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Geminin and its binding partner Cdt1 are essential for the regulation of DNA replication. Here we show that the CULLIN3 E3 ubiquitin ligase adaptor protein SPOP binds Geminin at endogenous level and regulates DNA replication. SPOP promotes K27-linked non-degradative poly-ubiquitination of Geminin at lysine residues 100 and 127. This poly-ubiquitination of Geminin prevents DNA replication over-firing by indirectly blocking the association of Cdt1 with the MCM protein complex, an interaction required for DNA unwinding and replication. SPOP is frequently mutated in certain human cancer types and implicated in tumorigenesis. We show that cancer-associated SPOP mutations impair Geminin K27-linked poly-ubiquitination and induce replication origin over-firing and re-replication. The replication stress caused by SPOP mutations triggers replication catastrophe and cell death upon ATR inhibition. Our results reveal a tumor suppressor role of SPOP in preventing DNA replication over-firing and genome instability and suggest that SPOP-mutated tumors may be susceptible to ATR inhibitor therapy., (© 2021. The Author(s).)

Ubiquitin-specific protease 1 (USP1) acts together with the cofactor UAF1 during DNA repair processes to specifically remove monoubiquitin signals. One substrate of the USP1-UAF1 complex is the monoubiquitinated FANCI-FANCD2 heterodimer, which is involved in the repair of DNA interstrand crosslinks via the Fanconi anemia pathway. Here we determine structures of human USP1-UAF1 with and without ubiquitin and bound to monoubiquitinated FANCI-FANCD2. The crystal structures of USP1-UAF1 reveal plasticity in USP1 and key differences to USP12-UAF1 and USP46-UAF1, two related proteases. A cryo-EM reconstruction of USP1-UAF1 in complex with monoubiquitinated FANCI-FANCD2 highlights a highly orchestrated deubiquitination process, with USP1-UAF1 driving conformational changes in the substrate. An extensive interface between UAF1 and FANCI, confirmed by mutagenesis and biochemical assays, provides a molecular explanation for the requirement of both proteins, despite neither being directly involved in catalysis. Overall, our data provide molecular details of USP1-UAF1 regulation and substrate recognition.

The Wnt/β-catenin pathway plays vital roles in diverse biological processes, including cell differentiation, proliferation, migration, and insulin sensitivity. A recent study reported that the DNA-binding transcriptional factor SIX3 is essential during embryonic development in vertebrates and capable of downregulating target genes of the Wnt/β-catenin pathway in lung cancer, indicating negative regulation of Wnt/β-catenin activation. However, regulation of the SIX3-Wnt/β-catenin pathway axis remains unknown. We measured the expression of TRIM27 and SIX3 as well as investigated whether there was a correlation between them in lung cancer tissue samples. Herein, we found that the E3 ubiquitin ligase, TRIM27, ubiquitinates, and degrades SIX3. TRIM27 induces non-small cell lung cancer (NSCLC) cell proliferation and metastasis, and the expression of β-catenin, S100P, TGFB3, and MMP-9 were significantly inhibited by SIX3. Furthermore, XAV939 is a selective β-catenin-mediated transcription inhibitor that inhibited TRIM27- and SIX3-mediated NSCLC cell proliferation, migration, and invasion. Clinically, lung tissue samples of cancer patients showed increased TRIM27 expression and decreased SIX3 expression. Taken together, these data demonstrate that TRIM27 acts as an oncogene regulating cell proliferation and metastasis in NSCLC through SIX3-β-catenin signaling.

Among the hallmarks of melanoma are impaired proteostasis and rapid development of resistance to targeted therapy that represent a major clinical challenge. However, the molecular machinery that links these processes is unknown. Here we describe that by stabilizing key melanoma oncoproteins, the ubiquitin ligase RNF4 promotes tumorigenesis and confers resistance to targeted therapy in melanoma cells, xenograft mouse models, and patient samples. In patients, RNF4 protein and mRNA levels correlate with poor prognosis and with resistance to MAPK inhibitors. Remarkably, RNF4 tumorigenic properties, including therapy resistance, require the translation initiation factor initiation elongation factor alpha (eIF2α). RNF4 binds, ubiquitinates, and stabilizes the phosphorylated eIF2α (p-eIF2α) but not activating transcription factor 4 or C/EBP homologous protein that mediates the eIF2α-dependent integrated stress response. In accordance, p-eIF2α levels were significantly elevated in high-RNF4 patient-derived melanomas. Thus, RNF4 and p-eIF2α establish a positive feed-forward loop connecting oncogenic translation and ubiquitin-dependent protein stabilization in melanoma., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Tubular damage initiated by inflammatory response and ischemic/hypoxic stress is a hallmark of septic acute kidney injury (AKI), albeit the molecular mechanism coupling the two events remains unclear. We investigated the intrinsic nature of tubular damage with respect to inflammatory/hypoxic stress during septic AKI. Methods: The apoptotic response of tubular cells to LPS stimuli was analyzed before and after hypoxia exposure. Cellular ubiquitination, co-immunoprecipitation, GST-pulldown, in vitro protein kinase assay, immunofluorescence and CRISPR technology were adopted to determine the molecular mechanism underlying this process. In vivo characterization was performed in wild-type and DAPK1 -/- mice models of cecal ligation and puncture (CLP). Results: We found that the MyD88-dependent inflammatory response couples to tubular damage during LPS stimuli under hypoxia in a Fn14/SCF Fbxw7α -dispensable manner via recruitment of caspase-8 with TRIF-RIP1 signalosome mediated by DAPK1, which directly binds to and phosphorylates Pellino1 at Ser39, leading to Pellino1 poly-ubiquitination and turnover. Either pharmacological deactivation or genetic ablation of DAPK1 makes tubular cells refractory to the LPS-induced damage in the context of hypoxia, while kinase activity of DAPK1 is essential for ruin execution. Targeting DAPK1 effectively protects mice against septic AKI and potentiates the efficacy of a MyD88 homodimerization inhibitor, ST2825. Conclusion: Our findings provide a rationale for the mechanism whereby inflammation intersects with hypoxic tubular damage during septic AKI through a previously unappreciated role of DAPK1-inducible Ser39 phosphorylation in Pellino1 turnover and underscore that combined targeting DAPK1 and MyD88 might be a feasible strategy for septic AKI management., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Structure-specific endonucleases (SSEs) play key roles in DNA replication, recombination, and repair. SSEs must be tightly regulated to ensure genome stability but their regulatory mechanisms remain incompletely understood. Here, we show that in the fission yeast Schizosaccharomyces pombe, the activities of two SSEs, Dna2 and Rad16 (ortholog of human XPF), are temporally controlled during the cell cycle by the CRL4Cdt2 ubiquitin ligase. CRL4Cdt2 targets Pxd1, an inhibitor of Dna2 and an activator of Rad16, for degradation in S phase. The ubiquitination and degradation of Pxd1 is dependent on CRL4Cdt2, PCNA, and a PCNA-binding degron motif on Pxd1. CRL4Cdt2-mediated Pxd1 degradation prevents Pxd1 from interfering with the normal S-phase functions of Dna2. Moreover, Pxd1 degradation leads to a reduction of Rad16 nuclease activity in S phase, and restrains Rad16-mediated single-strand annealing, a hazardous pathway of repairing double-strand breaks. These results demonstrate a new role of the CRL4Cdt2 ubiquitin ligase in genome stability maintenance and shed new light on how SSE activities are regulated during the cell cycle., Competing Interests: The authors have declared that no competing interests exist.

