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Proteasomes are highly sophisticated protease complexes that degrade non-lysosomal proteins, and their proper regulation ensures various biological functions such as spermatogenesis. The proteasome-associated proteins, PA200 and ECPAS, are predicted to function during spermatogenesis; however, male mice lacking each of these genes sustain fertility, raising the possibility that these proteins complement each other. To address this issue, we explored these possible roles during spermatogenesis by producing mice lacking these genes (double-knockout mice; dKO mice). Expression patterns and quantities were similar throughout spermatogenesis in the testes. In epididymal sperm, PA200 and ECPAS were expressed but were differentially localized to the midpiece and acrosome, respectively. Proteasome activity was considerably reduced in both the testes and epididymides of dKO male mice, resulting in infertility. Mass spectrometric analysis revealed LPIN1 as a target protein for PA200 and ECPAS, which was confirmed via immunoblotting and immunostaining. Furthermore, ultrastructural and microscopic analyses demonstrated that the dKO sperm displayed disorganization of the mitochondrial sheath. Our results indicate that PA200 and ECPAS work cooperatively during spermatogenesis and are essential for male fertility.

Hypoxia-inducible factor 1α (HIF1α) is a transcription factor that regulates angiogenesis under hypoxic conditions. To investigate the posttranscriptional regulatory mechanism of HIF1α, we performed a cell-based screening to reveal potential cis-elements and the regulatory RNA-binding proteins that act as trans-factors. We found that LIN28A promoted HIF1α protein expression independently of the downregulation of microRNA let-7, which is also directly mediated by LIN28A. Transcriptome analysis and evaluation of RNA stability using RNA-seq and SLAM-seq analyses, respectively, revealed that LIN28A upregulates HIF1A expression via mRNA stabilization. To investigate the physical association of LIN28A with HIF1A mRNA, we performed enhanced crosslinking immunoprecipitation in 293FT cells and integrally analyzed the transcriptome. We observed that LIN28A associates with HIF1A mRNA via its cis-element motif "UGAU". The "UGAU" motifs are recognized by the cold shock domain of LIN28A, and the introduction of a loss-of-function mutation to the cold shock domain diminished the upregulatory activities performed by LIN28A. Finally, the microvessel density assay showed that the expression of LIN28A promoted angiogenesis in vivo. In conclusion, our study elucidated the role of LIN28A in enhancing the HIF1α axis at the posttranscription layer.

Healthy limb joints are important for maintaining health and attaining longevity. Endochondral ossification (the replacement of cartilage with bone, occurring during skeletal development) is essential for bone formation, especially in long-axis bones. In contrast to endochondral ossification, chondrocyte populations in articular cartilage persist and maintain joint tissue into adulthood. Articular cartilage, a connective tissue consisting of chondrocytes and their surrounding extracellular matrices, plays an essential role in the mechanical cushioning of joints in postnatal locomotion. Osteoarthritis (OA) pathology relates to disruptions in the balance between anabolic and catabolic signals, that is, the loss of chondrocyte homeostasis due to aging or overuse of cartilages. The onset of OA increases with age, shortening a person’s healthy life expectancy. Although many people with OA experience pain, the mainstay of treatment is symptomatic therapy, and no fundamental treatment has yet been established. To establish regenerative or preventative therapies for cartilage diseases, further understanding of the mechanisms of cartilage development, morphosis, and homeostasis is required. In this review, we describe the general development of cartilage and OA pathology, followed by a discussion on anabolic and catabolic signals in cartilage homeostasis, mainly microRNAs.

Neat1 is a long noncoding RNA (lncRNA) that serves as an architectural component of the nuclear bodies known as paraspeckles. Two isoforms of Neat1, the short isoform Neat1_1 and the long isoform Neat1_2, are generated from the same gene locus by alternative 3’ processing. Neat1_1 is the most abundant and the best conserved isoform expressed in various cell types, whereas Neat1_2 is expressed in a small population of particular cell types, including the tip cells of the intestinal epithelium. To investigate the physiological significance of isoform switching, we created mutant mice that solely expressed Neat1_2 by deleting the upstream polyadenylation (poly-A) signal (PAS) required for the production of Neat1_1. We observed the loss of Neat1_1 and strong upregulation of Neat1_2 in various tissues and cells and the subsequent hyperformation of paraspeckles, especially in cells that normally express Neat1_2. However, the mutant mice were born at the expected Mendelian ratios and did not exhibit obvious external and histological abnormalities. These observations suggested that the hyperformation of paraspeckles does not interfere with the development and growth of these animals under normal laboratory conditions.

Programmed death-ligand 1 (PD-L1) is a co-inhibitory molecule expressed on tumor cells. Immune checkpoint inhibitors focusing on the PD-L1 mechanism are now being studied for the treatment of various cancer types. However, the regulatory mechanism of PD-L1 is yet to be fully clarified, and a high-throughput system for comparing the abilities of small compounds in regulating PD-L1 has not yet been established. Therefore, we created a HiBiT-tagged lung adenocarcinoma cell line, PC9-KI, for easier and faster detection of changes in PD-L1 protein expression. Using PC9-KI cells, we screened 1,280 chemical compounds from the Library of Pharmacologically Active Compounds (LOPAC) and identified microtubule polymerization inhibitors and thapsigargin as PD-L1 upregulators and a p97 inhibitor as a PD-L1 downregulator.

Let‐7 is an evolutionary conserved microRNA that mediates post‐transcriptional gene silencing to regulate a wide range of biological processes, including development, differentiation, and tumor suppression. Let‐7 biogenesis is tightly regulated by several RNA‐binding proteins, including Lin28A/B, which represses let‐7 maturation. To identify new regulators of let‐7, we devised a cell‐based functional screen of RNA‐binding proteins using a let‐7 sensor luciferase reporter and identified the tRNA pseudouridine synthase, TruB1. TruB1 enhanced maturation specifically of let‐7 family members. Rather than inducing pseudouridylation of the miRNAs, high‐throughput sequencing crosslinking immunoprecipitation (HITS‐CLIP) and biochemical analyses revealed direct binding between endogenous TruB1 and the stem‐loop structure of pri‐let‐7, which also binds Lin28A/B. TruB1 selectively enhanced the interaction between pri‐let‐7 and the microprocessor DGCR8, which mediates miRNA maturation. Finally, TruB1 suppressed cell proliferation, which was mediated in part by let‐7. Altogether, we reveal an unexpected function for TruB1 in promoting let‐7 maturation.

Promyelocytic leukaemia (PML) is a tumor suppressor protein covalently conjugated with SUMO family proteins, leading to the formation of PML nuclear bodies (NBs). PML-NBs provide a platform for efficient posttranslational modification of targets and protein-protein interaction, contributing to the adjustment of gene expression and chromatin integrity. Although PML SUMOylation is thought to play important roles in diverse cellular functions, the control mechanisms of adequate modification levels have remained unsolved. Here, we report that Cullin-related protein CACUL1/CAC1 (CACUL1) inhibits PML posttranslational modification. CACUL1 interacts with PML and suppresses PML SUMOylation, leading to the regulation of PML-NB size in the nucleus. We also found that Ubc9, a SUMO-conjugating enzyme, binds to CACUL1 and antagonizes the interaction between CACUL1 and PML. Furthermore, CACUL1 attenuates p53 transcriptional activity. These data suggest that CACUL1 is a novel regulator that negatively controls p53 activity through the regulation of PML SUMOylation.

