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APE1 (Ref-1) is an essential human protein involved in DNA damage repair and regulation of transcription. Although the cellular functions and biochemical properties of APE1 are well characterized, the mechanism involved in regulation of the cellular levels of this important DNA repair/transcriptional regulation enzyme, remains poorly understood. Using an in vitro ubiquitylation assay, we have now purified the human E3 ubiquitin ligase UBR3 as a major activity that polyubiquitylates APE1 at multiple lysine residues clustered on the N-terminal tail. We further show that a knockout of the Ubr3 gene in mouse embryonic fibroblasts leads to an up-regulation of the cellular levels of APE1 protein and subsequent genomic instability. These data propose an important role for UBR3 in the control of the steady state levels of APE1 and consequently error free DNA repair.

We previously reported two modes of development of acquired TRAIL resistance: early phase and late phase [1]. In these studies, we observed that greater Akt activity and the expression of Bcl-xL were related mainly to the late phase of acquired TRAIL resistance. Recently we became aware of a possible mechanism of early phase TRAIL resistance development through internalization and degradation of TRAIL receptors (DR4 and DR5). Our current studies demonstrate that TRAIL receptors rapidly diminish at the membrane as well as the cytoplasm within 4h after TRAIL exposure, but recover completely after one or two days. Our studies also reveal that Cbl, a ubiquitously expressed cytoplasmic adaptor protein, is responsible for the rapid degradation of TRAIL receptors; Cbl binds to them and induces monoubiquitination of these receptors concurrent with their degeneration soon after TRAIL exposure, creating the early phase of acquired TRAIL resistance.

Intracellular signaling is often mediated by a family of functionally overlapping signal mediators that contain multiple sites interacting with other proteins or ligands with weak affinity (K(d)microM). Conjugation of multiple low-affinity ligands into a high-affinity multivalent molecule provides a means to control the entire protein family within a single intracellular pathway. The N-end rule pathway is a ubiquitin (Ub)-dependent proteolytic system where at least four Ub ligases, called N-recognins, have a common domain critical for binding to type 1 (basic) and type 2 (bulky hydrophobic) destabilizing N-terminal residues of substrates as degrons. The recent development of a heterodivalent inhibitor targeting type 1 and type 2 substrate binding sites of the N-recognin family provides new opportunities to manipulate this proteolytic pathway in biochemical and pathophysiological conditions. We overview the N-end rule pathway as an intracellular target for heterodivalent molecules and discuss the basis of thermodynamics and kinetics related to heterodivalent interactions.

Her-2/neu (ErbB2) is a transmembrane tyrosine kinase and acts as a co-receptor for the other EGFR family members. It is well known that high expression of Her-2/neu is associated with a poor prognosis in breast cancer. Quercetin, a flavonoid present in many vegetables and fruits, has been studied extensively as a chemoprevention agent in several cancer models. In this study, we observed that quercetin decreased the level of Her-2/neu protein in time- and dose-dependent manners and also inhibited the downstream survival PI3K-Akt signaling pathway in Her-2/neu-overexpressing breast cancer SK-Br3 cells. We also observed that quercetin induced polyubiquitination of Her-2/neu. When the proteasome pathway was blocked by MG-132 during quercetin treatment, accumulation of the NP-40 insoluble form of Her-2/neu occurred. Interestingly, data from immunocomplex studies revealed that quercetin promoted interaction between Her-2/neu and Hsp90 which is a molecular chaperone involved in stabilization of Her-2/neu. In this condition, inhibition of Hsp90 activity by a specific inhibitor, geldanamycin (GA), or intracellular ATP depletion caused dissociation of Hsp90 from Her-2/neu and promoted ubiquitination and down-regulation of Her-2/neu protein. In addition, the carboxyl terminus of Hsc70-interacting protein (CHIP), a chaperone-dependent E3 ubiquitin ligase, played a crucial role in the quercetin-induced ubiquitination of Her-2/neu. Inhibition of tyrosine kinase activity of Her-2/neu by quercetin could indicate an lateration in the Her-2/neu structure which promotes CHIP recruitments and down-regulation of Her-2/neu. We believe that by using quercetin, new therapeutic strategies can be developed to treat Her-2/neu overexpressing cancers.

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.

Previous studies have shown that repeated application of TRAIL induces acquired resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Using human prostate adenocarcinoma DU-145 and human pancreatic carcinoma MiaPaCa-2 cells as a model, we now demonstrate for the first time that two states of acquired TRAIL resistance can be developed after TRAIL treatment. Data from survival assay and Western blot analysis show that acquired TRAIL resistance was developed within 1 day and gradually decayed within 6 days after TRAIL treatment in both cell lines. After TRAIL treatment, the level of Bcl-xL increased and reached a maximum within 2 days and gradually decreased in both cell lines. Bcl-xL-mediated development of acquired TRAIL resistance was suppressed by knockdown of Bcl-xL expression. Protein interaction assay revealed that during the development of TRAIL resistance, Bcl-xL dissociated from Bad and then associated with Bax. Overexpression of mutant-type Bad (S136A), which prevents this dissociation, partially suppressed the development of acquired TRAIL resistance. Thus, our results suggest that (a) dissociation of Bad from Bcl-xL and (b) an increase in the intracellular level of Bcl-xL are responsible for development of acquired TRAIL resistance.

The ubiquitin-proteasome system and the autophagy lysosome system are the two major protein degradation machineries in eukaryotic cells. These two systems coordinate the removal of unwanted intracellular materials, but the mechanism by which they achieve this synchronization is largely unknown. The ubiquitination of substrates serves as a universal degradation signal for both systems. Our study revealed that the amino-terminal Arg, a canonical N-degron in the ubiquitin-proteasome system, also acts as a degradation signal in autophagy. We showed that many ER residents, such as BiP, contain evolutionally conserved arginylation permissive pro-N-degrons, and that certain inducers like dsDNA or proteasome inhibitors cause their translocation into the cytoplasm where they bind misfolded proteins and undergo amino-terminal arginylation by arginyl transferase 1 (ATE1). The amino-terminal Arg of BiP binds p62, which triggers p62 oligomerization and enhances p62-LC3 interaction, thereby stimulating autophagic delivery and degradation of misfolded proteins, promoting cell survival. This study reveals a novel ubiquitin-independent mechanism for the selective autophagy pathway, and provides an insight into how these two major protein degradation pathways communicate in cells to dispose the unwanted proteins. [BMB Reports 2015; 48(9): 487-488]