Noradrenaline belongs to the monoamine system and is involved in cognition and emotional behaviors. Phox2a and Phox2b play essential but non-redundant roles during development of the locus coeruleus (LC), the main noradrenergic (NA) neuron center in the mammalian brain. The ubiquitin E3 ligase Rnf220 and its cofactor Zc4h2 participate in ventral neural tube patterning by modulating Shh/Gli signaling, and ZC4H2 mutation is associated with intellectual disability, although the mechanisms for this remain poorly understood. Here, we report that Zc4h2 and Rnf220 are required for the development of central NA neurons in the mouse brain. Both Zc4h2 and Rnf220 are expressed in developing LC-NA neurons. Although properly initiated at E10.5, the expression of genes associated with LC-NA neurons is not maintained at the later embryonic stages in mice with a deficiency of either Rnf220 or Zc4h2 In addition, we show that the Rnf220/Zc4h2 complex monoubiquitylates Phox2a/Phox2b, a process required for the full transcriptional activity of Phox2a/Phox2b. Our work reveals a role for Rnf220/Zc4h2 in regulating LC-NA neuron development, and this finding may be helpful for understanding the pathogenesis of ZC4H2 mutation-associated intellectual disability., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

A significant fraction (∼10%) of cancer cells maintain their telomere length via a telomerase-independent mechanism known as alternative lengthening of telomeres (ALT). There are no known molecular, ALT-specific, therapeutic targets. We have identified TSPYL5 (testis-specific Y-encoded-like protein 5) as a PML body component, co-localizing with ALT telomeres and critical for ALT + cell viability. TSPYL5 was described as an inhibitor of the USP7 deubiquitinase. We report that TSPYL5 prevents the poly-ubiquitination of POT1-a shelterin component-and protects POT1 from proteasomal degradation exclusively in ALT + cells. USP7 depletion rescued POT1 poly-ubiquitination and loss, suggesting that the deubiquitinase activates POT1 E3 ubiquitin ligase(s). Similarly, PML depletion suppressed POT1 poly-ubiquitination, suggesting an interplay between USP7 and PML to trigger POT1 degradation in TSPYL5-depleted ALT + cells. We demonstrate that ALT telomeres need to be protected from POT1 degradation in ALT-associated PML bodies and identify TSPYL5 as an ALT + cancer-specific therapeutic target., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Coordination of the cellular response to DNA damage is organised by multi-domain 'scaffold' proteins, including 53BP1 and TOPBP1, which recognise post-translational modifications such as phosphorylation, methylation and ubiquitylation on other proteins, and are themselves carriers of such regulatory signals. Here we show that the DNA damage checkpoint regulating S-phase entry is controlled by a phosphorylation-dependent interaction of 53BP1 and TOPBP1. BRCT domains of TOPBP1 selectively bind conserved phosphorylation sites in the N-terminus of 53BP1. Mutation of these sites does not affect formation of 53BP1 or ATM foci following DNA damage, but abolishes recruitment of TOPBP1, ATR and CHK1 to 53BP1 damage foci, abrogating cell cycle arrest and permitting progression into S-phase. TOPBP1 interaction with 53BP1 is structurally complimentary to its interaction with RAD9-RAD1-HUS1, allowing these damage recognition factors to bind simultaneously to the same TOPBP1 molecule and cooperate in ATR activation in the G1 DNA damage checkpoint., Competing Interests: NB, MD, RB, FW, AO, LP No competing interests declared, (© 2019, Bigot et al.)

TP53INP2 positively regulates autophagy by binding to Atg8 proteins. Here, we uncover a novel role of TP53INP2 in death-receptor signaling. TP53INP2 sensitizes cells to apoptosis induced by death receptor ligands. In keeping with this, TP53INP2 deficiency in cultured cells or mouse livers protects against death receptor-induced apoptosis. TP53INP2 binds caspase-8 and the ubiquitin ligase TRAF6, thereby promoting the ubiquitination and activation of caspase-8 by TRAF6. We have defined a TRAF6-interacting motif (TIM) and a ubiquitin-interacting motif in TP53INP2, enabling it to function as a scaffold bridging already ubiquitinated caspase-8 to TRAF6 for further polyubiquitination of caspase-8. Mutations of key TIM residues in TP53INP2 abrogate its interaction with TRAF6 and caspase-8, and subsequently reduce levels of death receptor-induced apoptosis. A screen of cancer cell lines showed that those with higher protein levels of TP53INP2 are more prone to TRAIL-induced apoptosis, making TP53INP2 a potential predictive marker of cancer cell responsiveness to TRAIL treatment. These findings uncover a novel mechanism for the regulation of caspase-8 ubiquitination and reveal TP53INP2 as an important regulator of the death receptor pathway., (© 2019 The Authors.)

TRAF-binding domain (Trabid), one of deubiquitination enzymes, was recently reported to activate Wnt/ β-catenin signaling pathway. However, the role of Trabid in tumors including hepatocellular carcinoma (HCC) and the underlying mechanisms controlling its activity remain poorly understood. Here, we report that Trabid is significantly downregulated in HCC tumor samples and cell lines compared with normal controls and that its expression level is negatively correlated with HCC pathological grading, recurrence, and metastasis. The reintroduction of Trabid expression in tumor cells significantly decreases HCC progression as well as pulmonary metastasis. The effect of Trabid on HCC development occurs at least partially through regulation of Twist1 activity. Mechanistically, Trabid forms a complex with Twist1 and specifically cleaves RNF8-induced K63-linked poly-ubiquitin chains from Twist1, which enhances the association of Twist1 with β-TrCP1 and allows for subsequent K48-linked ubiquitination of Twist1. Knockdown of Trabid increases K63-linked ubiquitination, but abrogates K48-linked ubiquitination and degradation of Twist1, thus enhancing HCC growth and metastasis. Interestingly, Twist1 negatively regulates the promoter activity of Trabid, indicating that a double-negative feedback loop exists. Our findings also identify an essential role for activation of Trabid by AKT-mediated phosphorylation at Ser78/Thr117 in negatively regulating Twist1 signaling, which further provides insights into the mechanisms by which Trabid regulates Twist1 ubiquitination. Our results reveal that Trabid is a previously unrecognized inhibitor of HCC progression and metastasis, which sheds light on new strategies for HCC treatment.

