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As next-generation artificial enzymes, nanozymes have shown great promise for tumor catalytic therapy. In particular, their peroxidase-like activity has been employed to catalyze hydrogen peroxide (H2O2) to produce highly toxic hydroxyl radicals (•OH) to kill tumor cells. However, limited by the low affinity between nanozymes with H2O2 and the low level of H2O2 in the tumor microenvironment, peroxidase nanozymes usually produced insufficient •OH to kill tumor cells for therapeutic purposes. Herein, we present a pyrite peroxidase nanozyme with ultrahigh H2O2 affinity, resulting in a 4144- and 3086-fold increase of catalytic activity compared with that of classical Fe3O4 nanozyme and natural horseradish peroxidase, respectively. We found that the pyrite nanozyme also possesses intrinsic glutathione oxidase-like activity, which catalyzes the oxidation of reduced glutathione accompanied by H2O2 generation. Thus, the dual-activity pyrite nanozyme constitutes a self-cascade platform to generate abundant •OH and deplete reduced glutathione, which induces apoptosis as well as ferroptosis of tumor cells. Consequently, it killed apoptosis-resistant tumor cells harboring KRAS mutation by inducing ferroptosis. The pyrite nanozyme also exhibited favorable tumor-specific cytotoxicity and biodegradability to ensure its biosafety. These results indicate that the high-performance pyrite nanozyme is an effective therapeutic reagent and may aid the development of nanozyme-based tumor catalytic therapy.

Background miRNA, as a biological marker, had more and more attention in recent years due to the important role it plays in cancer. Currently, there are extensive studies on miRNAs, among which miR-330-3p is reported to be implicated in the pathophysiological processes of various cancers. However, little progress has been made in the mechanism of miR-330-3p in gastric cancer. Objective To explore the expression and relevant mechanism of miR-330-3p and PRRX1 in gastric cancer (GC). Methods Forty-five GC patients (study group), from whom paired GC and paracancerous tissues were collected, and another 45 healthy subjects (control group) who underwent physical examination during the same period were enrolled. In addition, GC cells and human gastric mucosa cells were purchased, and miR-330-3p-mimics, miR-330-3p-inhibitor, miR-NC, si-PRRX1, and sh-PRRX1 were transfected into MKN45, SGC7901 cell. QRT-PCR was employed to assess the miR-330-3p and PRRX1 expressions in the samples, and the cell expressions of PRRX1, GSK-3β, p-GSK-3β, β-catenin, p-β-catenin, cyclin D1, N-cadherin, E-cadherin and vimentin were evaluated by Western blot (WB). MTT, Transwell and wound-healing experiments were adopted to detect cell proliferation, invasion and migration. Results MiR-330-3p was under-expressed, while PRRX1 was highly expressed in the serum of patients, both of which had an area under the curve (AUC) of more than 0.9. MiR-330-3p and PRRX1 were associated with tumor diameter, TNM staging, lymph node metastasis and differentiation of GC patients. Overexpression of miR-330-3p and inhibition of PRRX1 expression could suppress epithelial-mesenchymal transition (EMT), proliferation, invasion and apoptosis of cells. What is more, WB assay showed that overexpressed miR-330-3p and inhibited PRRX1 could inhibit the expression levels of p-GSK-3β, β-catenin, cyclin D1, N-cadherin and vimentin proteins, while elevating GSK-3β, p-β-catenin and E-cadherin protein expressions. Dual-luciferase reporter assay confirmed that there was a targeting relation between miR-330-3p and PRRX1. Furthermore, rescue experiments revealed that the cell proliferation, invasion, migration did not differ significantly between co-transfected miR-330-3p-mimics+sh-PRRX1, miR-330-3p-inhibitor+si-PRRX1 groups of MKN45 and SGC7901 and the miR-NC group (without transfected sequences). Conclusion Overexpressed miR-330-3p can promote cell EMT, proliferation, invasion and apoptosis through inhibiting PRRX1-mediated Wnt/β-catenin signaling pathway, which is expected to be a potential therapeutic target for GC.

Collagen and calcium-binding EGF domain-1 (CCBE1) is essential for lymphatic vascular development as it promotes vascular endothelial growth factor C (VEGFC) proteolysis. A recent study reported that CCBE1 was overexpressed in epithelial colorectal cancer (CRC) cells; however, the role of CCBE1 in tumor lymphangiogenesis and the mechanism underlying dysregulated CCBE1 expression in CRC remain undefined. Methods: The role of CCBE1 in tumor lymphangiogenesis and lymphatic metastasis was investigated using human lymphatic endothelial cells (HLECs) model in vitro, and a hindfoot lymphatic metastasis model in vivo. Immunochemistry analysis was performed to assess CCBE1 expression, prognostic value and correlation with clinicopathological characteristics in CRC. The biochemical function and transcriptional regulatory mechanism of CCBE1 were explored by western blot, qPCR, and chromatin immunoprecipitation. Results: Cancer cell-derived CCBE1 enhances VEGFC proteolysis in vitro, facilitates tube formation and migration of HLECs in vitro, and promotes tumor lymphangiogenesis and lymphatic metastasis in vivo. In addition to CRC cells, tumor stroma within CRC tissue shows high CCBE1 expression, which is associated with high lymphatic vessel density, increased lymph node metastasis and poor prognosis. Cancer-associated fibroblasts (CAFs) express and secret CCBE1, thereby contributing to VEGFC maturation and tumor lymphangiogenesis in CRC. Transforming growth factor beta (TGF-β) downregulates the transcription and lymphangiogenic function of CCBE1 in CAFs and CRC cells through direct binding of SMADs to CCBE1 gene locus. Inactivation of the TGF-β pathway correlates with increased CCBE1 expression in CRC. Conclusion: Our results demonstrate the protumorigenic role of CCBE1 in promoting lymphangiogenesis and lymphatic metastasis in CRC, revealing a new mechanism by which loss of TGF-β signaling promotes CRC metastasis.

Topoisomerase (TOP) inhibitors were commonly used as chemotherapeutic agents in the treatment of cancers. In our present study, we found that etoposide (ETO), a topoisomerase 2 (TOP2) inhibitor, upregulated the production of Interleukin 10 (IL-10) in lipopolysaccharide (LPS)-stimulated macrophages. Besides, other TOP2 inhibitors including doxorubicin hydrochloride (DOX) and teniposide (TEN) were also able to augment IL-10 production. Meanwhile, the expression levels of pro-inflammatory factors, for example IL-6 and TNF-α, were also decreased accordingly by the treatment of the TOP2 inhibitors. Of note, ETO facilitated IL-10 secretion, which might be regulated by transcription factor Maf via PI3K/AKT pathway, as pharmaceutic blockage of kinase PI3K or AKT attenuated ETO-induced Maf and IL-10 expression. Further, in LPS-induced mice sepsis model, the enhanced generation of IL-10 was observed in ETO-treated mice, whereas pro-inflammatory cytokines were decreased, which significantly reduced the mortality of mice from LPS-induced lethal cytokine storm. Taken together, these results indicated that ETO may exhibit an anti-inflammatory role by upregulating the alteration of transcription factor Maf and promoting subsequential IL-10 secretion via PI3K/Akt pathway in LPS-induced macrophages. Therefore, ETO may serve as a potential anti-inflammatory agent and employed to severe pro-inflammatory diseases including COVID-19.

