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Supplementary Figure S1: Interaction between NoxO1 and Grb2 Supplementary Figure S2: Interaction domains between NoxO1 and Grb2 Supplementary Figure S3: Stability of Nox1 and NoxA1 Supplementary Figure S4: Target of ubiquitination is not p47phox Supplementary Figure S5: PKC-dependent NoxO1 phosphorylation Supplementary Figure S6: Subcellular localization of NoxO1 and NoxA1 Supplementary Figure S7: Effect of NoxO1 on Erk activation

The generation of reactive oxygen species (ROS) is required for proper cell signaling, but must be tightly regulated to minimize deleterious oxidizing effects. Activation of the NADPH oxidases (Nox) triggers ROS production and, thus, regulatory mechanisms exist to properly control Nox activity. In this study, we report a novel mechanism in which Nox1 activity is regulated through the proteasomal degradation of Nox organizer 1 (NoxO1). We found that through the interaction between NoxO1 and growth receptor–bound protein 2 (Grb2), the Casitas B–lineage lymphoma (Cbl) E3 ligase was recruited, leading to decreased NoxO1 stability and a subsequent reduction in ROS generation upon epidermal growth factor (EGF) stimulation. Additionally, we show that EGF-mediated phosphorylation of NoxO1 induced its release from Grb2 and facilitated its association with Nox activator 1 (NoxA1) to stimulate ROS production. Consistently, overexpression of Grb2 resulted in decreased Nox1 activity, whereas knockdown of Grb2 led to increased Nox1 activity in response to EGF. CRISPR/Cas9-mediated NoxO1 knockout in human colon cancer cells abrogated anchorage-independent growth on soft agar and tumor-forming ability in athymic nude mice. Moreover, the expression and stability of NoxO1 were significantly increased in human colon cancer tissues compared with normal colon. Taken together, these results support a model whereby Nox1 activity and ROS generation are regulated by Grb2/Cbl-mediated proteolysis of NoxO1 in response to EGF, providing new insight into the processes by which excessive ROS production may promote oncogenic signaling to drive colorectal tumorigenesis. Cancer Res; 76(4); 855–65. ©2016 AACR.

Supplementary Materials and Methods

Supplementary Figure S1: Interaction between NoxO1 and Grb2 Supplementary Figure S2: Interaction domains between NoxO1 and Grb2 Supplementary Figure S3: Stability of Nox1 and NoxA1 Supplementary Figure S4: Target of ubiquitination is not p47phox Supplementary Figure S5: PKC-dependent NoxO1 phosphorylation Supplementary Figure S6: Subcellular localization of NoxO1 and NoxA1 Supplementary Figure S7: Effect of NoxO1 on Erkactivation

The generation of reactive oxygen species (ROS) is required for proper cell signaling, but must be tightly regulated to minimize deleterious oxidizing effects. Activation of the NADPH oxidases (Nox) triggers ROS production and, thus, regulatory mechanisms exist to properly control Nox activity. In this study, we report a novel mechanism in which Nox1 activity is regulated through the proteasomal degradation of Nox organizer 1 (NoxO1). We found that through the interaction between NoxO1 and growth receptor–bound protein 2 (Grb2), the Casitas B–lineage lymphoma (Cbl) E3 ligase was recruited, leading to decreased NoxO1 stability and a subsequent reduction in ROS generation upon epidermal growth factor (EGF) stimulation. Additionally, we show that EGF-mediated phosphorylation of NoxO1 induced its release from Grb2 and facilitated its association with Nox activator 1 (NoxA1) to stimulate ROS production. Consistently, overexpression of Grb2 resulted in decreased Nox1 activity, whereas knockdown of Grb2 led to increased Nox1 activity in response to EGF. CRISPR/Cas9-mediated NoxO1 knockout in human colon cancer cells abrogated anchorage-independent growth on soft agar and tumor-forming ability in athymic nude mice. Moreover, the expression and stability of NoxO1 were significantly increased in human colon cancer tissues compared with normal colon. Taken together, these results support a model whereby Nox1 activity and ROS generation are regulated by Grb2/Cbl-mediated proteolysis of NoxO1 in response to EGF, providing new insight into the processes by which excessive ROS production may promote oncogenic signaling to drive colorectal tumorigenesis. Cancer Res; 76(4); 855–65. ©2016 AACR.

In response to DNA damage, PCNA is mono-ubiquitinated and triggers translesion DNA synthesis (TLS) by recruiting polymerase-η. However, it remained unknown how error-prone TLS is turned off after DNA lesion bypass to prevent mutagenesis. Here we showed that ISG15 modification (ISGylation) of PCNA plays a key role in TLS termination. Upon UV irradiation, EFP, an ISG15 E3 ligase, bound to mono-ubiquitinated PCNA and promoted its ISGylation. ISGylated PCNA then tethered USP10 for deubiquitination and in turn the release of polymerase-η from PCNA. Eventually, PCNA was deISGylated by UBP43 for reloading of replicative DNA polymerases and resuming normal DNA replication. However, ISGylation-defective Lys-to-Arg mutations in PCNA or knockdown of any of ISG15, EFP, or USP10 led to persistent recruitment of mono-ubiquitinated PCNA and polymerase-η to nuclear foci, causing an increase in mutation frequency. These findings establish a crucial role of PCNA ISGylation in termination of error-prone TLS for preventing excessive mutagenesis.

SIRT1, the NAD+-dependent protein deacetylase, controls cell-cycle progression and apoptosis by suppressing p53 tumour suppressor. Although SIRT1 is known to be phosphorylated by JNK1 upon oxidative stress and subsequently down-regulated, it still remains elusive how SIRT1 stability and activity are controlled. Here, we have unveiled that CHFR functions as an E3 Ub-ligase of SIRT1, responsible for its proteasomal degradation under oxidative stress conditions. CHFR interacts with and destabilizes SIRT1 by ubiquitylation and subsequent proteolysis. Such CHFR-mediated SIRT1 inhibition leads to the increase of p53 acetylation and its target gene transcription. Notably, CHFR facilitates SIRT1 destabilization when SIRT1 is phosphorylated by JNK1 upon oxidative stress, followed by prominent apoptotic cell death. Meanwhile, JNK inhibitor prevents SIRT1 phosphorylation, leading to elevated SIRT1 protein levels even in the presence of H2O2. Taken together, our results indicate that CHFR plays a crucial role in the cellular stress response pathway by controlling the stability and function of SIRT1.

