Nuclear endonuclease G controls cell proliferation in ovarian cancer.

Ovarian cancer is characterized by a high degree of genetic heterogeneity. Platinum-based chemotherapy and some gene-targeted therapies have shown limited treatment efficacy due to toxicity and recurrence, and thus, it is essential to identify additional therapeutic targets based on an understanding of the pathological mechanism. Here, we report that endonuclease G, which exhibits altered expression in ovarian cancer, does not function as a cell death effector that digests chromosomal DNA in ovarian cancer. Endonuclease G is modulated by intracellular reactive oxygen species dynamics and plays a role in cell proliferation in ovarian cancer, suggesting that targeting endonuclease G alone or in combination with other antitumor agents may have the potential for development into a treatment for endonuclease G-overexpressing cancers, including ovarian cancer.

Stabilization of C-terminal binding protein 2 by cellular inhibitor of apoptosis protein 1 via BIR domains without E3 ligase activity.

C-terminal binding protein 2 (CtBP2) is a transcriptional co-repressor that regulates many genes involved in normal cellular events. Because CtBP2 overexpression has been implicated in various human cancers, its protein levels must be precisely regulated. Previously, we reported that CtBP1 and CtBP1-mediated transcriptional repression are regulated by X-linked inhibitor of apoptosis protein (XIAP). In the present study, we sought to investigate whether CtBP2 is also regulated by XIAP or any other human IAP. We found that cIAP1 interacts with CtBP2 via through BIR domains to regulates the steady-state levels of CtBP2 protein in the nucleus. The levels of CtBP2 were gradually increased upon cIAP1 overexpression and downregulated upon cIAP1 depletion. Interestingly, the RING domain of cIAP1 responsible for E3 ligase activity was not required for this regulation. Finally, the levels of CtBP2 modulated by cIAP1 affected the transcription of CtBP2 target genes and subsequent cell migration. Taken together, our data demonstrate a novel function of cIAP1 which involves protecting CtBP2 from degradation to stabilize its steady-state level. These results suggest that cIAP1 might be a useful target in strategies aiming to downregulate the steady-state level of CtBP2 protein in treating human cancers.

Drp1-Zip1 Interaction Regulates Mitochondrial Quality Surveillance System.

Mitophagy, a mitochondrial quality control process for eliminating dysfunctional mitochondria, can be induced by a response of dynamin-related protein 1 (Drp1) to a reduction in mitochondrial membrane potential (MMP) and mitochondrial division. However, the coordination between MMP and mitochondrial division for selecting the damaged portion of the mitochondrial network is less understood. Here, we found that MMP is reduced focally at a fission site by the Drp1 recruitment, which is initiated by the interaction of Drp1 with mitochondrial zinc transporter Zip1 and Zn2+ entry through the Zip1-MCU complex. After division, healthy mitochondria restore MMP levels and participate in the fusion-fission cycle again, but mitochondria that fail to restore MMP undergo mitophagy. Thus, interfering with the interaction between Drp1 and Zip1 blocks the reduction of MMP and the subsequent mitophagic selection of damaged mitochondria. These results suggest that Drp1-dependent fission provides selective pressure for eliminating "bad sectors" in the mitochondrial network, serving as a mitochondrial quality surveillance system.

Ubiquitin-proteasome dependent regulation of Profilin2 (Pfn2) by a cellular inhibitor of apoptotic protein 1 (cIAP1).

The two major isoforms of the profilin (Pfn) family of proteins in mammals are Pfn1 and Pfn2. Pfn1 is a universal actin cytoskeletal regulator, while Pfn2 is an actin binding protein and mediator of synapse architecture, specific to neural tissues. However, it has recently been suggested that Pfn2 is also widely distributed in various tissues and involved in numerous cellular events as well as cytoskeletal regulation. In our previous study, we showed that Pfn1 is regulated by carboxyl terminus of Hsc70-Interacting Protein (CHIP) via an ubiquitin (Ub) proteasome system; although, the mechanism of regulation of Pfn2 is unknown. In this report, we demonstrate that Pfn2 is heavily ubiquitinated via differential Ub-linkages for degradation or as a regulatory signal. We also show that cellular inhibitor of apoptosis 1 (cIAP1) rather than CHIP, functions as an E3 ligase that targets Pfn2 for proteasomal degradation. Finally, we observed that Pfn2 levels, regulated by cIAP1, affected intracellular levels of reactive oxygen species. These results may provide a regulatory mechanism for cellular function of Pfn2 in various tissues.