The ubiquitin–proteasome system regulates a variety of cellular processes including growth, differentiation and apoptosis. While E1, E2, and E3 are responsible for the conjugation of ubiquitin to substrates, deubiquitinating enzymes (DUBs) reverse the process to remove ubiquitin and edit ubiquitin chains, which have profound effects on substrates’ degradation, localization, and activities. In the present study, we found that the deubiquitinating enzyme USP47 was markedly decreased in primary colorectal cancers (CRC). Its reduced expression was associated with shorter disease-free survival of CRC patients. In cultured CRC cells, knockdown of USP47 increased pyroptosis and apoptosis induced by chemotherapeutic doxorubicin. We found that USP47 was able to bind with transcription elongation factor a3 (TCEA3) and regulated its deubiquitination and intracellular level. While ectopic expression of USP47 increased cellular TCEA3 and resistance to doxorubicin, the effect was markedly attenuated by TCEA3 knockdown. Further analysis showed that the level of pro-apoptotic Bax was regulated by TCEA3. These results indicated that the USP47-TCEA3 axis modulates cell pyroptosis and apoptosis and may serve as a target for therapeutic intervention in CRC.

COVID-19 is often characterized by dysregulated inflammatory and immune responses. It has been shown that the Traditional Chinese Medicine formulation Qing-Fei-Pai-Du decoction (QFPDD) is effective in the treatment of the disease, especially for patients in the early stage. Our network pharmacology analyses indicated that many inflammation and immune-related molecules were the targets of the active components of QFPDD, which propelled us to examine the effects of the decoction on inflammation. We found in the present study that QFPDD effectively alleviated dextran sulfate sodium-induced intestinal inflammation in mice. It inhibited the production of pro-inflammatory cytokines IL-6 and TNFα, and promoted the expression of anti-inflammatory cytokine IL-10 by macrophagic cells. Further investigations found that QFPDD and one of its active components wogonoside markedly reduced LPS-stimulated phosphorylation of transcription factor ATF2, an important regulator of multiple cytokines expression. Our data revealed that both QFPDD and wogonoside decreased the half-life of ATF2 and promoted its proteasomal degradation. Of note, QFPDD and wogonoside down-regulated deubiquitinating enzyme USP14 along with inducing ATF2 degradation. Inhibition of USP14 with the small molecular inhibitor IU1 also led to the decrease of ATF2 in the cells, indicating that QFPDD and wogonoside may act through regulating USP14 to promote ATF2 degradation. To further assess the importance of ubiquitination in regulating ATF2, we generated mice that were intestinal-specific KLHL5 deficiency, a CUL3-interacting protein participating in substrate recognition of E3s. In these mice, QFPDD mitigated inflammatory reaction in the spleen, but not intestinal inflammation, suggesting CUL3-KLHL5 may function as an E3 for ATF2 degradation.

Background Peanut is a nutrient-rich popular food. However, it has been estimated that 1%–2% of the world's population are allergic to peanut, which can cause serious allergic reactions, including allergic shock and anaphylactic death in certain people. Thus, identifying peanut allergens and decreasing allergenicity have been serious and important food safety issues. Scope and approach Sixteen peanut allergens and their structure and IgE linear binding site are summarized in this review. Various techniques and means to reduce peanut allergenicity, including physical (heat, radiation, and high-pressure treatments), chemical (glycosylation, magnetic-bead adsorption, and acid treatment), and biological (microbial fermentation, enzyme catalysis, and genetic engineering) methods, are evaluated and compared. Key findings and conclusions Multiple allergens are present in peanuts. Their sequences, structures and allergenic characters have been examined extensively. Although Ara h 1, Ara h 2, Ara h 3, and Ara h 6 are the major allergens, individuals allergic to peanut often had IgE recognized multiple peanut allergens. Among the various methods capable of reducing peanut allergenicity, heating and high-pressure treatments appear to be practical and effective in general, and irradiation, acid treatment, and other physical and chemical may work under certain circumstances. Of note, fermentation has the advantage of improving tasting and reducing allergenicity at the same time. It is conceivable that combination of multiple methods will result in more effective means to reduce allergenicity of peanut and prevent the development of serious allergy.

Ubiquitin specific peptidase 5 (USP5) is a ubiquitous expressed deubiquitinating enzyme (DUB). It has been shown involved in DNA repair, apoptosis, inflammation, and tumor cell growth. However, the function and molecular mechanism of USP5 in colorectal cancer (CRC) are still unclear. In the present study, we asked how it affected the growth of colorectal cancer cells. Methods: A shRNA-based high-content screening was performed to identify DUBs affecting the growth of CRC cells. CCK-8 assay and xenografts were used to assess CRC cell growth, survival and tumorigenesis. RT-qPCR, immunoblotting and immunohistochemistry were carried out to quantitate USP5 expression in CRC tissues and cell lines. Immunoprecipitation and mass spectrometry analysis were performed to identify USP5-interacting proteins. Cycloheximide chase was performed to assess Tu translation elongation factor (TUFM) stability. Dual luciferase reporter assay was utilized for USP5 promoter analysis. Results: We found that USP5 was highly expressed in a group of primary CRC tissues, and the increased USP5 was correlated with clinical stages and shorter overall survival. While USP5 knockdown effectively inhibited CRC cell growth, overexpressed USP5 promoted the growth of CRC cells and made them more resistant to doxorubicin (DOX). TUFM was discovered as a substrate of USP5. USP5 deubiquitinated TUFM and increased its level in CRC cells. Enforced expression of TUFM was able to alleviate the growth inhibition induced by USP5 knockdown. Further analyses showed that EBF transcription factor 1 (EBF1) was a major regulator for USP5 transcription, and DOX inhibited EBF1-USP5-TUFM axis in CRC cells. Conclusions: USP5 was required for CRC cells and promoted their growth and resistance to chemotherapeutics. TUFM was a USP5 deubiquitinating substrate that mediated the cellular effects of USP5. The transcription of USP5 was regulated by EBF1. Thus, targeting EBF1-USP5-TUFM axis is a potential novel strategy for CRC treatment.