RATIONALE Protein ubiquitination plays a critical role in regulating many cellular events, such as protein localization and stability, cellular signal transduction and DNA repair. Recent studies have shown that polyubiquitin (polyUb) chains elongate through heterogeneous isopeptide linkages to K11, K29, K48 and K63. In this study we have investigated the usage of isopeptide linkages of polyUb chains in different molecular weight regions by using quantitative mass spectrometry. METHODS Recombinant Chfr protein was autoubiquitinated by E1 enzyme, E2 enzyme UbcH5 and ubiquitin (WT Ub, K11R Ub, K48R Ub and K63R Ub) in vitro, and different molecular weight regions of ubiquitinated Chfr were then subjected to liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) following sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and in-gel digestion. RESULTS Absolute QUantitative Analysis (AQUA) of polyUb chain formation with wild-type (WT) and point mutants of ubiquitin was performed, and the results suggested that the K11 polyUb chain was most frequently used in the high ubiquitin conjugates of WT Ub. Furthermore, the extent of polyUb chain formation with K11R Ub was decreased about 10-fold compared to polyUb chain formation with WT Ub through the entire molecular weight region. The present study suggests that the linkage through K11 plays crucial roles in polyUb chain formation. CONCLUSIONS Topologies of polyUb chains in the low and high Ub conjugates were studied using mass spectrometry. K48 and K63 were the primary ubiquitination sites of the low molecular weight Ub conjugates, whereas K11 was the critical site of polyUb chain formation in high molecular weight Ub conjugates. Copyright © 2012 John Wiley & Sons, Ltd.

Identification of the cellular mechanisms that mediate cancer cell chemosensitivity is important for developing new cancer treatment strategies. Several chemotherapeutic drugs increase levels of the posttranslational modifier ISG15, which suggests that ISGylation could suppress oncogenesis. However, how ISGylation of specific target proteins controls tumorigenesis is unknown. Here, we identified proteins that are ISGylated in response to chemotherapy. Treatment of a human mammary epithelial cell line with doxorubicin resulted in ISGylation of the p53 family protein p63. An alternative splice variant of p63, ΔNp63α, suppressed the transactivity of other p53 family members, and its expression was abnormally elevated in various human epithelial tumors, suggestive of an oncogenic role for this variant. We showed that ISGylation played an essential role in the downregulation of ΔNp63α. Anticancer drugs, including doxorubicin, induced ΔNp63α ISGylation and caspase-2 activation, leading to cleavage of ISGylated ΔNp63α in the nucleus and subsequent release of its inhibitory domain to the cytoplasm. ISGylation ablated the ability of ΔNp63α to promote anchorage-independent cell growth and tumor formation in vivo as well to suppress the transactivities of proapoptotic p53 family members. These findings establish ISG15 as a tumor suppressor via its conjugation to ΔNp63α and provide a molecular rationale for therapeutic use of doxorubicin against ΔNp63α-mediated cancers.

Apoptosis inducing factor (AIF) is a mitochondrial oxidoreductase that scavenges reactive oxygen species under normal conditions. Under certain stresses, such as exposure to N-methyl-N′-nitro-N′-nitrosoguanidine (MNNG), AIF is truncated and released from the mitochondria and translocated into the nucleus, where the truncated AIF (tAIF) induces caspase-independent cell death. However, it is unknown how cells decide to kill themselves or operate ways to survive when they encounter stresses that induce the release of tAIF. Here, we demonstrated that USP2 and CHIP contribute to the control of tAIF stability. USP2 deubiquitinated and stabilized tAIF, thus promoting AIF-mediated cell death. In contrast, CHIP ubiquitinated and destabilized tAIF, thus preventing the cell death. Consistently, CHIP-deficient cells showed an increased sensitivity to MNNG. On the other hand, knockdown of USP2 attenuated MNNG-induced cell death. Moreover, exposure to MNNG caused a dramatic decrease in CHIP level, but not that of USP2, concurrent with cell shrinkage and chromatin condensation. These findings indicate that CHIP and USP2 show antagonistic functions in the control of AIF-mediated cell death, and implicate the role of the enzymes as a switch for cells to live or die under stresses that cause tAIF release.

Summary DUOX, a member of the NADPH oxidase family, acts as the first line of defense against enteric pathogens by producing microbicidal reactive oxygen species. DUOX is activated upon enteric infection, but the mechanisms regulating DUOX activity remain incompletely understood. Using Drosophila genetic tools, we show that enteric infection results in "pro-catabolic" signaling that initiates metabolic reprogramming of enterocytes toward lipid catabolism, which ultimately governs DUOX homeostasis. Infection induces signaling cascades involving TRAF3 and kinases AMPK and WTS, which regulate TOR kinase to control the balance of lipogenesis versus lipolysis. Enhancing lipogenesis blocks DUOX activity, whereas stimulating lipolysis via ATG1-dependent lipophagy is required for DUOX activation. Drosophila with altered activity in TRAF3-AMPK/WTS-ATG1 pathway components exhibit abolished infection-induced lipolysis, reduced DUOX activation, and enhanced susceptibility to enteric infection. Thus, this work uncovers signaling cascades governing inflammation-induced metabolic reprogramming and provides insight into the pathophysiology of immune-metabolic interactions in the microbe-laden gut epithelia.