In human genome, there are approximately 1,500 RNA-binding proteins (RBPs). They can regulate mRNA stability or translational efficiency via ribosomes and these processes are known as ‘post-transcriptional regulation’. Accumulating evidences indicate that post-transcriptional regulation is the determinant of the accurate levels of cytokines mRNAs. While transcriptional regulation of cytokines mRNAs has been well studied and found to be important for the rapid induction of mRNA and regulation of the acute phase of inflammation, post-transcriptional regulation by RBPs is essential for resolving inflammation in the later phase, and their dysfunction may lead to severe autoimmune diseases such as rheumatoid arthritis or systemic lupus erythematosus. For post-transcriptional regulation, RBPs recognize and directly bind to cis-regulatory elements in 3′ untranslated region of mRNAs such as AU-rich or constitutive decay elements and play various roles. In this review, we summarize the recent findings regarding the role of RBPs in the regulation of inflammation.

The cyclin-dependent kinase (CDK) inhibitor p21 is an unstructured protein regulated by multiple turnover pathways. p21 abundance is tightly regulated, and its defect causes tumor development. However, the mechanisms that underlie the control of p21 level are not fully understood. Here, we report a novel mechanism by which a component of the SCF ubiquitin ligase, Fbl12, augments p21 via the formation of atypical ubiquitin chains. We found that Fbl12 binds and ubiquitinates p21. Unexpectedly, Fbl12 increases the expression level of p21 by enhancing the mixed-type ubiquitination, including not only K48- but also K63-linked ubiquitin chains, followed by promotion of binding between p21 and CDK2. We also found that proteasome activator PA28γ attenuates p21 ubiquitination by interacting with Fbl12. In addition, UV irradiation induces a dissociation of p21 from Fbl12 and decreases K63-linked ubiquitination, leading to p21 degradation. These data suggest that Fbl12 is a key factor that maintains adequate intracellular concentration of p21 under normal conditions. Our finding may provide a novel possibility that p21's fate is governed by diverse ubiquitin chains.

The ubiquitin ligases, SCF complexes, consist of Cul1, Skp1, Rbx1 and the substrate recognition components F-box proteins. Previous studies have reported that one of these F-box proteins, Fbl12, which is produced by Fbxl12 gene, regulates both cell cycle and differentiation. In this paper, we show that the intronic region of Fbxl12 gene acts as an alternative promoter and induces expression of a short form of Fbl12 that lacks F-box domain (Fbl12ΔF). We also found that UV irradiation increases Fbl12ΔF mRNA in cells. Finally, Fbl12ΔF may promote the subcellular localization of Fbl12 from nucleus to cytoplasm through their binding. Our data provide the possibility that Fbl12ΔF induced by alternative promoter controls the SCFFbl12 activity in response to UV stimulation.

Kelch-like protein 7 (KLHL7) is a component of Cul3-based Cullin-RING ubiquitin ligase. Recent studies have revealed that mutations in klhl7 gene cause several disorders, such as retinitis pigmentosa (RP). Although KLHL7 is considered to be crucial for regulating the protein homeostasis, little is known about its biological functions. In this study, we report that KLHL7 increases terminal uridylyl transferase 1 (TUT1) ubiquitination involved in nucleolar integrity. TUT1 is normally localized in nucleolus; however, expression of KLHL7 facilitates a vulnerability of nucleolar integrity, followed by a decrease of TUT1 localization in nucleolus. On the other hand, pathogenic KLHL7 mutants, which causes an onset of RP, have little effect on both nucleolar integrity and TUT1 localization. Finally, KLHL7 increases TUT1 ubiquitination levels. Taken together, these results imply that KLHL7 is a novel regulator of nucleolus associated with TUT1 ubiquitination. Our study may provide a valuable information to elucidate a pathogenic mechanism of RP.

SRY-box 9 (SOX9) is a master transcription factor that regulates cartilage development. SOX9 haploinsufficiency resulting from breakpoints in a ∼1-Mb region upstream of SOX9 was reported in acampomelic campomelic dysplasia (ACD) patients, suggesting that essential enhancer regions of SOX9 for cartilage development are located in this long non-coding sequence. However, the cis-acting enhancer region regulating cartilage-specific SOX9 expression remains to be identified. To identify distant cartilage Sox9 enhancers, we utilized the combination of multiple CRISPR/Cas9 technologies including enrichment of the promoter-enhancer complex followed by next-generation sequencing and mass spectrometry (MS), SIN3A-dCas9-mediated epigenetic silencing, and generation of enhancer deletion mice. As a result, we could identify a critical far-upstream cis-element and Stat3 as a trans-acting factor, regulating cartilage-specific Sox9 expression and subsequent skeletal development. Our strategy could facilitate definitive ACD diagnosis and should be useful to reveal the detailed chromatin conformation and regulation.

In vitro immunization (IVI) possesses a number of advantages over conventional immunization. However, the number of positive clones derived from IVI is limited, and the affinity of the antibodies from derived clones is relatively low. Moreover, the majority of immunoglobulins produced in culture are IgMs instead of IgGs, which limits the application. Here, we report an improved protocol for IVI using mouse spleen cells. This protocol consists of multiple cycles of repeated antigen stimulation followed by cell expansion, which increases the frequency of plasma cells that produce antigen-specific IgG antibodies. The culture conditions, including the cell density, the type of stimulants, and the initial cell preparation, were found to be important for inducing the IgG response. In addition, an analysis of the genes and cytokines expressed during the IVI showed that the antigen-specific B cells were specifically activated via CD4-positive helper T cells. As evidence for this concept, our IVI protocol enabled us to establish an IgG antibody against keyhole limpet hemocyanin with a dissociation constant in the order of 10(-7)M.

The transcriptional factor Nrf1 (NF-E2-related factor 1) sustains protein homeostasis (proteostasis) by regulating the expression of proteasome genes. Under physiological conditions, the transcriptional activity of Nrf1 is repressed by its sequestration into the endoplasmic reticulum (ER) and furthermore by two independent ubiquitin-proteasome pathways, comprising Hrd1 and β-TrCP in the cytoplasm and nucleus, respectively. However, the molecular mechanisms underlying Nrf1 activation remain unclear. Here, we report that USP15 (Ubiquitin-Specific Protease 15) activates Nrf1 in the nucleus by stabilizing it through deubiquitination. We first identified USP15 as an Nrf1-associated factor through proteome analysis. USP15 physically interacts with Nrf1, and it markedly stabilizes Nrf1 by removing its ubiquitin moieties. USP15 activates the Nrf1-mediated expression of a proteasome gene luciferase reporter and endogenous proteasome activity. The siRNA-mediated knockdown of USP15 diminishes the Nrf1-induced proteasome gene expression in response to proteasome inhibition. These results uncover a new regulatory mechanism that USP15 activates Nrf1 against the β-TrCP inhibition to maintain proteostasis.