BACKGROUND We explored the dynamic expression of platelet-derived growth factor-D (PDGF-D) and phosphorylated platelet-derived growth factor receptor-β (p-PDGFR-β) after traumatic brain injury in rats to provide theoretical basis for selecting therapeutic target and intervention time after traumatic brain injury. METHODS This study prepared the weight drop/impact acceleration-induced traumatic brain injury (TBI) model in rats, including sham group and TBI groups at different observation time points (6 hours, 12 hours, 24 hours, 3 days, and 7 days after TBI). The dynamic expression of PDGF-D and p-PDGFR-β after TBI were detected by western blot and immunofluorescence staining. RESULTS The expression level of PDGF-D and p-PDGFR-β after TBI was detected by western blot. The PDGF-D level was increased (p < 0.05) 6 hours after TBI and remained at the high level until 3 days after TBI (p < 0.05). The p-PDGFR-β level was increased (p < 0.05) 12 hours after TBI and remained at the high level until 3 days after TBI (p < 0.05). PDGF-D and p-PDGFR-β in brain tissues were found by immunofluorescence in the perihematoma area 24 hours after TBI. CONCLUSIONS This study revealed the expression phenomenon of PDGF-D and p-PDGFR-β after TBI in rats, suggesting that PDGF-D participates in the process of secondary brain injury after TBI through specific binding with PDGFR-β, which provides a theoretical basis for further research on selecting therapeutic targets and intervention times after TBI.

Mutations and altered expression of deubiquitinating enzymes (DUBs) have been found associated with many human diseases including cancers. In this study, Ubiquitin specific protease 1 (USP1) expression was found significantly increased in some colorectal cancers (CRC). The elevated USP1 level was associated with short overall survival of patients and with advanced stages of cancers. In cultured CRC cells, knockdown of USP1 induced growth arrest at G2/M of cell cycle and reduced the expression of anti-apoptotic proteins Bcl-2 and Mcl-1. Its knockdown also led to reduction of DNA-repair related substrates FANCD2 and ID1. Further investigations found that small molecular inhibitor of USP1 ML323 sensitized CRC cells to DNA-targeting chemotherapeutics, including doxorubicin, TOPI/II inhibitors, and PARP inhibitor, but not to 5-Fu. These results indicate that USP1 plays a critical in colorectal cancer cell survival and is a promising target for anti-colorectal cancer chemotherapy. Targeting USP1 may represent an effective strategy to regulate the DNA-repairing system.

Although proteasome inhibitors such as bortezomib had significant therapeutic effects in multiple myeloma and mantel cell lymphoma, they exhibited minimal clinical activity as a monotherapy for solid tumors, including colorectal cancer. We found in this study that proteasome inhibition induced a remarkable nuclear exportation of ubiquitinated proteins. Inhibition of CRM1, the nuclear export carrier protein, hampered protein export and synergistically enhanced the cytotoxic action of bortezomib on colon cancer cells containing wild-type p53, which underwent G2–M cell-cycle block and apoptosis. Further analysis indicated that tumor suppressor p53 was one of the proteins exported from nuclei upon proteasome inhibition, and in the presence of CRM1 inhibitor KPT330, nuclear p53, and expression of its target genes were increased markedly. Moreover, knockdown of p53 significantly reduced the synergistic cytotoxic action of bortezomib and KPT330 on p53+/+ HCT116 cells. In mice, KPT330 markedly augmented the antitumor action of bortezomib against HCT116 xenografts as well as patient-derived xenografts that harbored functional p53. These results indicate that nuclear p53 is a major mediator in the synergistic antitumor effect of bortezomib and KPT330, and provides a rationale for the use of proteasome inhibitor together with nuclear export blocker in the treatment of colorectal cancer. It is conceivable that targeting nuclear exportation may serve as a novel strategy to overcome resistance and raise chemotherapeutic efficacy, especially for the drugs that activate the p53 system. Mol Cancer Ther; 16(4); 717–28. ©2016 AACR.

A nanohybrid probe was fabricated from manganese dioxide nanosheets (MnO2 NSs), molybdenum disulfide quantum dots (MoS2 QDs) and o-phenylenediamine (OPD) for ratiometric detection of glutathione (GSH) in aqueous solutions and living cells. The MoS2 QDs act as the fluorescent “turn off-on” units. The MnO2 NSs have 3 functions, viz. (a) as fluorescence quencher, (b) as fluorescence initiator for oxidized OPD (ox OPD) and (c) as selective recognizer of GSH. The quenched blue fluorescence of the MoS2 QDs can be restored by introducing GSH that reduces the MnO2 NSs. However, the green fluorescence of ox OPD is decreased through the loss of peroxidase activity of MnO2 NSs in the presence of GSH. Therefore, the ratio of the fluorescence intensities at 560 and 400 nm (from ox OPD and MoS2 QDs, respectively) linearly decreases with increasing concentrations of GSH. Under the optimal conditions, the detection limit for GSH is as low as 90 nM. The method was successfully applied to the determination of GSH in human serum samples. This nanohybrid also is shown to be membrane-permeable and to have low cytotoxicity. This paved the way to intracellular sensing of GSH in living normal HFF and cancerous HeLa cells. Additionally, by combining with logic gate, this assay was successfully applied to visually discriminate changes in the intracellular GSH. The combination of ratiometric fluorometry and peroxidase mimicking can provide a wide range of application in bioanalysis and intracellular imaging.

In the effort to identify natural products that regulate immunity and inflammation, we found that nitidine chloride (NC), an alkaloid from herb Zanthoxylum nitidum, enhanced IL-10 production in lipopolysaccharide (LPS)-stimulated myeloid cells. While NC was shown to be capable of inhibiting topoisomerase I (TOP1), NC analogs that could not inhibit TOP1 failed to increase IL-10 production. Moreover, medicinal TOP1 inhibitors TPT and SN-38 also augmented IL-10 production significantly, whereas knockdown of TOP1 prevented NC, TPT, and SN-38 from enhancing IL-10 expression. Thus, NC promoted IL-10 production by inhibiting TOP1. In LPS-induced endotoxemic mice, NC and TOP1 inhibitors increased IL-10 production, suppressed inflammatory responses, and reduced mortality remarkably. The anti-inflammatory activities of TOP1 inhibition were markedly reduced by IL-10-neutralizing antibody and largely absent in IL-10-deficient mice. In LPS-stimulated RAW264.7 cells and in peritoneal macrophages from endotoxemic mice, NC and TOP1 inhibitors significantly enhanced the activation of Akt, a critical signal transducer for IL-10 production, and inhibition of Akt prevented these compounds from enhancing IL-10 production and ameliorating endotoxemia. These data indicated that NC and TOP1 inhibitors are able to exert anti-inflammatory action through enhancing Akt-mediated IL-10 production and may assist with the treatment of inflammatory diseases.