Cancer stem cells (CSCs) are capable of initiation and metastasis of tumors. Therefore, understanding the biology of CSCs and the interaction between CSCs and their counterpart non-stem cells is crucial for developing a novel cancer therapy. We used CSC-like and non-stem breast cancer MDA-MB-231 and MDA-MB-453 cells to investigate mammosphere formation. We investigated the role of the epithelial cadherin (E-cadherin)-extracellular signal-regulated kinase (Erk) axis in anoikis. Data from E-cadherin small hairpin RNA assay and mitogen-activated protein kinase kinase (MEK) inhibitor study show that activation of Erk, but not modulation of E-cadherin level, may play an important role in anoikis resistance. Next, the two cell subtypes were mixed and the interaction between them during mammosphere culture and xenograft tumor formation was investigated. Unlike CSC-like cells, increased secretion of interleukin-6 (IL-6) and growth-related oncogene (Gro) chemokines was detected during mammosphere culture in non-stem cells. Similar results were observed in mixed cells. Interestingly, CSC-like cells protected non-stem cells from anoikis and promoted tumor growth. Our results suggest bystander effects between CSC-like cells and non-stem cells. J. Cell. Biochem. 117: 2289-2301, 2016. © 2016 Wiley Periodicals, Inc.

It is well known that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis can be initially triggered by surface death receptors (the extrinsic pathway) and subsequently amplified through mitochondrial dysfunction (the intrinsic pathway). However, little is known about signaling pathways activated by the TRAIL-induced endoplasmic reticulum (ER) stress response. In this study, we report that TRAIL-induced apoptosis is associated with the endoplasmic reticulum (ER) stress response. Human colorectal carcinoma HCT116 cells were treated with TRAIL and the ER stress-induced signal transduction pathway was investigated. During TRAIL treatment, expression of ER stress marker genes, in particular the BiP (binding immunoglobulin protein) gene, was increased and activation of the PERK (PKR-like ER kinase)-eIF2α (eukaryotic initiation factor 2α)-ATF4 (activating transcription factor 4)-CHOP (CCAAT-enhancer-binding protein homologous protein) apoptotic signal transduction pathway occurred. Experimental data from use of a siRNA (small interfering RNA) technique, caspase inhibitor, and caspase-3-deficient cell line revealed that TRAIL-induced caspase activation is a prerequisite for the TRAIL-induced ER stress response. TRAIL-induced ER stress was triggered by caspase-8-mediated cleavage of BAP31 (B cell receptor-associated protein 31). The involvement of the proapoptotic PERK-CHOP pathway in TRAIL-induced apoptosis was verified by using a PERK knockout (PERK(-/-)) mouse embryo fibroblast (MEF) cell line and a CHOP(-/-) MEF cell line. These results suggest that TRAIL-induced the activation of ER stress response plays a role in TRAIL-induced apoptotic death.

A structure-activity relationship (SAR) study was conducted using a series of PEGylated Fmoc-amino acid conjugates (PFA) as a simple model to gain more insight into carrier-drug interaction. Among the eight PEG2000-Fmoc conjugates with different neighboring structures of Fmoc motif, PEG2000-Fmoc-Lys (Cbz) (PFA2)-based nanomicelles exhibited the smallest particle size distribution, lowest critical micelle concentration (CMC) value, and highest loading capacity with paclitaxel (PTX). These biophysical properties were largely attributed to the strengthened carrier-carrier and carrier-drug interaction, including π-π stacking, hydrophobic, and hydrogen bonding interaction, as confirmed by fluorescence quenching and (13)C NMR study. In vitro and in vivo evaluation further demonstrated the effectiveness of PFA2 as a nanocarrier for efficient delivery of PTX to achieve improved antitumor activity. Importantly, PFA2 was also effective in formulating eight other model drugs of diverse structures, indicating a broad application potential. This work may shed insights into the molecular basis for the structural optimization of nanocarriers for improved delivery efficacy.

We show that ATE1-encoded Arg-transfer RNA transferase (R-transferase) of the N-end rule pathway mediates N-terminal arginylation of multiple endoplasmic reticulum (ER)-residing chaperones, leading to their cytosolic relocalization and turnover. N-terminal arginylation of BiP (also known as GRP78), protein disulphide isomerase and calreticulin is co-induced with autophagy during innate immune responses to cytosolic foreign DNA or proteasomal inhibition, associated with increased ubiquitylation. Arginylated BiP (R-BiP) is induced by and associated with cytosolic misfolded proteins destined for p62 (also known as sequestosome 1, SQSTM1) bodies. R-BiP binds the autophagic adaptor p62 through the interaction of its N-terminal arginine with the p62 ZZ domain. This allosterically induces self-oligomerization and aggregation of p62 and increases p62 interaction with LC3, leading to p62 targeting to autophagosomes and selective lysosomal co-degradation of R-BiP and p62 together with associated cargoes. In this autophagic mechanism, Nt-arginine functions as a delivery determinant, a degron and an activating ligand. Bioinformatics analysis predicts that many ER residents use arginylation to regulate non-ER processes.

TRAIL has been shown to induce apoptosis in cancer cells, but in some cases, certain cancer cells are resistant to this ligand. In this study, we explored the ability of representative HSP90 (heat shock protein 90) inhibitor NVP-AUY922 to overcome TRAIL resistance by increasing apoptosis in colorectal cancer (CRC) cells. The combination of TRAIL and NVP-AUY922 induced synergistic cytotoxicity and apoptosis, which was mediated through an increase in caspase activation. The treatment of NVP-AUY922 dephosphorylated JAK2 and STAT3 and decreased Mcl-1, which resulted in facilitating cytochrome c release. NVP-AUY922-mediated inhibition of JAK2/STAT3 signaling and down-regulation of their target gene, Mcl-1, occurred in a dose and time-dependent manner. Knock down of Mcl-1, STAT3 inhibitor or JAK2 inhibitor synergistically enhanced TRAIL-induced apoptosis. Taken together, our results suggest the involvement of the JAK2-STAT3-Mcl-1 signal transduction pathway in response to NVP-AUY922 treatment, which may play a key role in NVP-AUY922-mediated sensitization to TRAIL. By contrast, the effect of the combination treatments in non-transformed colon cells was minimal. We provide a clinical rationale that combining HSP90 inhibitor with TRAIL enhances therapeutic efficacy without increasing normal tissue toxicity in CRC patients.