The intracellular negatively regulatory mechanism which affects IL-1beta-induced MUC8 gene expression remains unclear. We found that SOCS3 overexpression suppressed IL-1beta-induced MUC8 gene expression in NCI-H292 cells, whereas silencing of SOCS3 restored IL-1beta-induced MUC8 gene expression. Sequentially activated ERK1/2, RSK1, and CREB by IL-1beta were not affected by SOCS3, indicating that SOCS3 has an independent mechanism of action. Using immunoprecipitaion and nano LC mass analysis, we found that SOCS3 bound NonO (non-POU-domain containing, octamer-binding domain protein) in the absence of IL-1beta, whereas IL-1beta treatment dissociated the direct binding of SOCS3 and NonO. A dominant-negative SOCS3 mutant (Y204F/Y221F) did not bind to NonO. Interestingly, SOCS3 overexpression dramatically suppressed MUC8 gene expression in cells transfected with wild-type or siRNA of NonO. Moreover, silencing of SOCS3 dramatically increased NonO-mediated MUC8 gene expression caused by IL-1beta compared to NonO overexpression alone, suggesting that SOCS3 acts as a suppressor by regulating the action of NonO.

The Hippo pathway plays crucial roles in regulating organ size and stem cell homeostasis. Although the signalling cascade of the core Hippo kinases is relatively well understood, little is known about the mechanisms that modulate the activity of the Hippo pathway. Here, we report identification of NEDD4, a HECT-type E3 ubiquitin ligase, as a regulatory component of the Hippo pathway. We demonstrate that NEDD4 ubiquitylates and destabilizes WW45 and LATS kinase, both of which are required for active Hippo signalling. Interestingly, MST1 protects WW45, but not LATS2, against NEDD4. We also provide evidence indicating that NEDD4 inactivation at high cell density is a prerequisite for the elevated Hippo activity linked to contact inhibition. Moreover, NEDD4 promotes intestinal stem cell renewal in Drosophila by suppressing Hippo signalling. Collectively, we present a regulatory mechanism by which NEDD4 controls the Hippo pathway leading to coordinated cell proliferation and apoptosis.

Small ubiquitin-like modifier (SUMO)-specific proteases (SENPs) that reverse protein modification by SUMO are involved in the control of numerous cellular processes, including transcription, cell division, and cancer development. However, the physiological function of SENPs in energy metabolism remains unclear. Here, we investigated the role of SENP2 in fatty acid metabolism in C2C12 myotubes and in vivo. In C2C12 myotubes, treatment with saturated fatty acids, like palmitate, led to nuclear factor-κB–mediated increase in the expression of SENP2. This increase promoted the recruitment of peroxisome proliferator–activated receptor (PPAR)δ and PPARγ, through desumoylation of PPARs, to the promoters of the genes involved in fatty acid oxidation (FAO), such as carnitine-palmitoyl transferase-1 (CPT1b) and long-chain acyl-CoA synthetase 1 (ACSL1). In addition, SENP2 overexpression substantially increased FAO in C2C12 myotubes. Consistent with the cell culture system, muscle-specific SENP2 overexpression led to a marked increase in the mRNA levels of CPT1b and ACSL1 and thereby in FAO in the skeletal muscle, which ultimately alleviated high-fat diet–induced obesity and insulin resistance. Collectively, these data identify SENP2 as an important regulator of fatty acid metabolism in skeletal muscle and further implicate that muscle SENP2 could be a novel therapeutic target for the treatment of obesity-linked metabolic disorders.

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.

Retinoid X receptor alpha (RXRalpha) belongs to a family of ligand-activated transcription factors that regulate many aspects of metazoan life. Here we demonstrate that RXRalpha is a target substrate of a small ubiquitin-related modifier (SUMO)-specific protease, SUSP1, which is capable of controlling the transcriptional activity of RXRalpha. RXRalpha was modified by SUMO-1 in vivo as well as in vitro, and the Lys-108 residue within the IKPP sequence of RXRalpha AF-1 domain was identified as the major SUMO-1 acceptor site. Prevention of SUMO modification by Lys-to-Arg mutation led to an increase not only in the transcriptional activity of RXRalpha but also in the activity of its heterodimeric complex with retinoic acid receptor-alpha or peroxisome proliferator-activated receptor-gamma (PPARgamma). SUSP1 co-localized with RXRalpha in the nucleus and removed SUMO-1 from RXRalpha but not from androgen receptor or PPARgamma. Moreover, overexpression of SUSP1 caused an increase in the transcriptional activity of RXRalpha, whereas small hairpin RNA-mediated knockdown of endogenous SUSP1 led to a decrease in RXRalpha activity. These results suggest that SUSP1 plays an important role in the control of the transcriptional activity of RXRalpha and thus in the RXRalpha-mediated cellular processes.

In the Drosophila gut, reactive oxygen species (ROS)-dependent immunity is critical to host survival. This is in contrast to the NF-kappaB pathway whose physiological function in the microbe-laden epithelia has yet to be convincingly demonstrated despite playing a critical role during systemic infections. We used a novel in vivo approach to reveal the physiological role of gut NF-kappaB/antimicrobial peptide (AMP) system, which has been 'masked' in the presence of the dominant intestinal ROS-dependent immunity. When fed with ROS-resistant microbes, NF-kappaB pathway mutant flies, but not wild-type flies, become highly susceptible to gut infection. This high lethality can be significantly reduced by either re-introducing Relish expression to Relish mutants or by constitutively expressing a single AMP to the NF-kappaB pathway mutants in the intestine. These results imply that the local 'NF-kappaB/AMP' system acts as an essential 'fail-safe' system, complementary to the ROS-dependent gut immunity, during gut infection with ROS-resistant pathogens. This system provides the Drosophila gut immunity the versatility necessary to manage sporadic invasion of virulent pathogens that somehow counteract or evade the ROS-dependent immunity.

Skp1p-cullin-F-box protein (SCF) complexes are ubiquitin-ligases composed of a core complex including Skp1p, Cdc53p, Hrt1p, the E2 enzyme Cdc34p, and one of multiple F-box proteins which are thought to provide substrate specificity to the complex. Here we show that the F-box protein Rcy1p is required for recycling of the v-SNARE Snc1p in Saccharomyces cerevisiae. Rcy1p localized to areas of polarized growth, and this polarized localization required its CAAX box and an intact actin cytoskeleton. Rcy1p interacted with Skp1p in vivo in an F-box-dependent manner, and both deletion of its F box and loss of Skp1p function impaired recycling. In contrast, cells deficient in Cdc53p, Hrt1p, or Cdc34p did not exhibit recycling defects. Unlike the case for F-box proteins that are known to participate in SCF complexes, degradation of Rcy1p required neither its F box nor functional 26S proteasomes or other SCF core subunits. Importantly, Skp1p was the only major partner that copurified with Rcy1p. Our results thus suggest that a complex composed of Rcy1p and Skp1p but not other SCF components may play a direct role in recycling of internalized proteins.

