The mammalian N-end rule pathway: new insights into its components and physiological roles

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The mammalian N-end rule pathway: new insights into its components and physiological roles Takafumi Tasaki and Yong Tae Kwon Center for Pharmacogenetics and Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261, USA

The N-end rule pathway is a ubiquitin-dependent proteolytic system, in which destabilizing N-terminal residues of short-lived proteins function as an essential determinant of an N-terminal degradation signal (N-degron). An N-degron can be created from a pre-N-degron through specific N-terminal modifications, providing a means conditionally to destabilize otherwise stable polypeptides. The pathway has been found in all organisms examined, from prokaryotes to eukaryotes. Recent biochemical and proteomic studies identified many components of the mammalian N-end rule pathway, including a family of substrate recognition ubiquitin ligases and their substrates. The genetic dissection in animals and humans revealed its essential role in various vital physiological processes, ranging from cardiovascular development and meiosis to the pathogenesis of human genetic diseases. These discoveries have provided new insights into the components, functions and mechanics of this unique proteolytic system. The N-end rule pathway A substrate of the ubiquitin (Ub) system is ubiquitylated through the action of three enzymes, the Ub-activating enzyme (E1), the Ub-conjugating enzyme (E2) and the Ub ligase (E3), for degradation by the 26S proteasome [1–4]. The selectivity of ubiquitylation is determined mainly by the E3 Ub ligase that recognizes a degradation signal (degron) of the target protein. The N-end rule pathway is an Ub-dependent proteolytic system, in which N-terminal residues of short-lived proteins function as an essential component of degrons called N-degrons [1– 4] (Figure 1). The pathway was originally discovered by the laboratory of Alex Varshavsky, based on an unexpected observation that a stable chimeric protein such as Escherichia coli b-galactosidase, bearing an E. coli Lac repressorderived N-terminal extension, can be destabilized in Saccharomyces cerevisiae cells when its N-terminal methionine (Met) is mutated to other residues [1]. This finding defines a set of N-terminal amino acids as a degradation signal, the first to be identified, of the Ub system. Subsequent genetic screening in S. cerevisiae for proteins involved in N-degron-dependent proteolysis yielded an enzymatic cascade that sequentially modifies Corresponding author: Kwon, Y.T. ([email protected]). Available online 25 October 2007. www.sciencedirect.com

pre-N-degrons and recognizes N-degrons for protein degradation [4]. This proteolytic system was found to have an important role in peptide import and chromosome stability in S. cerevisiae [5–7]. However, S. cerevisiae mutant strains deficient in the pathway are viable, with apparently mild phenotypes, and, as such, its overall importance in physiological processes has been often debated. Many proteins involved in the mammalian N-end rule pathway have recently been identified by a mixture of biochemical and proteomic studies, including a family of Ub ligases and their substrates. Genetic studies on animals and humans deficient in specific components of the pathway have also revealed physiological functions of the pathway in various important processes, including cardiac development, angiogenesis, meiosis and pathogenesis of human genetic diseases. We describe here the current understanding of the mammalian N-end rule pathway, with an emphasis on the components and hierarchical structure, the creation and recognition of N-degrons, and physiological functions. The hierarchical structure, components and substrates of the mammalian N-end rule pathway In the N-end rule pathway, a set of destabilizing N-terminal residues function as an essential determinant of N-degrons through their recognition and binding by N-recognins. A functional N-degron can be created by N-terminal modifications (deamidation, oxidation, and/or arginylation) of a pre-N-degron. The proteolytic system that creates and recognizes N-degrons is present both in prokaryotes and eukaryotes, even though prokaryotes lack the Ub–proteasome system. Conversion of a pre-N-degron to an N-degron through N-terminal modifications In eukaryotes, N-terminal Asn and Gln can function as tertiary destabilizing residues of the N-end rule pathway through deamidation by N-terminal amidohydrolases (Ntamidases) into the secondary destabilizing N-terminal residues Asp and Glu, respectively (Figure 1). In S. cerevisiae, a single enzyme called Nt-amidase (NTA1) deamidates N-terminal Asn and Gln [8]. In contrast to S. cerevisiae, in mammals two distinct enzymes seem to deamidate N-terminal Asn and Gln, respectively, because knockout of NTAN1 (NtN-amidase) selectively abolishes