Spatial management of stress-induced protein aggregation is an integral part of the proteostasis network. Protein modification by the ubiquitin-like molecule NEDD8 increases upon proteotoxic stress and it is characterised by the formation of hybrid NEDD8/ubiquitin conjugates. However, the biological significance of this response is unclear. Combination of quantitative proteomics with biological analysis shows that, during proteotoxic stress, NEDDylation promotes nuclear protein aggregation, including ribosomal proteins as a major group. This correlates with protection of the nuclear Ubiquitin Proteasome System from stress-induced dysfunction. Correspondingly, we show that NEDD8 compromises ubiquitination and prevents targeting and processing of substrates by the proteasome. Moreover, we identify HUWE1 as a key E3-ligase that is specifically required for NEDDylation during proteotoxic stress. The study reveals a specific role for NEDD8 in nuclear protein aggregation upon stress and is consistent with the concept that transient aggregate formation is part of a defence mechanism against proteotoxicity.

Mutations in the tumor suppressor SPOP (speckle-type POZ protein) cause prostate, breast, and other solid tumors. SPOP is a substrate adaptor of the cullin3-RING ubiquitin ligase and localizes to nuclear speckles. Although cancer-associated mutations in SPOP interfere with substrate recruitment to the ligase, mechanisms underlying assembly of SPOP with its substrates in liquid nuclear bodies and effects of SPOP mutations on assembly are poorly understood. Here, we show that substrates trigger phase separation of SPOP in vitro and co-localization in membraneless organelles in cells. Enzymatic activity correlates with cellular co-localization and in vitro mesoscale assembly formation. Disease-associated SPOP mutations that lead to the accumulation of proto-oncogenic proteins interfere with phase separation and co-localization in membraneless organelles, suggesting that substrate-directed phase separation of this E3 ligase underlies the regulation of ubiquitin-dependent proteostasis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Histone acetylation influences protein interactions and chromatin accessibility and plays an important role in the regulation of transcription, replication, and DNA repair. Conversely, DNA damage affects these crucial cellular processes and induces changes in histone acetylation. However, a comprehensive overview of the effects of DNA damage on the histone acetylation landscape is currently lacking. To quantify changes in histone acetylation, we developed an unbiased quantitative mass spectrometry analysis on affinity-purified acetylated histone peptides, generated by differential parallel proteolysis. We identify a large number of histone acetylation sites and observe an overall reduction of acetylated histone residues in response to DNA damage, indicative of a histone-wide loss of acetyl modifications. This decrease is mainly caused by DNA damage-induced replication stress coupled to specific proteasome-dependent loss of acetylated histones. Strikingly, this degradation of acetylated histones is independent of ubiquitylation but requires the PA200-proteasome activator, a complex that specifically targets acetylated histones for degradation. The uncovered replication stress-induced degradation of acetylated histones represents an important chromatin-modifying response to cope with replication stress., (© 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, retarded growth, and malabsorption syndrome. The ARV p10 protein, a viroporin responsible for the induction of cell syncytium formation and apoptosis, is rapidly degraded in host cells. Our previous report demonstrated that cellular lysosome-associated membrane protein 1 (LAMP-1) interacted with p10 and was involved in its degradation. However, the molecular mechanism underlying LAMP-1-mediated p10 degradation remains elusive. We report here that the E3 ubiquitin ligase seven in absentia homolog 1 (Siah-1) is critical for p10 ubiquitylation. Our data show that Siah-1 ubiquitylated p10 and targeted it for proteasome degradation. Furthermore, the ubiquitylation of p10 by Siah-1 required the participation of LAMP-1 by forming a multicomponent complex. Thus, LAMP-1 promotes the proteasomal degradation of p10 via interacting with both p10 and the E3 ligase Siah-1. These data establish a novel host defense mechanism where LAMP-1 serves as a scaffold for both Siah-1 and p10 that allows the E3 ligase targeting p10 for ubiquitylation and degradation to suppress ARV infection. IMPORTANCE Avian reovirus (ARV) is an important poultry pathogen causing viral arthritis, chronic respiratory diseases, retarded growth, and malabsorption syndrome, leading to considerable economic losses to the poultry industry across the globe. The ARV p10 protein is a virulence factor responsible for the induction of cell syncytium formation and apoptosis and is rapidly degraded in host cells. We previously found that cellular lysosome-associated membrane protein 1 (LAMP-1) interacts with p10 and is involved in its degradation. Here we report that the E3 ubiquitin ligase seven in absentia homolog 1 (Siah-1) ubiquitylated p10 and targeted it for proteasomal degradation. Furthermore, the ubiquitylation of p10 by Siah-1 required the participation of LAMP-1 by forming a multicomponent complex. Thus, LAMP-1 serves as an adaptor to allow Siah-1 to target p10 for degradation, thereby suppressing ARV growth in host cells., (Copyright © 2018 American Society for Microbiology.)

Androgen receptor (AR) activation is critical for prostate cancer (PCa) development and progression, including castration resistance. The nuclear export signal of AR (NES AR ) has an important role in AR intracellular trafficking and proteasome-dependent degradation. Here, we identified the RNA helicase DHX15 as a novel AR co-activator using a yeast mutagenesis screen and revealed that DHX15 regulates AR activity by modulating E3 ligase Siah2-mediated AR ubiquitination independent of its ATPase activity. DHX15 and Siah2 form a complex with AR, through NES AR . DHX15 stabilized Siah2 and enhanced its E3 ubiquitin-ligase activity, resulting in AR activation. Importantly, DHX15 was upregulated in PCa specimens and its expression was correlated with Gleason scores and prostate-specific antigen recurrence. Furthermore, DHX15 immunostaining correlated with Siah2. Finally, DHX15 knockdown inhibited the growth of C4-2 prostate tumor xenografts in mice. Collectively, our data argue that DHX15 enhances AR transcriptional activity and contributes to PCa progression through Siah2.

IRF5 is a signature transcription factor that induces M1 macrophage polarization. However, little is known regarding cytosolic proteins that induce IRF5 activation for M1 polarization. Here, we report the interaction between ubiquitin E3 ligase Pellino-1 and IRF5 in the cytoplasm, which increased nuclear translocation of IRF5 by K63-linked ubiquitination in human and mouse M1 macrophages. LPS and/or IFN-γ increased Pellino-1 expression, and M1 polarization was attenuated in Pellino-1-deficient macrophages in vitro and in vivo. Defective M1 polarization in Pellino-1-deficient macrophages improved glucose intolerance in mice fed a high-fat diet. Furthermore, macrophages in adipose tissues from obese humans exhibited increased Pellino-1 expression and IRF5 nuclear translocation compared with nonobese subjects, and these changes are associated with insulin resistance index. This study demonstrates that cytosolic Pellino-1-mediated K63-linked ubiquitination of IRF5 in M1 macrophages regulates glucose intolerance in obesity, suggesting a cytosolic mediator function of Pellino-1 in TLR4/IFN-γ receptor-IRF5 axis during M1 polarization., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

RanBPM (Ran-binding protein M, also called RanBP9) is an evolutionarily conserved, ubiquitous protein which localizes to both nucleus and cytoplasm. RanBPM has been implicated in the regulation of a number of signalling pathways to regulate several cellular processes such as apoptosis, cell adhesion, migration as well as transcription, and plays a critical role during development. In addition, RanBPM has been shown to regulate pathways implicated in cancer and Alzheimer's disease, implying that RanBPM has important functions in both normal and pathological development. While its functions in these processes are still poorly understood, RanBPM has been identified as a component of a large complex, termed the CTLH (C-terminal to LisH) complex. The yeast homologue of this complex functions as an E3 ubiquitin ligase that targets enzymes of the gluconeogenesis pathway. While the CTLH complex E3 ubiquitin ligase activity and substrates still remain to be characterized, the high level of conservation between the complexes in yeast and mammals infers that the CTLH complex could also serve to promote the degradation of specific substrates through ubiquitination, therefore suggesting the possibility that RanBPM's various functions may be mediated through the activity of the CTLH complex., (© 2017 The Authors.)