Exit from mitosis in budding yeast requires a group of essential proteins—including the GTPase Tem1 and the protein phosphatase Cdc14—that downregulate cyclin-dependent kinase activity. We identified a mutation, net1-1, that bypasses the lethality of tem1Δ. NET1 encodes a novel protein, and mass spectrometric analysis reveals that it is a key component of a multifunctional complex, denoted RENT (for re gulator of n ucleolar silencing and t elophase), that also contains Cdc14 and the silencing regulator Sir2. From G1 through anaphase, RENT localizes to the nucleolus, and Cdc14 activity is inhibited by Net1. In late anaphase, Cdc14 dissociates from RENT, disperses throughout the cell in a Tem1-dependent manner, and ultimately triggers mitotic exit. Nucleolar sequestration may be a general mechanism for the regulation of diverse biological processes.

An unusual lon mutation (called lonR9) is dominant over the wild-type gene, which encodes the ATP-dependent protease La (Lon) in Escherichia coli, when present in multicopy plasmids. Here, we cloned and sequenced lonR9, and showed that the mutant gene carries a single point mutation in its open reading frame, which leads to replacement of Glu614 by Lys. The LonR9 protein and its poly-His-tagged form were purified to apparent homogeneity. Both of the purified proteins were capable of inhibiting the ATP-dependent proteolysis and the protein-activated ATP hydrolysis by protease La. Furthermore, the His-tagged LonR9 protein was found to form mixed oligomeric complexes with protease La, upon analysis by chromatography on a metal-chelating column. These results suggest that the phenotypic dominance of the lonR9 mutant is due to the formation of mixed oligomeric complexes between LonR9 and protease La, in which the defective components prevent the function of the wild-type subunits.

HslVU is an ATP-dependent protease consisting of two multimeric components: the HslU ATPase and the HslV peptidase. To gain an insight into the role of ATP hydrolysis in protein breakdown, we determined the insulin B-chain-degrading activity and assembly of HslVU in the presence of ATP and its nonhydrolyzable analogs. While beta,gamma-methylene-ATP could not support the proteolytic activity, beta,gamma-imido-ATP supported it to an extent less than 10% of that seen with ATP. Surprisingly, however, HslVU degraded insulin B-chain even more rapidly in the presence of ATPgammaS than with ATP. Furthermore, the ability of ATP and its analogs in supporting the proteolytic activity was closely correlated with their ability in supporting the oligomerization of HslU and the formation of the HslVU complex. However, ADP, which is capable of supporting the HslU oligomerization, could not support the HslVU complex formation or the proteolytic activity, suggesting that the conformation of the ADP-bound HslU oligomer is different from that of ATP-bound form. Thus, it appears that ATP-binding, but not its hydrolysis, is essential for assembly and proteolytic activity of HslVU.

CHFR ubiquitin ligase plays an important role in cell cycle progression and tumorigenesis. CHFR tumor suppressor function is highly associated with its protein level. We recently reported that CHFR protein levels are negatively regulated by SUMOylation-mediated proteasomal degradation. In the present study, we uncover a detailed molecular mechanism how SUMOylation promotes CHFR destabilization. We demonstrate that SUMO modification of CHFR promotes its ubiquitylation and subsequent proteasomal degradation. However, SUMOylation of CHFR does not affect its auto-ubiquitylation, which generally serves as a maintenance mechanism for most ubiquitin ligases. Moreover, the E3 ubiquitin ligase activity of CHFR is dispensable for this SUMOylation-mediated ubiquitylation and degradation. Conversely, SENP2 deSUMOylating enzyme reduces SUMOylation-induced ubiquitylation of CHFR, leading to elevated CHFR protein levels. Taken together, our results present a new regulatory mechanism for CHFR that sequential post-translational modifications of CHFR by SUMO and ubiquitin coordinately regulates its stability.

HslVU in E. coli is a new type of ATP-dependent protease composed of two heat shock proteins, the HslU ATPase and the HslV peptidase related to certain β-type subunits of the 20S proteasome. Here we show that the ATP-dependent hydrolysis of N -carbobenzoxy-Gly-Gly-Leu-7-amido-4-methylcoumarin by the HslVU protease can be markedly stimulated by poly-L-lysine, that is known to activate the casein-degrading activity of the 20S proteasome. However, poly-L-lysine showed little or no effect on the peptidase activity of HslV itself. Instead, it stimulated the hydrolysis of ATP by HslU several-fold. Histone that could stimulate the ATPase activity of HslU also increased the rate of the ATP-dependent peptide hydrolysis by HslV, although to a much lesser extent than by poly-L-lysine. Thus, the poly-L-lysine-mediated increase in the ATPase activity of HslU appears to be responsible for the dramatic activation of the ATP-dependent peptide hydrolysis by HslV. These results suggest that, in the reconstituted HslVU complex, the peptide hydrolysis by HslV occurs in a tightly coupled process with the cleavage of ATP by HslU.

We have isolated a new type of ATP-dependent protease from Escherichia coli. It is the product of the heat-shock locus hslVU that encodes two proteins: HslV, a 19-kDa protein similar to proteasome beta subunits, and HslU, a 50-kDa protein related to the ATPase ClpX. In the presence of ATP, the protease hydrolyzes rapidly the fluorogenic peptide Z-Gly-Gly-Leu-AMC and very slowly certain other chymotrypsin substrates. This activity increased 10-fold in E. coli expressing heat-shock proteins constitutively and 100-fold in cells expressing HslV and HslU from a high copy plasmid. Although HslV and HslU could be coimmunoprecipitated from cell extracts of both strains with an anti-HslV antibody, these two components were readily separated by various types of chromatography. ATP stimulated peptidase activity up to 150-fold, whereas other nucleoside triphosphates, a nonhydrolyzable ATP analog, ADP, or AMP had no effect. Peptidase activity was blocked by the anti-HslV antibody and by several types of inhibitors of the eukaryotic proteasome (a threonine protease) but not by inhibitors of other classes of proteases. Unlike eukaryotic proteasomes, the HslVU protease lacked tryptic-like and peptidyl-glutamyl-peptidase activities. Electron micrographs reveal ring-shaped particles similar to en face images of the 20S proteasome or the ClpAP protease. Thus, HslV and HslU appear to form a complex in which ATP hydrolysis by HslU is essential for peptide hydrolysis by the proteasome-like component HslV.

