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 ϳ70-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 ATE1-encoded Arg-transferase mediates conjugation of Arg to N-terminal Asp, Glu, and Cys of certain eukaryotic proteins, yielding N-terminal Arg that can act as a degradation signal for the ubiquitin-dependent N-end rule pathway. We have previously shown that mouse ATE1 ؊/؊ embryos die with defects in heart development and angiogenesis. Here, we report that the ATE1 Arg-transferase mediates the in vivo degradation of RGS4 and RGS5, which are negative regulators of specific G proteins whose functions include cardiac growth and angiogenesis. The proteolysis of these regulators of G protein signaling (RGS) proteins was perturbed either by hypoxia or in cells lacking ubiquitin ligases UBR1 and͞or UBR2. Mutant RGS proteins in which the conserved Cys-2 residue could not become N-terminal were long-lived in vivo. We propose a model in which the sequential modifications of RGS4, RGS5, and RGS16 (N-terminal exposure of their Cys-2, its oxidation, and subsequent arginylation) act as a licensing mechanism in response to extracellular and intracellular signals before the targeting for proteolysis by UBR1 and UBR2. We also show that ATE1 ؊/؊ embryos are impaired in the activation of extracellular signal-regulated kinase mitogen-activated protein kinases and in the expression of G protein-induced downstream effectors such as Jun, cyclin D1, and ␤-myosin heavy chain. These results establish RGS4 and RGS5 as in vivo substrates of the mammalian N-end rule pathway and also suggest that the O2-ATE1-UBR1͞UBR2 proteolytic circuit plays a role in RGS-regulated G protein signaling in the cardiovascular system. ATE1 R-transferase ͉ G protein signaling ͉ oxidation ͉ ubiquitin ͉ UBR T he ubiquitin (Ub)-dependent N-end rule pathway relates the in vivo half-life of a protein to the identity of its N-terminal residue (1) (Fig. 1A). We previously identified the mouse ATE1 gene encoding Arg-transferase, which conjugates Arg to Nterminal Asp, Glu, and Cys of engineered N-end rule substrates (2, 3), yielding N-terminal Arg that can act as an essential component of N-degron (N-terminal degradation signal). Ndegrons can be recognized by Ub ligases (E3s) for protein ubiquitylation. Mouse ATE1 Ϫ/Ϫ embryos died with cardiovascular defects, including ventricular hypoplasia, ventricular septal defect, and impaired late angiogenesis (3), suggesting that the ATE1-dependent proteolysis of unknown substrate(s) is a crucial regulatory mechanism for myocardial growth and blood vessel integrity͞maturation. One aim of this study was to identify in vivo ATE1 substrates that are important for cardiovascular functions.We previously reported that RGS4, a G protein-specific GTPase-activating protein (4), is N-terminally arginylated and degraded by the Ub system in ATP-supplemented reticulocyte extract and that its in vitro degradation is inhibited by the Arg-Ala dipeptide inhibitor of the N-end rule pathway (5). However, previous attempts to verify the functional connection between RGS4 and the N-end rule pathway in vivo (in mammalian cells) were unsuccessful....

The S. cerevisiae proteasome comprises a 19-subunit regulatory particle (RP) and 28-subunit core particle (CP). To be degraded, substrates must cross the CP-RP interface, a site of complex conformational changes and regulatory events. This interface includes two aligned heteromeric rings: the six ATPase (Rpt) subunits of the RP and the seven α subunits of the CP. Rpt C-termini bind intersubunit cavities of the α ring, thus directing CP gating and proteasome assembly. We used crosslinking to map the Rpt C-termini to the α subunit pockets. This reveals an unexpected asymmetry: one side of the ring shows 1:1 contacts of Rpt2–α4, Rpt6–α3, and Rpt3–α2, whereas, on the opposite side, the Rpt1, Rpt4, and Rpt5 tails each crosslink to multiple α pockets. Rpt-CP crosslinks are all sensitive to nucleotide, implying that ATP hydrolysis drives dynamic alterations at the CP-RP interface.