Background We and others have previously shown that the STAT3 signaling pathway is activated in some esophageal squamous cell carcinoma (ESCC) cells and is required for the survival and growth of these primary ESCC-derived xenografts. It has also been shown that the natural polyphenol curcumin is an effective anti-tumor agent. Methods Luciferase assay and immunoblotting were performed to examine whether curcumin suppressed STAT3 signaling. CCK-8 assay and xenografts were utilized for analyzing ESCC cell growth in culture and mice. Soft agar assay was carried out to determine the colony formation ability of ESCC cells in the presence or absence of curcumin. Cell death and cell cycle were assessed by In CELL Analyzer 2000. Immunohistochemistry and TUNEL assay were used for detecting apoptosis in ESCC tisuses. Molecular docking was performed to evaluate the interaction of curcumin with JAK2. JAK2 activity was assessed using an in vitro cell-free system. HE staining was used to evaluate the ESCC tissues. Results The natural polyphenol curcumin inhibited STAT3 phosphorylation rapidly and blocked STAT3-mediated signaling in ESCC cells. It also induced growth arrest and apoptosis in cultured ESCC cells, which were attenuated by enforced expression of STAT3. Furthermore, curcumin preferentially blocked the growth of primary ESCC-derived xenografts that harbored activated STAT3. Conclusions Curcumin is able to exert anti-tumor action through inhibiting the STAT3 signaling pathway. Giving its wide use in traditional medicines with low toxicity and few adverse reactions, it is conceivable that curcumin might be further explored as a unique STAT3 inhibitor for anti-cancer therapies.

Ferritin, a widely expressed iron binding protein, has emerged as a promising drug delivery vehicle due to its unique architecture, intrinsic tumor targeting property, and excellent biocompatibility. However, the translation studies of ferritin drug carrier are impeded by the low efficiency and low yield of the drug loading process, which typically employed Urea and pH dependent dis-assembly/reassembly methods. Here, based on crystal structure and protein mutation analyses, we identified a natural drug entry channel existed on the shell of recombinant human H-ferritin (HFn). Moreover, the opening state of this drug entry channel is sensitive to temperature changes. Based on these findings, we develop a simple channel-based drug loading strategy that avoids the denaturation of HFn protein cage with denaturing agents. Loading doxorubicin (Dox) into the HFn protein cage (HFn-Dox) by the channel-based strategy yields significantly higher drug loading efficiency, increased HFn recovery rate, and better stability than that of the denaturation-based methods. Importantly, animal experiments showed that the channel-loaded HFn-Dox had excellent biosafety and significantly improved antitumor activity when compared with the HFn-Dox prepared by the denaturation-based methods. We also found that the drug entry channel appears unique for HFn protein cage and is accessible to multiple small molecule anticancer drugs. Thus, the drug entry channel of HFn protein cage identified in this study provides a novel and highly effective drug loading approach for preparing HFn-drug formulations, which will greatly facilitate the translational study of ferritin drug carriers in cancer-targeting therapies.

Enzyme-mediated activation of prodrug in tumor cells is an attractive strategy for targeted cancer therapy. Of note, natural peroxidase and indole-3-acetic acid (IAA) have been shown as a promising enzyme-prodrug combination. However, insufficient endogenous peroxidase activity and the difficulty in selectively expressing exogenous peroxidase in tumor cells have severely limited its antitumor efficiency. The discovery of nanomaterials with peroxidase-like activity (nanozyme), particularly the metal-free nanozymes that possesses high stability and good biocompatibility, bring a new opportunity to overcome these challenges. Here, under the guidance of theoretical calculations, we developed a metal-free phosphorous and nitrogen dual-doped porous hollow carbon sphere nanozyme (PNCNzyme) with robust peroxidase-like activity in acidic environment. Based on this metal-free nanozyme, we developed a nanozyme-IAA activation strategy that activated IAA to produce abundant ROS and trigger tumor cell apoptosis. We also introduced folate (FA) onto the nanozyme to enhance its tumor targeting and endocytosis efficacy by tumor cells. Upon administrated to mice, the FA-PNCNzymes@IAA effectively accumulated in the xenografts derived from cervical cancer cells, catalyzed the production of ROS, and induced apoptosis. Taken together, these data demonstrate that the nanozyme-IAA combination is an effective enzyme-prodrug activation strategy for tumor targeted catalytic therapy.

Although it is generally believed that nicotine accounts for the beneficial effect of smoking on ulcerative colitis, the underlying mechanisms remain not well understood. Our previous finding that nicotine inhibits inflammatory responses through inducing miR-124 prompted us to ask whether the miRNA is involved in the protective action of nicotine against UC. Our present study found that miR-124 expression is upregulated in colon tissues from UC patients and DSS colitis mice. Nicotine treatment further augmented miR-124 expression in lymphocytes isolated from human ulcerative colonic mucosa and ulcerative colon tissues from DSS mice, both in infiltrated lymphocytes and epithelial cells. Moreover, knockdown of miR-124 significantly diminished the beneficial effect of nicotine on murine colitis and IL-6-treated Caco-2 colon epithelial cells. Further analysis indicated that nicotine inhibited STAT3 activation in vivo and in IL-6 treated Caco-2 cells and Jurkat human T lymphocytes, in which miR-124 knockdown led to increased activation of STAT3. Blocking STAT3 activity alone is beneficial for DSS colitis and also abolished nicotine's protective effect in this model. These data indicate that nicotine exerts its protective action in UC through inducing miR-124 and inhibiting STAT3, and suggest that the miR-124/STAT3 system is a potential target for the therapeutic intervention of UC.Nicotine upregulates miR-124 expression in ulcerative colon tissues and cells. MiR-124 is required for the protective role of nicotine in DSS colitis mice and epithelial cells. The protective effect of nicotine in murine DSS colitis depends on blocking STAT3 activation. MiR-124 mediates the inhibitory role of nicotine on STAT3/p-STAT3. Targeting miR-124 and STAT3 represents a novel approach for treating ulcerative colitis.