Contains fulltext : 48537.pdf (Publisher’s version ) (Closed access) Johanson-Blizzard syndrome (OMIM 243800) is an autosomal recessive disorder that includes congenital exocrine pancreatic insufficiency, multiple malformations such as nasal wing aplasia, and frequent mental retardation. We mapped the disease-associated locus to chromosome 15q14-21.1 and identified mutations, mostly truncating ones, in the gene UBR1 in 12 unrelated families with Johanson-Blizzard syndrome. UBR1 encodes one of at least four functionally overlapping E3 ubiquitin ligases of the N-end rule pathway, a conserved proteolytic system whose substrates include proteins with destabilizing N-terminal residues. Pancreas of individuals with Johanson-Blizzard syndrome did not express UBR1 and had intrauterine-onset destructive pancreatitis. In addition, we found that Ubr1(-/-) mice, whose previously reported phenotypes include reduced weight and behavioral abnormalities, had an exocrine pancreatic insufficiency, with impaired stimulus-secretion coupling and increased susceptibility to pancreatic injury. Our findings indicate that deficiency of UBR1 perturbs the pancreas' acinar cells and other organs, presumably owing to metabolic stabilization of specific substrates of the N-end rule pathway.

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.

Although proteasome inhibitors (PIs) are used as anticancer drugs to treat various cancers, their relative therapeutic efficacy on stem cells vs. bulk cancers remains unknown. Here, we show that stem cells derived from gliomas, GSCs, are up to 1,000-fold more sensitive to PIs (IC50, 27-70 nM) compared with their differentiated controls (IC50, 47->>100 µM). The stemness of GSCs correlates to increased ubiquitination, whose misregulation readily triggers apoptosis. PI-induced apoptosis of GSCs is independent of NFκB but involves the phosphorylation of JNK as well as the transcriptional activation of endoplasmic reticulum (ER) stress-associated proapoptotic mediators. In contrast to the general notion that ER stress-associated apoptosis is signaled by prolonged unfolded protein response (UPR), GSC-selective apoptosis is instead counteracted by the UPR. ATF3 is a key mediator in GSC-selective apoptosis. Pharmaceutical uncoupling of the UPR from its downstream apoptosis sensitizes GSCs to PIs in vitro and during tumorigenesis in mice. Thus, a combinational treatment of a PI with an inhibitor of UPR-coupled apoptosis may enhance targeting of stem cells in gliomas. Key words: glioma; glioma stem cells; proteasome inhibitors; ubiquitin proteasome system; apoptosis; c-Jun N-terminal kinase (JNK) Citation Format: Dae-Hee Lee, Sang Cheul Oh, Yong Tae Kwon, Yong J. Lee, Young Dong Yoo. Glioma derived cancer stem cells are hypersensitive to proteasomal inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5327. doi:10.1158/1538-7445.AM2017-5327

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.

The ubiquitin-proteolytic pathway regulates numerous cellular processes, including signal transduction, cell-cycle progression, DNA repair, apoptosis, antigen processing, and gene expression (Varshavsky 1997). The N-end-rule pathway, one subset of the ubiquitin pathway, relates the in vivo half-life of a protein to the identity of its N-terminal residue (Bachmair et al. 1986). In mice, N-terminal Asn and Gln of the N-end-rule substrates are deamidated to Asp and Glu by NTAN1 and NTAQ1, respectively (Grigoryev et al. 1996; Kwon et al. 2000). Amino-terminal Asp and Glu are conjugated with Arg by ATE1, which exists as two distinct species, ATE1–1 and ATE1–2, the products of alternative splicing (Kwon et al. 1999a). Target proteins bearing Arg (or other primary destabilizing residues) are recognized and ubiquitylated by UBR1/E3α (the E3 component of the N-end-rule pathway) with the help of a ubiquitin-conjugating enzyme (E2) and are processively degraded by 26S proteasomes. UBR1 recognizes N-end-rule substrates through its type 1 and type 2 substrate-binding sites (Varshavsky 1996; Kwon et al. 1999b). Mouse UBR1/E3α cDNA has been recently cloned (Kwon 1998), and the mice lacking UBR1 have been recently constructed (Y.T. Kwon and A. Varshavsky, unpubl.). Recently, mice lacking NTAN1 have been constructed (Kwon et al. 2000). These mice specifically lack the asparagine branch of the N-end-rule pathway and were found to be less active in an open field and impaired on several spatial memory tasks (Kwon et al. 2000). One of these spatial tasks was the Lashley III. This maze contains cul-de-sacs that must be avoided and T-choices at which an animal must learn whether to turn right or left (see Fig. ​Fig.1).1). When given one trial per day for 5 d, NTAN1−/− mice were less competent than wild-type mice in learning. Further, when retested ∼8 wk later, NTAN1−/− mice exhibited poorer retention (Kwon et al. 2000). Thus, with an intertrial interval (ITI) of 24 hr, wild-type mice were better learners than NTAN1−/− mice in this maze. Figure 1 Overhead view of Lashley III maze. Arrows labelled C indicate a forward-going cul-entry error. Arrows labelled T indicate a forward-going T-choice error. It is well established that memory is a time-dependent process that requires the consolidation and storage of newly acquired information (McGaugh 1966). In addition, it is well known that the temporal parameters employed during training affect recall (Warden 1923; Travis 1937; Thompson and Pennington 1957; Kamin 1957, 1963; Frieder and Allweis 1982a,b; Yongue and Roy 1985; Bernabeu et al. 1997; Bourtchouladze et al. 1998; Heyser et al. 1999). Research has revealed that memory can be divided into at least two main phases: short-term, which lasts only minutes and requires the modification of preexisting proteins, and long-term, which lasts for days or longer and is accompanied by neural growth and the synthesis of protein (Barondes and Cohen 1966; Cohen and Barondes 1968; Davis and Squire 1984; Bailey et al. 1996). It is important to note, however, that other researchers have further dissected memory into three phases (Ng et al. 1991; Rosenzweig et al. 1991) or four phases (Frieder and Allweis 1982a). Regardless of which system one relies upon, it is generally assumed that an ITI of 24 hr requires the utilization of a long-term presumably protein-dependent memory system (Mark and Watts 1971; Flood et al. 1973; Abel et al. 1997; Bourtchouladze et al. 1998). Inactivation of the NTAN1 gene in mice resulted in impaired long-term memory in the Lashley III maze (Kwon et al. 2000). One interpretation of this result is that a normally short-lived regulatory protein that is targeted for degradation by the N-end-rule pathway through the protein's N-terminal Asn becomes long-lived in NTAN1-deficient mice. The resulting increase in the steady-state concentration of this protein alters the functioning of relevant neural networks in the brain. However, physiological substrates of NTAN1 remain to be identified. The role of NTAN1 in short-term (and intermediate-term) memory systems is still not known. Therefore, in the current set of experiments, wild-type and NTAN1−/− mice were given five trials in the Lashley III maze all on one day with ITIs varying from 2 to 180 min in anticipation that there would be a time-dependent dimension to the NTAN1−/− performance, as a result of the stabilization of proteins. The range of ITI values was selected so as to measure time points preceding, including, and following the ∼84-min half-life of asparagine-bearing proteins.