Parkinson's disease (PD) is a debilitating movement disorder typically associated with the accumulation of intracytoplasmic aggregate prone protein deposits. Over recent years, increasing evidence has led to the suggestion that the mutations underlying certain forms of PD impair autophagy. Autophagy is a degradative pathway that delivers cytoplasmic content to lysosomes for degradation and represents a major route for degradation of aggregated cellular proteins and dysfunctional organelles. Autophagy up-regulation is a promising therapeutic strategy that is being explored for its potential to protect cells against the toxicity of aggregate-prone proteins in neurodegenerative diseases. Here, we describe how the mutations in different subtypes of PD can affect different stages of autophagy.

An intronic hexanucleotide repeat expansion (HRE) mutation in the C9ORF72 gene is the most common cause of familial ALS and frontotemporal dementia (FTD) and is found in ∼7% of individuals with apparently sporadic disease. Several different diamino acid peptides can be generated from the HRE by noncanonical translation (repeat-associated non-ATG translation, or RAN translation), and some of these peptides can be toxic. Here, we studied the effects of two arginine containing RAN translation products [proline/arginine repeated 20 times (PR20) and glycine/arginine repeated 20 times (GR20)] in primary rat spinal cord neuron cultures grown on an astrocyte feeder layer. We find that PR20 kills motor neurons with an LD50 of 2 µM, but in contrast to the effects of other ALS-causing mutant proteins (i.e., SOD or TDP43), PR20 does not evoke the biochemical signature of mitochondrial dysfunction, ER stress, or mTORC down-regulation. PR20 does result in a time-dependent build-up of ubiquitylated substrates, and this is associated with a reduction of flux through both autophagic and proteasomal degradation pathways. GR20, however, does not have these effects. The effects of PR20 on the proteasome are likely to be direct because (1) PR20 physically associates with proteasomes in biochemical assays, and (2) PR20 inhibits the degradation of a ubiquitylated test substrate when presented to purified proteasomes. Application of a proteasomal activator (IU1) blocks the toxic effects of PR20 on motor neuron survival. This work suggests that proteasomal activators have therapeutic potential in individuals with C9ORF72 HRE.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. A subset of degradation signals recognized by the N-end rule pathway comprises the signals, called N-degrons, whose determinants include destabilizing N-terminal residues. Our previous work identified a family of at least four mammalian E3 ubiquitin ligases, including UBR1 and UBR2, that share the UBR box and recognize N-degrons. These E3 enzymes mediate the multifunctional N-end rule pathway, but their individual roles are just beginning to emerge. Mutations of UBR1 in humans are the cause of Johanson-Blizzard syndrome. UBR1 and UBR2 are 46% identical and appear to be indistinguishable in their recognition of N-degrons. UBR1 ؊/؊ mice are viable but have defects that include pancreatic insufficiency, similarly to UBR1 ؊/؊ human patients with Johanson-Blizzard syndrome. UBR2 ؊/؊ mice are inviable in some strain backgrounds and are defective in male meiosis. To examine functional relationships between UBR1 and UBR2, we constructed mouse strains lacking both of these E3s. We report here that UBR1 ؊/؊ UBR2 ؊/؊ embryos die at midgestation, with defects in neurogenesis and cardiovascular development. These defects included reduced proliferation as well as precocious migration and differentiation of neural progenitor cells. The expression of regulators such as D-type cyclins and Notch1 was also altered in UBR1 ؊/؊ UBR2 ؊/؊ embryos. We conclude that the functions of UBR1 and UBR2 are significantly divergent, in part because of differences in their expression patterns and possibly also because of differences in their recognition of protein substrates that contain degradation signals other than N-degrons. ubiquitylation ͉ proteolysis ͉ N-recognin ͉ UBR box ͉ arginylation

