for supporting conjugation mediated by this pathway during fasting.

A test of fusion protein stability in the plant Arabidopsis thaliana reveals degradation signals from ACC synthase and from the plant N-end rule pathway

We tested three different protein domains for their ability to confer a short half life onto the otherwise stable test protein dihydrofolate reductase (DHFR) in the plant Arabidopsis thaliana. E-DHFR, a protein with Glu as the first residue, is metabolically unstable in Arabidopsis, indicating that Glu is a destabilizing residue of the plant N-end rule pathway. In contrast, the degradation signal deg1 from Saccharomyces cerevisiae transcription factor Matα2p is not recognized when present at the amino-terminus of DHFR. Finally, we show that the amino-terminal 93 amino acids of the plant protein 1-aminocyclopropane-1-carboxylic acid synthase contain a degradation signal that is also active in yeast.

An Essential Role of N-Terminal Arginylation in Cardiovascular Development

The enzymatic conjugation of arginine to the N-termini of proteins is a part of the ubiquitin-dependent N-end rule pathway of protein degradation. In mammals, three N-terminal residues-aspartate, glutamate, and cysteine-are substrates for arginylation. The mouse ATE1 gene encodes a family of Arg-tRNA-protein transferases (R-transferases) that mediate N-terminal arginylation. We constructed ATE1-lacking mouse strains and found that ATE1-/- embryos die with defects in heart development and in angiogenic remodeling of the early vascular plexus. Through biochemical analyses, we show that N-terminal cysteine, in contrast to N-terminal aspartate and glutamate, is oxidized before its arginylation by R-transferase, suggesting that the arginylation branch of the N-end rule pathway functions as an oxygen sensor.

Construction and Analysis of Mouse Strains Lacking the Ubiquitin Ligase UBR1 (E3α) of the N-End Rule Pathway

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In the yeast Saccharomyces cerevisiae, the UBR1-encoded ubiquitin ligase (E3) of the N-end rule pathway mediates the targeting of substrate proteins in part through binding to their destabilizing N-terminal residues. The functions of the yeast N-end rule pathway include fidelity of chromosome segregation and the regulation of peptide import. Our previous work described the cloning of cDNA and a gene encoding the 200-kDa mouse UBR1 (E3alpha). Here we show that mouse UBR1, in the presence of a cognate mouse ubiquitin-conjugating (E2) enzyme, can rescue the N-end rule pathway in ubr1Delta S. cerevisiae. We also constructed UBR1(-/-) mouse strains that lacked the UBR1 protein. UBR1(-/-) mice were viable and fertile but weighed significantly less than congenic +/+ mice. The decreased mass of UBR1(-/-) mice stemmed at least in part from smaller amounts of the skeletal muscle and adipose tissues. The skeletal muscle of UBR1(-/-) mice apparently lacked the N-end rule pathway and exhibited abnormal regulation of fatty acid synthase upon starvation. By contrast, and despite the absence of the UBR1 protein, UBR1(-/-) fibroblasts contained the N-end rule pathway. Thus, UBR1(-/-) mice are mosaics in regard to the activity of this pathway, owing to differential expression of proteins that can substitute for the ubiquitin ligase UBR1 (E3alpha). We consider these UBR1-like proteins and discuss the functions of the mammalian N-end rule pathway.

N-end Rule Specificity within the Ubiquitin/Proteasome Pathway Is Not an Affinity Effect