The signaling adaptor MAVS forms prion-like aggregates to activate an innate antiviral immune response after viral infection. However, the molecular mechanisms that regulate MAVS aggregation are poorly understood. Here we identified TRIM31, an E3 ubiquitin ligase of the TRIM family of proteins, as a regulator of MAVS aggregation. TRIM31 was recruited to mitochondria after viral infection and specifically regulated antiviral signaling mediated by RLR pattern-recognition receptors. TRIM31-deficient mice were more susceptible to infection with RNA virus than were wild-type mice. TRIM31 interacted with MAVS and catalyzed the Lys63 (K63)-linked polyubiquitination of Lys10, Lys311 and Lys461 on MAVS. This modification promoted the formation of prion-like aggregates of MAVS after viral infection. Our findings reveal new insights in the molecular regulation of MAVS aggregation and the cellular antiviral response through TRIM31-mediated K63-linked polyubiquitination of MAVS.

Human CtIP is a decisive factor in DNA double-strand break repair pathway choice by enabling DNA-end resection, the first step that differentiates homologous recombination (HR) from non-homologous end-joining (NHEJ). To coordinate appropriate and timely execution of DNA-end resection, CtIP function is tightly controlled by multiple protein-protein interactions and post-translational modifications. Here, we identify the Cullin3 E3 ligase substrate adaptor Kelch-like protein 15 (KLHL15) as a new interaction partner of CtIP and show that KLHL15 promotes CtIP protein turnover via the ubiquitin-proteasome pathway. A tripeptide motif (FRY) conserved across vertebrate CtIP proteins is essential for KLHL15-binding; its mutation blocks KLHL15-dependent CtIP ubiquitination and degradation. Consequently, DNA-end resection is strongly attenuated in cells overexpressing KLHL15 but amplified in cells either expressing a CtIP-FRY mutant or lacking KLHL15, thus impacting the balance between HR and NHEJ. Collectively, our findings underline the key importance and high complexity of CtIP modulation for genome integrity.

Staphylococcal nuclease and tudor domain containing 1 (SND1) is overexpressed in multiple cancers, including hepatocellular carcinoma (HCC), and functions as an oncogene. This study was carried out to identify novel SND1-interacting proteins to better understand its molecular mechanism of action. SND1-interacting proteins were identified by a modified yeast two-hybrid assay. Protein-protein interaction was confirmed by co-immunoprecipitation analysis. Monoglyceride lipase (MGLL) expression was analyzed by quantitative RT-PCR, Western blot, and immunohistochemistry. MGLL-overexpressing clones were analyzed for cell proliferation and cell cycle analysis and in vivo tumorigenesis in nude mice. MGLL was identified as an SND1-interacting protein. Interaction of SND1 with MGLL resulted in ubiquitination and proteosomal degradation of MGLL. MGLL expression was detected in normal human hepatocytes and mouse liver, although it was undetected in human HCC cell lines. An inverse correlation between SND1 and MGLL levels was identified in a human HCC tissue microarray as well as in the TCGA database. Forced overexpression of MGLL in human HCC cells resulted in marked inhibition in cell proliferation with a significant delay in cell cycle progression and a marked decrease in tumor growth in nude mouse xenograft assays. MGLL overexpression inhibited Akt activation that is independent of enzymatic activity of MGLL and overexpression of a constitutively active Akt rescued cells from inhibition of proliferation and restored normal cell cycle progression. This study unravels a novel mechanism of SND1 function and identifies MGLL as a unique tumor suppressor for HCC. MGLL might function as a homeostatic regulator of Akt restraining its activation., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Monoubiquitination of proliferating cell nuclear antigen (PCNA) is a critical regulator of post replication repair (PRR). The depletion of BAF180, a unique subunit of the PBAF chromatin remodeling complex in human cells results in reduced PCNA ubiquitination leading to less efficient fork progression following DNA damage, but little is known about the mechanism. Here, we report that the expression of exogenous BAF180 in cells promotes PCNA ubiquitination during S-phase after UV irradiation and it persists for many hours. No correlation was observed between the protein level of ubiquitin-specific protease 1 (USP1) and ubiquitinated PCNA in BAF180 expressing cells. Analysis of cells expressing BAF180 deletion mutants showed that the bromo-adjacent homology (BAH) domains are responsible for this effect. Surprisingly, a deletion construct encoding only the BAH domain region is able to increase the level of ubiquitinated PCNA, even though it is unable to be assembled into the PBAF complex. These results suggest that the ATPase-dependent chromatin remodeling activity of PBAF is not necessary, but instead the BAH domains are sufficient to promote PCNA ubiquitination., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Spinocerebellar ataxia type 1 (SCA1), an autosomal-dominant neurodegenerative disorder, is caused by expansion of the polyglutamine tract within ataxin-1 (ATXN1). The AXH domain of ATXN1 can mediate neurodegeneration through its interaction with other proteins. We have previously showed that the ubiquitin-conjugating enzyme UbcH6 modulates the transcriptional repression activity of ATXN1 through ubiquitylation. In the present study, we sought to identify sites in the AXH domain that are ubiquitylated by UbcH6. Systematic replacement of each lysine residue in the AXH domain revealed that the lysine at 589 (K589) of ATXN1 is essential for its ubiquitylation by UbcH6. Mass spectrometry studies further confirmed the ubiquitylation site. Interestingly, protein aggregation was significantly enhanced in mutant AXH K589R, implying that the aggregation is strongly associated with the level of ATXN1 expression. Our study may suggest a therapeutic potential of UbcH6 in the treatment of SCA1., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Vitamin C is considered as an important anticancer therapeutic agent although this view is debatable. In this study, we introduce a physiological mechanism demonstrating how vitamin C exerts anticancer activity that induces cell cycle arrest and apoptosis. Our previous and current data reveal that p53 tumor suppressor is the prerequisite factor for stronger anticancer effects of vitamin C. In addition, vitamin C-mediated cancer cell cytotoxicity appears to be achieved at least partly through the downregulation of the p34SEI-1 oncoprotein. Our previous study showed that p34SEI-1 increases the survival of various types of cancer cells by inhibiting their apoptosis. Present data suggest that vitamin C treatment decreases the p34SEI-1 expression at the protein level and therefore alleviates its anti-apoptotic activity. Of note, SIAH1, E3 ubiquitin ligase, appears to be responsible for the p34SEI-1 polyubiquitination and its subsequent degradation, which is dependent on p53. In summary, vitamin C increases cancer cell death by inducing SIAH1-mediated polyubiquitination/degradation of the p34SEI-1 oncoprotein in a p53-dependent manner.

