Yili Yang

TNF-RII and c-IAP1 mediate ubiquitination and degradation of TRAF2

Tumour necrosis factor-α (TNF-α) 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-α-mediated activation of c-Jun N-terminal kinase (JNK), contributes to activation of NF-κB, 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-α 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-α-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.

Inhibitors of Ubiquitin-Activating Enzyme (E1), a New Class of Potential Cancer 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-kappaB activation. This correlates with inhibition of nonproteasomal (Lys-63) ubiquitylation of TRAF6, which is essential to IkappaB kinase activation. PYR-41 also prevents the downstream ubiquitylation and proteasomal degradation of IkappaBalpha. 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.

Regulating the p53 system through ubiquitination

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.

Fusion peptide of HIV-1 as a site of vulnerability to neutralizing antibody

An antibody to block viral fusion A small fraction of HIV-1–infected individuals develop broad and potent antibodies that bind the HIV-1 envelope protein (Env). These antibodies recognize a limited set of conserved epitopes on Env, such as Envs host receptor-binding site. Kong et al. now report a neutralizing antibody isolated from an HIV-1–infected individual that binds to the fusion peptide of Env. This is unexpected because viruses often try to mask such key components of their cell entry machinery from antibody attack. Crystal structures of the antibody bound to the fusion peptide and to Env itself define the epitope, provide insight into the specific mechanism of antibody binding, and may inform HIV-1 vaccine design. Science, this issue p. 828 A neutralizing antibody against HIV-1 unexpectedly targets a key component of the virus’ cell entry machinery. The HIV-1 fusion peptide, comprising 15 to 20 hydrophobic residues at the N terminus of the Env-gp41 subunit, is a critical component of the virus-cell entry machinery. Here, we report the identification of a neutralizing antibody, N123-VRC34.01, which targets the fusion peptide and blocks viral entry by inhibiting conformational changes in gp120 and gp41 subunits of Env required for entry. Crystal structures of N123-VRC34.01 liganded to the fusion peptide, and to the full Env trimer, revealed an epitope consisting of the N-terminal eight residues of the gp41 fusion peptide and glycan N88 of gp120, and molecular dynamics showed that the N-terminal portion of the fusion peptide can be solvent-exposed. These results reveal the fusion peptide to be a neutralizing antibody epitope and thus a target for vaccine design.

Promotion and Inhibition of Activation-Induced Apoptosis in T-Cell Hybridomas by Oncogenes and Related Signals

The Two Signal: Death/Survival Model suggests that cellular proliferation and physiological cell death should be intimately associated such that, in the absence of external influences, a normal cell departing from rest will have an equal probability of undergoing either process. The c-Myc protooncogene product has been implicated in cell cycle progression and in the control of gene expression, and more recently c-Myc has also been seen to promote apoptotic cell death. As predicted from the model, c-Myc-induced apoptosis is inhibited by growth factors or other anti-apoptotic signals including those provided by some oncogenes. Here, we discuss experiments that test the Two Signal: Death/Survival Model in the phenomenon of activation-induced apoptosis in T-cell hybridomas. Ligation of the antigen receptor on these cells leads to activation, resulting in cytokine production and apoptosis. Inhibition of c-Myc expression by addition of antisense oligodeoxynucleotides or transforming growth factor beta inhibits this form of apoptosis. Because c-Myc is known to bind to several cellular proteins, including Max, we further examined the effects of expression of a dominant negative Max on activation-induced apoptosis. We found that this Max mutant, which interferes with the function of the Myc/Max heterodimer, inhibits the induction of apoptosis by antigen receptor ligation. Thus, both Myc and Max play roles in activation-induced apoptosis, presumably via control of gene expression. Further, as predicted, signals generated from growth factor receptors or the v-Abl oncogene interfere with activation-induced apoptosis. In contrast, the anti-apoptotic effects of Bcl-2 are not active in this form of apoptosis. Finally, a role for Fas/Fas-ligand interactions in activation-induced apoptosis is considered.

Identification of Inhibitors for MDM2 Ubiquitin Ligase Activity from Natural Product Extracts by a Novel High-Throughput Electrochemiluminescent Screen

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. (Journal of Biomolecular Screening 2008:229-237)

Targeting tumor cells expressing p53 with a water-soluble inhibitor of Hdm2

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]

Model cell lines for the study of apoptosis in vitro.

Publisher Summary This chapter provides information about the use of six different cell lines—Jurkat, 2B4.11, DOl1.10, MCF-7, PC12, and C2C12—for the study of apoptosis. The Jurkat cell line, originally derived from a patient with acute lymphoblastic T-cell leukemia, and the clone E6-1 described in this chapter, originates from the Jurkat-FHCRC cell line. Morphologically, the cells are round to racquet shaped and express the T-cell antigen receptor. They produce interleukin (IL)-2 after stimulation with phorbol esters and lectins or monoclonal antibodies against the TCR. These cells can undergo apoptosis in response to a wide variety of well-defined physiological, pharmacological, and pathological triggers. 2B4 is a mouse lymphoid cell line model for the study of apoptosis in vitro. It was generated by fusing lymph node T cells from pigeon cytochrome c (PPC)-immunized B10. DO 11.10 is a mouse T-cell hybridoma derived from a fusion between BALB/c splenocytes and the thymic leukemic cell line BW51479. The C2C12 cell line was derived from the C2 mouse muscle cell line as a fast-fusing subclone and has the characteristics of a very proliferative muscle satellite cell. It is considered the standard model for studies on skeletal muscle differentiation, function, and death.

Increased TEAD4 expression and nuclear localization in colorectal cancer promote epithelial-mesenchymal transition and metastasis in a YAP-independent manner.

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.

Cyclic depsipeptides as potential cancer therapeutics.

Cyclic depsipeptides are polypeptides in which one or more amino acid is replaced by a hydroxy acid, resulting in the formation of at least one ester bond in the core ring structure. Many natural cyclic depsipeptides possessing intriguing structural and biological properties, including antitumor, antifungal, antiviral, antibacterial, anthelmintic, and anti-inflammatory activities, have been identified from fungi, plants, and marine organisms. In particular, the potent effects of cyclic depsipeptides on tumor cells have led to a number of clinical trials evaluating their potential as chemotherapeutic agents. Although many of the trials have not achieved the desired results, romidepsin (FK228), a bicyclic depsipeptide that inhibits histone deacetylase, has been shown to have clinical efficacy in patients with refractory cutaneous T-cell lymphoma and has received Food and Drug Administration approval for use in treatment. In this review, we discuss antitumor cyclic depsipeptides that have undergone clinical trials and focus on their structural features, mechanisms, potential applications in chemotherapy, and pharmacokinetic and toxicity data. The results of this study indicate that cyclic depsipeptides could be a rich source of new cancer therapeutics.