0968-0004/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2007.08.010

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Figure 1. The hierarchical structure of the mammalian N-end rule pathway. N-terminal residues are indicated by single-letter abbreviations for amino acids. The orange ovals denote the remaining portion of a protein substrate. In the mammalian N-end rule pathway, the tertiary destabilizing N-terminal residues Asn and Gln are deamidated by two distinct enzymes, NTAN1 and a currently hypothetical enzyme termed NTAQ1, into Asp and Glu, respectively [9–11]. NTAN1-deficient mouse fibroblasts lack N-terminal Asn-specific deamidation but fully retain N-terminal Gln-specific deamidation [10]. The identity of NTAQ1, which specifically deamidates N-terminal Gln but not Asn, remains unknown. The secondary destabilizing N-terminal residues Asp and Glu are arginylated by ATE1 R-transferases [14,17,19]. N-terminal Cys is a tertiary destabilizing residue in mammals but not in S. cerevisiae, where it is a stabilizing residue [14–17,19]. The destabilizing activity of N-terminal Cys requires its oxidation before arginylation by ATE1 (see main text). It seems that normally growing S. cerevisiae cells lack N-terminal Cys oxidation activity, although yeast ATE1 has the ability to arginylate N-terminal Cys when expressed in mammalian cells [19]. C* denotes the oxidized N-terminal Cys residue, either CysO2(H) or CysO3(H). In mammals, alternative splicing of the ATE1 gene produces at least six isoforms (ATE1–1 through to ATE1–6); ATE1–1 through to ATE1–4 contain a unique set of regions encoded by two pairs of alternative exons [13,17,18]. The resulting N-terminal Arg, together with other type 1 (blue box) and type 2 (green box) destabilizing N-terminal residues, are directly bound by a set of N-recognins characterized by the UBR (ubiquitin recognition) box [2,3,33–35]. Proteins capable of binding to destabilizing N-terminal residues include UBR1, UBR2, UBR4 and UBR5 [3,33–35]. In mammals, either of two highly similar E2 enzymes, HR6A or HR6B, can be a component of E2–E3 complexes that mediate ubiquitylation of UBR1, UBR2 and UBR3 [33–35]. UBR box proteins can target substrates through recognition of the internal degron (I-degron; yellow box) embedded in the body of the substrate.

the deamidation activity for N-terminal Asn [9–11]. The identity of the hypothetical enzyme termed NTAQ1 (NtQamidase), responsible for deamidation of N-terminal Gln, remains unknown. NTAN1-deficient mice were found to be impaired in spontaneous activity, spatial memory and a socially conditioned exploratory behavior [10]. Drosophila inhibitor of apoptosis 1 (DIAP1), which inhibits apoptosis by binding to and neutralizing active caspases, is the only known substrate of N-terminal deamidation to date [12]. DIAP1 can be cleaved by caspase, creating a C-terminal fragment bearing N-terminal Asn, which is subsequently targeted through the NTAN1–ATE1–UBR pathway (Figure 1). Perturbation of DIAP1 degradation resulted in the failure of DIAP1 to regulate Rpr and Hid-mediated apoptosis [12]. Thus, the NTAN1-dependent DIAP1 degradation by the N-end rule pathway, initiated by a caspase-mediated cleavage of DIAP1, is important for the regulation of apoptosis. In eukaryotes, N-terminal Asp and Glu can function as secondary destabilizing residues through arginylation by ATE1-encoded Arg-tRNA-protein transferase (R-transferase), creating the primary destabilizing residue Arg at the N-terminus [13,14] (Figure 1). N-terminal Cys is stabilizing in S. cerevisiae but destabilizing in mammals through its serial modifications, including ATE1-dependent www.sciencedirect.com

arginylation [14–16]. N-terminal arginylation, a universal eukaryotic protein modification, requires Arg from ArgtRNAArg of the protein synthesis machinery and thereby defines a unique tRNA-dependent Ub proteolytic system [4]. In mammals, alternative splicing of ATE1 produces at least six isoforms with differential cellular localization and tissue distribution and also enzymatic activities for N-terminal Asp, Glu and Cys [14,17,18]. ATE1 seems to be solely responsible for N-terminal arginylation [19] because ATE1-knockout mice are deficient in all known N-terminal arginylation activities. Recent genetic studies identified physiological functions of N-terminal arginylation in the control of cardiac development and angiogenesis in mammals, apoptosis in the fly Drosophila melanogaster and senescence in the plant Arabidopsis thaliana [12,19–21]. It has been demonstrated that a set of mammalian regulator of G protein signaling (RGS) proteins (RGS4, RGS5 and RGS16) are ubiquitylated through N-terminal arginylation [15–17,22]. These structurally related RGS proteins, which function as GTPase-activating proteins (GAPs) for Ga subunits [23], commonly bear an N-terminal Met-Cys sequence. The degradation of these RGS proteins is initiated by cleavage of N-terminal Met by Met-aminopeptidases (MetAPs), exposing Cys-2 at the N-terminus. Remarkably, the N-terminally exposed