Chemokine‐like factor (CKLF)‐like, MAL and related proteins for vesicle trafficking and membrane link (MARVEL) transmembrane domain‐containing family proteins (CMTMs) have significant roles in the immune system, in male reproduction, as well as in tumorigenesis. Previous studies have shown that CMTM family member 7 (CMTM7) was broadly expressed in various normal tissues, but not in lung, gastric, esophageal, pancreas, and cervix cancers. To explore its relationship with liver cancer, we examined the expression of CMTM7 in liver cancers and its correlation with clinical and pathological conditions. We found that CMTM7 expression was markedly reduced in liver cancer tissues, and negatively correlated with TNM staging and tumor metastasis. In vitro studies showed that enforced expression of CMTM7 inhibited the cell growth and migration of liver cancer cells. Further analysis revealed that CMTM7 suppressed AKT signaling and induced cell cycle arrest at the G0/G1 phase in the liver cancer cells, likely as the consequent of decreased levels of cyclin D1, cyclin‐dependent kinase 4 (CDK4), and CDK6, and increased p27 expression. Thus, CMTM7 functions as a tumor suppressor in liver cancer through suppressing cell cycle progression.

Dysregulation of the Hippo pathway occurs in a variety of cancers and often correlates with a poor prognosis. To further explore the potential role of Hippo pathway dysregulation in tumor development and progression, we investigated its downstream transcription factor TEAD4 in colorectal cancer (CRC). Increased expression and nuclear localization of TEAD4 were found in a significant portion of CRC tissues, in association with metastasis and a poor prognosis. In CRC cells, TEAD4 knockdown induced the mesenchymal-epithelial transition and decreased cell mobility in vitro and metastasis in vivo. Microarray analysis revealed that TEAD4 promoted cell adhesion and upregulated the epithelial-mesenchymal transition-related transcriptome in CRC cells. Vimentin was identified as a new direct target gene mediating TEAD4 function in CRC cells, whereby forced vimentin expression markedly reversed TEAD4-knockdown-induced cell morphological changes and decreased mobility. Interestingly, rescued expression of both WT TEAD4 and a Y429H mutant can reverse the mesenchymal-epithelial transition and increase vimentin expression, cell mobility and metastatic potential in TEAD4-knockdown CRC cells. The discrepant expression of YAP and TEAD4 in CRC tissues, the rescue ability of TEAD4 mutant defect in YAP binding and no effect on vimentin expression by YAP knockdown in CRC cells, all implicated a YAP-independent manner of TEAD4 function in CRC. Furthermore, vimentin positively correlated and CDH1 reversely correlated with the level of TEAD4 in CRC tissues and xenograft tumors. Our results suggest that TEAD4 nuclear expression can serve as a biomarker for CRC progression and poor prognosis. The transcription factor TEAD4 regulates a pro-metastasis transcription program in a YAP-independent manner in CRC, thus providing a novel mechanism of TEAD4 transcriptional regulation and its oncogenic role in CRC, independently of the Hippo pathway.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) have been used to treat non-small cell lung carcinoma (NSCLC) patients that have EGFR-activating mutations. EGFR-TKI monotherapy in most NSCLC patients with EGFR mutations who initially respond to EGFR-TKIs results in the development of acquired resistance. We investigated the role of fibroblasts in stromal cell-mediated resistance to gefitinib-induced apoptosis in EGFR-mutant NSCLC cells. While gefitinib induced apoptosis in EGFR-mutant NSCLC cells, apoptosis induction was diminished under stromal co-culture conditions. Protection appeared to be mediated in part by Aurora-A kinase (AURKA) upregulation. The protective effect of stromal cells was significantly reduced by pre-exposure to AURKA-shRNA. We suggest that combinations of AURKA antagonists and EGFR inhibitors may be effective in clinical trials targeting mutant EGFR NSCLCs.

The vagus nerve can control inflammatory response through a 'cholinergic anti-inflammatory pathway', which is mediated by the α7-nicotinic acetylcholine receptor (α7nAChR) on macrophages. However, the intracellular mechanisms that link α7nAChR activation and pro-inflammatory cytokine production remain not well understood. In this study, we found that miR-124 is upregulated by cholinergic agonists in LPS-exposed cells and mice. Utilizing miR-124 mimic and siRNA knockdown, we demonstrated that miR-124 is a critical mediator for the cholinergic anti-inflammatory action. Furthermore, our data indicated that miR-124 modulates LPS-induced cytokine production by targeting signal transducer and activator of transcription 3 (STAT3) to decrease IL-6 production and TNF-α converting enzyme (TACE) to reduce TNF-α release. These results also indicate that miR-124 is a potential therapeutic target for the treatment of inflammatory diseases.

Understanding and managing fatigue is a significant challenge in clinic and society. In attempting to explore how the body responds to and regulates fatigue, we found in rodent fatigue models that orosomucoid 1 (ORM1) was significantly increased in multiple tissues, including blood and muscle. Interestingly, administration of exogenous ORM1 increased muscle glycogen and enhanced muscle endurance, whereas ORM1 deficiency resulted in a significant decrease of muscle endurance both in vivo and in vitro, which could largely be restored by exogenous ORM1. Further studies demonstrated that ORM1 can bind to C-C chemokine receptor type 5 (CCR5) on muscle cells and deletion of the receptor abolished the effect of ORM1. Thus, fatigue upregulates the level of ORM1, which in turn functions as an anti-fatigue protein to enhance muscle endurance via the CCR5 pathway. Modulation of the level of ORM1 and CCR5 signaling could be a novel strategy for the management of fatigue.

We have shown previously that activation of STAT1 contributes to the pathogenesis of Wilms tumor. This neoplasm caricatures metanephric development and is believed to originate from embryonic renal mesenchymal progenitors that lose their ability to undergo mesenchymal–epithelial transition (MET). Therefore, we hypothesized that STAT1 is also activated and functional during metanephric development. Here we have demonstrated that both STAT1 and STAT3 are activated during normal development of the embryonic kidney. Furthermore, activation of STAT1 stimulated the proliferation of metanephric mesenchymal cells, but it prevented MET and tubulogenesis induced by leukemia inhibitory factor, which preferentially activates STAT3. Consistent with its negative regulation of metanephric mesenchymal differentiation, inhibition of STAT1 activation with protein kinase CK2 inhibitor TBB or RNAi-mediated knockdown of STAT1 promoted differentiation of metanephric progenitors and abolished the effect of cytokine-induced STAT1 activation in these cells. Additionally, a cell-permeable peptide that inhibits STAT1-mediated transactivation by targeting the STAT1 N-domain also blocked cytokine-induced STAT1-dependent proliferation in metanephric progenitors and promoted LIF-induced MET and tubulogenesis. Finally, the STAT1 peptide inhibitor caused the down regulation of survival/anti-apoptotic factors, Mcl-1 and Hsp-27, and induced apoptosis in renal tumor cells with constitutively active STAT1, indicating that STAT1 is required for these cells to survive. These findings show that both metanephric progenitors and renal tumor cells utilize a STAT1-dependent mechanism for growth or survival.

