Takafumi Tasaki

A Family of Mammalian E3 Ubiquitin Ligases That Contain the UBR Box Motif and Recognize N-Degrons

ABSTRACT A subset of proteins targeted by the N-end rule pathway bear degradation signals called N-degrons, whose determinants include destabilizing N-terminal residues. Our previous work identified mouse UBR1 and UBR2 as E3 ubiquitin ligases that recognize N-degrons. Such E3s are called N-recognins. We report here that while double-mutant UBR1−/− UBR2 −/− mice die as early embryos, the rescued UBR1 −/− UBR2 −/− fibroblasts still retain the N-end rule pathway, albeit of lower activity than that of wild-type fibroblasts. An affinity assay for proteins that bind to destabilizing N-terminal residues has identified, in addition to UBR1 and UBR2, a huge (570 kDa) mouse protein, termed UBR4, and also the 300-kDa UBR5, a previously characterized mammalian E3 known as EDD/hHYD. UBR1, UBR2, UBR4, and UBR5 shared a ∼70-amino-acid zinc finger-like domain termed the UBR box. The mammalian genome encodes at least seven UBR box-containing proteins, which we propose to call UBR1 to UBR7. UBR1 −/− UBR2 −/− fibroblasts that have been made deficient in UBR4 as well (through RNA interference) were significantly impaired in the degradation of N-end rule substrates such as the Sindbis virus RNA polymerase nsP4 (bearing N-terminal Tyr) and the human immunodeficiency virus type 1 integrase (bearing N-terminal Phe). Our results establish the UBR box family as a unique class of E3 proteins that recognize N-degrons or structurally related determinants for ubiquitin-dependent proteolysis and perhaps other processes as well.

The N-End Rule Pathway

The N-end rule pathway is a proteolytic system in which N-terminal residues of short-lived proteins are recognized by recognition components (N-recognins) as essential components of degrons, called N-degrons. Known N-recognins in eukaryotes mediate protein ubiquitylation and selective proteolysis by the 26S proteasome. Substrates of N-recognins can be generated when normally embedded destabilizing residues are exposed at the N terminus by proteolytic cleavage. N-degrons can also be generated through modifications of posttranslationally exposed pro-N-degrons of otherwise stable proteins; such modifications include oxidation, arginylation, leucylation, phenylalanylation, and acetylation. Although there are variations in components, degrons, and hierarchical structures, the proteolytic systems based on generation and recognition of N-degrons have been observed in all eukaryotes and prokaryotes examined thus far. The N-end rule pathway regulates homeostasis of various physiological processes, in part, through interaction with small molecules. Here, we review the biochemical mechanisms, structures, physiological functions, and small-molecule-mediated regulation of the N-end rule pathway.

Female Lethality and Apoptosis of Spermatocytes in Mice Lacking the UBR2 Ubiquitin Ligase of the N-End Rule Pathway

ABSTRACT Substrates of the ubiquitin-dependent N-end rule pathway include proteins with destabilizing N-terminal residues. UBR1−/− mice, which lacked the pathways ubiquitin ligase E3α, 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.

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 systems 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.

The substrate recognition domains of the N-end rule pathway.

The N-end rule pathway is a ubiquitin-dependent system where E3 ligases called N-recognins, including UBR1 and UBR2, recognize type-1 (basic) and type-2 (bulky hydrophobic) N-terminal residues as part of N-degrons. We have recently reported an E3 family (termed UBR1 through UBR7) characterized by the 70-residue UBR box, among which UBR1, UBR2, UBR4, and UBR5 were captured during affinity-based proteomics with synthetic degrons. Here we characterized substrate binding specificity and recognition domains of UBR proteins. Pull-down assays with recombinant UBR proteins suggest that 570-kDa UBR4 and 300-kDa UBR5 bind N-degron, whereas UBR3, UBR6, and UBR7 do not. Binding assays with 24 UBR1 deletion mutants and 31 site-directed UBR1 mutations narrow down the degron-binding activity to a 72-residue UBR box-only fragment that recognizes type-1 but not type-2 residues. A surface plasmon resonance assay shows that the UBR box binds to the type-1 substrate Arg-peptide with Kd of ∼3.4 μm. Downstream from the UBR box, we identify a second substrate recognition domain, termed the N-domain, required for type-2 substrate recognition. The ∼80-residue N-domain shows structural and functional similarity to 106-residue Escherichia coli ClpS, a bacterial N-recognin. We propose a model where the 70-residue UBR box functions as a common structural element essential for binding to all known destabilizing N-terminal residues, whereas specific residues localized in the UBR box (for type 1) or the N-domain (for type 2) provide substrate selectivity through interaction with the side group of an N-terminal amino acid. Our work provides new insights into substrate recognition in the N-end rule pathway.