Cancer genome characterization has revealed driver mutations in genes that govern ubiquitylation; however, the mechanisms by which these alterations promote tumorigenesis remain incompletely characterized. Here, we analyzed changes in the ubiquitin landscape induced by prostate cancer-associated mutations of SPOP, an E3 ubiquitin ligase substrate-binding protein. SPOP mutants impaired ubiquitylation of a subset of proteins in a dominant-negative fashion. Of these, DEK and TRIM24 emerged as effector substrates consistently up-regulated by SPOP mutants. We highlight DEK as a SPOP substrate that exhibited decreases in ubiquitylation and proteasomal degradation resulting from heteromeric complexes of wild-type and mutant SPOP protein. DEK stabilization promoted prostate epithelial cell invasion, which implicated DEK as an oncogenic effector. More generally, these results provide a framework to decipher tumorigenic mechanisms linked to dysregulated ubiquitylation., (Copyright © 2014, American Association for the Advancement of Science.)

MID1 catalyzes the ubiquitination of the protein alpha4 and the catalytic subunit of protein phosphatase 2A. Mutations within the MID1 Bbox1 domain are associated with X-linked Opitz G syndrome (XLOS). Our functional assays have shown that mutations of Ala130 to Val or Thr, Cys142 to Ser and Cys145 to Thr completely disrupt the polyubiquitination of alpha4. Using NMR spectroscopy, we characterize the effect of these mutations on the tertiary structure of the Bbox1 domain by itself and in tandem with the Bbox2 domain. The mutation of either Cys142 or Cys145, each of which is involved in coordinating one of the two zinc ions, results in the collapse of signal dispersion in the HSQC spectrum of the Bbox1 domain indicating that the mutant protein structure is unfolded. Each mutation caused the coordination of both zinc ions, which are ∼ 13 Å apart, to be lost. Although Ala130 is not involved in the coordination of a zinc ion, the Ala130Thr mutant Bbox1 domain yields a poorly dispersed HSQC spectrum similar to those of the Cys142Ser and Cys145Thr mutants. Interestingly, neither cysteine mutation affects the structure of the adjacent Bbox2 domain when the two Bbox domains are engineered in their native tandem Bbox1-Bbox2 protein construct. Dynamic light scattering measurements suggest that the mutant Bbox1 domain has an increased propensity to form aggregates compared to the wild type Bbox1 domain. These studies provide insight into the mechanism by which mutations observed in XLOS affect the structure and function of the MID1 Bbox1 domain.

Monoubiquitination of histone H2B lysine 123 regulates methylation of histone H3 lysine 4 (H3K4) and 79 (H3K79) and the lack of H2B ubiquitination in Saccharomyces cerevisiae coincides with metacaspase-dependent apoptosis. Here, we discovered that loss of H3K4 methylation due to depletion of the methyltransferase Set1p (or the two COMPASS subunits Spp1p and Bre2p, respectively) leads to enhanced cell death during chronological aging and increased sensitivity to apoptosis induction. In contrast, loss of H3K79 methylation due to DOT1 disruption only slightly affects yeast survival. SET1 depleted cells accumulate DNA damage and co-disruption of Dot1p, the DNA damage adaptor protein Rad9p, the endonuclease Nuc1p, and the metacaspase Yca1p, respectively, impedes their early death. Furthermore, aged and dying wild-type cells lose H3K4 methylation, whereas depletion of the H3K4 demethylase Jhd2p improves survival, indicating that loss of H3K4 methylation is an important trigger for cell death in S. cerevisiae. Given the evolutionary conservation of H3K4 methylation this likely plays a role in apoptosis regulation in a wide range of organisms.

After acute DNA damage, the cell arrests S-phase progression by inhibiting origin initiation and fork progression to repair damaged DNA. The intra-S-phase checkpoint kinase Chk1 phosphorylates Cdc25A to target the latter for degradation by CRL1(β-TrCP) and so inhibit origin firing. The mechanism for inhibiting fork progression, however, has not been identified. Here, we show that degradation of p12, the fourth subunit of DNA polymerase δ, is critical for inhibiting fork progression. CRL4(Cdt2) is an E3 ligase that ubiquitinates and degrades p12 after UV treatment. Cells expressing a stable form of p12 exhibit UV-resistant DNA synthesis. DNA fiber assay and alkaline-sucrose gradient assay demonstrate that the impairment of fork progression after DNA damage requires p12 degradation. These results suggest that ubiquitination of p12 through CRL4(Cdt2) and subsequent degradation form one mechanism by which a cell responds to DNA damage to inhibit fork progression.

Protein ubiquitination in eukaryotic cells is mediated by diverse E3 ligase enzymes that each target specific substrates. The cullin E3 ligase complexes are the most abundant class of E3 ligases; they contain various cullin components that serve as scaffolds for interaction with substrate-recruiting adaptor proteins. SPOP is a BTB-domain adaptor of the cullin-3 E3 ligase complexes; it selectively recruits substrates via its N-terminal MATH domain, whereas its BTB domain mediates dimerization and interactions with cullin-3. It has recently been recognized that the high-order oligomerization of SPOP enhances the ubiquitination of substrates. Here, a dimerization interface in the SPOP C-terminus is identified and it is shown that the dimerization interfaces of the BTB domain and of the C-terminus act independently and in tandem to generate high-order SPOP oligomers. The crystal structure of the dimeric SPOP C-terminal domain is reported at 1.5 Å resolution and it is shown that Tyr353 plays a critical role in high-order oligomerization. A model of the high-order SPOP oligomer is presented that depicts a helical organization that could enhance the efficiency of substrate ubiquitination.

The receptor for advanced glycation endproduct (RAGE) is involved in diabetic complications and chronic inflammation, conditions known to affect the sensitivity towards apoptosis. Here, we studied the effect of genetically depleting RAGE on the susceptibility towards apoptosis. In murine osteoblastic cells, RAGE knockout increased both spontaneous and induced apoptosis. Decreased levels of B-cell lymphoma 2 protein and increased intrinsic apoptosis were observed in Rage(-/-) cells. Furthermore, loss of RAGE increased expression of the death receptor CD95 (Fas, Apo-1), CD95-dependent caspase activation and extrinsic apoptosis, whereas NF-kB-p65 nuclear translocation was diminished. Importantly, depletion of RAGE reduced the ubiquitination and degradation of p53 and p73 and increased their nuclear translocation. The increase of p53 and p73 transactivational activity was essential for the RAGE-dependent regulation of apoptosis, because knockdown of p53 and p73 significantly decreased apoptosis in RAGE-deficient but not in RAGE-expressing cells. Thus, the RAGE-mediated posttranslational regulation of p53 and p73 orchestrates a sequence of events culminating in control of intrinsic and extrinsic apoptosis signaling pathways.