A multitude of regulatory circuits involve conditionally or constitutively short-lived proteins (26, 27, 44, 48, 49, 64). Features of proteins that confer metabolic instability are called degradation signals, or degrons (37, 63). The essential component of one degradation signal, termed the N-degron, is a destabilizing N-terminal residue of a protein (3). A set of N-degrons containing different N-terminal residues which are destabilizing in a given cell yields a rule, termed the N-end rule, which relates the in vivo half-life of a protein to the identity of its N-terminal residue. An N-end rule pathway is present in all organisms examined, from mammals and plants to fungi and prokaryotes (63). In eukaryotes, an N-degron comprises two determinants: a destabilizing N-terminal residue and an internal lysine of a substrate protein (4, 32, 60). The Lys residue is the site of formation of a substrate-linked multiubiquitin chain (15, 49). The N-end rule pathway is thus one pathway of the ubiquitin (Ub) system (25–27). Ub is a 76-residue eukaryotic protein that exists in cells either free or covalently conjugated to many other proteins. The Ub system plays a role in a vast range of processes, including cell growth, division, differentiation, and responses to stress. In most of these processes, Ub acts through routes that involve the degradation of Ub-protein conjugates by the 26S proteasome, an ATP-dependent multisubunit protease (10, 17, 20, 51). (Throughout the text, the names of mouse genes are in italics, with the first letter uppercase. The names of human and Saccharomyces cerevisiae genes are also in italics, all uppercase. If human and mouse genes are named in the same sentence, the mouse gene notation is used. The names of S. cerevisiae proteins are roman, with the first letter uppercase and an extra lowercase “p” at the end. The names of mouse and human proteins are the same, except that all letters but the last “p” are uppercase. The latter usage is a modification of the existing convention [58], to facilitate simultaneous discussions of yeast, mouse, and human proteins. In some citations, the abbreviated name of a species precedes the gene's name.) The N-end rule has a hierarchic structure. In the yeast S. cerevisiae, Asn and Gln are tertiary destabilizing N-terminal residues in that they function through their conversion, by the NTA1-encoded N-terminal amidohydrolase (Nt-amidase), into the secondary destabilizing N-terminal residues Asp and Glu (6). Destabilizing activity of N-terminal Asp and Glu requires their conjugation, by the S. cerevisiae ATE1-encoded Arg-tRNA protein transferase (R-transferase) (8, 41), to Arg, one of the primary destabilizing residues (Fig. ​(Fig.1A).1A). In mammals, the deamidation step is mediated by two Nt-amidases, NtN-amidase and NtQ-amidase, which are specific, respectively, for N-terminal Asn and Gln (Fig. ​(Fig.1A)1A) (24, 59). The mammalian counterpart of the yeast R-transferase Ate1p exists as two distinct species, ATE1-1p and ATE1p-2, which are produced through alternative splicing of Ate1 pre-mRNA (34). In vertebrates, the set of secondary destabilizing residues contains not only Asp and Glu but also Cys, which is a stabilizing residue in yeast (Fig. ​(Fig.1A)1A) (18, 23). The primary destabilizing N-terminal residues are bound directly by the UBR1-encoded N-recognin, the targeting (E3) component of the N-end rule pathway. In S. cerevisiae, Ubr1p is a 225-kDa protein which recognizes potential N-end rule substrates through its type 1 and type 2 substrate-binding sites. The type 1 site binds the basic N-terminal residues Arg, Lys, and His. The type 2 site binds the bulky hydrophobic N-terminal terminal residues Phe, Leu, Trp, Tyr, and Ile (35, 63). Ubr1p contains yet another substrate-binding site that targets proteins such as Cup9p and Gpa1p, which bear internal (non-N-terminal) degrons (12, 54). The Ubr1 genes encoding mouse and human N-recognins, also called E3α, have been cloned (36), and mouse strains lacking Ubr1 have recently been constructed (Y. T. Kwon and A. Varshavsky, unpublished data). FIG. 1 Deletion-disruption of the mouse Ntan1 gene. (A) Comparison of enzymatic reactions that underlie the activity of tertiary and secondary destabilizing residues in the yeast S. cerevisiae and the mouse. N-terminal residues are indicated by single-letter ... The known functions of the N-end rule pathway include the control of peptide import in S. cerevisiae, through the degradation of Cup9p, a transcriptional repressor of PTR2, which encodes the peptide transporter (1, 12); a mechanistically undefined role in regulating the Sln1p-dependent phosphorylation cascade that mediates osmoregulation in S. cerevisiae (47); the degradation of alphaviral RNA polymerases and other viral proteins in infected metazoan cells (19, 38); and the degradation of Gpa1p, a Gα protein of S. cerevisiae (43, 54). Physiological N-end rule substrates were also identified among the proteins secreted into the mammalian cell's cytosol by intracellular parasites such as the bacterium Listeria monocytogenes. Short half-lives of these bacterial proteins are required for the efficient presentation of their peptides to the immune system (56). Inhibition of the N-end rule pathway was reported to interfere with mammalian cell differentiation (28) and to delay limb regeneration in amphibians (61). Studies of the Ub-dependent proteolysis of endogenous proteins in muscle extracts suggested that the N-end rule pathway plays a role in catabolic states that result in muscle atrophy (39, 57). A crush injury to the rat sciatic nerve was reported to result in a ∼10-fold increase in the rate of arginine conjugation to the N termini of proteins in the nerve's region upstream of the crush site, suggesting an injury-induced increase in the concentration of R-transferase substrates and/or an enhanced activity of the N-end rule pathway (65). Physiological substrates of either yeast or metazoan Nt-amidases and R-transferases are unknown. Engineered N-end rule substrates, including substrates of Nt-amidases and R-transferases, can be produced in vivo through the Ub fusion technique, in which a Ub-X reporter fusion is cleaved by deubiquitylating enzymes (DUBs) (66) after the last residue of Ub, yielding a reporter bearing the desired N-terminal residue X (3, 63). The mouse Asn-specific NtN-amidase is encoded by the 17-kb Ntan1 gene. The 1.6-kb Ntan1 mRNA specifies the 310-residue NtN-amidase (24). In the present work, we characterized the expression and intracellular localization of NtN-amidase. We also constructed mouse strains bearing a homozygous deletion-disruption of Ntan1 and showed that these mice lacked both NtN-amidase and the Asn-specific branch of the N-end rule pathway. The Ntan1−/− mice were fertile and outwardly normal but were found to differ from their congenic wild-type counterparts in spontaneous activity and spatial memory. Among these differences was a socially conditioned exploratory phenotype of Ntan1−/− mice that has not been previously described, to our knowledge, with other mouse strains.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The N-end rule pathway is one proteolytic pathway of the ubiquitin system. The recognition component of this pathway, called N-recognin or E3, binds to a destabilizing N-terminal residue of a substrate protein and participates in the formation of a substrate-linked multiubiquitin chain. We report the cloning of the mouse and human Ubr1 cDNAs and genes that encode a mammalian N-recognin called E3α. Mouse UBR1p (E3α) is a 1,757-residue (200-kDa) protein that contains regions of sequence similarity to the 225-kDa Ubr1p of the yeast Saccharomyces cerevisiae . Mouse and human UBR1p have apparent homologs in other eukaryotes as well, thus defining a distinct family of proteins, the UBR family. The residues essential for substrate recognition by the yeast Ubr1p are conserved in the mouse UBR1p. The regions of similarity among the UBR family members include a putative zinc finger and RING-H2 finger, another zinc-binding domain. Ubr1 is located in the middle of mouse chromosome 2 and in the syntenic 15q15-q21.1 region of human chromosome 15. Mouse Ubr1 spans ≈120 kilobases of genomic DNA and contains ≈50 exons. Ubr1 is ubiquitously expressed in adults, with skeletal muscle and heart being the sites of highest expression. In mouse embryos, the Ubr1 expression is highest in the branchial arches and in the tail and limb buds. The cloning of Ubr1 makes possible the construction of Ubr1 -lacking mouse strains, a prerequisite for the functional understanding of the mammalian N-end rule pathway.