Synthetic heterovalent inhibitors targeting recognition E3 components of the N-end rule pathway

Multivalent binding allows high selectivity and affinity in a ligand–protein interaction. The N-end rule pathway is a ubiquitin (Ub)-dependent proteolytic system in which specific E3s, called N-recognins, mediate ubiquitylation through the recognition of types 1 and 2, destabilizing N-terminal residues of substrates. We recently identified a set of E3 Ub ligases (named UBR1–UBR7) containing the 70-residue UBR box, and we demonstrated that UBR1, UBR2, UBR4, and UBR5 can bind to destabilizing N-terminal residues. To explore a model of heterovalent interaction to the N-recognin family, we synthesized the small-molecule compound RF-C11, which bears two heterovalent ligands designed to target N-recognins, together with control molecules with two homovalent ligands. We demonstrate that heterovalent ligands of RF-C11 selectively and cooperatively bind cognate-binding sites of multiple N-recognins and thereby inhibit both types 1 and 2 N-end rule activities. Furthermore, the efficacy of heterovalent RF-C11 was substantially higher than homovalent inhibitors, which can target either a type 1 or type 2 site, providing the molecular basis of designing multivalent inhibitors for the control of specific intracellular pathways. In addition, RF-C11 exhibited higher efficacy and stability, compared with dipeptides bearing destabilizing N-terminal residues, which are known competitive inhibitors of the pathway. We also used the heterovalent compound to study the function of N-recognins in cardiac signaling. Using mouse and rat cardiomyocytes, we demonstrate that the N-end rule pathway has a cell-autonomous function in cardiac proliferation and hypertrophy, explaining our earlier results implicating the pathway in cardiac development and proteolysis of multiple cardiovascular regulators.

Biochemical and Genetic Studies of UBR3, a Ubiquitin Ligase with a Function in Olfactory and Other Sensory Systems

Our previous work identified E3 ubiquitin ligases, termed UBR1-UBR7, that contain the ∼70-residue UBR box, a motif important for the targeting of N-end rule substrates. In this pathway, specific N-terminal residues of substrates are recognized as degradation signals by UBR box-containing E3s that include UBR1, UBR2, UBR4, and UBR5. The other E3s of this set, UBR3, UBR6, and UBR7, remained uncharacterized. Here we describe the cloning and analyses of mouse UBR3. The similarities of UBR3 to the UBR1 and UBR2 E3s of the N-end rule pathway include the RING and UBR domains. We show that HR6A and HR6B, the E2 enzymes that bind to UBR1 and UBR2, also interact with UBR3. However, in contrast to UBR1 and UBR2, UBR3 does not recognize N-end rule substrates. We also constructed UBR3-lacking mouse strains. In the 129SvImJ background, UBR3-/- mice died during embryogenesis, whereas the C57BL/6 background UBR3-/- mice exhibited neonatal lethality and suckling impairment that could be partially rescued by litter size reduction. The adult UBR3-/- mice had female-specific behavioral anosmia. Cells of the olfactory pathway were found to express β-galactosidase (LacZ) that marked the deletion/disruption UBR3- allele. The UBR3-specific LacZ expression was also prominent in cells of the touch, vision, hearing, and taste systems, suggesting a regulatory role of UBR3 in sensory pathways, including olfaction. By analogy with functions of the UBR domain in the N-end rule pathway, we propose that the UBR box of UBR3 may recognize small compounds that modulate the targeting, by this E3, of its currently unknown substrates.