Hybridoma formation is an indispensable step in the production of monoclonal antibodies. Obtaining highly efficient fusion of an antibody-producing cell to the myeloma cell to form the hybridoma is an important step in this process. The electrofusion method is superior to chemical fusion methods such as the polyethylene glycol (PEG) method due to its high fusion efficiency. However, this method requires cell activation prior to electrofusion, a process that is time-consuming and tends to cause cell death. In this study, we achieved much higher fusion efficiency by stimulating B cells with CpG oligodeoxynucleotide (CpG ODN) over shorter periods. Splenocytes were isolated from immunized mice and cultured in the presence of a CpG ODN for 1 or 2 days. This CpG ODN stimulation evokes about one order of magnitude higher fusion efficiency than other stimulators. CpG ODN stimulation not only increases the fusion efficiency but also the number of antibody-producing cells. This leads to a substantial increase in the number of positive clones obtained. This highly efficient fusion method was used to produce a functional antibody against Gaussia luciferase. This method was found to produce greater numbers of hybridomas and to enable direct screening for antibodies with functional characteristics such as inhibition of the luminescence activity of an antigen. We were able to establish a functional antibody against Gaussia luciferase after a single fusion experiment using our electrofusion method.

NEDD8 is a small ubiquitin-like protein that modifies target proteins in a reaction similar to ubiquitination. In this reaction, three enzymes are required and sufficient: NEDD8 activating E1-like enzyme (APP-BP1/Uba3), NEDD8-specific E2 enzyme (Ubc12) and RING-finger protein ROC1 (NEDD8 E3 ligase). Unlike ubiquitin, which is well known to form poly-ubiquitin chain, little is known about the formation of poly-NEDD8 chain. Here we show the mechanism of poly-NEDD8 chain formation on Cullin-1 using a complete in vitro reconstituted NEDD8 conjugation system. Intriguingly, poly-NEDD8 chain was build up on catalytic Cysteine residue of Ubc12. Furthermore, Ubc12 formed poly-NEDD8 chain without the activity of ROC1. Rather ROC1 mutant, defective for ubiquitin ligase activity, dramatically enhanced the poly-NEDD8 chain formation. In turn, ROC1 was essential for the transfer of poly-NEDD8 chain from Ubc12 to Cul-1. These results suggest the important regulatory role of ROC1 for poly-NEDD8 chain formation.

Heat shock protein 90 (hsp90) and the proteasome activator PA28 stimulate major histocompatibility complex (MHC) class I antigen processing. It is unknown whether hsp90 influences the proteasome activity to produce T cell epitopes, although association of PA28 with the 20 S proteasome stimulates the enzyme activity. Here, we show that hsp90 is essential in assembly of the 26 S proteasome and as a result, is involved in epitope production. Addition of recombinant hsp90alpha to cell lysate enhanced chymotrypsin-like activity of the 26 S proteasome in an ATP-dependent manner as determined by an in-gel hydrolysis assay. We successfully pulled down histidine-tagged hsp90alpha- and PA28alpha-induced, newly assembled 26 S proteasomes from the cell extracts for in vitro epitope production assay, and we found these structures to be sensitive to geldanamycin, an hsp90 inhibitor. We found a cleaved epitope unique to the proteasome pulled down by both hsp90alpha and PA28alpha, whereas two different epitopes were identified in the hsp90alpha- and PA28alpha-pulldowns, respectively. Processing of these respective peptides in vivo was enhanced faithfully by the protein combinations used for the proteasome pulldowns. Inhibition of hsp90 in vivo by geldanamycin partly disrupted the 26 S proteasome structure, consistent with down-regulated MHC class I expression. Our results indicate that hsp90 facilitates MHC class I antigen processing through epitope production in a complex of the 26 S proteasome.

Transforming growth factor-beta1 (TGF-beta1) has many physiological functions and inhibits the differentiation of osteoblasts. Previously, we reported that TGF-beta1 stimulation induces the degradation of p57(KIP2) in osteoblasts. p57(KIP2) proteolysis depends on the ubiquitin-proteasome pathway and SMAD-mediated transcription; however, the molecular mechanism underlying p57(KIP2) degradation has been largely unknown. Here, we show that FBL12, a new F-box protein expressed in the limb bud of developing embryos, is involved in TGF-beta1-induced degradation of p57(KIP2). FBL12 formed an SCF(FBL12) complex and directly ubiquitinated p57(KIP2) in a phosphorylation-dependent manner. Inhibition of FBL12 by RNA interference suppressed the degradation of p57(KIP2) and a dominant-negative mutant of FBL12 (FBL12DeltaF) increased the steady-state level of p57(KIP2). Furthermore, wild-type FBL12 inhibited and FBL12DeltaF promoted the differentiation of primary osteoblasts. As overexpression of p57(KIP2) promoted osteoblast differentiation, these results indicate the importance of FBL12 and the degradation of p57(KIP2) in the regulation of osteoblast cell differentiation.

During pathophysiological muscle wasting, a family of ubiquitin ligases, including muscle RING-finger protein-1 (MuRF1), has been proposed to trigger muscle protein degradation via ubiquitination. Here, we characterized skeletal muscles from wild-type (WT) and MuRF1 knockout (KO) mice under amino acid (AA) deprivation as a model for physiological protein degradation, where skeletal muscles altruistically waste themselves to provide AAs to other organs. When WT and MuRF1 KO mice were fed a diet lacking AA, MuRF1 KO mice were less susceptible to muscle wasting, for both myocardium and skeletal muscles. Under AA depletion, WT mice had reduced muscle protein synthesis, while MuRF1 KO mice maintained nonphysiologically elevated levels of skeletal muscle protein de novo synthesis. Consistent with a role of MuRF1 for muscle protein turnover during starvation, the concentrations of essential AAs, especially branched-chain AAs, in the blood plasma significantly decreased in MuRF1 KO mice under AA deprivation. To clarify the molecular roles of MuRF1 for muscle metabolism during wasting, we searched for MuRF1-associated proteins using pull-down assays and mass spectrometry. Muscle-type creatine kinase (M-CK), an essential enzyme for energy metabolism, was identified among the interacting proteins. Coexpression studies revealed that M-CK interacts with the central regions of MuRF1 including its B-box domain and that MuRF1 ubiquitinates M-CK, which triggers the degradation of M-CK via proteasomes. Consistent with MuRF1's role of adjusting CK activities in skeletal muscles by regulating its turnover in vivo, we found that CK levels were significantly higher in the MuRF1 KO mice than in WT mice. Glucocorticoid modulatory element binding protein-1 and 3-hydroxyisobutyrate dehydrogenase, previously identified as potential MuRF1-interacting proteins, were also ubiquitinated MuRF1-dependently. Taken together, these data suggest that, in a multifaceted manner, MuRF1 participates in the regulation of AA metabolism, including the control of free AAs and their supply to other organs under catabolic conditions, and in the regulation of ATP synthesis under metabolic-stress conditions where MuRF1 expression is induced.