Ubiquitination of histones provides an important mechanism regulating chromatin remodeling and gene expression. Recent studies have revealed ubiquitin ligases involved in histone ubiquitination, yet the responsible enzymes and the function of histone ubiquitination in spermatogenesis remain unclear. We have previously shown that mice lacking the ubiquitin ligase UBR2, one of the recognition E3 components of the N-end rule proteolytic pathway, are infertile associated with meiotic arrest at prophase I. We here show that UBR2 localizes to meiotic chromatin regions, including unsynapsed axial elements linked to chromatin inactivation, and mediates transcriptional silencing via the ubiquitination of histone H2A. UBR2 interacts with the ubiquitin conjugating enzyme HR6B and its substrate H2A and promotes the HR6B-H2A interaction and the HR6B-to-H2A transfer of ubiquitin. UBR2 and ubiquitinated H2A (uH2A) spatiotemporally mark meiotic chromatin regions subject to transcriptional silencing, and UBR2-deficient spermatocytes fail to induce the ubiquitination of H2A during meiosis. UBR2-deficient spermatocytes are profoundly impaired in chromosomewide transcriptional silencing of genes linked to unsynapsed axes of the X and Y chromosomes. Our findings suggest that insufficiency in UBR2-dependent histone ubiquitination triggers a pachytene checkpoint system, providing a new insight into chromatin remodeling and gene expression regulation. A pre-N-degron of a stable protein can be converted into an Ndegron through N-terminal modifications, such as deamidation, oxidation, and arginylation (3-5). UBR1 and UBR2 are really interesting new gene (RING) finger E3s that share size (200 kDa), sequence (46% identity), and enzymatic specificities to type-1 (Arg, Lys, and His; basic) and type-2 (Phe, Leu, Trp, Tyr, and Ile; bulky hydrophobic) destabilizing N-terminal residues (3, 5, 6). Regardless of the similarity in biochemical properties, deficiency of UBR1 and UBR2 causes completely different null phenotypes in mammals. Mutations of human UBR1 cause Johanson-Blizzard syndrome (JBS), an autosomal recessive disorder with multiple developmental abnormalities including exocrine pancreatic insufficiency; knockout of mouse UBR1 also results in metabolic abnormalities such as JBS-like exocrine pancreatic defects (reviewed in ref. 2). UBR2 −∕− male mice are infertile associated with germ cell apoptosis and arrest of spermatocytes at meiotic prophase I, whereas UBR2 −∕− female mice show reduced fertility and partial lethality (5). Besides these functions, the N-end rule pathway plays a role in a variety of processes, including cardiovascular system, DNA repair, neurogenesis, olfaction, sensing oxygen and nitric oxide, G-protein signaling, chromosome segregation, peptide import, selective degradation of misfolded proteins, and apoptosis (reviewed in ref. 2; 3-8).Spermatogenesis involves one of the most dramatic chromatinremodeling processes, including synapsis and transcriptional silencing. During the pachytene stage of meiosis, the X and Y ...

Pentaminomycins C–E (1–3) were isolated from the culture of the Streptomyces sp. GG23 strain from the guts of the mealworm beetle, Tenebrio molitor. The structures of the pentaminomycins were determined to be cyclic pentapeptides containing a modified amino acid, N5-hydroxyarginine, based on 1D and 2D NMR and mass spectroscopic analyses. The absolute configurations of the amino acid residues were assigned using Marfey’s method and bioinformatics analysis of their nonribosomal peptide biosynthetic gene cluster (BGC). Detailed analysis of the BGC enabled us to propose that the structural variations in 1–3 originate from the low specificity of the adenylation domain in the nonribosomal peptide synthetase (NRPS) module 1, and indicate that macrocyclization can be catalyzed noncanonically by penicillin binding protein (PBP)-type TE. Furthermore, pentaminomycins C and D (1 and 2) showed significant autophagy-inducing activities and were cytoprotective against oxidative stress in vitro.