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Cys-2 was found to be oxidized into Cys-sulfinic acid [CysO2(H)] or Cys-sulfonic acid [CysO3(H)] before arginylation by ATE1, and the arginylation was demonstrated to require nitric oxide (NO) in addition to oxygen (O2) or its derivatives [15,16,19] (Figure 1). Cys-sulfinic acid is structurally similar to Asp, one of the previously known N-terminal residues that can be arginylated by ATE1, suggesting that the oxidation of N-terminal Cys is a specific modification to create a secondary destabilizing residue. ATE1 conjugates Arg to the N-terminus not only for Ub-dependent proteolysis, but also for non-proteolytic processes. N-terminal Met (and sometimes the second residue) of b-actin can be removed, exposing Asp-2 or Asp-3 at the N-terminus [24]. Notably, recent evidence suggests that 40% of b-actin in mouse embryonic fibroblasts is N-terminally arginylated by ATE1 without b-actin proteolysis, a process that regulates actin filament properties, b-actin localization and lamella formation in moving cells [25]. In addition to b-actin, several other proteins, including calreticulin, neurotensin and ornithine decarboxylase, have been reported to be arginylated at the N-terminus [25–29]. It has been found that amino acids can be post-translationally incorporated into

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proteins in bacteria and eukaryotes [30–32]. Therefore, the N-terminal arginylation branch of the N-end rule pathway might be part of this global post-translational modification, whose physiological meaning remains poorly understood. UBR box proteins as recognition components of the N-end rule pathway N-terminal degradation determinants of the N-end rule pathway are divided into type 1 (basic; Arg, Lys and His) and type 2 (bulky hydrophobic; Phe, Tyr, Trp, Leu and Ile) destabilizing residues. These N-terminal residues are recognized and bound by two cognate and mutually exclusive binding sites of specific Ub ligases called N-recognins [2–4] (Figure 1). In S. cerevisiae, a single N-recognin called UBR1 is solely responsible for ubiquitylation of substrates bearing destabilizing N-terminal residues [4]. Likewise, mammalian UBR1 (also known as E3a), with a size of 200 kDa, has long been considered as the sole N-recognin [2,33,34]. However, current evidence indicates that ubiquitylation in the N-end rule pathway is mediated by multiple N-recognins that contain a 70-residue zinc-fingerlike domain termed the UBR box [3,34] (Figure 2). The

Figure 2. The UBR box motif defines the E3 Ub ligase recognition components of the N-end rule pathway. (a) Schematic diagram of the mammalian UBR box protein family, showing the signature domains of substrate recognition proteins in the Ub system, including ubiquitylation domains (RING, HECT and PHD) or E3-substrate adaptor domain (F-box). Abbreviations: AI (in orange), autoinhibitory domain; CRD (in yellow), cysteine-rich domain; F-box (in green), F-box domain; HECT (in violet), HECT domain; PHD (in gray), plant homeodomain finger; RING (in turquoise), RING finger; UBR (in red), UBR box. (b) Sequence alignment of the UBR boxes from four species. Shown are the 70 amino acid regions where conserved Cys and His residues are highlighted (cyan). The Cys residue of Arabidopsis UBR4 (also called BIG) whose missense mutation perturbs auxin transport [67], is highlighted in orange. Note that different organisms contain different sets of UBR box proteins, hence the absence of, for example, UBR2 from D. melanogaster. Abbreviations: a, A. thaliana; d, D. melanogaster; m, Mus musculus; sc, S. cerevisiae. www.sciencedirect.com

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mammalian genome encodes at least seven UBR box-containing proteins, named UBR1 through to UBR7, and different organisms contain different sets of UBR box proteins [3]. UBR box proteins are generally heterogeneous in size and sequence but contain, with the exception of UBR4, specific signatures unique to E3 Ub ligases or a substrate recognition subunit of the E3 complex: the RING domain in UBR1, UBR2 and UBR3; the HECT domain in UBR5; the F-box in UBR6 and the plant homeodomain (PHD) finger in UBR7 [3] (Figure 2). The ability of UBR1, UBR2, UBR4 and UBR5 to bind to destabilizing N-terminal amino acids in proteins or short peptides defines them as N-recognins, whereas the biochemical properties and enzymatic specificities of UBR3, UBR6 and UBR7 as candidate N-recognins remain unclear [3,34,35]. It is currently unknown whether there is a common substrate recognition domain that specifically recognizes type 1 and type 2 destabilizing N-terminal residues. Given that the UBR box is the only domain conserved in all known N-recognins [3], it remains to be tested whether the UBR box is a general substrate recognition domain of the N-end rule pathway. Characterization of knockout mice demonstrated that mammalian UBR1 and UBR2 are functionally overlapping N-recognins whose biochemical properties are similar to each other and also to yeast UBR1 [2,33,36]. UBR1deficient mice are viable but they display various metabolic phenotypes, ranging from exocrine pancreatic abnormalities [reminiscent of Johanson–Blizzard syndrome (JBS) in human patients] [37] to hypoglycemia and altered fat metabolism [33]. The mutants also exhibit impairment in Ub-dependent proteolysis of endogenous skeletal muscle proteins [33]. Similarly, pharmacological dipeptide inhibitors of the N-end rule pathway inhibit Ub conjugation in skeletal muscle extracts from rats in normal and pathological (tumor, septic or diabetic) conditions [38–40]. These lines of evidence suggest that the N-end rule pathway, together with other Ub machinery, such as the muscle RING finger-1 (MuRF-1) E3 Ub ligase and the muscle atrophy F-box protein [MAFbx; a component of the Skp1–cullin–F-box (SCF) complex] [41], has a role in muscle atrophy, a pathophysiological condition in cancer. UBR2-deficient mice exhibit phenotypes (male-specific infertility and female-specific lethality) that are distinct from those of UBR1-lacking mice [34]. Mouse embryos lacking both UBR1 and UBR2 die at midgestation, indicating a functional interaction between these two E3s during mammalian development [42]. In addition, embryos deficient in both UBR1 and UBR2 suffer from impaired neurogenesis during mouse development [42]: in the forebrain of embryos deficient in both UBR1 and UBR2, the balance between proliferation and differentiation of neural stem cells shifted toward reduced proliferation (a stem cell-like self-renewal) and accelerated differentiation. One possible circuit underlying UBR1- and UBR2-dependent neurogenesis is the Notch signaling pathway [42]. Embryos deficient in both UBR1 and UBR2 also exhibit cardiovascular defects, which remain to be further characterized [42]. UBR1 and UBR2 substrates that are targeted through N-degrons include mammalian RGS proteins, the Separase-cleaved yeast SCC1 (a cohesin complex subunit) www.sciencedirect.com