The hslVU operon in Escherichia coli encodes two heat shock proteins, HslV, a 19-kDa protein homologous to β-type subunits of the 20 S proteasomes, and HslU, a 50-kDa protein related to the ATPase ClpX. We have recently shown that HslV and HslU can function together as a novel ATP-dependent protease, the HslVU protease. We have now purified both proteins to apparent homogeneity from extracts of E. coli carrying the hslVU operon on a multicopy plasmid. HslU by itself cleaved ATP, and pure HslV is a weak peptidase degrading certain hydrophobic peptides. HslU dramatically stimulated peptide hydrolysis by HslV when ATP is present. With a 1:4 molar ratio of HslV to HslU, approximately a 200-fold increase in peptide hydrolysis was observed. HslV stimulated the ATPase activity of HslU 2–4-fold, but had little influence on the affinity of HslU to ATP. The nonhydrolyzable ATP analog, β,γ-methylene-ATP, did not support peptide hydrolysis. Other nucleotides (CTP, dATP) that were slowly hydrolyzed by HslU allowed some peptide hydrolysis. Therefore, ATP cleavage appears essential for the HslV activity. Upon gel filtration on a Sephacryl S-300 column, HslV behaved as a 250-kDa oligomer (i.e. 12–14 subunits), and HslU behaved as a 100-kDa protein (i.e. a dimer) in the absence of ATP, but as a 450-kDa multimer (8–10 subunits) in its presence. Therefore ATP appears necessary for oligomerization of HslU. Thus the HslVU protease appears to be a two-component protease in which HslV harbors the peptidase activity, while HslU provides an essential ATPase activity.

The ATP-dependent protease Ti (Clp) consists of two distinct components, ClpP containing the serine active sites for proteolysis and ClpA having two ATP-binding sites. A ClpA variant (ClpAT) carrying Thr in place of Met 169 is highly soluble but indistinguishable from the wild-type ClpA in its ability to hydrolyze ATP and to support the ClpP-mediated proteolysis. Here we show that ATP hydrolysis is essential for assembly of ClpAT/ClpP complex upon analysis of the mixture of its components by gel filtration followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate Either ADP or adenosine 5′-(β,γ-imido)-triphosphate could not support the complex formation. Furthermore, ClpAT/K501T which carries a mutation in the second ATP-binding site and therefore is unable to cleave ATP could not interact with ClpP. On the other hand, ClpAT/K220T carrying a mutation in the first site and ClpP could be assembled into a complex at 2 mM ATP but not at 0.5 mM, at which concentration the trimeric mutant protein can not form a hexamer. These results indicate that assembly of protease Ti requires hydrolysis of ATP by ClpA in addition to its binding for hexamer formation.

The negatively regulating zinc finger protein (NZFP) is an essential transcription repressor required for early development during gastrulation in Xenopus laevis. In this study, we found that NZFP interacts with the small ubiquitin-like modifier (SUMO) conjugation E2 enzyme, Ubc9, and contains three putative SUMO conjugation sites. Studies with NZFP mutants containing mutations at the putative SUMO conjugation sites showed that these sites were able to be modified independently with SUMO. NZFP was found to be localized in the same nuclear bodies with SUMO-1. However, sumoylation of NZFP did not play a role either in the translocation of NZFP into the nucleus or on nuclear body formation. While wild type NZFP showed significant transcriptional repression, SUMO-conjugation site mutants manifested a decrease in transcriptional repression activity which is reversely proportional to the amount of sumoylation. The sumoylation defective mutant lost its TBP binding activity, while wild type NZFP interacted with TBP and inhibited transcription complex formation. These results strongly suggest that the sumoylation of NZFP facilitates NZFP to bind to TBP and the NZFP/TBP complex then represses the transcription of the target gene by inhibiting basal transcription complex formation.

Protein ubiquitination plays a critical role in regulating many cellular events, such as protein localization and stability, cellular signal transduction and DNA repair. Recent studies have shown that polyubiquitin (polyUb) chains elongate through heterogeneous isopeptide linkages to K11, K29, K48 and K63. In this study we have investigated the usage of isopeptide linkages of polyUb chains in different molecular weight regions by using quantitative mass spectrometry.Recombinant Chfr protein was autoubiquitinated by E1 enzyme, E2 enzyme UbcH5 and ubiquitin (WT Ub, K11R Ub, K48R Ub and K63R Ub) in vitro, and different molecular weight regions of ubiquitinated Chfr were then subjected to liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) following sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and in-gel digestion.Absolute QUantitative Analysis (AQUA) of polyUb chain formation with wild-type (WT) and point mutants of ubiquitin was performed, and the results suggested that the K11 polyUb chain was most frequently used in the high ubiquitin conjugates of WT Ub. Furthermore, the extent of polyUb chain formation with K11R Ub was decreased about 10-fold compared to polyUb chain formation with WT Ub through the entire molecular weight region. The present study suggests that the linkage through K11 plays crucial roles in polyUb chain formation.Topologies of polyUb chains in the low and high Ub conjugates were studied using mass spectrometry. K48 and K63 were the primary ubiquitination sites of the low molecular weight Ub conjugates, whereas K11 was the critical site of polyUb chain formation in high molecular weight Ub conjugates.