A novel function of cIAP1 as a mediator of CHIP-driven eIF4E regulation.

eIF4E is an initiator protein in cap-dependent translation. Its overexpression is linked to tumorigenesis in various human cancers, suggesting that the levels of eIF4E must be under tight control in normal cells. Although several eIF4E regulatory mechanisms have been demonstrated, the intracellular mechanisms controlling eIF4E protein levels remain poorly understood. Here, we report that eIF4E is efficiently regulated by dual mechanisms, both involving human inhibitor of apoptosis family protein cIAP1. cIAP1 itself ubiquitinates eIF4E as an E3 ligase, and interestingly, cIAP1 also functions as a mediator to present eIF4E to another E3 ligase, CHIP. This collaborative activity of cIAP1 and CHIP directs eIF4E toward degradation, controlling its levels and suppressing tumorigenesis. Our results provide the first evidence for a mediator function of cIAP1 and collaborative activity of cIAP1 and CHIP, suggesting that maintaining balanced levels of these E3 ligases might be beneficial for normal cell growth.

Regulation of fragile X mental retardation 1 protein by C-terminus of Hsc70-interacting protein depends on its phosphorylation status.

The fragile X mental retardation 1 (FMR1) protein binds mRNA and acts as a negative regulator of translation. Lack of FMR1 causes the most common neurological disorder, fragile X syndrome, while its overexpression is associated with metastasis of breast cancer. Its activity has been well-studied in nervous tissue, but recent evidence as well as its role in cancer indicates that it also acts in other tissues. We have investigated the expression of FMR1 in brain and other tissues of mouse and examined its regulation. We detected expression of FMR1 in liver and heart tissues of mice as well as in brain tissue, supporting other contentions that it acts in non-nervous tissue. Expression of FMR1 inversely correlated with expression of the C-terminus of Hsc70-interacting protein (CHIP) and, based on the known activity of CHIP in protein homeostasis, we suggest that CHIP regulates expression of FMR1. CHIP ubiquitinated FMR1 for proteasomal degradation in a molecular chaperone-independent manner. FMR1 expression was reduced following treatment with okadaic acid, a phosphatase inhibitor, but not in CHIP-depleted cells. Also, a non-phospho FMR1 mutant was much less efficiently ubiquitinated by CHIP and had a longer half-life compared to either wild-type FMR or a phospho-mimic mutant. Taken together, our results demonstrate that CHIP regulates the levels of FMR1 as an E3 ubiquitin ligase in phosphorylation-dependent manner, suggesting that CHIP regulates FMR1-mediated translational repression by regulating the levels of FMR1.

Cellular inhibitor of apoptosis protein 1 ubiquitinates endonuclease G but does not affect endonuclease G-mediated cell death.

Inhibitors of Apoptosis Proteins (IAPs) are evolutionarily well conserved and have been recognized as the key negative regulators of apoptosis. Recently, the role of IAPs as E3 ligases through the Ring domain was revealed. Using proteomic analysis to explore potential target proteins of DIAP1, we identified Drosophila Endonuclease G (dEndoG), which is known as an effector of caspase-independent cell death. In this study, we demonstrate that human EndoG interacts with IAPs, including human cellular Inhibitor of Apoptosis Protein 1 (cIAP1). EndoG was ubiquitinated by IAPs in vitro and in human cell lines. Interestingly, cIAP1 was capable of ubiquitinating EndoG in the presence of wild-type and mutant Ubiquitin, in which all lysines except K63 were mutated to arginine. cIAP1 expression did not change the half-life of EndoG and cIAP1 depletion did not alter its levels. Expression of dEndoG 54310, in which the mitochondrial localization sequence was deleted, led to cell death that could not be suppressed by DIAP1 in S2 cells. Moreover, EndoG-mediated cell death induced by oxidative stress in HeLa cells was not affected by cIAP1. Therefore, these results indicate that IAPs interact and ubiquitinate EndoG via K63-mediated isopeptide linkages without affecting EndoG levels and EndoG-mediated cell death, suggesting that EndoG ubiquitination by IAPs may serve as a regulatory signal independent of proteasomal degradation.

C-Terminus of Hsc70-interacting protein regulates profilin1 and breast cancer cell migration.

Profilin1 (Pfn1) is a key mediator of actin polymerization and regulates cell migration. Low expression of Pfn1 is implicated in tumorigenesis of various cancers, including breast cancer. The regulatory mechanism behind Pfn1 levels has not yet been elucidated. In the present study, we find that Pfn1 is poly-ubiquitinated in human cell lines, and a portion of poly-ubiquitinated Pfn1 is regulated in a proteasome-dependent manner. C-terminus of Hsc70-interacting protein (CHIP), a co-chaperone E3 ligase, interacts with and ubiquitinates Pfn1, targeting it for proteasome-dependent degradation. Depletion of CHIP stabilizes Pfn1, suggesting that CHIP functions as a major E3 ligase for Pfn1. Stable expression of wild-type CHIP in the breast cancer cell line MDA-MB231 yielded downregulation of Pfn1 and enhanced cell migration. Pfn1 overexpression in MDA-MB231 cells expressing wild-type CHIP suppressed the enhanced cell migration. Taken together, our results demonstrate that CHIP regulates Pfn1 levels as an E3 ligase, and possibly plays a role in cell migration and metastasis of breast cancer.