Gender differences in fatigue manifest as females being more prone to feel exhaustion and having lower muscle endurance. However, the mechanisms of these effects remain unclear. We investigated whether orosomucoid, an endogenous anti-fatigue protein that enhances muscle endurance, is involved in this regulation. Female rats exhibited lower muscle endurance, and this gender difference disappeared in orosomucoid-1-deficient mice. Female rats also exhibited weaker orosomucoid induction in serum, liver and muscle in response to fatigue compared with male rats. Ovariectomy elevated orosomucoid levels and increased swimming time, and estrogen replenishment reversed these effects. Exogenous estrogen treatment in male and female mice produced opposite effects. Estrogen decreased orosomucoid expression and its promoter activity in C2C12 muscle and Chang liver cells in vitro, and estrogen receptor or p38 mitogen-activated protein kinase blockade abolished this effect. Therefore, estrogen negatively regulates orosomucoid expression that is responsible for the weaker muscle endurance in females.

Compounds that stabilize p53 could suppress tumors providing a additional tool to fight cancer. Mdm2, and the human ortholog, Hdm2 serve as ubiquitin E3 ligases and target p53 for ubiquitylation and degradation. Inhibition of Hdm2 stabilizes p53, inhibits cell proliferation and induces apoptosis. Using HTS to discover inhibitors, we identified three new alkaloids, isolissoclinotoxin B, diplamine B, and lissoclinidine B from Lissoclinum cf. badium. Lissoclinidine B inhibited ubiquitylation and degradation of p53, and selectively killed transformed cells harboring wild-type p53, suggesting this compound could be used to develop new treatments.

The tumor suppressor protein p53 is a potent inducer of apoptosis in transformed cells. Hdm2 is an ubiquitin ligase (E3) that acts as a major regulator of p53 by promoting its ubiquitylation and proteasomal degradation. For this reason, inhibiting the E3 activity of Hdm2 has been proposed as a therapeutic approach for cancers expressing wild-type p53. We previously identified a family of small molecules (HLI98s, 7-nitro-10-aryl-5-deazaflavins) that inhibit the E3 activity of Hdm2, increase cellular p53, and selectively kill transformed cells expressing wild-type p53. However, issues of both potency and solubility in aqueous solution limit the utility of the HLI98s. Here, we report that a highly soluble derivative of the HLI98s, which has a 5-dimethylaminopropylamino side chain but lacks the 10-aryl group (HLI373), has greater potency than the HLI98s in stabilizing Hdm2 and p53, activating p53-dependent transcription, and inducing cell death. Furthermore, we show that HLI373 is effective in inducing apoptosis of several tumor cells lines that are sensitive to DNA-damaging agents. These results suggest that HLI373 could serve as a potential lead for developing cancer therapeutics based on inhibition of the ubiquitin ligase activity of Hdm2. [Mol Cancer Ther 2008;7(8):2445–54]

High-throughput screening technologies have revolutionized the manner in which potential therapeutics are identified. Although they are the source of lead compounds for ~65% of anticancer and antimicrobial drugs approved by the Food and Drug Administration between 1981 and 2002, natural products have largely been excluded from modern screening programs. This is due, at least in part, to the inherent difficulties in testing complex extract mixtures, which often contain nuisance compounds, in modern bioassay systems. In this article, the authors present a novel electrochemiluminescent assay system for inhibition of MDM2 activity that is suitable for testing natural product extracts in high-throughput screening systems. The assay was used to screen more than 144,000 natural product extracts. The authors identified 1 natural product, sempervirine, that inhibited MDM2 auto-ubiquitination, MDM2-mediated p53 degradation, and led to accumulation of p53 in cells. Sempervirine preferentially induced apoptosis in transformed cells expressing wild-type p53, suggesting that it could be a potential lead for anticancer therapeutics.

The conjugation of proteins with ubiquitin plays numerous regulatory roles through both proteasomal-dependent and nonproteasomal-dependent functions. Alterations in ubiquitylation are observed in a wide range of pathologic conditions, including numerous malignancies. For this reason, there is great interest in targeting the ubiquitin-proteasome system in cancer. Several classes of proteasome inhibitors, which block degradation of ubiquitylated proteins, are widely used in research, and one, Bortezomib, is now in clinical use. Despite the well-defined and central role of the ubiquitin-activating enzyme (E1), no cell permeable inhibitors of E1 have been identified. Such inhibitors should, in principle, block all functions of ubiquitylation. We now report 4[4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester (PYR-41) as the first such inhibitor. Unexpectedly, in addition to blocking ubiquitylation, PYR-41 increased total sumoylation in cells. The molecular basis for this is unknown; however, increased sumoylation was also observed in cells harboring temperature-sensitive E1. Functionally, PYR-41 attenuates cytokine-mediated nuclear factor-κB activation. This correlates with inhibition of nonproteasomal (Lys-63) ubiquitylation of TRAF6, which is essential to IκB kinase activation. PYR-41 also prevents the downstream ubiquitylation and proteasomal degradation of IκBα. Furthermore, PYR-41 inhibits degradation of p53 and activates the transcriptional activity of this tumor suppressor. Consistent with this, it differentially kills transformed p53-expressing cells. Thus, PYR-41 and related pyrazones provide proof of principle for the capacity to differentially kill transformed cells, suggesting the potential for E1 inhibitors as therapeutics in cancer. These inhibitors can also be valuable tools for studying ubiquitylation. [Cancer Res 2007;67(19):9472–81]

The acute-phase protein orosomucoid (ORM) exhibits a variety of activities in vitro and in vivo, notably modulation of immunity and transportation of drugs. We found in this study that mice lacking ORM1 displayed aberrant energy homeostasis characterized by increased body weight and fat mass. Further investigation found that ORM, predominantly ORM1, is significantly elevated in sera, liver, and adipose tissues from the mice with high-fat diet (HFD)–induced obesity and db/db mice that develop obesity spontaneously due to mutation in the leptin receptor (LepR). Intravenous or intraperitoneal administration of exogenous ORM decreased food intake in C57BL/6, HFD, and leptin-deficient ob/ob mice, which was absent in db/db mice and was significantly reduced in mice with arcuate nucleus (ARC) LepR knockdown, whereas enforced expression of ORM1 in ARC significantly decreased food intake, body weight, and serum insulin level. Furthermore, we found that ORM is able to bind directly to LepR and activate the receptor-mediated JAK2–STAT3 signaling in hypothalamus tissue and GT1-7 cells, which was derived from hypothalamic tumor. These data indicated that ORM could function through LepR to regulate food intake and energy homeostasis in response to nutrition status. Modulating the expression of ORM is a novel strategy for the management of obesity and related metabolic disorders.