The clpA gene that encodes the ATPase subunit of the ATP-dependent protease Ti (Clp) in Escherichia coli contains a putative internal translational initiation site. Here we show that mutagenesis of its 5'-end AUG codon resulted in an exclusive synthesis of the 65-kDa protein (ClpA65), while mutation at the internal 169th AUG codon (Met) to ACG (Thr) produced only the 84-kDa protein (ClpA84T). On the other hand, the cells carrying the wild-type clpA gene produced both the 84- and 65-kDa proteins (ClpA84/65). While the purified ClpA84T and ClpA84/65 hydrolyzed ATP nearly as well as the 84-kDa ClpA alone (ClpA84), ClpA65 cleaved ATP at a rate less than 5% of that by ClpA84. Unlike ClpA84 and ClpA84T, ClpA65 could not support the casein-degrading activity of ClpP. Furthermore, ClpA65 inhibited the proteolysis by the mixture of ClpP with ClpA84 or ClpA84T but not that with ClpA84/65, which could support the proteolytic activity of ClpP only about 40% as well as ClpA84. Nevertheless, ClpA65 showed little or no effect on the basal or protein-activated ATPase activity of ClpA84, ClpA84T, or ClpA84/65 alone or in the presence of ClpP. These results suggest that ClpA65 may interfere the interaction of ClpA84 or ClpA84T with ClpP and, hence, impair their assembly into an active form of the ATP-dependent protease Ti.

ClpX, an alternative ATP-binding subunit for protease Ti (also called Clp), has been shown to support the ATP-dependent hydrolysis of λO-protein by ClpP. clpX has also been reported to be in an operon with clpP , and therefore both are co-transcribed in a single mRNA using the promoter proximal to clpP , Here, we show that clpX can be expressed independently from clpP using its own promoter. The cells carrying clpX alone on a multicopy plasmid successively produced the 46-kDa ClpX protein, Moreover, in vitro translation analysis revealed that the recombinant plasmid containing clpX generates the 46-kDa protein that can be immunoprecipitated with anti-ClpX antibody. In addition, it has recently been reported that ClpX, but not ClpP, is required for normal replication of bacteriophage Mu. Thus, it appears that clpX can be expressed alone and/or co-expressed with clpP in cells depending on physiological conditions.

The importance of the P1 reactive site for the specificity of ecotin on target proteases was examined by site-directed mutagenesis. The replacement of Met at the P1 site with Ile, Arg, Glu, or Tyr showed little or no effect on the ability of ecotin to inhibit trypsin. Similar results were obtained for chymotrypsin, except that its replacement with Glu caused about 40% reduction of the inhibitory activity of ecotin. On the other hand, the replacement of the Met residue with Arg, Tyr, or Glu dramatically reduced its ability to inhibit elastase, while that with Ile showed little or no effect. Nevertheless, elastase could be completely inhibited upon incubation with excess amounts of the mutant ecotin containing Arg, Glu, or Tyr. Moreover, all the mutant forms of ecotin could be cleaved at the mutated P1 site upon incubation with trypsin at pH 3.75. In addition, the replacement of a Cys residue in the disulfide bridge with Ser showed little or no effect on the ability of ecotin to inhibit trypsin, chymotrypsin, or elastase. However, the mutant ecotin containing Ser was more sensitive to inactivation by heating at 100 degrees C than the wild-type inhibitor. Furthermore, the wild-type ecotin whose disulfide bond had been reduced and alkylated was also more easily inactivated by heat treatment than the untreated control. These results strongly suggest that the P1 site of ecotin is not crucial for its specificity on target proteases and that the disulfide bridge in ecotin appears to play an important role in maintenance of its structural stability.

CHFR functions as a mitotic checkpoint by delaying entry into metaphase in response to mitotic stress. CHFR is frequently silenced by hypermethylation in human cancers, indicating that CHFR is a tumor suppressor. To further elucidate the role of CHFR in tumorigenesis, we studied the relationship between CHFR and a novel CHFR-interacting protein, HLTF, helicase-like transcription factor. Here we show that CHFR binds to and ubiquitinates HLTF, leading to its degradation. HLTF modulates basal expression of PAI-1 involved in regulation of cell migration. Consistently, overexpression of CHFR inhibits cell migration, resulting from reduced HLTF followed by decreased PAI-1 expression. HLTF expression is also higher in human breast cancer cells where CHFR is not expressed. Taken together, this is the first report identifying the regulatory mechanism of HLTF by CHFR, suggesting that CHFR-mediated downregulation of HLTF may help protect against cancer.

HslVU is a bacterial ATP-dependent protease distantly related to eukaryotic proteasomes consisting of hexameric HslU ATPase and dodecameric HslV protease. As a homolog of the 20 S proteasome beta-subunits, HslV also uses the N-terminal threonine as the active site residue. However, unlike the proteasome that has only 6 active sites among the 14 beta-subunits, HslV has 12 active sites that could potentially contribute to proteolytic activity. Here, by using a series of HslV dodecamers containing different numbers of active sites, we demonstrate that like the proteasome, HslV with only approximately 6 active sites is sufficient to support full catalytic activity. However, a further reduction of the number of active sites leads to a proportional decrease in activity. Using proteasome inhibitors, we also demonstrate that substrate-mediated stabilization of the HslV-HslU interaction remains unchanged until the number of the active sites is decreased to approximately 6 but is gradually compromised upon further reduction. These results with a mathematical model suggest HslVU utilizes no more than 6 active sites at any given time, presumably because of the action of HslU. These results also suggest that each ATP-bound HslU subunit activates one HslV subunit and that substrate bound to the HslV active site stimulates the HslU ATPase activity by stabilizing the HslV-HslU interaction. We propose this mechanism plays an important role in supporting complete degradation of substrates while preventing wasteful ATP hydrolysis in the resting state by controlling the interaction between HslV and HslU through the catalytic engagement of the proteolytic active sites.