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and the caspase-cleaved DIAP1 [6,12,15,16]. In addition, UBR1 can target substrates through the internal degron (I-degron) embedded in the body of the substrate. This class of UBR1 substrates includes S. cerevisiae CUP9 (a transcriptional repressor of the peptide transporter PTR2), S. cerevisiae GPA1 (a Ga subunit that controls signal transduction during mating) and mammalian c-Fos [a component of the activator protein-1 (AP-1) transcription factor] [7,43–45] (Box 1). In addition to the ability to recognize N-degrons and I-degrons, N-recognins might have other biological functions outside of the Nterminus-dependent proteolysis, at least in part accounting for various phenotypes observed in mutants of components involved in the N-end rule pathway. Sequence similarity, functional domains and specificities to E2 Ub-conjugating enzymes indicate that UBR1, UBR2 and UBR3 can be classified as the RING domain–UBR subfamily [35]. Conserved domains in these

Box 1. Other physiological substrates of the N-end rule pathway In addition to the N-end rule substrates described in the main text, several other proteins are known targets of the N-end rule pathway. The functions of the mammalian transcription factor c-Fos, a component of the AP-1 transcription complex, are tightly controlled by phosphorylation and ubiquitylation. UBR1 is transcriptionally induced by signal transducer and activation of transcription (STAT) 3 and subsequently ubiquitylates c-Fos in the cytoplasm for degradation through recognition of an I-degron [44]. UBR1-dependent c-Fos ubiquitylation is negatively regulated through extracellular-signal-regulated kinase (ERK) 5-dependent phosphorylation of two c-Fos residues: Thr-232 in the nuclear export signal (to inhibit the nuclear export of c-Fos) and Ser32 (to disrupt the c-Fos–UBR1 interaction). The S. cerevisiae homeodomain protein CUP9 inhibits the import of extracellular dipeptides and tripeptides through transcriptional repression of the transporter PTR2. The function of CUP9 in PTR2-dependent peptide import is regulated by its UBR1dependent proteolysis through recognition of an I-degron [7,45]. UBR1-dependent CUP9 degradation is allosterically activated by small peptides bearing destabilizing N-terminal residues, a positive feedback loop in which imported peptides bind to UBR1 and accelerate UBR1-dependent CUP9 proteolysis, leading to accelerated peptide import [46]. Thus, peptide import in S. cerevisiae is tightly controlled by CUP9 proteolysis through the allosteric activation of UBR1 by small peptides. S. cerevisiae GPA1, a Ga subunit of the heterotrimeric G protein, regulates signal transduction and cell differentiation in response to mating pheromones. The level of GPA1 is, at least in part, controlled by UBR1-dependent ubiquitylation, and the enhanced degradation of GPA1 by UBR1 overexpression inhibits the growth of haploid but not diploid cells [43]. The N-end rule pathway targets viral proteins, including Sindbis RNA polymerase nsP4 and HIV-1 integrase [3,55]. The nsP4 polymerase is produced by an endoproteolytic cleavage of the precursor polyprotein nsP1234, whereas HIV-1 integrase is produced by cleavage of the 160 kDa Gag-Pol precursor and subsequently catalyzes the irreversible insertion of viral genome into the host chromosome [55,69]. The nsP4 and integrase proteins, bearing type 2 N-terminal residues Tyr and Phe, respectively, are degraded in mammalian cells in a manner dependent upon type 2 N-degrons [3,55]. Notably, all of the examined lentiviral integrases (e.g. HIV-1 integrase) and alphaviral RNA polymerases (e.g. Sindbis polymerase) were found to bear type 2 N-terminal residues [3], indicating that other viral proteins of these groups might also be targeted by the pathway. Thus, the virus might use the N-end rule pathway as a host proteolytic system that effectively eliminates the free, unincorporated viral proteins bearing N-degrons.