The RING finger family of proteins possess ubiquitin ligase activity and play pivotal roles in protein degradation and receptor-mediated endocytosis. In this study, we examined whether the breast cancer-associated gene 2 (BCA2), a novel RING domain protein, has E3 ubiquitin ligase activity and investigated its expression status in breast tumors. The full-length BCA2 gene was cloned from the human breast cancer cell line MDA-MB-468. It encodes an open reading frame of 304 amino acids and contains a RING-H2 domain. BCA2 maps to chromosome 1q21.1, a region known to harbor cytogenetic aberrations in breast cancers. We found that the BCA2 protein has an intrinsic autoubiquitination activity, the hallmark of E3 ligases, whereas mutant RING protein is not autoubiquitinated. This indicates that the BCA2 ubiquitin ligase activity is dependent on the RING-H2 domain. Using tissue microarrays and immunohistochemistry, we found strong to intermediate BCA2 staining in 56% of 945 invasive breast cancers cases, which was significantly correlated with positive estrogen receptor status [odds ratio (OR), 1.51; P = 0.004], negative lymph node status (OR, 0.73; P = 0.02), and an increase in disease-free survival for regional recurrence (OR, 0.45; P = 0.03). Overexpression of BCA2 increased proliferation and small interfering RNA inhibited growth of T47D human breast cancer cells and NIH3T3 mouse cells. The autoubiquitination activity of BCA2 indicates that it is a novel RING-type E3 ligase. Its association with clinical measures and its effects on cell growth indicate that BCA2 may be important for the ubiquitin modification of proteins crucial to breast carcinogenesis and growth.

The tumor suppressor p53 is tightly controlled at low levels in cells by constant ubiquitination and proteasomal degradation. In response to stresses, ubiquitination of p53 is inhibited through diverse pathways, depending on the nature of the stimulus and cell type. This leads to the accumulation and activation of p53, which induces cell cycle arrest and/or apoptosis to prevent cells from transformation. Many studies have indicated that defects of the p53 system are present in most, if not all, human tumor cells. Meanwhile, significant progress has been made in understanding the molecular mechanisms of p53 ubiquitination and the regulation of the p53 system. Therefore, it is possible now to consider targeting ubiquitination as a means to regulate and reactivate p53 in tumors. Emerging evidence suggests that inhibiting the E3 activity of Mdm2, blocking the interaction of p53 and Mdm2, and restoring the function of mutated p53 are potential effective strategies to kill certain tumor cells selectively. It is conceivable that new chemotherapeutic agents based on these studies will be generated in the not-so-distant future.

IAPs (inhibitors of apoptosis) are a family of proteins containing one or more characteristic BIR domains. These proteins have multiple biological activities that include binding and inhibiting caspases, regulating cell cycle progression, and modulating receptor-mediated signal transduction. Our recent studies found the IAP family members XIAP and c-IAP1 are ubiquitinated and degraded in proteasomes in response to apoptotic stimuli in T cells, and their degradation appears to be important for T cells to commit to death. In addition to three BIR domains, each of these IAPs also contains a RING finger domain. We found this region confers ubiquitin protease ligase (E3) activity to IAPs, and is responsible for the auto-ubiquitination and degradation of IAPs after an apoptotic stimulus. Given the fact that IAPs can bind a variety of proteins, such as caspases and TRAFs, it will be of interest to characterize potential substrates of the E3 activity of IAPs and the effects of ubiquitination by IAPs on signal transduction, cell cycle, and apoptosis.

To determine why proteasome inhibitors prevent thymocyte death, we examined whether proteasomes degrade anti-apoptotic molecules in cells induced to undergo apoptosis. The c-IAP1 and XIAP inhibitors of apoptosis were selectively lost in glucocorticoid- or etoposide-treated thymocytes in a proteasome-dependent manner before death. IAPs catalyzed their own ubiquitination in vitro, an activity requiring the RING domain. Overexpressed wild-type c-IAP1, but not a RING domain mutant, was spontaneously ubiquitinated and degraded, and stably expressed XIAP lacking the RING domain was relatively resistant to apoptosis-induced degradation and, correspondingly, more effective at preventing apoptosis than wild-type XIAP. Autoubiquitination and degradation of IAPs may be a key event in the apoptotic program.

CC3 is a metastasis suppressor that inhibits metastasis of the variant small cell lung carcinoma (v-SCLC) by predisposing cells to apoptosis. The same protein was also reported as a cellular cofactor, TIP30, which stimulates HIV-1 Tat-activated transcription by interacting with both Tat and RNA polymerase II. We report here that TIP30/CC3 is a novel serine/threonine kinase. It phosphorylates the heptapeptide repeats of the C-terminal domain (CTD) of the largest RNA polymerase II subunit in a Tat-dependent manner. Amino acid substitutions in the putative ATP binding motif that abolish the TIP30 kinase activity also inhibit the ability of TIP30 to enhance Tat-activated transcription or to sensitize NIH 3T3 and v-SCLC cells to apoptosis. Furthermore, ectopic expression of TIP30/CC3 in v–SCLC cells induces expression of a number of genes that include the apoptosis-related genes Bad and Siva, as well as metastasis suppressor NM23-H2. These data demonstrate a molecular mechanism for TIP30/CC3 function and suggest a novel pathway for regulating apoptosis.

Extract of Tripterygium wilfordii Hook. f (TWHf) has immunosuppressive activity and has been used as anti-inflammatory agent in traditional Chinese medicine for centuries. Recent studies have demonstrated that triptolide is the major active component in the extract that inhibits antigen or mitogen-induced T cell proliferation. In attempting to investigate its effect on activation of T lymphocytes, we found triptolide induces apoptotic death of T cell hybridomas and peripheral T cells but not that of thymocytes. The triptolide-induced apoptosis is accompanied by increase of DEVD-cleavable caspases activity and degradation of caspase substrate poly (ADP-ribose) polymerase (PARP). A specific inhibitor of caspases, zVAD-FMK, prevents triptolide-induced PARP degradation and DNA fragmentation but not growth arrest. Furthermore, enforced expression of Bcl-2 inhibited triptolide-induced degradation of PARP and apoptosis. These results indicate that triptolide induces T cell apoptosis through activating caspases, and suggest the growth arrest and apoptotic effect of triptolide may contribute to the immunosuppressive activity of TWHf extract.