The N-end rule pathway contributes significantly to accelerated muscle proteolysis mediated by the ubiquitin-proteasome pathway in various catabolic conditions. UBR2 (aka E3α-II) is the only known E3 ubiquitin ligase of the N-end rule pathway that is up-regulated by cachectic stimuli including proinflammatory cytokines and tumors. However, the signaling mechanism through which UBR2 is up-regulated remains undetermined. Here we identify a signaling pathway that mediates tumor cell-induced up-regulation of UBR2. UBR2 expression in C2C12 myotubes was up-regulated by conditioned medium from Lewis lung carcinoma cells or C26 colon adenocarcinoma cells, which was blocked by a pharmacological inhibitor of p38α/β mitogen-activated protein kinase (MAPK), SB202190. Similarly, SB202190 administration (i.p.) abolished UBR2 up-regulation in the tibialis anterior of LLC tumor-bearing mice. Genetic gain and loss of function assays in C2C12 myotubes indicated that tumor-induced activation of the p38β isoform is sufficient and necessary for UBR2 up-regulation. In addition, UBR2 up-regulation required p38β-mediated phosphorylation of CCAAT/enhancer binding protein (C/EBP)-β Thr-188, which was critical to C/EBPβ binding to the UBR2 promoter. Furthermore, luciferase reporter assay revealed that the C/EBPβ binding motif in the UBR2 promoter is a functional C/EBPβ-responsive cis-element that enhances the promoter activity on activation by p38β. Finally, genetic ablation of C/EBPβ blocked UBR2 up-regulation in LLC tumor-bearing mice. These results suggest that UBR2 up-regulation in cachectic muscle is mediated by the p38β-C/EBPβ signaling pathway responsible for the bulk of tumor-induced muscle proteolysis.—Zhang, G., Lin, R.-K., Kwon, Y. T., Li, Y.-P. Signaling mechanism of tumor cell-induced up-regulation of E3 ubiquitin ligase UBR2.

The third author's name was spelled incorrectly. The correct name is: N.R. Thimmegowda. The correct citation is: Kim S-O, Sakchaisri K, Thimmegowda NR, Soung NK, Jang J-H, et al. (2013) STK295900, a Dual Inhibitor of Topoisomerase 1 and 2, Induces G2 Arrest in the Absence of DNA Damage. PLoS ONE 8(1): e53908. doi:10.1371/journal.pone.0053908