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Ub ligases include the autoinhibitory domain unique to mammalian proteins. In S. cerevisiae, type 1 and type 2 dipeptides allosterically activate UBR1 and subsequently accelerate the degradation of CUP9 bearing an I-degron [7,45], a positive feedback loop essential for the homeostasis of peptide import. The N-terminal region of UBR1, containing substrate-binding sites, is normally closed through intramolecular interaction with the C-terminal autoinhibitory domain. The binding of type 1 and type 2 dipeptides to UBR1 disrupts this inhibitory interaction and prompts UBR1-dependent CUP9 degradation and subsequent uptake of extracellular peptides [46]. Thus, it seems that one function of the RING UBR subfamily is the ability to be controlled by small molecule modulators (e.g. dipeptides or structurally similar molecules). The RING–UBR subfamily forms an E2–E3 complex with either of the Ub-conjugating enzymes HR6A and HR6B [33–35]. HR6A and HR6B, sharing 95% identity each other, are functional homologs of S. cerevisiae UBC2 (also called RAD6). The yeast RAD6 has been implicated not only in N-end rule-dependent protein degradation, but also in non-N-end rule dependent processes, including postreplication repair, damage-induced mutagenesis, sporulation and modulating chromatin structure through histone ubiquitylation [47]. In contrast to UBR1 and UBR2, UBR3 does not bind to known destabilizing N-terminal residues, indicating that this 213 kDa-protein is not an N-recognin [35] and instead has some other function. It was recently found that UBR3-deficient mice are impaired in olfaction, and UBR3 is prominently expressed in sensory cells crucial for the five major senses (smell, touch, vision, hearing and taste) [35], suggesting that UBR3 controls a general circuit underlying different sensory nervous systems. Mice deficient in both UBR1 and UBR2 retain residual activities for the N-end rule pathway [3]. Recent affinitybased proteomic screening revealed additional N-recognins, termed UBR4 and UBR5, which are unrelated to UBR1 and UBR2 in sequence and size [3]. Mammalian UBR4, an extraordinarily large protein with a size of 570 kDa, binds to both type 1 and type 2 residues of synthetic peptides, whereas mammalian and insect UBR5, with a size of 300 kDa, preferentially bind to type 1 residues [3]. Cells deficient in UBR1, UBR2 and UBR4 are almost completely impaired in the type 2 pathway but not in the type 1 pathway [3]. Biochemical properties and physiological functions of UBR4 and UBR5 as N-recognins are to be further investigated; known functions and interacting proteins of UBR4 and UBR5 are summarized in Box 2. The 90 kDa-UBR6 protein is an F-box protein, which is usually a component of the SCF-type E3 complex. F-box proteins contain an F-box motif that binds SKP1 (suppressor of kinetochore protein 1) for assembly into the SKP1– CUL1 (cullin-1) complex and a C-terminal domain that recognizes substrates [48]. UBR7 is a 50-kDa protein containing the PHD finger, a conserved zinc finger that resembles the RING finger domain and therefore is thought to be the functional domain for E3 Ub ligase [49]. The biochemical properties of UBR6 and UBR7 as candidate N-recognins remain unclear. www.sciencedirect.com