C-terminus of Hsc70-interacting protein regulates C-terminal binding protein 2 and the expression of its target genes.

C-terminal binding protein 2 (CtBP2) is a transcriptional co-repressor involved in cell migration, apoptosis, and tumorization. Up-regulation of CtBP2 is implicated in various human cancers including breast cancers. In our present study, we found that the C-terminus of Hsc70-interacting protein (CHIP) regulated the steady-state level of CtBP2. CHIP interacted with CtBP2 via a tetratricopeptide domain in the nucleus and ubiquitinated CtBP2, marking it for proteasomal degradation. Depletion of CHIP resulted in stabilization of the CtBP2 protein, which was not affected by ultraviolet irradiation. Finally, CHIP expression regulated the expression of CtBP2 target genes. Taken together, our data demonstrate that CHIP regulates the steady-state level of CtBP2 as an E3 ubiquitin ligase and determines the expression levels of CtBP2 target genes. Regulating CtBP2 levels with CHIP may be a useful therapeutic strategy in human cancers.

C-terminal binding protein-mediated transcriptional repression is regulated by X-linked inhibitor of apoptosis protein.

Inhibitors of Apoptosis Proteins (IAPs) are known as the key negative regulators of apoptosis. To explore new functions of IAPs, we sought to identify proteins that interact with Diap1 in insect S2 cells. We found that Diap1 bound to Drosophila C-terminal binding protein (dCtBP), which is a transcriptional co-repressor. CtBP1 also interacted with X-linked inhibitor of apoptosis protein (XIAP) in human cells. CtBPs were ubiquitinated by IAPs and targeted for proteasome-mediated degradation. Finally, the expression of CtBP1 target genes was regulated by XIAP expression. This is the first report to demonstrate that XIAP specifically regulates CtBP1, suggesting that XIAP may play a role in regulating CtBP1-mediated transcriptional repression by regulating the level of CtBP1.

Chfr is linked to tumour metastasis through the downregulation of HDAC1.

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.

Expression of Kir2.1 channels in astrocytes under pathophysiological conditions.

Astrocyte ion channels participate in ionic homeostasis in the brain. Inward rectifying potassium channels (Kir channels) in astrocytes have been particularly implicated in K(+) homeostasis because of their high open probability at resting potential and their increased conductance at high concentrations of extracellular K(+). We examined the expression of the Kir2.1 subunit, one of the Kir channel subunits, in the mouse brain by immunohistochemistry. Kir2.1 channels were widely distributed throughout the brain, with high expression in the olfactory bulb and the cerebellum. Interestingly, they were abundantly expressed in astrocytes of the olfactory bulb, while astrocytes in other brain regions including the hippocampus did not show any detectable expression. However, Kir2.1 channel-expressing cells were dramatically increased in the hippocampus by kainic acid-induced seizure and the cells were glial fibrillary acidic protein (GFAP)-positive, which confirms that astrocytes in the hippocampus express Kir2.1 channels under pathological conditions. Our results imply that Kir2.1 channels in astrocyte may be involved in buffering K(+) against accumulated extracellular K(+) caused by neuronal hyperexcitability under phathophysiological conditions.

Association of BAF53 with mitotic chromosomes.

The conversion of mitotic chromosome into interphase chromatin consists of at least two separate processes, the decondensation of the mitotic chromosome and the formation of the higher-order structure of interphase chromatin. Previously, we showed that depletion of BAF53 led to the expansion of chromosome territories and decompaction of the chromatin, suggesting that BAF53 plays an essential role in the formation of higher-order chromatin structure. We report here that BAF53 is associated with mitotic chromosomes during mitosis. Immunostaining with two different anti-BAF53 antibodies gave strong signals around the DNA of mitotic preparations of NIH3T3 cells and mouse embryo fibroblasts (MEFs). The immunofluorescent signals were located on the surface of mitotic chromosomes prepared by metaphase spread. BAF53 was also found in the mitotic chromosome fraction of sucrose gradients. Association of BAF53 with mitotic chromosomes would allow its rapid activation on the chromatin upon exit from mitosis.

Deubiquitination of Chfr, a checkpoint protein, by USP7/HAUSP regulates its stability and activity.

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.

The Drosophila inhibitor of apoptosis (IAP) DIAP2 is dispensable for cell survival, required for the innate immune response to gram-negative bacterial infection, and can be negatively regulated by the reaper/hid/grim family of IAP-binding apoptosis inducers.