We characterized the cellular properties of cancer stem-like cells (CSLCs) isolated from immortalized MDA-MB453 human breast cancer cells in culture. We showed that although the expression of Octamer-binding transcription factor-4 (OCT4) correlates to stemness in these CSLCs, OCT4 knockdown does not induce their differentiation. Our results suggest that the differentiation program in MDA-MB453 CSLCs is blocked at a step upstream of the transcription of the OCT4 promoter, allowing CSLCs to maintain their population through asymmetric cell division during many repeated passages. Comparative expression analysis indicates that only a subset of genes and signaling pathways known to be associated with survival and maintenance of CSCs are selectively expressed in CSLCs, as compared with non-CSLCs fractionated from the same parental MDA-MB453 cells. These results suggest that selective expression of a limited number of genes may be sufficient for establishment and maintenance of CSLCs with high tumorigenicity.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In both fungi and mammals, the tertiary destabilizing N-terminal residues asparagine and glutamine function through their conversion, by enzymatic deamidation, into the secondary destabilizing residues aspartate and glutamate, whose destabilizing activity requires their enzymatic conjugation to arginine, one of the primary destabilizing residues. We report the isolation and analysis of a mouse cDNA and the corresponding gene (termed Ntan1) that encode a 310-residue amidohydrolase (termed NtN-amidase) specific for N-terminal asparagine. The ∼17-kilobase pair Ntan1 gene is located in the proximal region of mouse chromosome 16 and contains 10 exons ranging from 54 to 177 base pairs in length. The ∼1.4-kilobase pair Ntan1 mRNA is expressed in all of the tested mouse tissues and cell lines and is down-regulated upon the conversion of myoblasts into myotubes. The Ntan1 promoter is located ∼500 base pairs upstream of the Ntan1 start codon. The deduced amino acid sequence of mouse NtN-amidase is 88% identical to the sequence of its porcine counterpart, but bears no significant similarity to the sequence of the NTA1-encoded N-terminal amidohydrolase of the yeast Saccharomyces cerevisiae, which can deamidate either N-terminal asparagine or glutamine. The expression of mouse NtN-amidase in S. cerevisiae nta1Δ was used to verify that NtN-amidase retains its asparagine selectivity in vivo and can implement the asparagine-specific subset of the N-end rule. Further dissection of mouse Ntan1, including its null phenotype analysis, should illuminate the functions of the N-end rule, most of which are still unknown.

Previous studies have demonstrated that a small subset of cancer cells is capable of tumor initiation. The existence of tumor initiating cancer stem cells (CSCs) has several implications in terms of future cancer treatment and therapies. However, recently, several researchers proposed that differentiated cancer cells (non-CSCs) can convert to stem-like cells to maintain equilibrium. These results imply that removing CSCs may prompt non-CSCs in the tumor to convert into stem cells to maintain the equilibrium. Interleukin-6 (IL-6) has been found to play an important role in the inducible formation of CSCs and their dynamic equilibrium with non-stem cells. In this study, we used CSC-like human breast cancer cells and their alternate subset non-CSCs to investigate how IL-6 regulates the conversion of non-CSCs to CSCs. MDA-MB-231 and MDA-MB-453 CSC-like cells formed mammospheres well, whereas most of non-stem cells died by anoikis and only part of the remaining non-stem cells produced viable mammospheres. Similar results were observed in xenograft tumor formation. Data from cytokine array assay show that IL-6 was secreted from non-CSCs when cells were cultured in ultra-low attachment plates. IL-6 regulates CSC-associated OCT-4 gene expression through the IL-6-JAK1-STAT3 signal transduction pathway in non-CSCs. Inhibiting this pathway by treatment with anti-IL-6 antibody (1 μg/ml) or niclosamide (0.5–2 μM)/LLL12 (5–10 μM) effectively prevented OCT-4 gene expression. These results suggest that the IL-6-JAK1-STAT3 signal transduction pathway plays an important role in the conversion of non-CSCs into CSCs through regulation of OCT-4 gene expression.

Nuclear factor-κB (NF-κB) ligand (RANKL) was shown to induce osteoclast differentiation by increasing the expression of c-Fos, NFATc1 and TRAP. Salubrinal treatment to bone marrow macrophage (BMM) cells, however, significantly blocked NFATc1 expression and osteoclast differentiation by RANKL. Overexpression of NFATc1 further confirmed that NFATc1 is a key factor affected by salubrinal in osteoclast differentiation by RANKL. Unexpectedly, NFATc1 and c-Fos mRNA expressions were not affected by salubrinal, implicating that NFATc1 expression is regulated at a translational stage. In support of this, salubrinal increased the phosphorylation of a translation factor eIF2α, decreasing the global protein synthesis including NFATc1. In contrast, a phosphorylation mutant plasmid pLenti-eIF2α-S51A restored RANKL-induced NFATc1 expression and osteoclast differentiation even in the presence of salubrinal. Furthermore, knockdown of ATF4 significantly reduced salubrinal-induced osteoblast differentiation as evidenced by decreased calcium accumulation and lowered expressions of the osteoblast differentiation markers, alkaline phosphatase and RANKL in MC3T3-E1 osteoblast cells. Salubrinal treatment to co-cultured BMM and MC3T3-E1 cells also showed reduction of osteoclast differentiation. Finally, salubrinal efficiently blocked osteoporosis in mice model treated with RANKL as evidenced by elevated bone mineral density (BMD) and other osteoporosis factors. Collectively, our data indicate that salubrinal could affect the differentiation of both osteoblast and osteoclast, and be developed as an excellent anti-osteoporosis drug. In addition, modulation of ATF4 and NFATc1 expressions through eIF2α phosphorylation could be a valuable target for the treatment of osteoporosis.

The gene vapT, encoding VapT, one of the extracellular sodium dodecyl sulfate (SDS)-resistant alkaline serine proteases (Serp) from the Gram - Vibrio metschnikovii strain RH530 has been cloned in Escherichia coli. The recombinant E. coli produced a protease which co-migrated with VapT on gelatin polyacrylamide gels. The nucleotide (nt) sequence of the cloned vapT revealed a single open reading frame of 1641 bp encoding 547 amino acids (aa) (58 961 Da). Upon analysis of the N-terminal aa sequence, VapT was shown to be processed properly in recombinant E. coli and to consist of 428 aa (45 626 Da). The deduced aa sequence of VapT showed significant sequence homology to subtilisin Carlsberg from Bacillus licheniformis, particularly in the regions containing active site residues and calcium-binding sites. VapT had an intervening region of approx. 149 aa between the His and Ser residues of the active site, as compared with other Serp.