Despite fast protein degradation in muscles, protein concentrations remain constant during differentiation and maintenance of muscle tissues. Myogenin, a basic helix-loop-helix-type myogenic transcription factor, plays a critical role through transcriptional activation in myogenesis as well as muscle maintenance. TBP-interacting protein 120/cullin-associated neddylation-dissociated (TIP120/CAND) is known to bind to cullin and negatively regulate SCF (Skp1-Cullin1-F-box protein) ubiquitin ligase, although its physiological role has not been elucidated. We have identified a muscle-specific isoform of TIP120, named TIP120B/CAND2. In this study, we found that TIP120B is not only induced in association with myogenic differentiation but also actively accelerates the myogenic differentiation of C2C12 cells. Although myogenin is a short lived protein and is degraded by a ubiquitin-proteasome system, TIP120B suppressed its ubiquitination and subsequent degradation of myogenin. TIP120B bound to cullin family proteins, especially Cullin 1 (CUL1), and was associated with SCF complex in cells. It was demonstrated that myogenin was also associated with SCF and that CUL1 small interference RNA treatment inhibited ubiquitination of myogenin and stabilized it. TIP120B was found to break down the SCF-myogenin complex. Consequently suppression of SCF-dependent ubiquitination of myogenin by TIP120B, which leads to stabilization of myogenin, can account for the TIP120B-directed accelerated differentiation of C2C12 cells. TIP120B is proposed to be a novel regulator for myogenesis.

The Y chromosome plays a critical role in spermatogenesis. Formerly, it had been difficult to generate knockout mice with specific Y chromosome mutations using conventional gene-targeting strategies. Recently, a transcription activator-like effector nuclease (TALEN) was successfully used for editing a mouse Y chromosome-linked gene. Here, we report the generation of a mouse model with a mutation in Eif2s3y, a Y chromosome-linked gene, and analysis of its phenotype. The mouse carrying a targeted mutation of Eif2s3y was infertile and had hypoplastic testes. Histological and electron microscopic analyses showed that differentiation of spermatogonia was arrested at the stage of spermatogonial stem cells (undifferentiated spermatogonia) and that the progression of spermatogenesis was interrupted, resulting in azoospermia. Using TALEN, we verified that EIF2S3Y performs a key function in differentiation of spermatogonial stem cells.

The p53 tumour suppressor has a key role in the control of cell growth and differentiation, and in the maintenance of genome integrity1,2. p53 is kept labile under normal conditions, but in response to stresses, such as DNA damage, it accumulates in the nucleus for induction of cell-cycle arrest, DNA repair or apoptosis. Mdm2 is an ubiquitin ligase that promotes p53 ubiquitination and degradation3,4,5. Mdm2 is also self-ubiquitinated and degraded. Here, we identified a novel cascade for the increase in p53 level in response to DNA damage. A new SUMO-specific protease, SUSP4, removed SUMO-1 from Mdm2 and this desumoylation led to promotion of Mdm2 self-ubiquitination, resulting in p53 stabilization. Moreover, SUSP4 competed with p53 for binding to Mdm2, also resulting in p53 stabilization. Overexpression of SUSP4 inhibited cell growth, whereas knockdown of susp4 by RNA interference (RNAi) promoted of cell growth. UV damage induced SUSP4 expression, leading to an increase in p53 levels in parallel with a decrease in Mdm2 levels. These findings establish a new mechanism for the elevation of cellular p53 levels in response to UV damage.

Hsp90 participates in many distinct aspects of cellular functions and accomplishes these roles by interacting with multiple client proteins. To gain insight into the interactions between Hsp90 and its clients, here we have reduced the protein level of Hsp90 in avian cells by gene targeting in an attempt to elicit the otherwise undetectable (because of the vast amount of cellular Hsp90) Hsp90-interacting proteins. Hsp90beta-deficient cells can grow, albeit more slowly than wild-type cells. B cell antigen receptor signaling is multiply impaired in these mutant cells; in particular, the amount of immunoglobulin M heavy chain protein is markedly reduced. Furthermore, serum activation does not promote ERK phosphorylation in Hsp90beta-deficient cells. These multifaceted depressive effects seem to be provoked independently of each other and possibly recapitulate the proteome-wide in vivo functions of Hsp90. Reintroduction of the Hsp90beta gene efficiently restores all of the defects. Unexpectedly, however, introducing the Hsp90alpha gene is also effective in restoration; thus, these defects might be caused by a reduction in the total expression of Hsp90 rather than by loss of Hsp90beta-specific function.

MUT1 is an H-2Kb-restricted 8-mer CTL epitope expressed in Lewis lung carcinoma (3LL) tumor cells derived from C57BL/6 (B6) mice. We constructed a chimeric gene encoding ubiquitin-fused MUT1 (pUB-MUT1). By using a gene gun, B6 mice were immunized with the gene prior to challenge with 3LL tumor cells. Tumor growth and lung metastasis were prominently suppressed in mice immunized with pUB-MUT1 but only slightly in those immunized with the MUT1 gene (pMUT) alone. CD8+ T cells were confirmed to be the final effector by in vitro experiments and in vivo removal of the cells with a corresponding antibody. Anti-tumor immunity was profoundly suppressed in mice deficient in an immuno-subunit of proteasome, LMP7. Furthermore, mice deficient in a proteasome regulator, PA28alpha/beta, failed to acquire protective immunity. Thus, application of the ubiquitin-fusion degradation pathway was useful even in immunization with genes encoding a single CTL epitope for induction of specific and active CD8+ T cells.

Peroxisomes are degraded by autophagic machinery termed "pexophagy" in yeast; however, whether this is essential for peroxisome degradation in mammals remains unknown. Here we have shown that Atg7, an essential gene for autophagy, plays a pivotal role in the degradation of excess peroxisomes in mammals. Following induction of peroxisomes by a 2-week treatment with phthalate esters in control and Atg7-deficient livers, peroxisomal degradation was monitored within 1 week after discontinuation of phthalate esters. Although most of the excess peroxisomes in the control liver were selectively degraded within 1 week, this rapid removal was exclusively impaired in the mutant liver. Furthermore, morphological analysis revealed that surplus peroxisomes, but not mutant hepatocytes, were surrounded by autophagosomes in the control. Our results indicated that the autophagic machinery is essential for the selective clearance of excess peroxisomes in mammals. This is the first direct evidence for the contribution of autophagic machinery in peroxisomal degradation in mammals.