Chfr is a ubiquitin ligase that functions in the mitotic checkpoint by delaying entry into metaphase in response to mitotic stress. It has been suggested that Chfr is a tumour suppressor as Chfr is frequently silenced in human cancers. To better understand how Chfr activity relates to cell-cycle progression and tumorigenesis, we sought to identify Chfr-interacting proteins using affinity purification combined with mass spectrometry. Histone deacetylase 1 (HDAC1), which represses transcription by deacetylating histones, was newly isolated as a Chfr-interacting protein. Chfr binds and downregulates HDAC1 by inducing its polyubiquitylation, both in vitro and in vivo. Ectopic expression of Chfr in cancer cells that normally do not express it results in downregulation of HDAC1, leading to upregulation of the Cdk inhibitor p21^(CIP1/WAF1) and the metastasis suppressors KAI1 and E-cadherin. Coincident with these changes, cells arrest in the G1 phase of the cell cycle and become less invasive. Collectively, our data suggest that Chfr functions as a tumour suppressor by regulating HDAC1.

FLOWERING LOCUS T (FT) plays a key role as a mobile floral induction signal that initiates the floral transition. Therefore, precise control of FT expression is critical for the reproductive success of flowering plants. Coexistence of bivalent histone H3 lysine 27 trimethylation (H3K27me3) and H3K4me3 marks at the FT locus and the role of H3K27me3 as a strong FT repression mechanism in Arabidopsis have been reported. However, the role of an active mark, H3K4me3, in FT regulation has not been addressed, nor have the components affecting this mark been identified. Mutations in Arabidopsis thaliana Jumonji4 (AtJmj4) and EARLY FLOWERING6 (ELF6), two Arabidopsis genes encoding Jumonji (Jmj) family proteins, caused FT-dependent, additive early flowering correlated with increased expression of FT mRNA and increased H3K4me3 levels within FT chromatin. Purified recombinant AtJmj4 protein possesses specific demethylase activity for mono-, di-, and trimethylated H3K4. Tagged AtJmj4 and ELF6 proteins associate directly with the FT transcription initiation region, a region where the H3K4me3 levels were increased most significantly in the mutants. Thus, our study demonstrates the roles of AtJmj4 and ELF6 as H3K4 demethylases directly repressing FT chromatin and preventing precocious flowering in Arabidopsis.

The nucleotide sequence of a 876 bp region in E. coli chromosome that encodes Ecotin was determined. The proposed coding sequence for Ecotin is 486 nucleotides long, which would encode a protein consisting of 162 amino acids with a calculated molecular weight of 18 192 Da. The deduced primary sequence of Ecotin includes a 20-residue signal sequence, cleavage of which would give rise to a mature protein with a molecular weight of 16 099 Da. Ecotin does not contain any consensus reactive site sequences of known serine protease inhibitor families, suggesting that Ecotin is a novel inhibitor.

Summary The DNA encoding protease Do was isolated from an E. coli genomic DNA library in λgt11, and cloned into a Bluescript plasmid. The cells transformed with the recombinant plasmid were able to overproduce protease Do and grew normally. A mutant lacking the protease activity was also isolated by interrupting the chromosomal DNA with the kan gene. The mutant showed a prolonged lag period and reduced ability to degrade cell proteins as compared to its wild type. Moreover, they were unable to survive at high temperatures, similarly to the htrA mutants. These results suggest that protease Do may play an important role in the intracellular protein breakdown and is essential for survival at high temperatures. Identity of protease Do with the htrA gene product is discussed.

Chfr, a checkpoint with FHA and RING finger domains, plays an important role in cell cycle progression and tumor suppression. Chfr possesses the E3 ubiquitin ligase activity and stimulates the formation of polyubiquitin chains by Ub-conjugating enzymes, and induces the proteasome-dependent degradation of a number of cellular proteins, including Plk1 and Aurora A. While Chfr is a nuclear protein that functions within the cell nucleus, how Chfr is localized in the nucleus has not been clearly demonstrated. Here, we show that nuclear localization of Chfr is mediated by nuclear localization signal (NLS) sequences. To reveal the signal sequences responsible for nuclear localization, a short lysine-rich stretch (KKK) at amino acid residues 257-259 was replaced with alanine, which completely abolished nuclear localization. Moreover, we show that nuclear localization of Chfr is essential for its checkpoint function but not for its stability. Thus, our results suggest that NLS-mediated nuclear localization of Chfr leads to its accumulation within the nucleus, which may be important in the regulation of Chfr activation and Chfr-mediated cellular processes, including cell cycle progression and tumor suppression.

Escherichia coli HslVU is an ATP-dependent protease consisting of two heat shock proteins, the HslU ATPase and HslV peptidase. In the reconstituted enzyme, HslU stimulates the proteolytic activity of HslV by one to two orders of magnitude, while HslV increases the rate of ATP hydrolysis by HslU several-fold. Here we show that HslV alone can efficiently degrade certain unfolded proteins, such as unfolded lactalbumin and lysozyme prepared by complete reduction of disulfide bonds, but not their native forms. Furthermore, HslV alone cleaved a lactalbumin fragment sandwiched by two thioredoxin molecules, indicating that it can hydrolyze the internal peptide bonds of lactalbumin. Surprisingly, ATP inhibited the degradation of unfolded proteins by HslV. This inhibitory effect of ATP was markedly diminished by substitution of the Arg86 residue located in the apical pore of HslV with Gly, suggesting that interaction of ATP with the Arg residue blocks access of unfolded proteins to the proteolytic chamber of HslV. These results suggest that uncomplexed HslV is inactive under normal conditions, but may can degrade unfolded proteins when the ATP level is low, as it is during carbon starvation.

Chfr, a mitotic stress checkpoint, plays an important role in cell cycle progression, tumor suppression and the processes that require the E3 ubiquitin ligase activity mediated by the RING finger domain. Chfr stimulates the formation of polyubiquitin chains by ub-conjugating enzymes, and induces the proteasome-dependent degradation of a number of cellular proteins including Plk1 and Aurora A. In this study, we identified USP7 (also known as HAUSP), which is a member of a family of proteins that cleave polyubiquitin chains and/or ubiquitin precursors, as an interacting protein with Chfr by immunoaffinity purification and mass spectrometry, and their interaction greatly increases the stability of Chfr. In fact, USP7 can remove ubiquitin moiety from the autoubiquitinated Chfr both in vivo and in vitro, which results in the accumulation of Chfr in the cell. Thus, our finding suggests that USP7-mediated deubiquitination of Chfr leads to its accumulation, which might be a key regulatory step for Chfr activation and that USP7 may play an important role in the regulation of Chfr-mediated cellular processes including cell cycle progression and tumor suppression.