Centrosomal proteins play important roles in cell cycle. Among them, the centrosomal protein of 131kDa (CEP131) has been reported as a critical factor for cilia formation which is related with development, signaling, and various diseases, the malfunction of cilia leading to cancer. Specificity protein 1 (SP1), known as a centrosome regulator, is an essential transcription factor regulating the genes involved in multiple cellular processes such as cell cycle, apoptosis, and DNA damages. In this study, we explored the crucial role of SP1 in the regulation of CEP131 gene transcription. A deletion analysis of the CEP131 promoter region revealed dominant promoter elements within the sequence between -400bp and -200bp, which contained consensus binding sites for SP1. Electrophoretic mobility shift assay (EMSA) and chromatin immuno-precipitation (ChIP) assay further confirmed the direct binding of SP1 to the CEP131 promoter. On the other hand, CEP131 transcription could be inhibited by mithramycin (a GC-rich region inhibitor), but exogenous expression of SP1 could increase CEP131 expression as evidenced by a reporter gene assay. In addition, mutation of several SP1 binding sites revealed four SP1 binding sites at -244/-225, -258/-239, -304/-283 and -323/-304 that strongly affect CEP131 expression. Hence, it is suggested that SP1 is a pivotal transcription factor for the regulation of CEP131 expression, consequently leading the control of centrosome functions.

Among the functions of intracellular proteolysis are the elimination of abnormal proteins, the maintenance of amino acid pools in cells affected by stresses such as starvation, and the generation of protein fragments that act as hormones, antigens, or other effectors. Yet another function of proteolytic pathways is selective destruction of proteins whose concentrations must vary with time and alterations in the state of a cell. Metabolic instability is a property of many regulatory proteins. A short in vivo half-life* of a regulator provides a way to generate its spatial gradients and allows for rapid adjustments of its concentration (or subunit composition) through changes in the rate of its synthesis. A protein can also be conditionally unstable: long-lived or short-lived depending on the state of a cell. Conditionally short-lived regulators are often deployed as components of control circuits. One example is cyclins—a family of related proteins whose destruction at specific stages of the cell cycle regulates cell division and growth (Chapter 12; Murray and Hunt, 1993). In addition, many proteins are long-lived as components of larger complexes such as ribosomes and oligomeric proteins but are metabolically unstable as free subunits. The short in vivo lifetimes of free subunits allow for a less stringent control over the relative rates of their synthesis, as a subunit produced in excess would not accumulate to a significant level.

The N-end rule defines the protein-destabilizing activity of a given amino-terminal residue and its post-translational modification. Since its discovery 25 years ago, the pathway involved in the N-end rule has been thought to target only a limited set of specific substrates of the ubiquitin-proteasome system. Recent studies have provided insights into the components, substrates, functions and structural basis of substrate recognition. The N-end rule pathway is now emerging as a major cellular proteolytic system, in which the majority of proteins are born with or acquire specific N-terminal degradation determinants through protein-specific or global post-translational modifications.

The polymerase gene of the hepatitis B virus was fused with the E. coli Maltose Binding Protein (MBP) and expressed as a fusion protein. Reverse transcriptase activity was detected in purified P protein with the assay conditions of 50mM Mg concentration at 30 °C yielding the best results.

The gene (smp) encoding an extracellular protease (Smp) from Serratia marcescens ATCC21074 has been cloned and expressed in Escherichia coli HB101. The nucleotide (nt) sequence of the cloned smp gene revealed a single open reading frame of 1056 bp coding for 352 amino acids (aa) (38 479 Da). The N-terminal aa sequence of Smp excreted from the E. coli host cells revealed that mature Smp consists of 300 aa (32 515 Da). The deduced aa sequence of Smp showed high overall homology (43%) to the Erwinia carotovora metalloprotease, but low homology (15–20%) to other metallopro-teases, including the S. marcescens major metalloprotease. The location for three zinc ligands and the active site for Smp was predicted from other metalloproteases. The biochemical properties of Smp show that this enzyme is a metalloprotease whose activity is optimal at pH 8.0 and 50°C.

The N-end rule pathway is a proteolytic system in which recognition components (N-recognins) recognize a set of N-terminal residues as part of degradation signals (N-degrons). Two studies in this issue report the structures of Ubr boxes, a substrate recognition domain of eukaryotic N-recognins. We discuss how eukaryotic and prokaryotic N-recognins use a similar molecular principle to recognize a different set of N-degrons.

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 chromosome-wide 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.

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 approximately 3.4 microm. Downstream from the UBR box, we identify a second substrate recognition domain, termed the N-domain, required for type-2 substrate recognition. The approximately 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.

Quercetin is a flavonoid present in many vegetables, fruits, and beverages. Due to its anti-oxidant, anti-tumor, and anti-inflammatory activity, quercetin has been studied extensively as a chemoprevention agent in several cancer models. Since most of these studies used higher doses of quercetin than clinically achievable, we focused on the effectiveness of physiologically relevant doses of quercetin. A low dose of quercetin exerted cancer cell-specific inhibition of proliferation and this inhibition resulted from cell cycle arrest at the G(1) phase. Quercetin induced p21 CDK inhibitor with a concomitant decrease of phosphorylation of pRb, which inhibits the G(1)/S cell cycle progression by trapping E2F1. A low dose of quercetin induced mild DNA damage and Chk2 activation, which is the main regulator of p21 expression by quercetin. In addition, quercetin down-regulated the cyclin B1 and CDK1, essential components of G(2)/M cell cycle progression. Inhibition of the recruitment of key transcription factor NF-Y to cyclin B1 gene promoter by quercetin led to transcriptional inhibition. This study proved that the chemo-preventive efficacy of a physiologically relevant dose of quercetin can be achievable through the inhibition of cell cycle progression.

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.

Our previous work identified E3 ubiquitin ligases, termed UBR1-UBR7, that contain the approximately 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 beta-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.

Previous studies have shown that repeated application of TRAIL induces acquired resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Using human prostate adenocarcinoma DU-145 and human pancreatic carcinoma MiaPaCa-2 cells as a model, we now demonstrate for the first time that two states of acquired TRAIL resistance can be developed after TRAIL treatment. Data from survival assay and Western blot analysis show that acquired TRAIL resistance was developed within 1 day and gradually decayed within 6 days after TRAIL treatment in both cell lines. After TRAIL treatment, the level of Bcl-xL increased and reached a maximum within 2 days and gradually decreased in both cell lines. Bcl-xL-mediated development of acquired TRAIL resistance was suppressed by knockdown of Bcl-xL expression. Protein interaction assay revealed that during the development of TRAIL resistance, Bcl-xL dissociated from Bad and then associated with Bax. Overexpression of mutant-type Bad (S136A), which prevents this dissociation, partially suppressed the development of acquired TRAIL resistance. Thus, our results suggest that (a) dissociation of Bad from Bcl-xL and (b) an increase in the intracellular level of Bcl-xL are responsible for development of acquired TRAIL resistance.