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Box 2. UBR4 and UBR5: emerging N-recognins Mammalian UBR4 (also termed p600 in the literature), with a size of 570 kDa, interacts with the human papillomavirus type 16 (HPV-16) E7 oncoprotein, in addition to the retinoblastoma tumor suppressor protein pRB [70,71]. HPV E7 underlies the pathogenesis of cervical cancer, in part through its ability to bind to pRB and the p107 and p130 pocket proteins, leading to the release of the E2F transcription factor and the initiation of S phase progression [72]. It has been proposed that UBR4 has a role in anchorage-independent growth and cellular transformation in cancer cells [70,71], in addition to integrin-mediated ruffled membrane formation and integrinmediated survival pathways [73]. In Arabidopsis, auxin regulates vital physiological processes, including vascular differentiation, organogenesis, tropic growth, and root and shoot architecture. Mutations in the Arabidopsis UBR4 (also called BIG) gene were identified in two independent mutants with altered expression of light-regulated genes and reduced auxin transport [67], suggesting that UBR4 is a crucial player in auxin signaling. Originally identified as a calmodulin-binding protein, Drosophila PUSHOVER (i.e. the equivalent of UBR4 in mammals) has been implicated in male reproduction, non-recombinant chromosome segregation in female meiosis, synaptic transmission in photoreceptor cells and perineurial glial growth [74,75]. Thus, it seems that UBR4 regulates signaling pathways in response to extracellular signals (e.g. hormones and neurotransmitters). Mammalian UBR5 (also known as EDD, or E3 identified by differential display), with a size of 300 kDa, is a HECT domain E3 Ub ligase, primarily localized in the nucleus [76]. UBR5 has been implicated in progesterone-regulated cell proliferation, DNA damage responses and tumorigenesis [76,77]. UBR5 interacts with a diverse subset of proteins, including the progesterone receptor (to potentiate progestin-mediated gene transactivation), importin a5 (a component of the nuclear import complex), calcium and integrin binding protein (which associates with integrin, the DNA-dependent protein kinase and the polo-like kinases) and DNA topoisomerase IIb-binding protein 1 (TopBP1) [76–78]. TopBP1 is normally degraded by UBR5, stabilized in response to DNA damage through its phosphorylation and subsequently recruited to DNA damage sites together with g-H2AX (phosphorylated histone H2AX) [78]. In Drosophila, mutations in the UBR5/HYD gene result in imaginal disc hyperplasia associated with uncontrolled cell proliferation [79]. UBR5-deficient mouse embryos die during embryogenesis, as a result of impairments in yolk sac vascular development and chorioallantoic fusion, which were attributed to abnormal cell proliferation in extra-embryonic tissues [80].

Creation of the N-degron An N-degron is composed of a destabilizing N-terminal residue, an internal Lys residue (the site of poly-Ub chain formation) and a characteristic conformational feature that promotes ubiquitylation and degradation [4,50,51]. Because newly synthesized proteins bear a stabilizing residue (i.e. the N-terminal Met in eukaryotes and the formyl-methionine in prokaryotes), N-degrons must be created by post-translational modification of the proteins. In eukaryotes, a common feature in creating the N-degron, and thereby destabilizing a normally long-lived polypeptide, is the N-terminal exposure of an internal residue through either the removal of N-terminal Met or an endoproteolytic cleavage (Figure 3). The first way to create destabilizing N-terminal residues is through removal of the N-terminal Met, which exposes the second residue at the N-terminus. Removal of the N-terminal Met by mammalian MetAPs occurs when the second residue is either Val, Gly, Pro, Ala, Ser, Thr or Cys [16,52] (Figure 3). Both in eukaryotes and prokaryotes, MetAPs cannot create primary destabilizing N-terminal

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rule substrates could be because this type of N-degron is conditionally created following the cleavage of a precursor protein, which, in turn, depends on the activation of a specific endopeptidase (e.g. caspase or separase).

Figure 3. Creation of destabilizing N-terminal residues. All host cell proteins in mammals are synthesized with a Met residue at the N-terminus. Two different mechanisms can lead to the generation of destabilizing N-terminal residues, as indicated here. (a) In the first mechanism, the removal of N-terminal Met by MetAPs can expose the second residue at the N-terminus. Mammalian MetAPs remove N-terminal Met when the second residue is Val, Gly, Pro, Ala, Ser, Thr or Cys. Among these N-terminal residues, Val, Gly, Pro, Ala, Ser and Thr can be classified as stabilizing, whereas Cys is a tertiary destabilizing residue. Based on recent biochemical analyses, Ala, Ser and Thr (indicated by asterisks), which were originally classified as type 3 destabilizing residues [53,54], have now been reclassified as stabilizing [3,35]. (b) In the second mechanism, the endoproteolytic cleavage (e.g. through the action of caspases, separases or calpains) of a protein can create a C-terminal fragment bearing a tertiary or secondary destabilizing N-terminal residue (i.e. Asn, Gln, Cys, Asp or Glu in mammals) or a primary destabilizing residue (i.e. Arg, Lys, His, Leu, Phe, Trp, Tyr or Ile in mammals).