We previously demonstrated that the histone deacetylase (HDAC) inhibitor, 4b, which preferentially targets HDAC1 and HDAC3, ameliorates Huntington's disease (HD)-related phenotypes in different HD model systems. In the current study, we investigated extensive behavioral and biological effects of 4b in N171-82Q transgenic mice and further explored potential molecular mechanisms of 4b action. We found that 4b significantly prevented body weight loss, improved several parameters of motor function and ameliorated Huntingtin (Htt)-elicited cognitive decline in N171-82Q transgenic mice. Pathways analysis of microarray data from the mouse brain revealed gene networks involving post-translational modification, including protein phosphorylation and ubiquitination pathways, associated with 4b drug treatment. Using real-time qPCR analysis, we validated differential regulation of several genes in these pathways by 4b, including Ube2K, Ubqln, Ube2e3, Usp28 and Sumo2, as well as several other related genes. Additionally, 4b elicited increases in the expression of genes encoding components of the inhibitor of kappaB kinase (IKK) complex. IKK activation has been linked to phosphorylation, acetylation and clearance of the Htt protein by the proteasome and the lysosome, and accordingly, we found elevated levels of phosphorylated endogenous wild-type (wt) Htt protein at serine 16 and threonine 3, and increased AcK9/pS13/pS16 immunoreactivity in cortical samples from 4b-treated mice. We further show that HDAC inhibitors prevent the formation of nuclear Htt aggregates in the brains of N171-82Q mice. Our findings suggest that one mechanism of 4b action is associated with the modulation of the ubiquitin-proteasomal and autophagy pathways, which could affect accumulation, stability and/or clearance of important disease-related proteins, such as Htt.

Tumor suppressors are frequently downregulated in human cancers and understanding of the mechanisms through which tumor cells restrict the expression of tumor suppressors is important for the prognosis and intervention of diseases. The promyelocytic leukemia (PML) protein plays a critical role in multiple tumor suppressive functions, such as growth inhibition, apoptosis, replicative senescence, suppression of oncogenic transformation, and inhibition of migration and angiogenesis. These tumor suppression functions are recapitulated in several mouse models. The expression of PML protein is frequently downregulated in diverse types of human tumors and this downregulation often correlates with tumor progression. Recent evidence has emerged that PML is aberrantly degraded in various types of tumors through ubiquitination-dependent mechanisms. Here, we summarize our current understanding of the PML ubiquitination/degradation pathways in human cancers. We point out that multiple pathways lead to PML ubiquitination and degradation. Furthermore, the PML ubiquitination processes are often dependent on other types of posttranslational modifications, such as phosphorylation, prolylisomerization, and sumoylation. Such feature indicates a highly regulated nature of PML ubiquitination in different cellular conditions and cell contexts, thus providing many avenues of opportunity to intervene PML ubiquitination pathways. We discuss the potential of targeting PML ubiquitination pathways for anti-cancer therapeutic strategies.

Nucleophosmin (NPM1/B23) and the activating transcription factor 5 (ATF5) are both known to subject to cell type-dependent regulation. NPM1 is expressed weakly in hepatocytes and highly expressed in hepatocellular carcinomas (HCC) with a clear correlation between enhanced NPM1 expression and increased tumor grading and poor prognosis, whereas in contrast, ATF5 is expressed abundantly in hepatocytes and down-regulated in HCC. Re-expression of ATF5 in HCC inhibits cell proliferation. We report here that using an unbiased approach, tandem affinity purification (TAP) followed with mass spectrometry (MS), we identified NPM1 as a novel ATF5-interacting protein. Unlike many other NPM1-interacting proteins that interact with the N-terminal oligomerization domain of NPM1, ATF5 binds via its basic leucine zipper to the C-terminal region of NPM1 where its nucleolar localization signal is located. NPM1 association with ATF5, whose staining patterns partially overlap in the nucleoli, promotes ATF5 protein degradation through proteasome-dependent and caspase-dependent pathways. NPM1-c, a mutant NPM1 that is defective in nucleolar localization, failed to stimulate ATF5 polyubiquitination and was unable to down-regulate ATF5. NPM1 interaction with ATF5 displaces HSP70, a known ATF5-interacting protein, from ATF5 protein complexes and antagonizes its role in stabilization of ATF5 protein. NPM1-promoted ATF5 down-regulation diminished ATF5-mediated repression of cAMP-responsive element-dependent gene transcription and abrogates ATF5-induced G(2)/M cell cycle blockade and inhibition of cell proliferation in HCC cells. Our study establishes a mechanistic link between elevated NPM1 expression and depressed ATF5 in HCC and suggests that regulation of ATF5 by NPM1 plays an important role in the proliferation and survival of HCC.

Super elongation complexes (SECs) contain two different transcription elongation factors, P-TEFb and ELL1/2, linked by the scaffolding protein AFF4 or AFF1. They stimulate the expression of both normal and disease-related genes, especially those of HIV or those involved in leukemogenesis. Among all SEC subunits, ELL2 is stoichiometrically limiting and uniquely regulated at the level of protein stability. Here we identify the RING domain protein Siah1, but not the homologous Siah2, as the E3 ubiquitin ligase for ELL2 polyubiquitination and proteasomal degradation. Siah1 cannot access and ubiquitinate ELL2 bound to AFF4, although, at high concentrations, it also degrades AFF4/1 to destroy SECs. Prostratin and HMBA, two well-studied activators of HIV transcription and latency, enhance ELL2 accumulation and SECs formation largely through decreasing Siah1 expression and ELL2 polyubiquitination. Given its importance in formation of SECs, the Siah1 ubiquitination pathway provides a fresh avenue for developing strategies to control disease-related transcription., (Copyright © 2012 Elsevier Inc. All rights reserved.)

p53 plays an important role in the regulation of the cell cycle, DNA repair, and apoptosis and is an attractive cancer therapeutic target. Mdm2 and Mdmx are recognized as the main p53 negative regulators. Although it remains unclear why Mdm2 and Mdmx are both required for p53 degradation, a model has been proposed whereby these two proteins function independent of one another; Mdm2 acts as an E3 ubiquitin ligase that catalyzes the ubiquitination of p53 for degradation, whereas Mdmx inhibits p53 by binding to and masking the transcriptional activation domain of p53, without causing its degradation. However, Mdm2 and Mdmx have been shown to function collaboratively. In fact, recent studies have pointed to a more important role for an Mdm2/Mdmx co-regulatory mechanism of p53 regulation than previously thought. In this review, we summarize current progress in the field about the functional and physical interaction between Mdm2 and Mdmx, their individual and collaborative roles in controlling p53, and inhibitors that target Mdm2 and Mdmx as a novel class of anticancer therapeutics.