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-kappaB 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.

Translation initiation factor 4E (eIF4E) is regulated by cell death inhibitor, Diap1.

Translation initiation factor 4E (eIF4E) is a key regulator of protein synthesis. Abnormal regulation of eIF4E is closely linked to oncogenic transformation. Several regulatory mechanisms affecting eIF4E are discussed, including transcriptional regulation, phosphorylation and binding of an inhibitor protein. However it is not clear how the level of eIF4E protein is regulated under basal conditions. Here we demonstrate that Diap1 (Drosophila Inhibitor of Apoptosis Protein), a cell death inhibitor, binds directly to eIF4E and poly-ubiquitinates it via its E3 ligase activity, promoting its proteasome-dependent degradation. Expression of Diap1 caused a reduction of Cyclin D1 protein level and inhibited the growth stimulation induced by overexpression of eIF4E. Taken together, our results suggest that the level of eIF4E protein is regulated by Diap1, and that IAPs may play a role in cap-dependent translation by regulating the level of eIF4E protein.

The Drosophila caspase Ice is important for many apoptotic cell deaths and for spermatid individualization, a nonapoptotic process.

Caspase family proteases play important roles in the regulation of apoptotic cell death. Initiator caspases are activated in response to death stimuli, and they transduce and amplify these signals by cleaving and thereby activating effector caspases. In Drosophila, the initiator caspase Nc (previously Dronc) cleaves and activates two short-prodomain caspases, Dcp-1 and Ice (previously Drice), suggesting these as candidate effectors of Nc killing activity. dcp-1-null mutants are healthy and possess few defects in normally occurring cell death. To explore roles for Ice in cell death, we generated and characterized an Ice null mutant. Animals lacking Ice show a number of defects in cell death, including those that occur during embryonic development, as well as during formation of adult eyes, arista and wings. Ice mutants exhibit subtle defects in the destruction of larval tissues, and do not prevent destruction of salivary glands during metamorphosis. Cells from Ice animals are also markedly resistant to several stresses, including X-irradiation and inhibition of protein synthesis. Mutations in Ice also suppress cell death that is induced by expression of Rpr, Wrinkled (previously Hid) and Grim. These observations demonstrate that Ice plays an important non-redundant role as a cell death effector. Finally, we demonstrate that Ice participates in, but is not absolutely required for, the non-apoptotic process of spermatid differentiation.

Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin.

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.

Analysis of gene expression induced by microtubule-disrupting agents in HeLa cells using microarray.

Microtubules are important cytoskeletal elements that have been shown to play a major role in many cellular processes because of their mechanical properties and/or their participation in various cell signaling pathways. Nocodazole is used as an effective microtubule-disrupting agent, and many recent studies have used it for the activation of spindle checkpoint. In this study, we sought to identify the potential nocodazole target genes by profiling the gene expression pattern in HeLa cells. When treated with 0.1ug/ml of nocodazole, cells were efficiently arrested in the mitotic phase. HeLa cells also showed a higher proportion of apoptosis after drug treatment for a prolonged period. By DNA chip assay, we discovered that 50 genes changed their expressions in the nocodazole-treated cells with a minimal 2.0-fold change at 18h post-treatment. The majority of the differentially expressed genes belonged to two functional groups--genes involved in transcription regulation and in cellular signaling. These observations could have significant implications for our understanding of the physiological and pathophysiological regulation of spindle disrupting agents in human cells.

Grim stimulates Diap1 poly-ubiquitination by binding to UbcD1.

Diap1 is an essential Drosophila cell death regulator that binds to caspases and inhibits their activity. Reaper, Grim and Hid each antagonize Diap1 by binding to its BIR domain, activating the caspases and eventually causing cell death. Reaper and Hid induce cell death in a Ring-dependent manner by stimulating Diap1 auto-ubiquitination and degradation. It was not clear that how Grim causes the ubiquitination and degradation of Diap1 in Grim-dependent cell death. We found that Grim stimulates poly-ubiquitination of Diap1 in the presence of UbcD1 and that it binds to UbcD1 in a GST pull-down assay, so presumably promoting Diap1 degradation. The possibility that dBruce is another E2 interacting with Diap1 was examined. The UBC domain of dBruce slightly stimulated poly-ubiquitination of Diap1 in Drosophila extracts but not in the reconstitution assay. However Grim did not stimulate Diap1 poly-ubiquitination in the presence of the UBC domain of dBruce. Taken together, these results suggest that Grim stimulates the poly-ubiquitination and presumably degradation of Diap1 in a novel way by binding to UbcD1 but not to the UBC domain of dBruce as an E2.