Parkin, a product of the gene responsible for autosomal recessive juvenile parkinsonism (AR-JP), is an important player in the pathogenic process of Parkinson's disease (PD). Despite numerous studies including search for the substrate of parkin as an E3 ubiquitin-protein ligase, the mechanism by which loss-of-function of parkin induces selective dopaminergic neuronal death remains unclear. Related to this issue, here we show that antisense knockdown of parkin causes apoptotic cell death of human dopaminergic SH-SY5Y cells associated with caspase activation and accompanied by accumulation of oxidative dopamine (DA) metabolites due to auto-oxidation of DOPA and DA. Forced expression of alpha-synuclein (alpha-SN), another familial PD gene product, prevented accumulation of oxidative DOPA/DA metabolites and cell death caused by parkin loss. Our findings indicate that both parkin and alpha-SN share a common pathway in DA metabolism whose abnormality leads to accumulation of oxidative DA metabolites and subsequent cell death.

Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to UV irradiation and high incidence of skin cancer caused by inherited defects in DNA repair. Mutational malfunction of damaged-DNA binding protein 2 (DDB2) causes the XP complementation group E (XP-E). DDB2 together with DDB1 comprises a heterodimer called DDB complex, which is involved in damaged-DNA binding and nucleotide excision repair. Interestingly, by screening for a cellular protein(s) that interacts with Cullin 4A (Cul4A), a key component of the ubiquitin ligase complex, we identified DDB1. Immunoprecipitation confirmed that Cul4A interacts with DDB1 and also associates with DDB2. To date, it has been reported that DDB2 is rapidly degraded after UV irradiation and that overproduction of Cul4A stimulates the ubiquitylation of DDB2 in the cells. However, as biochemical analysis using pure Cul4A-containing E3 is missing, it is still unknown whether the Cul4A complex directly ubiquitylates DDB2 or not. We thus purified the Cul4A-containing E3 complex to near homogeneity and attempted to ubiquitylate DDB2 in vitro. The ubiquitylation of DDB2 was reconstituted using this pure E3 complex, indicating that DDB–Cul4A E3 complex in itself can ubiquitylate DDB2 directly. We also showed that an amino acid substitution, K244E, in DDB2 derived from a XP-E patient did not affect its ubiquitylation.

Cancer vaccine that targets 'self'-antigens expressed at high levels in tumor cells is a potentially useful immunotherapy, but immunological tolerance often defeats this strategy. Here, we describe the use of a naked DNA vaccine encoding a self tumor antigen, tyrosinase-related protein 2, to whose N-terminus ubiquitin is fused in a 'nonremovable' fashion. Unlike conventional DNA vaccines, this vaccine broke the tolerance and induced protective immunity to melanoma in C57BL/6 mice, as evaluated by tumor growth, survival rate and lung metastasis. The protective immunity was cancelled in the proteasome activator PA28alpha/beta knockout mice. Moreover, this vaccination exhibited therapeutic effects on melanoma implanted before vaccination. Our findings provide evidence for the first time that naked DNA vaccines encoding a ubiquitin-fused self-antigen preferentially induce the main effector CD8+ T cells through efficient proteolysis mediated by the ubiquitin-proteasome pathway, and lead the way to strategies aimed at targeting tissue differentiation antigens expressed by tumors.

Antitumour immunity against murine melanoma B16 was achieved by genetic immunization with a naked chimeric DNA encoding a fusion protein linking green fluorescent protein (GFP) to the N-terminus of a major CD8(+) cytotoxic T lymphocyte (CTL) epitope of tyrosinase-related protein 2 (TRP-2(181-188)) of murine melanoma, designated as pGFP-TRP-2. Tumour growth was profoundly suppressed in C57BL/6 mice immunized with pGFP-TRP-2, while mice vaccinated with pTRP-2 showed rapid tumour growth and died within 40 days after tumour challenge. Splenocytes of mice immunized with pGFP-TRP-2 showed high CTL activity specific for TRP-2(181-188). GFP-TRP-2 expressed in COS-7 cells was rapidly degradated in vitro and the degradation was almost completely prevented by adding a proteasome inhibitor, MG-132, in the culture. Furthermore, the antimelanoma immunity induced by genetic immunization with pGFP-TRP-2 was completely cancelled in mice deficient in proteasome activator PA28alpha/beta. Taken together, GFP-TRP-2 processed by cytosolic proteasome played a central role in breaking peripheral tolerance to a melanoma/melanocyte antigen, TRP-2(181-188), by activating CD8(+) CTL specific for TRP-2(181-188). TRP-2(181-188) fused to GFP may be readily cut off from GFP by the ubiquitin-fusion degradation (UFD) pathway and efficiently presented to major histocompatibility complex class I molecules, resulting in effective induction of CD8(+) T cells specific for the CTL epitope. Furthermore, CD4(+) T cells specific for GFP were shown to play a crucial role in the antimelanoma immunity, probably potentiating activity of TRP-2-specific CTL and/or the "ubiquitin-proteasome pathway". It is noteworthy to document that genetic immunization with pGFP plus pTRP-2(181-188) failed to exert the antitumour immunity.

Transcription factor Nrf2 is a major regulator of genes encoding phase 2 detoxifying enzymes and antioxidant stress proteins in response to electrophilic agents and oxidative stress. In the absence of such stimuli, Nrf2 is inactive owing to its cytoplasmic retention by Keap1 and rapid degradation through the proteasome system. We examined the contribution of Keap1 to the rapid turnover of Nrf2 (half-life of less than 20 min) and found that a direct association between Keap1 and Nrf2 is required for Nrf2 degradation. In a series of domain function analyses of Keap1, we found that both the BTB and intervening-region (IVR) domains are crucial for Nrf2 degradation, implying that these two domains act to recruit ubiquitin-proteasome factors. Indeed, Cullin 3 (Cul3), a subunit of the E3 ligase complex, was found to interact specifically with Keap1 in vivo. Keap1 associates with the N-terminal region of Cul3 through the IVR domain and promotes the ubiquitination of Nrf2 in cooperation with the Cul3-Roc1 complex. These results thus provide solid evidence that Keap1 functions as an adaptor of Cul3-based E3 ligase. To our knowledge, Nrf2 and Keap1 are the first reported mammalian substrate and adaptor, respectively, of the Cul3-based E3 ligase system.

Several studies have examined the importance of ubiquitin-like posttranslational modifiers (which consist of an unexpectedly large family). Of these, NEDD8 (also called Rub1, related to ubiquitin 1) with a high homology to ubiquitin is covalently linked to all members of cullin (Cul)-family proteins through an enzymatic cascade analogous to ubiquitylation. Cul-family proteins are scaffold proteins for a wide series of ubiquitin-protein ligase complexes, such as SCFs (Skp1, Cul-1, Roc1, and F-box proteins), which regulate the degradation of broad range of cellular proteins. Unlike ubiquitin, which mostly acts as a degradation signal for the target proteins, NEDD8 acts as an activation signal for Cul-family proteins; i.e., Cul-based ubiquitin-protein ligases. Accordingly, the NEDD8 conjugation pathway regulating Cul-protein function is responsible for a diverse array of biologically important processes, such as the cell cycle progression, signalling cascades and developmental programs. Furthermore, recent studies have revealed that the COP9/Signalosome complex interacts physically and genetically with Cul-family proteins, and catalyzes deconjugation of NEDD8 ligated to Cul-family proteins. This review summarizes recent advances in biochemical and genetic studies on how the NEDD8-modifying system regulates Cul-family proteins and their physiology.