B lymphoma Mo-MLV insertion region 1 (Bmi1) is a Polycomb Group (PcG) protein important in gene silencing. It is a component of Polycomb Repressive Complex 1 (PRC1), which is required to maintain the transcriptionally repressive state of many genes. Bmi1 was initially identified as an oncogene that regulates cell proliferation and transformation, and is important in hematopoiesis and the development of nervous systems. Recently, it was reported that Bmi1 is a potential marker for intestinal stem cells. Because Wnt signaling plays a key role in intestinal stem cells, we analyzed the effects of Wnt signaling on Bmi1 expression. We found that Wnt signaling indeed regulates the expression of Bmi1 in colon cancer cells. In addition, the expression of Bmi1 in human colon cancers is significantly associated with nuclear β-catenin, a hallmark for the activated Wnt signaling. Krüppel-like factor 4 (KLF4) is a zinc finger protein highly expressed in the gut and skin. We recently found that KLF4 cross-talks with Wnt/β-catenin in regulating intestinal homeostasis. We demonstrated that KLF4 directly inhibits the expression of Bmi1 in colon cancer cells. We also found that Bmi1 regulates histone ubiquitination and is required for colon cancer proliferation in vitro and in vivo. Our findings further suggest that Bmi1 is an attractive target for cancer therapeutics.

Eukaryotic gene expression involves tight coordination between transcription and pre-mRNA splicing; however, factors responsible for this coordination remain incompletely defined. Here, we explored the genetic, functional, and biochemical interactions of a likely coordinator, Npl3, an SR-like protein in Saccharomyces cerevisiae that we recently showed is required for efficient co-transcriptional recruitment of the splicing machinery. We surveyed the NPL3 genetic interaction space and observed a significant enrichment for genes involved in histone modification and chromatin remodeling. Specifically, we found that Npl3 genetically interacts with both Bre1, which mono-ubiquitinates histone H2B as part of the RAD6 Complex, and Ubp8, the de-ubiquitinase of the SAGA Complex. In support of these genetic data, we show that Bre1 physically interacts with Npl3 in an RNA-independent manner. Furthermore, using a genome-wide splicing microarray, we found that the known splicing defect of a strain lacking Npl3 is exacerbated by deletion of BRE1 or UBP8, a phenomenon phenocopied by a point mutation in H2B that abrogates ubiquitination. Intriguingly, even in the presence of wild-type NPL3, deletion of BRE1 exhibits a mild splicing defect and elicits a growth defect in combination with deletions of early and late splicing factors. Taken together, our data reveal a connection between Npl3 and an extensive array of chromatin factors and describe an unanticipated functional link between histone H2B ubiquitination and pre-mRNA splicing., Competing Interests: The authors have declared that no competing interests exist.

NOD2, the nucleotide-binding domain and leucine-rich repeat containing gene family (NLR) member 2 is involved in mediating antimicrobial responses. Dysfunctional NOD2 activity can lead to severe inflammatory disorders, but the regulation of NOD2 is still poorly understood. Recently, proteins of the tripartite motif (TRIM) protein family have emerged as regulators of innate immune responses by acting as E3 ubiquitin ligases. We identified TRIM27 as a new specific binding partner for NOD2. We show that NOD2 physically interacts with TRIM27 via the nucleotide-binding domain, and that NOD2 activation enhances this interaction. Dependent on functional TRIM27, ectopically expressed NOD2 is ubiquitinated with K48-linked ubiquitin chains followed by proteasomal degradation. Accordingly, TRIM27 affects NOD2-mediated pro-inflammatory responses. NOD2 mutations are linked to susceptibility to Crohn's disease. We found that TRIM27 expression is increased in Crohn's disease patients, underscoring a physiological role of TRIM27 in regulating NOD2 signaling. In HeLa cells, TRIM27 is partially localized in the nucleus. We revealed that ectopically expressed NOD2 can shuttle to the nucleus in a Walker A dependent manner, suggesting that NOD2 and TRIM27 might functionally cooperate in the nucleus.We conclude that TRIM27 negatively regulates NOD2-mediated signaling by degradation of NOD2 and suggest that TRIM27 could be a new target for therapeutic intervention in NOD2-associated diseases.

Regulation of PCNA ubiquitylation plays a key role in the tolerance to DNA damage in eukaryotes. Although the evolutionary conserved mechanism of PCNA ubiquitylation is well understood, the deubiquitylation of ubPCNA remains poorly characterized. Here, we show that the histone H2B(K123) ubiquitin protease Ubp10 also deubiquitylates ubPCNA in Saccharomyces cerevisiae. Our results sustain that Ubp10-dependent deubiquitylation of the sliding clamp PCNA normally takes place during S phase, likely in response to the simple presence of ubPCNA. In agreement with this, we show that Ubp10 forms a complex with PCNA in vivo. Interestingly, we also show that deletion of UBP10 alters in different ways the interaction of PCNA with DNA polymerase ζ-associated protein Rev1 and with accessory subunit Rev7. While deletion of UBP10 enhances PCNA-Rev1 interaction, it decreases significantly Rev7 binding to the sliding clamp. Finally, we report that Ubp10 counteracts Rad18 E3-ubiquitin ligase activity on PCNA at lysine 164 in such a manner that deregulation of Ubp10 expression causes tolerance impairment and MMS hypersensitivity., Competing Interests: The authors have declared that no competing interests exist.

p73, a homolog of the tumor suppressor p53, transactivates many p53 target genes, leading to apoptosis or cell-cycle arrest. p73 has recently been reported to play an important role in tumor suppression in a mouse model. Here, we show that Pirh2 physically interacted with p73 and downregulated p73 function through its E3 ligase activity. Pirh2 promoted p73 ubiquitination in vivo and in vitro. Intriguingly, Pirh2 primarily used K63-linked chains to ubiquitinate p73 in vitro, but in vivo, Pirh2 utilized K11-, K29-, K48-, and K63-linked chains to promote p73 ubiquitination. Depletion of Pirh2 by siRNA significantly reduced the ubiquitination of p73 in p53 null cells. Ectopic expression of Pirh2 repressed p73-dependent transcriptional activity, but the levels of p73 were not decreased. We consistently showed that ablation of endogenous Pirh2 restored p73-mediated transactivational activity. We found that Pirh2 repressed p73 transcriptional activity by directly inhibiting the p73 transcript, and p73 repression by Pirh2 was required for p73-dependent transcriptional activity and G(1) arrest but not for apoptosis. This study provides evidence that the ubiquitination of p73 mediated by Pirh2 represents an important pathway for controlling the suppressive function of p73. Furthermore, the data suggest a link between the transcriptional activity of p73 and its ubiquitination.