residues. Among the above residues, Val, Gly and Pro are known to be stabilizing residues in mammals. By contrast, Ala, Ser and Thr have been classified as type 3 destabilizing residues that can be targeted by a specific Ub ligase whose molecular identity has remained elusive [53,54]. However, based on biochemical analyses, Ala, Ser and Thr were recently reclassified to be stabilizing [3,35]. Thus, in mammals, the removal of N-terminal Met can create N-degrons only when the second residue is Cys, as exemplified by certain RGS proteins that are destabilized by the exposure of Cys-2 at the N-terminus [15,16]. By searching the International Protein Index (Version 3.32) with a program developed in our laboratory and written in the Python language, we found that mouse genome encodes 500 proteins bearing an N-terminal Met-Cys sequence. Although this unpublished result needs to be verified by experimentation, it will be interesting to see which of these proteins produce N-degrons after exposing Cys-2 at the N-terminus. Another way to create an N-degron is through endoproteolytic cleavage of a long-lived polypeptide, yielding a short-lived C-terminal fragment of the protein which consequently bears a destabilizing N-terminal residue (Figure 3). Intracellular endopeptidases (e.g. caspases, separases and calpains) can create a C-terminal fragment bearing a tertiary or secondary destabilizing N-terminal residue (Asn, Gln, Cys, Asp or Glu in mammals) or a primary destabilizing residue (Arg, Lys, His, Leu, Phe, Trp, Tyr or Ile in mammals). Although, in theory, endoproteolysis would produce a vast number of substrates, the only substrates known to be created through this mechanism are the yeast SCC1, the fly DIAP1 and a few viral substrates [3,6,12,55] (Box 1). The scarcity of known N-end www.sciencedirect.com

Physiological functions of proteins involved in the mammalian N-end rule pathway The genetic dissection in S. cerevisiae demonstrated that the N-end rule pathway regulates the import of peptides through ubiquitylation of CUP9, a transcriptional repressor of the peptide transporter PTR2 [7,45] (Box 1). It was also discovered that the pathway has an essential role in chromosome stability through ubiquitylation of the separase-produced SCC1 fragment bearing N-terminal Arg [6]. These findings suggest that the N-end rule pathway is important for certain physiological processes. However, S. cerevisiae N-end rule pathway mutants were found to be all viable [4], and, as such, the biochemical and physiological importance of the N-terminus in Ub-dependent proteolysis has been often debated [56]. Recent identification of mammalian components and construction of knockout mice revealed functions of this proteolytic pathway in many important cellular processes, some of which directly or potentially participate in the pathogenesis of human diseases. Cardiac signaling and development Although N-terminal arginylation was initially characterized nearly half a century ago [57], the physiological functions of this universal eukaryotic protein modification remained unclear until ATE1-deficient mouse embryos were found to die at midgestation, associated with defects in cardiac development and angiogenesis [19]. ATE1 cardiac phenotypes include myocardial hypoplasia, ventricular septal defect (VSD, a defect characterized by a blood flow between the septum and endocardial cushion) and persistent truncus arteriosus (an incomplete septation of outflow tracts), whereas vascular phenotypes include impaired angiogenic processes such as the growth, remodeling and branching of both small and large vessels [19]. These results suggest that ATE1-dependent protein arginylation has an important role in cardiovascular development and homeostasis. UBR1 and UBR2 are functionally overlapping downstream Ub ligases of ATE1dependent arginylation [2,33]. Whereas UBR1 / and UBR2 / mice do not exhibit cardiovascular impairment [33,34], embryos lacking both UBR1 and UBR2 die at midgestation with defects in cardiac development and angiogenesis [42]. It remains to be determined whether cardiovascular phenotypes of these two mutant strains are related with the same substrates. A recent proteomic approach identified RGS4, RGS5 and RGS16 as substrates of of the ATE1–UBR1/UBR2 proteolytic pathway [15,16] (Figure 4). The negative regulation of Gq/Gi-activated signaling by these RGS proteins is crucial for the control of myocardial growth and vascular maturation and integrity. Several lines of evidence indicate that RGS4 and RGS5 have a crucial role in Gq-dependent proliferation and signaling in cardiomyocytes and vascular smooth muscle cells, respectively [58–60]. Further, the proteolysis of these RGS proteins requires not only ATE1, but also NO and O2 (or its derivatives) [15,16] (Figure 4), indicating

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X chromosome-encoded HR6A gene exhibit female-specific infertility [61,62]. It remains unclear whether these knockout phenotypes are functionally related. In S. cerevisiae, the cleavage of the cohesin component SCC1 by separase is required to initiate sister chromatid separation at the metaphase-to-anaphase transition during mitosis [63]; the resulting SCC1 fragment bearing an N-terminal Arg is degraded by UBR1, a process essential for chromosome stability [6]. Analogous processes in humans and mice produce an SCC1 fragment with N-terminal Glu that can function as a secondary destabilizing residue through ATE1-dependent arginylation [64]. Given that SCC1 has a role in meiosis in addition to mitosis [65], it will be interesting to determine whether mammalian SCC1 is targeted by UBR2 in meiotic spermatocytes and by UBR1 in mitotic spermatocytes. Remarkably, ATE1 was found to be strongly expressed in meiotic spermatocytes in adult testes in a pattern almost identical to that of UBR2 [34], suggesting that ATE1 has a role in spermatogenesis, possibly associated with UBR2 function.