PML-RARalpha, a fusion protein of promyelocytic leukemia (PML) and the retinoic acid receptor-alpha (RARalpha), causes acute promyelocytic leukemias (APL). Although the role of nuclear PML-RARalpha has been extensively studied, a significant amount of PML-RARalpha is in the cytoplasm. The role cytoplasmic PML-RARalpha plays in leukemogenesis is unknown. Here we report that PML-RARalpha induces the N-CoR accumulation in the endoplasmic reticulum (ER), leading to the induction of ER stress and the processing of activating transcription factor 6 (ATF6), the unfolded protein response. PML-RARalpha stimulates the ubiquitylation of N-CoR via Ubc6 that is involved in the protein quality control. This ER-associated degradation (ERAD) of N-CoR reduces the soluble N-CoR protein levels in the nucleus. The two N-CoR-interacting sites in PML-RARalpha are required for the ERAD of N-CoR, suggesting the aberrant binding of PML-RARalpha to N-CoR may induce the ERAD of N-CoR. Overexpression of N-CoR induces the differentiation of APL-derived NB4 cells, suggesting that the low levels of N-CoR in the nucleus may contribute at least partly to PML-RARalpha-mediated leukemogenesis.

Ubiquitin (Ub) ligation is implicated in active protein metabolism and subcellular trafficking and its impairment is involved in various neurologic diseases. In rat brain, we identified two novel Ub ligases, Momo and Sakura, carrying double zinc finger motif and RING finger domain. Momo expression is enriched in the brain gray matter and testis, and Sakura expression is more widely detected in the brain white matter as well as in many peripheral organs. Both proteins associate with the cell membranes of neuronal and/or glial cells. We examined their Ub ligase activity in vivo and in vitro using viral expression vectors carrying myc-tagged Momo and Sakura. Overexpression of either Momo or Sakura in mixed cortical cultures increased total polyubiquitination levels. In vitro ubiquitination assay revealed that the combination of Momo and UbcH4 and H5c, or of Sakura and UbcH4, H5c and H6 is required for the reaction. Deletion mutagenesis suggested that the E3 Ub ligase activity of Momo and Sakura depended on their C-terminal domains containing RING finger structure, while their N-terminal domains influenced their membrane association. In agreement, Sakura associating with the membrane was specifically palmitoylated. Although the molecular targets of their Ub ligation remain to be identified, these findings imply a novel function of the palmitoylated E3 Ub ligase(s).

The 26 S proteasome is an evolutionarily conserved ATP-dependent protease complex that degrades poly-ubiquitinated proteins and plays essential roles in a critical part of cellular regulation. In vertebrates, the roles of the proteasome have been widely studied by use of specific inhibitors, but not genetically. Here, we generated a cell line Z(-/-/-)/Z-HA, in which the expression of the catalytic subunit of the proteasome, Z (beta2) could be manipulated. This cell line expresses exogenous Z protein under the control of a tetracycline-repressible promoter in a Z-nullizygous genetic background. Treatment of these cells with doxycycline inhibited Z expression and, hence, the function of the proteasome. The latter resulted in accumulation of poly-ubiquitinated proteins and concomitant induction of molecular chaperones Hsp70 and Hsp40. These results suggest a synergistic role for the proteasome with these molecular chaperones to eliminate misfolded or damaged proteins in vivo. Furthermore, knockdown of the proteasome induced apoptotic cell death following cell-cycle arrest at G(2)/M phase. Our Z(-/-/-)/Z-HA cell line would be useful for evaluating proteolytic processes catalyzed by the proteasome in many biological events in vertebrate cells.

Major histocompatibility complex (MHC) class I ligands are mainly produced by the proteasome. Herein, we show that the processing of antigens is regulated by two distinct pathways, one requiring PA28 and the other hsp90. Both hsp90 and PA28 enhanced the antigen processing of ovalbumin (OVA). Geldanamycin, an inhibitor of hsp90, almost completely suppressed OVA antigen presentation in PA28alpha(-/-)/beta(-/-) lipopolysaccharide blasts, but not in wild-type cells, indicating that hsp90 compensates for the loss of PA28 and is essential in the PA28-independent pathway. In contrast, treatment of cells with interferon (IFN)-gamma, which induces PA28 expression, abrogated the requirement of hsp90, suggesting that IFN-gamma enhances the PA28-dependent pathway, whereas it diminishes hsp90-dependent pathway. Importantly, IFN-gamma did not induce MHC class I expressions in PA28-deficient cells, indicating a prominent role for PA28 in IFN-gamma-stimulated peptide supply. Thus, these two pathways operate either redundantly or specifically, depending on antigen species and cell type.

The mammalian Y chromosome plays a critical role in spermatogenesis. However, the exact functions of each gene in the Y chromosome have not been completely elucidated, partly owing to difficulties in gene targeting analysis of the Y chromosome. Zfy was first proposed to be a sex determination factor, but its function in spermatogenesis has been recently elucidated. Nevertheless, Zfy gene targeting analysis has not been performed thus far. Here, we adopted the highly efficient CRISPR/Cas9 system to generate individual Zfy1 or Zfy2 knockout (KO) mice and Zfy1 and Zfy2 double knockout (Zfy1/2-DKO) mice. While individual Zfy1 or Zfy2-KO mice did not show any significant phenotypic alterations in fertility, Zfy1/2-DKO mice were infertile and displayed abnormal sperm morphology, fertilization failure, and early embryonic development failure. Mass spectrometric screening, followed by confirmation with western blot analysis, showed that PLCZ1, PLCD4, PRSS21, and HTT protein expression were significantly deceased in spermatozoa of Zfy1/2-DKO mice compared with those of wild-type mice. These results are consistent with the phenotypic changes seen in the double-mutant mice. Collectively, our strategy and findings revealed that Zfy1 and Zfy2 have redundant functions in spermatogenesis, facilitating a better understanding of fertilization failure and early embryonic development failure., Author Summary The Y chromosome is known to play a critical role in spermatogenesis based on human genetic evidence. However, specific Y chromosome genes responsible for spermatogenesis have not been fully elucidated yet, because the targeted deletion of Y chromosome genes is difficult owing to its unique structure. In this study, using our highly optimized CRISPR/Cas9 system, we disrupted Zfy1 and Zfy2 ampliconic genes located on the Y chromosome in double-deletion mutant mice. By analyzing the mice, we revealed that Zfy1 and Zfy2 have redundant functions, and that a lack of both Zfy1 and Zfy2 leads to male infertility. Zfy1 and Zfy2 double-deletion mutant mice showed severe abnormalities in their sperm, including defects in morphology, motility, capacitation, acrosome reaction, and oocyte activation, as well as chromosomal aberrations, indicating that Zfy1 and Zfy2 are required for multiple aspects of spermatogenesis. Elucidating the mechanisms that underlie Zfy-dependent spermatogenesis will shed light on the failure of fertilization and early embryonic development in mammals.