Wnt4 and β-catenin are both required for nephrogenesis, but studies using TCF-reporter mice suggest that canonical Wnt signaling is not activated in metanephric mesenchyme (MM) during its conversion to the epithelia of the nephron. To better define the role of Wnt signaling, we treated rat metanephric mesenchymal progenitors directly with recombinant Wnt proteins. These studies revealed that Wnt4 protein, which is required for nephron formation, induces tubule formation and differentiation markers Lim1 and E-cadherin in MM cells, but does not activate a TCF reporter or up regulate expression of canonical Wnt target gene Axin-2 and has little effect on the stabilization of β-catenin or phosphorylation of disheveled-2. Furthermore, Wnt4 causes membrane localization of ZO-1 and occludin in tight junctions. To directly examine the role of β-catenin/TCF-dependent transcription, we developed synthetic cell-permeable analogs of β-catenin's helix C, which is required for transcriptional activation, in efforts to specifically inhibit canonical Wnt signaling. One inhibitor blocked TCF-dependent transcription and induced degradation of β-catenin but did not affect tubule formation and stimulated the expression of Lim1 and E-cadherin. Since a canonical mechanism appears not to be operative in tubule formation, we assessed the involvement of the non-canonical Ca2+-dependent pathway. Treatment of MM cells with Wnt4 induced an influx of Ca2+ and caused phosphorylation of CaMKII. Moreover, Ionomycin, a Ca2+-dependent pathway activator, stimulated tubule formation. These results demonstrate that the canonical Wnt pathway is not responsible for mesenchymal–epithelial transition (MET) in nephron formation and suggest that the non-canonical calcium/Wnt pathway mediates Wnt4-induced tubulogenesis in the kidney.

The ubiquitin-proteasome system plays a critical role in controlling the level, activity, and location of various cellular proteins. Significant progress has been made in investigating the molecular mechanisms of ubiquitination, particularly in understanding the structure of the ubiquitination machinery and identifying ubiquitin protein ligases, the primary specificity-determining enzymes. Therefore, it is now possible to target specific molecules involved in the ubiquitination and proteasomal degradation to regulate many cellular processes such as signal transduction, proliferation and apoptosis. In particular, alterations in ubiquitination are observed in most, if not all, cancer cells. This is manifested by destabilization of tumor suppressors, such as p53, and overexpression of oncogenes such as c-Myc and c-Jun. In addition to the development and clinical validation of proteasome inhibitor Bortezomib in myeloma therapy, recent studies have demonstrated that it is possible to develop inhibitors for specific ubiquitination and deubiquitination enzymes. With the help of structural studies, rational design, and chemical synthesis, it is conceivable that we will be able to use “druggable” inhibitors of the ubiquitin system to evaluate their effects in animal tumor models in the not-so-distant future.

A concept that has arisen over the last decade is that proteins can, in general, be covalently modified by polypeptides, resulting in alterations in their fate and function. The first-identified and most well studied of these modifying polypeptides is ubiquitin. Although targeting for proteasomal degradation is the best studied outcome of ubiquitylation, we now understand that modification of proteins with ubiquitin has numerous other cellular roles that alter protein function and that are unrelated to proteasomal degradation. Ubiquitylation is a complex process that is regulated at the level of both addition and removal of ubiquitin from target proteins. This unit includes a number of different basic protocols that will facilitate the study of components of the ubiquitin system and substrate ubiquitylation both in vitro and in cells. Because another protein modifier, NEDD8, itself regulates aspects of the ubiquitin system, basic protocols on neddylation are also included in this unit.

RING finger proteins represent the largest class of potential ubiquitin ligases. This chapter describes methods used to express and assess the activity of proteins containing RING fingers based on our experience with a number of different family members. In addition to general protocols for assessing activity, specific protocols are provided for evaluating the ubiquitylation of p53 by the RING finger E3 Hdm2/Mdm2. Use of these methods may help identify new E3s, dissect factors involved in ubiquitylation of substrates, and screen for molecules that affect ubiquitylation.

SummaryThe p53 tumor suppressor protein is regulated by its interaction with HDM2, which serves as a ubiquitin ligase (E3) to target p53 for degradation. We have identified a family of small molecules (HLI98) that inhibits HDM2’s E3 activity. These compounds show some specificity for HDM2 in vitro, although at higher concentrations effects on unrelated RING and HECT domain E3s are detectable, which could be due, at least in part, to effects on E2-ubiquitin thiol-ester levels. In cells, the compounds allow the stabilization of p53 and HDM2 and activation of p53-dependent transcription and apoptosis, although other p53-independent toxicity was also observed.

Ubiquitin is a ubiquitously expressed 76 amino acid protein that can be covalently attached to target proteins, leading to their ubiquitination. Many ubiquitinated proteins are degraded by the proteasome, a 2000 kDa ATP-dependent proteolytic complex. Numerous studies have demonstrated that the ubiquitination and proteasome system plays an important role in controlling the levels of various cellular proteins and therefore regulates basic cellular processes such as cell cycle progression, signal transduction, and cell transformation. Ubiquitination also directly affects the function and location of target proteins. Recent studies found that ubiquitination-mediated degradation and change in activity regulate many molecules of the cell death machinery, such as p53, caspases, and Bcl-2 family members. Ring finger-containing members of the IAP (inhibitor of apoptosis) family proteins themselves can function as ubiquitin protein ligases to ubiquitinate their target proteins or promote autoubiquitination. It has been demonstrated that degradation of the IAP proteins is required for apoptosis to occur in some systems, indicating apoptosis proceeds by activating death pathways as well as eliminating "roadblocks" through ubiquitination. These new findings also suggest that ubiquitination is one of the major mechanisms that regulate apoptotic cell death and could be a unique target for therapeutic intervention.

Tumour necrosis factor-alpha (TNF-alpha) is a proinflammatory mediator that exerts its biological functions by binding two TNF receptors (TNF-RI and TNF-RII), which initiate biological responses by interacting with adaptor and signalling proteins. Among the signalling components that associate with TNF receptors are members of the TNF-R-associated factor (TRAF) family. TRAF2 is required for TNF-alpha-mediated activation of c-Jun N-terminal kinase (JNK), contributes to activation of NF-kappaB, and mediates anti-apoptotic signals,. TNF-RI and TNF-RII signalling complexes also contain the anti-apoptotic ('inhibitor of apoptosis') molecules c-IAP1 and c-IAP2 (refs 5, 6), which also have RING domain-dependent ubiquitin protein ligase (E3) activity. The function of IAPs in TNF-R signalling is unknown. Here we show that binding of TNF-alpha to TNF-RII induces ubiquitination and proteasomal degradation of TRAF2. Although c-IAP1 bound TRAF2 and TRAF1 in vitro, it ubiquitinated only TRAF2. Expression of wild-type c-IAP1, but not an E3-defective mutant, resulted in TRAF2 ubiquitination and degradation. Moreover, E3-defective c-IAP1 prevented TNF-alpha-induced TRAF2 degradation and inhibited apoptosis. These findings identify a physiologic role for c-IAP1 and define a mechanism by which TNF-RII-regulated ubiquitin protein ligase activity can potentiate TNF-induced apoptosis.