Rationale: The stress-induced growth arrest and DNA damage-inducible a (GADD45a) gene is up-regulated by mechanical stress with GADD45a knockout (GADD45a(-/-)) mice demonstrating both increased susceptibility to ventilator-induced lung injury (VILI) and reduced levels of the cell survival and vascular permeability signaling effector (Akt). However, the functional role of GADD45a in the pathogenesis of VILI is unknown., Objectives: We sought to define the role of GADD45a in the regulation of Akt activation induced by mechanical stress., Methods: VILI-challenged GADD45a(-/-) mice were administered a constitutively active Akt1 vector and injury was assessed by bronchoalveolar lavage cell counts and protein levels. Human pulmonary artery endothelial cells (EC) were exposed to 18% cyclic stretch (CS) under conditions of GADD45a silencing and used for immunoprecipitation, Western blotting or immunofluoresence. EC were also transfected with mutant ubiquitin vectors to characterize site-specific Akt ubiquitination. DNA methylation was measured using methylspecific polymerase chain reaction assay., Measurements and Main Results: Studies exploring the linkage of GADD45a with mechanical stress and Akt regulation revealed VILI challenged GADD45a(-/-) mice to have significantly reduced lung injury on overexpression of Akt1 transgene. Increased mechanical stress with 18% CS in EC induced Akt phosphorylation via E3 ligase tumor necrosis factor receptor–associated factor 6 (TRAF6)–mediated Akt K63 ubiquitination resulting in Akt trafficking and activation at the membrane. GADD45a is essential to this process because GADD45a silenced endothelial cells and GADD45a(-/-) mice exhibited increased Akt K48 ubiquitination leading to proteasomal degradation. These events involve loss of ubiquitin carboxyl terminal hydrolase 1(UCHL1), a deubiquitinating enzyme that normally removes K48 polyubiquitin chains bound to Akt thus promoting Akt K63 ubiquitination. Loss of GADD45a significantly reduces UCHL1 expression via UCHL1 promoter methylation resulting in increased Akt K48 ubiquitination and reduced Akt levels., Conclusions: These studies highlight a novel role for GADD45a in the regulation of site-specific Akt ubiquitination and activation and implicate a significant functional role for GADD45a in the clinical predisposition to VILI.

Ribosomal proteins play a critical role in tightly coordinating p53 signaling with ribosomal biogenesis. Several ribosomal proteins have been shown to induce and activate p53 via inhibition of MDM2. Here, we report that S27a, a small subunit ribosomal protein synthesized as an 80-amino acid ubiquitin C-terminal extension protein (CEP80), functions as a novel regulator of the MDM2-p53 loop. S27a interacts with MDM2 at the central acidic domain of MDM2 and suppresses MDM2-mediated p53 ubiquitination, leading to p53 activation and cell cycle arrest. Knockdown of S27a significantly attenuates the p53 activation in cells in response to treatment with ribosomal stress-inducing agent actinomycin D or 5-fluorouracil. Interestingly, MDM2 in turn ubiquitinates S27a and promotes proteasomal degradation of S27a in response to actinomycin D treatment, thus forming a mutual-regulatory loop. Altogether, our results reveal that S27a plays a non-redundant role in mediating p53 activation in response to ribosomal stress via interplaying with MDM2.

Infection with human T-cell leukemia virus induces cellular genomic instability mediated through the viral oncoprotein Tax. Here we present evidence that Tax undermines the cellular DNA damage response by sequestration of damage response factors. We show by confocal microscopy that Tax forms damage-independent nuclear foci that contain DNA-PK, BRCA1, and MDC1. Tax sequesters MDC1 to chromatin sites distinct from classic ionizing radiation-induced foci. The recruitment of MDC1 is competitive between the two foci. The N-terminal region of Tax is sufficient for foci localization, and the C-terminal half is critical for binding to MDC1 and recruitment of additional response factors. Tax expression and DNA damage response factor recruitment repressed the formation of ionizing radiation-induced Nbs1-containing foci. The Tax-induced "pseudo" DNA damage response results in phosphorylation and monoubiquitylation of H2AX, which is ablated by siRNA suppression of MDC1. These data support a model for virus-induced genomic instability in which viral oncogene-induced damage-independent foci compete with normal cellular DNA damage response.

Oxygen deprivation (hypoxia) results in reprogrammed gene expression patterns that induce multifaceted cellular responses. Here we identify a regulated interaction between the serine/threonine kinase HIPK2 and the ubiquitin E3 ligase Siah2 as a mechanism controlling the hypoxic response. Under normoxic conditions, several mechanisms ensure HIPK2 stability: only a fraction of HIPK2 is found in association with Siah2, whereas HIPK2-mediated phosphorylation of this E3 ligase at positions 26, 28 and 68 weakens mutual binding and destabilizes its phosphorylated interaction partner. Hypoxic conditions allow a markedly increased HIPK2/Siah2 interaction and result in efficient polyubiquitylation and proteasomal degradation of the kinase. Accordingly, hypoxia-induced HIPK2 elimination is markedly reduced in Siah2-deficient cells. As HIPK2 has an important role as a negative regulator of gene expression, its elimination from promoter-associated repressor complexes allows the induction of a substantial fraction of hypoxia-induced genes.

Huntington's disease (HD) is caused by the expansion of a polyglutamine tract in the N-terminal region of huntingtin (htt) and is characterized by selective neurodegeneration. In addition to forming nuclear aggregates, mutant htt accumulates in neuronal processes as well as synapses and affects synaptic function. However, the mechanism for the synaptic toxicity of mutant htt remains to be investigated. We targeted fluorescent reporters for the ubiquitin-proteasome system (UPS) to presynaptic or postsynaptic terminals of neurons. Using these reporters and biochemical assays of isolated synaptosomes, we found that mutant htt decreases synaptic UPS activity in cultured neurons and in HD mouse brains that express N-terminal or full-length mutant htt. Given that the UPS is a key regulator of synaptic plasticity and function, our findings offer insight into the selective neuronal dysfunction seen in HD and also establish a method to measure synaptic UPS activity in other neurological disease models.

P62 has been proposed to mark ubiquitinated protein bodies for autophagic degradation. We report that the Drosophila melanogaster p62 orthologue, Ref(2)P, is a regulator of protein aggregation in the adult brain. We demonstrate that Ref(2)P localizes to age-induced protein aggregates as well as to aggregates caused by reduced autophagic or proteasomal activity. A similar localization to protein aggregates is also observed in D. melanogaster models of human neurodegenerative diseases. Although atg8a autophagy mutant flies show accumulation of ubiquitin- and Ref(2)P-positive protein aggregates, this is abrogated in atg8a/ref(2)P double mutants. Both the multimerization and ubiquitin binding domains of Ref(2)P are required for aggregate formation in vivo. Our findings reveal a major role for Ref(2)P in the formation of ubiquitin-positive protein aggregates both under physiological conditions and when normal protein turnover is inhibited.

The NKX3.1 gene located at 8p21.2 encodes a homeodomain-containing transcription factor that acts as a haploinsufficient tumor suppressor in prostate cancer. Diminished protein expression of NKX3.1 has been observed in prostate cancer precursors and carcinomas. TOPORS is a ubiquitously expressed E3 ubiquitin ligase that can ubiquitinate tumor suppressor p53. Here we report interaction between NKX3.1 and TOPORS. NKX3.1 can be ubiquitinated by TOPORS in vitro and in vivo, and overexpression of TOPORS leads to NKX3.1 proteasomal degradation in prostate cancer cells. Conversely, small interfering RNA-mediated knockdown of TOPORS leads to an increased steady-state level and prolonged half-life of NKX3.1. These data establish TOPORS as a negative regulator of NKX3.1 and implicate TOPORS in prostate cancer progression.