NEDD8/Rub1 is a ubiquitin (Ub)-like molecule that covalently ligates to target proteins through an enzymatic cascade analogous to ubiquitylation. This modifier is known to target all cullin (Cul) family proteins. The latter are essential components of Skp1/Cul-1/F-box protein (SCF)–like Ub ligase complexes, which play critical roles in Ub-mediated proteolysis. To determine the role of the NEDD8 system in mammals, we generated mice deficient in Uba3 gene that encodes a catalytic subunit of NEDD8-activating enzyme. Uba3−/− mice died in utero at the periimplantation stage. Mutant embryos showed selective apoptosis of the inner cell mass but not of trophoblastic cells. However, the mutant trophoblastic cells could not enter the S phase of the endoreduplication cycle. This cell cycle arrest was accompanied with aberrant expression of cyclin E and p57Kip2. These results suggested that the NEDD8 system is essential for both mitotic and the endoreduplicative cell cycle progression. β-Catenin, a mediator of the Wnt/wingless signaling pathway, which degrades continuously in the cytoplasm through SCF Ub ligase, was also accumulated in the Uba3−/− cytoplasm and nucleus. Thus, the NEDD8 system is essential for the regulation of protein degradation pathways involved in cell cycle progression and morphogenesis, possibly through the function of the Cul family proteins.

NEDD8/Rub1 is a ubiquitin (Ub)-like post-translational modifier that is covalently linked to cullin (Cul)-family proteins in a manner analogous to ubiquitylation. NEDD8 is known to enhance the ubiquitylating activity of the SCF complex (composed of Skp1, Cul-1, ROC1 and F-box protein), but the mechanistic role is largely unknown. Using an in vitro reconstituted system, we report here that NEDD8 modification of Cul-1 enhances recruitment of Ub-conjugating enzyme Ubc4 (E2) to the SCF complex (E3). This recruitment requires thioester linkage of Ub to Ubc4. Our findings indicate that the NEDD8-modifying system accelerates the formation of the E2-E3 complex, which stimulates protein polyubiquitylation.

Smads are signal mediators for the members of the transforming growth factor-β (TGF-β) superfamily. Upon phosphorylation by the TGF-β receptors, Smad3 translocates into the nucleus, recruits transcriptional coactivators and corepressors, and regulates transcription of target genes. Here, we show that Smad3 activated by TGF-β is degraded by the ubiquitin–proteasome pathway. Smad3 interacts with a RING finger protein, ROC1, through its C-terminal MH2 domain in a ligand-dependent manner. An E3 ubiquitin ligase complex ROC1-SCFFbw1aconsisting of ROC1, Skp1, Cullin1, and Fbw1a (also termed βTrCP1) induces ubiquitination of Smad3. Recruitment of a transcriptional coactivator, p300, to nuclear Smad3 facilitates the interaction with the E3 ligase complex and triggers the degradation process of Smad3. Smad3 bound to ROC1-SCFFbw1ais then exported from the nucleus to the cytoplasm for proteasomal degradation. TGF-β/Smad3 signaling is thus irreversibly terminated by the ubiquitin–proteasome pathway.

Smad7 is an inhibitory Smad that acts as a negative regulator of signaling by the transforming growth factor-beta (TGF-beta) superfamily proteins. Smad7 is induced by TGF-beta, stably interacts with activated TGF-beta type I receptor (TbetaR-I), and interferes with the phosphorylation of receptor-regulated Smads. Here we show that Smurf1, an E3 ubiquitin ligase for bone morphogenetic protein-specific Smads, also interacts with Smad7 and induces Smad7 ubiquitination and translocation into the cytoplasm. In addition, Smurf1 associates with TbetaR-I via Smad7, with subsequent enhancement of turnover of TbetaR-I and Smad7. These results thus reveal a novel function of Smad7, i.e. induction of degradation of TbetaR-I through recruitment of an E3 ligase to the receptor.

The 26S proteasome is a multisubunit protein- destroying machinery that degrades ubiquitin-tagged proteins. To date only a single species of Rpn10, which possibly functions as a multiubiquitin chain-binding subunit, has been identified in various organisms. Here we report that mouse Rpn10 mRNAs occur in at least five distinct forms, named Rpn10a to Rpn10e, and that they are generated from a single gene by developmentally regulated, alternative splicing. Rpn10a is ubiquitously expressed, whereas Rpn10e is expressed only in embryos, with the highest levels of expression in the brain. Both forms of Rpn10 are components of the 26S proteasome, with an apparently similar affinity for multiubiquitylated [(125)I]lysozyme in vitro. However, they exert markedly divergent effects on the destruction of B-type cyclin in Xenopus egg extracts. Thus, the 26S proteasome occurs in at least two functionally distinct forms: one containing a ubiquitously expressed Rpn10a and the other a newly identified, embryo-specific Rpn10e. While the former is thought to perform proteolysis constitutively in a wide variety of cells, the latter may play a specialized role in early embryonic development.

A ubiquitin-like modifier, NEDD8, is covalently attached to cullin-family proteins, but its physiological role is poorly understood. Here we report that the NEDD8-modifying pathway is essential for cell viability and function of Pcu1 (cullin-1 orthologue) in fission yeast. Pcu1 assembled on SCF ubiquitin-ligase was completely modified by NEDD8. Pcu1(K713R) defective for NEDD8 conjugation lost the ability to complement lethality due to pcu1 deletion. Forced expression of Pcu1(K713R) or depletion of NEDD8 in cells resulted in impaired cell proliferation and marked stabilization of the cyclin-dependent kinase inhibitor Rum1, which is a substrate of the SCF complex. Based on these findings, we propose that covalent modification of cullin-1 by the NEDD8 system plays an essential role in the function of SCF in fission yeast.

We found previously that overexpression of an F-box protein betaTrCP1 and the structurally related betaTrCP2 augments ubiquitination of phosphorylated IkappaBalpha (pIkappaBalpha) induced by tumor necrosis factor-alpha (TNF-alpha), but the relationship of the two homologous betaTrCP proteins remains unknown. Herein we reveal that deletion mutants of betaTrCP1 and betaTrCP2 lacking the F-box domain suppressed ubiquitination and destruction of pIkappaBalpha as well as transcriptional activation of NF-kappaB. The ectopically expressed betaTrCP1 and betaTrCP2 formed both homodimer and heterodimer complexes without displaying the trimer complex. Dimerization of betaTrCP1 and/or betaTrCP2 takes place at their conserved NH(2)-terminal regions, termed a "D-domain" (for dimerization domain), located upstream of the F-box domain. The D-domain was necessary and sufficient for the dimer formation. Intriguingly, the betaTrCP homodimer, but not the heterodimer, was selectively recruited to pIkappaBalpha induced by TNF-alpha. These results indicate that not only betaTrCP1 but also betaTrCP2 participates in the ubiquitination-dependent destruction of IkappaBalpha by forming SCF(betaTrCP1-betaTrCP1) and SCF(betaTrCP2-betaTrCP2) ubiquitin-ligase complexes.