Substrates of the N-end rule pathway include proteins with destabilizing N-terminal residues. Three of them, Asp, Glu, and (oxidized) Cys, function through their conjugation to Arg, one of destabilizing N-terminal residues that are recognized directly by the pathway's ubiquitin ligases. The conjugation of Arg is mediated by arginyltransferase, encoded by ATE1. Through its regulated degradation of specific proteins, the arginylation branch of the N-end rule pathway mediates, in particular, the cardiovascular development, the fidelity of chromosome segregation, and the control of signaling by nitric oxide. We show that mouse ATE1 specifies at least six mRNA isoforms, which are produced through alternative splicing, encode enzymatically active arginyltransferases, and are expressed at varying levels in mouse tissues. We also show that the ATE1 promoter is bidirectional, mediating the expression of both ATE1 and an oppositely oriented, previously uncharacterized gene. In addition, we identified GRP78 (glucose-regulated protein 78) and protein-disulfide isomerase as putative physiological substrates of arginyltransferase. Purified isoforms of arginyltransferase that contain the alternative first exons differentially arginylate these proteins in extract from ATE1(-/-) embryos, suggesting that specific isoforms may have distinct functions. Although the N-end rule pathway is apparently confined to the cytosol and the nucleus, and although GRP78 and protein-disulfide isomerase are located largely in the endoplasmic reticulum, recent evidence suggests that these proteins are also present in the cytosol and other compartments in vivo, where they may become N-end rule substrates.

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.

Unresectable colorectal liver metastases remain a major unresolved issue and more effective novel regimens are urgently needed. While screening synergistic drug combinations for colon cancer therapy, we identified a novel multidrug treatment for colon cancer: chemotherapeutic agent melphalan in combination with proteasome inhibitor bortezomib and mTOR (mammalian target of rapamycin) inhibitor rapamycin. We investigated the mechanisms of synergistic antitumor efficacy during the multidrug treatment. All experiments were performed with highly metastatic human colon cancer CX-1 and HCT116 cells, and selected critical experiments were repeated with human colon cancer stem Tu-22 cells and mouse embryo fibroblast (MEF) cells. We used immunochemical techniques to investigate a cross-talk between apoptosis and autophagy during the multidrug treatment. We observed that melphalan triggered apoptosis, bortezomib induced apoptosis and autophagy, rapamycin caused autophagy and the combinatorial treatment-induced synergistic apoptosis, which was mediated through an increase in caspase activation. We also observed that mitochondrial dysfunction induced by the combination was linked with altered cellular metabolism, which induced adenosine monophosphate-activated protein kinase (AMPK) activation, resulting in Beclin-1 phosphorylated at Ser 93/96. Interestingly, Beclin-1 phosphorylated at Ser 93/96 is sufficient to induce Beclin-1 cleavage by caspase-8, which switches off autophagy to achieve the synergistic induction of apoptosis. Similar results were observed with the essential autophagy gene, autophagy-related protein 7, -deficient MEF cells. The multidrug treatment-induced Beclin-1 cleavage was abolished in Beclin-1 double-mutant (D133A/D146A) knock-in HCT116 cells, restoring the autophagy-promoting function of Beclin-1 and suppressing the apoptosis induced by the combination therapy. These observations identify a novel mechanism for AMPK-induced apoptosis through interplay between autophagy and apoptosis.

The N-end rule pathway of protein degradation targets proteins with destabilizing N-terminal residues. Ubr2 is one of the E3 ubiquitin ligases of the mouse N-end rule pathway. We have previously shown that Ubr2−/− male mice are infertile, owing to the arrest of spermatocytes between the leptotene/zygotene and pachytene of meiosis I, the failure of chromosome pairing, and subsequent apoptosis. Here, we report that mouse fibroblast cells derived from Ubr2−/− embryos display genome instability. The frequency of chromosomal bridges and micronuclei were much higher in Ubr2−/− fibroblasts than in +/+ controls. Metaphase chromosome spreads from Ubr2−/− cells revealed a high incidence of spontaneous chromosomal gaps, indicating chromosomal fragility. These fragile sites were generally replicated late in S phase. Ubr2−/− cells were hypersensitive to mitomycin C, a DNA cross-linking agent, but displayed normal sensitivity to gamma-irradiation. A reporter assay showed that Ubr2−/− cells are significantly impaired in the homologous recombination repair of a double strand break. In contrast, Ubr2−/− cells appeared normal in an assay for non-homologous end joining. Our results therefore unveil the role of the ubiquitin ligase Ubr2 in maintaining genome integrity and in homologous recombination repair.

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 and/or 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 β-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 O 2 -ATE1-UBR1/UBR2 proteolytic circuit plays a role in RGS-regulated G protein signaling in the cardiovascular system.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to be selective in the induction of apoptosis in cancer cells with minimal toxicity to normal tissues. However, not all cancers are sensitive to TRAIL-mediated apoptosis. Thus, TRAIL-resistant cancer cells must be sensitized first to become responsive to TRAIL. In this study, we observed that pretreatment by acetylsalicylic acid (ASA) augmented TRAIL-induced apoptotic death in human prostate adenocarcinoma LNCaP and human colorectal carcinoma CX-1 cells. Western blot analysis showed that pretreatment of ASA followed by TRAIL treatment activated caspases (8, 9, and 3) and cleaved poly(ADP-ribose) polymerase, the hallmark feature of apoptosis. Most interestingly, at least 12 h of pretreatment with ASA was prerequisite for promoting TRAIL-induced apoptosis and was related to down-regulation of BCL-2. Biochemical analysis revealed that ASA inhibited NF-kappaB activity, which is known to regulate BCL-2 gene expression, by dephosphorylating IkappaB-alpha and inhibiting IKKbeta activity but not by affecting the HER-2/neu phosphatidylinositol 3-kinase-Akt signal pathway. Overexpression of BCL-2 suppressed the promotive effect of ASA on TRAIL-induced apoptosis and changes in mitochondrial membrane potential. Taken together, our studies suggested that ASA-promoted TRAIL cytotoxicity is mediated through down-regulating BCL-2 and by decreasing mitochondrial membrane potential.