Figure 4. The proteolysis of RGS proteins and its role in cardiovascular signaling and homeostasis. The cellular concentrations of RGS4, RGS5 and RGS16 (in yellow) are tightly controlled by the N-end rule pathway [15,16,22]. Proteolysis of these RGS proteins starts as a result of the removal of the N-terminal Met by MetAPs, exposing Cys-2 at the N-terminus. The exposed N-terminal Cys-2 seems to be oxidized into CysO2(H) or CysO3(H), which requires NO as well as O2 or its derivatives [15,16,22] (Figure 1). The oxidized Cys-2 residue is conjugated with Arg by ATE1 R-transferase. N-terminally arginylated RGS proteins are ubiquitylated by UBR1 and UBR2 for degradation, perhaps with the help of the E2 enzymes HR6A and/or HR6B. RGS proteins are GAPs for Ga subunits and negatively regulate the GPCR (in blue) signaling [23]. Following activation by an agonist-occupied receptor, the heterotrimeric Gq protein dissociates into individual Gaq (in green) and Gbg (in gray) subunits. GTP-bound Gaq activates phospholipase C (PLC), resulting in inositol trisphosphate-mediated calcium release and diacylglycerolmediated activation of protein kinase C (PKC). Dissociated Gbg has the potential to activate the small GTP-binding protein Ras and initiate a tyrosine kinase cascade, leading to activation of mitogen-activated protein kinases (MAPKs). Gaq can also activate MAPKs, independently of Gbg, through a PKC-dependent mechanism. RGS4 and RGS5 have been implicated as negative regulators of Gq-dependent signaling in the heart and blood vessels, respectively, whereas RGS16 has been shown to control the chemokine-stimulated signaling and migration of megakaryocytes [58–60,68].

that the ATE1 pathway might be a sensor for NO and O2 in cardiovascular signaling and homeostasis. Thus, one function of the N-end rule pathway is the proteolysis of RGS4, RGS5 and RGS16 through the pathway involving NO, O2, ATE1, UBR1 and UBR2, which seems to regulate Gq/Gidependent signaling in hearts and blood vessels (Figure 4). Meiosis, mitosis and other chromosomal processes UBR2-deficient mice exhibit male-specific infertility and female-specific lethality [34]. Spermatocytes lacking UBR2 are arrested at the transition between the leptotene–zygotene and pachytene stages associated with defects in homologous chromosome pairing during meiotic prophase I; arrested spermatocytes undergo apoptosis, resulting in vacuolized tubules and testes degeneration [34]. UBR2 functions with the E2 enzymes HR6A and HR6B to execute the N-end rule pathway [34]. Mice lacking HR6B also show male-specific infertility associated with defects in meiosis and spermiogenesis, whereas mice lacking the www.sciencedirect.com

JBS JBS (OMIM 243800) is an autosomal recessive disorder that encompasses congenital exocrine pancreatic insufficiency, multiple physical malformations (e.g. scalp defects, imperforate anus, deafness and hypothyroidism) and mental retardation [66]. Mutations, mostly truncations, in the UBR1 gene, were recently identified in 12 unrelated families with JBS [37]. Whereas a high level of UBR1 protein was detected in acinar cells in normal pancreatic tissues, no UBR1 protein was detected in the pancreas of JBS patients [37]. Notably, UBR1-deficient mice exhibit a mild version of the symptoms of JBS in human patients, including pancreatic exocrine insufficiency [37]. Zymogens, such as trypsinogen and proelastase, are normally synthesized but fail to be secreted properly in the mutant animals. In addition, UBR1-deficient mice exhibit hypersensitivity to pancreatitis induced by a high concentration of secretagogue [37]. Thus, UBR1 is an important regulator in the secretion of pancreatic enzymes. Concluding remarks and future perspectives Biochemical and genetic studies over the past two decades have revealed most of the essential components of the N-end rule pathway, both in S. cerevisiae and in mammals, and the hierarchical structure and mechanistic details of the pathway are now fairly well understood. The characterization of mutants lacking specific N-end rule components, both in S. cerevisiae and in mammals, led to the finding that this unique proteolytic system is crucial in a variety of physiological processes, ranging from peptide import and chromosome stability in yeast to the cardiovascular system and meiosis in mammals. Despite these advances, several questions remain unanswered. Additional N-end rule substrates are yet to be identified, and better identification methods are needed. The list of physiological functions of the N-end rule pathway is far from complete, and the exact molecular mechanisms underlying known phenotypes of the mutant animals of the pathway are yet to be understood. The continuing biochemical and genetic dissections of the mammalian

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pathway are needed to address these questions. It is also becoming increasingly necessary to identify or synthesize efficient small-molecule inhibitors, which might some day be used to treat pathophysiological conditions that are regulated by the N-end rule pathway. Acknowledgements We thank members of the Kwon laboratory for helpful reading of the manuscript. This work was supported by the NIH grants (GM69482, GM074000 and HL083365) and the American Heart Association grant to Y.T.K..

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