The proteasome activator PA28 binds to both ends of the central catalytic machine, known as the 20 S proteasome, in opposite orientations to form the enzymatically active proteasome. The PA28 family is composed of three members designated alpha, beta, and gamma; PA28alpha and PA28beta form the heteropolymer mainly located in the cytoplasm, whereas PA28gamma forms a homopolymer that predominantly occurs in the nucleus. Available evidence indicates that the heteropolymer of PA28alpha and PA28beta is involved in the processing of intracellular antigens, but the function of PA28gamma remains elusive. To investigate the role of PA28gamma in vivo, we generated mice deficient in the PA28gamma gene. The PA28gamma-deficient mice were born without appreciable abnormalities in all tissues examined, but their growth after birth was retarded compared with that of PA28gamma(+/-) or PA28gamma(+/+) mice. We also investigated the effects of the PA28gamma deficiency using cultured embryonic fibroblasts; cells lacking PA28gamma were larger and displayed a lower saturation density than their wild-type counterparts. Neither the expression of PA28alpha/beta nor the subcellular localization of PA28alpha was affected in PA28gamma(-/-) cells. These results indicate that PA28gamma functions as a regulator of cell proliferation and body growth in mice and suggest that neither PA28alpha nor PA28beta compensates for the PA28gamma deficiency.

Activation of the transcriptional factor NF-kappaB is triggered by signal-dependent degradation of its inhibitor protein IkappaB through the ubiquitin (Ub)-proteasome pathway. We found here that a phosphorylated IkappaBalpha immunoprecipitated (IP-pIkappaBalpha) from the crude extract of HeLa cells which had been treated with tumor necrosis factor-alpha (TNFalpha) caused a dramatic ubiquitination of itself, termed autoubiquitination, when incubated with ATP, Ub, and E1-activating and E2-conjugating enzymes. IP-pIkappaBalpha also catalyzed ubiquitination of an in vitro synthesized 35S-IkappaBalpha previously phosphorylated by IkappaB-kinase (IKK) which is referred to as transubiquitination. No appreciable activity of auto- and transubiquitination was observed in an unphosphorylated IP-IkappaBalpha. Moreover, the putative IkappaBalpha-Ub ligase (IkappaBalpha-E3) present in HeLa cell cytosol associated in vitro with an IKK-phosphorylated recombinant IkappaBalpha, a process independent of NF-kappaB binding to IkappaBalpha or TNFalpha stimulation. Replacement of the two Ser residues at positions 32 and 36 corresponding to IKK phosphorylation sites by Ala resulted in almost complete prevention of binding of an IkappaBalpha-E3 to IkappaBalpha. These results indicate that phosphorylation of IkappaBalpha is necessary and sufficient for recruitment of this IkappaBalpha-E3 to associate with IkappaBalpha.

Destruction of the transcriptional inhibitor IkappaB by the ubiquitin (Ub) system is required for signal-dependent activation of the multifunctional transcriptional factor NF-kappaB, but details of this ubiquitination are largely unknown. We report here that the IkappaBalpha-ubiquitin ligase (IkappaBalpha-E3) is an SCF-like complex containing Skp1, cullin-1, and two homologous F-box/WD40-repeat proteins, betaTrCP1 and betaTrCP2. Intriguingly, all these components are cooperatively recruited to bind to a phosphorylated IkappaBalpha (pIkappaBalpha) produced by tumor necrosis factor-alpha (TNF-alpha) stimulation. IkappaBalpha-E3 bound to pIkappaBalpha catalyzed in vitro ubiquitination of pIkappaBalpha in the presence of ATP, Ub, and E1-activating and E2-conjugating enzymes. Forced expression of betaTrCP1 and betaTrCP2 resulted in dramatic augmentation of the in vitro polyubiquitination activity of IkappaBalpha-E3. These results indicate that the long-sought IkappaBalpha-E3 is an SCF-like complex consisting of multiple proteins which are coordinately assembled during phosphorylation of IkappaBalpha in response to external signals.

The induction of phase II detoxifying enzymes is an important defense mechanism against intake of xenobiotics. While this group of enzymes is believed to be under the transcriptional control of antioxidant response elements (AREs), this contention is experimentally unconfirmed. Since the ARE resembles the binding sequence of erythroid transcription factor NF-E2, we investigated the possibility that the phase II enzyme genes might be regulated by transcription factors that also bind to the NF-E2 sequence. The expression profiles of a number of transcription factors suggest that an Nrf2/small Maf heterodimer is the most likely candidate to fulfill this role in vivo. To directly test these questions, we disrupted the murine nrf2 gene in vivo. While the expression of phase II enzymes (e.g., glutathione S-transferase and NAD(P)H: quinone oxidoreductase) was markedly induced by a phenolic antioxidant in vivo in both wild type and heterozygous mutant mice, the induction was largely eliminated in the liver and intestine of homozygous nrf2-mutant mice. Nrf2 was found to bind to the ARE with high affinity only as a heterodimer with a small Maf protein, suggesting that Nrf2/small Maf activates gene expression directly through the ARE. These results demonstrate that Nrf2 is essential for the transcriptional induction of phase II enzymes and the presence of a coordinate transcriptional regulatory mechanism for phase II enzyme genes. The nrf2-deficient mice may prove to be a very useful model for the in vivo analysis of chemical carcinogenesis and resistance to anti-cancer drugs.

Nucleolus is a dynamic structure that controls biogenesis of ribosomal RNA and senses cellular stresses. Nucleolus contains a number of proteins including ribosomal proteins that conduct cellular stresses to downstream signaling such as p53 pathway. Recently, it has been reported that modification by a ubiquitin-like molecule, Nedd8, regulates subnuclear localization of ribosomal protein L11. Most of L11 is normally localized and neddylated in nucleolus. However, cellular stress triggers deneddylation and redistribution of L11, and subsequent activation of p53. Although Nedd8 modification is thought to be important for L11 localization, the mechanism of how neddylation of L11 is regulated remains largely unknown. Here, we show that Myeloma overexpressed 2 (Myeov2) controls L11 localization through down-regulation of Nedd8 modification. Expression of Myeov2 reduced neddylation of proteins including L11. We also found that Myeov2 associates with L11 and withholds L11 in nucleoplasm. Although Myeov2 interacted with a Nedd8 deconjugation enzyme COP9 signalosome, L11 deneddylation was mediated by another deneddylase Nedp1, independently of Myeov2. Finally, p53 transcriptional activity is upregulated by Myeov2 expression. These data demonstrate that Myeov2 hampers L11 neddylation through their interactions and confines L11 to nucleoplasm to modulate nucleolar integrity. Our findings provide a novel link between oncogenic stress and p53 pathway and may shed light on the protective mechanism against cancer.