Ubiquitin-fold modifier 1 (Ufm1) is a recently identified new ubiquitin-like protein, whose tertiary structure displays a striking resemblance to ubiquitin. Similar to ubiquitin, it has a Gly residue conserved across species at the C-terminal region with extensions of various amino acid sequences that need to be processed in vivo prior to conjugation to target proteins. Here we report the isolation, cloning, and characterization of two novel mouse Ufm1-specific proteases, named UfSP1 and UfSP2. UfSP1 and UfSP2 are composed of 217 and 461 amino acids, respectively, and they have no sequence homology with previously known proteases. UfSP2 is present in most, if not all, of multicellular organisms including plant, nematode, fly, and mammal, whereas UfSP1 could not be found in plant and nematode upon data base search. UfSP1 and UfSP2 cleaved the C-terminal extension of Ufm1 but not that of ubiquitin or other ubiquitin-like proteins, such as SUMO-1 and ISG15. Both were also capable of releasing Ufm1 from Ufm1-conjugated cellular proteins. They were sensitive to inhibition by sulfhydryl-blocking agents, such as N-ethylmaleimide, and their active site Cys could be labeled with Ufm1-vinylmethylester. Moreover, replacement of the conserved Cys residue by Ser resulted in a complete loss of the UfSP1 and UfSP2 activities. These results indicate that UfSP1 and UfSP2 are novel thiol proteases that specifically process the C terminus of Ufm1.

Many inhibitor of apoptosis (IAP) family proteins inhibit apoptosis. IAPs contain N-terminal baculovirus IAP repeat domains and a C-terminal RING ubiquitin ligase domain. Drosophila IAP DIAP1 is essential for the survival of many cells, protecting them from apoptosis by inhibiting active caspases. Apoptosis initiates when proteins such as Reaper, Hid, and Grim bind a surface groove in DIAP1 baculovirus IAP repeat domains via an N-terminal IAP-binding motif. This evolutionarily conserved interaction disrupts DIAP1-caspase interactions, unleashing apoptosis-inducing caspase activity. A second Drosophila IAP, DIAP2, also binds Rpr and Hid and inhibits apoptosis in multiple contexts when overexpressed. However, due to a lack of mutants, little is known about the normal functions of DIAP2. We report the generation of diap2 null mutants. These flies are viable and show no defects in developmental or stress-induced apoptosis. Instead, DIAP2 is required for the innate immune response to Gram-negative bacterial infection. DIAP2 promotes cytoplasmic cleavage and nuclear translocation of the NF-{kappa}B homolog Relish, and this requires the DIAP2 RING domain. Increasing the genetic dose of diap2 results in an increased immune response, whereas expression of Rpr or Hid results in down-regulation of DIAP2 protein levels. Together these observations suggest that DIAP2 can regulate immune signaling in a dose-dependent manner, and this can be regulated by IBM-containing proteins. Therefore, diap2 may identify a point of convergence between apoptosis and immune signaling pathways.

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by the degeneration of dopaminergic neurons in the substantia nigra. Mitochondrial dysfunction has been implicated as an important trigger for Parkinson's disease-like pathogenesis because exposure to environmental mitochondrial toxins leads to Parkinson's disease-like pathology. Recently, multiple genes mediating familial forms of Parkinson's disease have been identified, including PTEN-induced kinase 1 (PINK1 ; PARK6 ) and parkin (PARK2 ), which are also associated with sporadic forms of Parkinson's disease. PINK1 encodes a putative serine/threonine kinase with a mitochondrial targeting sequence. So far, no in vivo studies have been reported for pink1 in any model system. Here we show that removal of Drosophila PINK1 homologue (CG4523; hereafter called pink1) function results in male sterility, apoptotic muscle degeneration, defects in mitochondrial morphology and increased sensitivity to multiple stresses including oxidative stress. Pink1 localizes to mitochondria, and mitochondrial cristae are fragmented in pink1 mutants. Expression of human PINK1 in the Drosophila testes restores male fertility and normal mitochondrial morphology in a portion of pink1 mutants, demonstrating functional conservation between human and Drosophila Pink1. Loss of Drosophila parkin shows phenotypes similar to loss of pink1 function. Notably, overexpression of parkin rescues the male sterility and mitochondrial morphology defects of pink1 mutants, whereas double mutants removing both pink1 and parkin function show muscle phenotypes identical to those observed in either mutant alone. These observations suggest that pink1 and parkin function, at least in part, in the same pathway, with pink1 functioning upstream of parkin. The role of the pink1–parkin pathway in regulating mitochondrial function underscores the importance of mitochondrial dysfunction as a central mechanism of Parkinson's disease pathogenesis.

Daxx is a multifunctional protein that regulates a variety of cellular processes, including transcription, cell cycle, and apoptosis. SPOP is a BTB (Bric-a-brac/Tramtrack/Broad complex) protein that constitutes Cul3-based ubiquitin ligases. Here we show that SPOP serves as an adaptor of Daxx for the ubiquitination by Cul3-based ubiquitin ligase and subsequent degradation by the proteasome. Expression of SPOP with Cul3 markedly reduced Daxx level, and this degradation was blocked by SPOP-specific short hairpin RNAs. Inhibition of the proteasome by MG132 caused the prevention of Daxx degradation in parallel with the accumulation of ubiquitinated Daxx. Expression of SPOP with Cul3 reversed Daxx-mediated repression of ETS1- and p53-dependent transcription, and short hairpin RNA-mediated knock down of SPOP blocked the recovery of their transcriptional activation. Furthermore, Daxx degradation led to the cleavage of poly(ADP-ribose) polymerase and the increase in the number of terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein nick end-labeling-positive apoptotic cells. These results suggest that SPOP/Cul3-ubiquitin ligase plays an essential role in the control of Daxx level and, thus, in the regulation of Daxx-mediated cellular processes, including transcriptional regulation and apoptosis.