UBR box N-recognin-4 (UBR4), an N-recognin of the N-end rule pathway, and its role in yolk sac vascular development and autophagy

The N-end rule pathway is a proteolytic system in which destabilizing N-terminal residues of short-lived proteins act as degradation determinants (N-degrons). Substrates carrying N-degrons are recognized by N-recognins that mediate ubiquitylation-dependent selective proteolysis through the proteasome. Our previous studies identified the mammalian N-recognin family consisting of UBR1/E3α, UBR2, UBR4/p600, and UBR5, which recognize destabilizing N-terminal residues through the UBR box. In the current study, we addressed the physiological function of a poorly characterized N-recognin, 570-kDa UBR4, in mammalian development. UBR4-deficient mice die during embryogenesis and exhibit pleiotropic abnormalities, including impaired vascular development in the yolk sac (YS). Vascular development in UBR4-deficient YS normally advances through vasculogenesis but is arrested during angiogenic remodeling of primary capillary plexus associated with accumulation of autophagic vacuoles. In the YS, UBR4 marks endoderm-derived, autophagy-enriched cells that coordinate differentiation of mesoderm-derived vascular cells and supply autophagy-generated amino acids during early embryogenesis. UBR4 of the YS endoderm is associated with a tissue-specific autophagic pathway that mediates bulk lysosomal proteolysis of endocytosed maternal proteins into amino acids. In cultured cells, UBR4 subpopulation is degraded by autophagy through its starvation-induced association with cellular cargoes destined to autophagic double membrane structures. UBR4 loss results in multiple misregulations in autophagic induction and flux, including synthesis and lipidation/activation of the ubiquitin-like protein LC3 and formation of autophagic double membrane structures. Our results suggest that UBR4 plays an important role in mammalian development, such as angiogenesis in the YS, in part through regulation of bulk degradation by lysosomal hydrolases.

The N-end rule proteolytic system in autophagy

The N-end rule pathway is a cellular proteolytic system that utilizes specific N-terminal residues as degradation determinants, called N-degrons. N-degrons are recognized and bound by specific recognition components (N-recognins) that mediate polyubiquitination of low-abundance regulators and selective proteolysis through the proteasome. Our earlier work identified UBR4/p600 as one of the N-recognins that promotes N-degron-dependent proteasomal degradation. In this study, we show that UBR4 is associated with cellular cargoes destined to autophagic vacuoles and is degraded by the lysosome. UBR4 loss causes multiple misregulations in autophagic pathways, including an increased formation of LC3 puncta. UBR4-deficient mice die during embryogenesis primarily due to defective vascular development in the yolk sac (YS), wherein UBR4 is associated with a bulk lysosomal degradation system that absorbs maternal proteins from the YS cavity and digests them into amino acids. Our results suggest that UBR4 plays a role not only in selective proteolysis of short-lived regulators through the proteasome, but also bulk degradation through the lysosome. Here, we discuss a possible mechanism of UBR4 as a regulatory component in the delivery of cargoes destined to interact with the autophagic core machinery.

The N-end rule and retroviral infection: no effect on integrase

BackgroundIntegration of double stranded viral DNA is a key step in the retroviral life cycle. Virally encoded enzyme, integrase, plays a central role in this reaction. Mature forms of integrase of several retroviruses (i.e. HIV-1 and MLV) bear conserved destabilizing N-terminal residues of the N-end rule pathway - a ubiquitin dependent proteolytic system in which the N-terminal residue of a protein determines its half life. Substrates of the N-end rule pathway are recognized by E3 ubiquitin ligases called N-recognins. We have previously shown that the inactivation of three of these N-recognins, namely UBR1, UBR2 and UBR4 in mouse embryonic fibroblasts (MEFs) leads to increased stability of ectopically expressed HIV-1 integrase. These findings have prompted us to investigate the involvement of the N-end rule pathway in the HIV-1 life cycle.ResultsThe infectivity of HIV-1 but not MLV was decreased in N-recognin deficient cells in which three N-recognins (UBR1, UBR2 and UBR4) were depleted. HIV-1 integrase mutants of N-terminal amino acids (coding for stabilizing or destabilizing residues) were severely impaired in their infectivity in both human and mouse cells. Quantitative PCR analysis revealed that this inhibition was mainly caused by a defect in reverse transcription. The decreased infectivity was independent of the N-end rule since cells deficient in N-recognins were equally refractory to infection by the integrase mutants. MLV integrase mutants showed no difference in their infectivity or intravirion processing of integrase.ConclusionsThe N-end rule pathway impacts the early phase of the HIV-1 life cycle; however this effect is not the result of the direct action of the N-end rule pathway on the viral integrase. The N-terminal amino acid residue of integrase is highly conserved and cannot be altered without causing a substantial decrease in viral infectivity.