The N-end rule relates the amino terminus to the rate of degradation through the ubiquitin/26 S proteasome pathway. Proteins bearing basic (type 1) or large hydrophobic (type 2) amino termini are assumed to be targeted through this pathway by their higher affinity for binding to the responsible E3 ligase compared with proteins bearing other residues (type 3). Paradoxically, a significant fraction of eukaryotic protein degradation occurs through the N-end rule pathway, although the majority of cellular proteins are type 3 substrates. We have exploited specific interactions between ubiquitin carrier proteins (E2/Ubc) and their cognate E3 ligases to purify for the first time the mammalian N-end rule ligase E3alpha/Ubr1 to near homogeneity. In vitro studies show that E3alpha forms lysine 48-linked polyubiquitin degradation signals on type 1-3 substrates and is absolutely dependent on Ubc2/Rad6 orthologs. Biochemically defined kinetic studies show that the basis of N-end rule specificity is a k(cat) rather than the K(m) effect originally proposed, since all three substrate classes show similar binding affinities (K(m) approximately 5 microm) but V(max) values that are 100- and 50-fold greater for type 1 and 2 versus type 3 model substrates, respectively. In addition, the N-end rule dipeptides lysylalanine and phenylalanylalanine are general noncompetitive inhibitors for E3alpha-catalyzed ubiquitination of type 1-3 substrates rather than type-specific competitive inhibitors as predicted. These observations are consistent with a model in which the N-end rule effect reflects substrate binding-induced transitions in E3alpha to a catalytically competent conformer, the equilibrium for which depends on the identity of the amino terminus or the presence of basic or hydrophobic surface features. The model reconciles conflicts between specific predictions and empirical observations relating N-end rule targeting in addition to explicating the efficacy of selected dipeptides as potent in vivo inhibitors of this pathway.

Degradation of a cohesin subunit by the N-end rule pathway is essential for chromosome stability.

Cohesion between sister chromatids is established during DNA replication and depends on a protein complex called cohesin. At the metaphase-anaphase transition in the yeast Saccharomyces cerevisiae, the ESP1-encoded protease separin cleaves SCC1, a subunit of cohesin with a relative molecular mass of 63,000 (Mr 63K). The resulting 33K carboxy-terminal fragment of SCC1 bears an amino-terminal arginine-a destabilizing residue in the N-end rule. Here we show that the SCC1 fragment is short-lived (t1/2 approximately 2 min), being degraded by the ubiquitin/proteasome-dependent N-end rule pathway. Overexpression of a long-lived derivative of the SCC1 fragment is lethal. In ubr1Delta cells, which lack the N-end rule pathway, we found a highly increased frequency of chromosome loss. The bulk of increased chromosome loss in ubr1Delta cells is caused by metabolic stabilization of the ESP1-produced SCC1 fragment. This fragment is the first physiological substrate of the N-end rule pathway that is targeted through its N-terminal residue. A number of yeast proteins bear putative cleavage sites for the ESP1 separin, suggesting other physiological substrates and functions of the N-end rule pathway.

Facilitated stimulus-response associative learning and long-term memory in mice lacking the NTAN1 amidase of the N-end rule pathway

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Inactivation of the NTAN1 gene encoding the asparagine-specific N-terminal amidase in mice results in impaired spatial memory [26]. The studies described here were designed to further characterize the effects upon learning and memory of inactivating the NTAN1 gene. NTAN1-deficient mice were found to be better than wild-type mice on black–white and horizontal–vertical discrimination learning. They were also better at 8-week Morris maze retention testing when a reversal trial was not included in the testing procedures. In all three tasks NTAN1-deficient mice appeared to use a strong win–stay strategy. It is concluded that inactivating the asparagine-specific branch of the N-end rule pathway in mice results in impaired spatial learning with concomitant compensatory restructuring of the nervous system in favor of non-spatial (stimulus-response) learning.

Muscle Cachexia: Current Concepts of Intracellular Mechanisms and Molecular Regulation

To review present knowledge of intracellular mechanisms and molecular regulation of muscle cachexia. Muscle cachexia, mainly reflecting degradation of myofibrillar proteins, is an important clinical feature in patients with severe injury, sepsis, and cancer. The catabolic response in skeletal muscle may result in muscle wasting and weakness, delaying or preventing ambulation and rehabilitation in these patients and increasing the risk for pulmonary complications. Muscle cachexia, induced by severe injury, sepsis, and cancer, is associated with increased gene expression and activity of the calcium/calpain- and ubiquitin/proteasome-proteolytic pathways. Calcium/calpain-regulated release of myofilaments from the sarcomere is an early, and perhaps rate-limiting, component of the catabolic response in muscle. Released myofilaments are ubiquitinated in the N-end rule pathway, regulated by the ubiquitin-conjugating enzyme E2(14k) and the ubiquitin ligase E3 alpha, and degraded by the 26S proteasome. An understanding of the mechanisms regulating muscle protein breakdown is important for the development of therapeutic strategies aimed at treating or preventing muscle cachexia in patients with severe injury, sepsis, cancer, and perhaps other catabolic conditions as well.

A new screen for protein interactions reveals that the Saccharomyces cerevisiae high mobility group proteins Nhp6A/B are involved in the regulation of the GAL1 promoter.

The split-ubiquitin assay detects protein interactions in vivo. To identify proteins interacting with Gal4p and Tup1p, two transcriptional regulators, we converted the split-ubiquitin assay into a generally applicable screen for binding partners of specific proteins in vivo. A library of genomic Saccharomyces cerevisiae DNA fragments fused to the N-terminal half of ubiquitin was constructed and transformed into yeast strains carrying either Gal4p or Tup1p as a bait. Both proteins were C-terminally extended by the C-terminal half of ubiquitin followed by a modified Ura3p with an arginine in position 1, a destabilizing residue in the N-end rule pathway. The bait fusion protein alone is stable and enzymatically active. However, upon interaction with its prey, a native-like ubiquitin is reconstituted. RUra3p is then cleaved off by the ubiquitin-specific proteases and rapidly degraded by the N-end rule pathway. In both screens, Nhp6B was identified as a protein in close proximity to Gal4p as well as to Tup1p. Direct interaction between either protein and Nhp6B was confirmed by coprecipitation assays. Genetic analysis revealed that Nhp6B, a member of the HMG1 family of DNA-binding proteins, can influence transcriptional activation as well as repression at a specific locus in the chromosome of the yeast S. cerevisiae.

Design of toxins that can be activated by cell-specific proteases and their potential use in targeted cell killing

Protein toxins designed to eliminate specific cell types, e. g. disease-associated cells, have mainly made by linking the active domain of the toxin to a protein that only binds to certain cells. A different approach for the construction of toxins capable of killing disease-associated cells is suggested here, based on the knowledge that many of these cells express specific proteases that are not expressed in normal tissue. The construction of toxins that become activated through cleavage by the protease (HIV-1 PR) expressed by the HIV-1 virus is described. These toxins contain a signal for degradation by the N-end rule pathway, which is cleaved off by HIV-1 PR, resulting in increased toxicity. Alternative strategies for the construction of toxins that can be activated by proteases are discussed.

Degradation of HIV-1 Integrase by the N-end Rule Pathway

Human immunodeficiency virus type-1 (HIV-1) integrase catalyzes the irreversible insertion of the viral genome into host chromosomal DNA. We have developed a mammalian expression system for the synthesis of authentic HIV-1 integrase in the absence of other viral proteins. Integrase, which bears a N-terminal phenylalanine, was found to be a short-lived protein in human embryo kidney 293T cells. The degradation of integrase could be suppressed by proteasome inhibitors. N-terminal phenylalanine is recognized as a degradation signal by a ubiquitin-proteasome proteolytic system known as the N-end rule pathway. The replacement of N-terminal phenylalanine with methionine, valine, or glycine, which are stabilizing residues in the N-end rule, resulted in metabolically stabilized integrase proteins (half-life of N-terminal Met-integrase was at least 3 h). Conversely, the substitution of N-terminal phenylalanine with other destabilizing residues retained the metabolic instability of integrase. These findings indicate that the HIV-1 integrase is a physiological substrate of the N-end rule. We discuss a possible functional similarity to the better understood turnover of the bacteriophage Mu transposase and functions of integrase instability to the maintenance and integrity of the host cell genome.

Dipeptides: rulers of the N-end rule

There are many different E1–E2–E3 cascades leading to the ubiquitination and proteasome-dependent proteolysis of specific proteins. The specificity of each cascade is given by the ubiquitin ligase (E3), and to a certain extent by the ubiquitin-conjugating enzyme (E2), while the ubiquitin-activating enzyme (E1) is unspecific, common to all pathways. One of the best-studied ubiquitination pathways is the N-end rule, which involves the selective degradation of proteins carrying ‘destabilizing’ amino acids at their amino terminus. The N-end rule pathway was discovered through the degradation of engineered proteins with different N-terminal amino acids. In Saccharomyces cerevisiae, the E3 responsible for their ubiquitination was shown to be Ubr1p. The only known physiological function of Ubr1p is the regulation of dipeptide uptake, through degradation of Cup9p, a transcriptional repressor of the di-/tri-peptide transporter Ptr2p. Curiously, the Cup9p degradation signal is not an N-terminal amino acid but is a sequence located in the C-terminal half of the molecule.