Elena Lomonosova

BH3-only proteins in apoptosis and beyond: an overview.

BH3-only BCL-2 family proteins are effectors of canonical mitochondrial apoptosis. They discharge their pro-apoptotic functions through BH1–3 pro-apoptotic proteins such as BAX and BAK, while their activity is suppressed by BH1–4 anti-apoptotic BCL-2 family members. The precise mechanism by which BH3-only proteins mediate apoptosis remains unresolved. The existing data are consistent with three mutually non-exclusive models (1) displacement of BH1–3 proteins from complexes with BH1–4 proteins; (2) direct interaction with and conformational activation of BH1–3 proteins; and (3) membrane insertion and membrane remodeling. The BH3-only proteins appear to play critical roles in restraining cancer and inflammatory diseases such as rheumatoid arthritis. Molecules that mimic the effect of BH3-only proteins are being used in treatments against these diseases. The cell death activity of a subclass of BH3-only members (BNIP3 and BNIP3L) is linked to cardiomyocyte loss during heart failure. In addition to their established role in apoptosis, several BH3-only members also regulate diverse cellular functions in cell-cycle regulation, DNA repair and metabolism. Several members are implicated in the induction of autophagy and autophagic cell death, possibly through unleashing of the BH3-only autophagic effector Beclin 1 from complexes with BCL-2/BCL-xL. The Chapters included in the current Oncogene Review issues provide in-depth discussions on various aspects of major BH3-only proteins.

Hydroxylated Tropolones Inhibit Hepatitis B Virus Replication by Blocking Viral Ribonuclease H Activity

ABSTRACT Hepatitis B virus (HBV) remains a major human pathogen despite the development of both antiviral drugs and a vaccine, in part because the current therapies do not suppress HBV replication far enough to eradicate the virus. Here, we screened 51 troponoid compounds for their ability to suppress HBV RNaseH activity and HBV replication based on the activities of α-hydroxytropolones against HIV RNaseH, with the goal of determining whether the tropolone pharmacophore may be a promising scaffold for anti-HBV drug development. Thirteen compounds inhibited HBV RNaseH, with the best 50% inhibitory concentration (IC50) being 2.3 μM. Similar inhibition patterns were observed against HBV genotype D and C RNaseHs, implying limited genotype specificity. Six of 10 compounds tested against HBV replication in culture suppressed replication via blocking of viral RNaseH activity, with the best 50% effective concentration (EC50) being 0.34 μM. Eighteen compounds inhibited recombinant human RNaseH1, and moderate cytotoxicity was observed for all compounds (50% cytotoxic concentration [CC50] = 25 to 79 μM). Therapeutic indexes ranged from 3.8 to 94. Efficient inhibition required an intact α-hydroxytropolone moiety plus one or more short appendages on the tropolone ring, but a wide variety of constituents were permissible. These data indicate that troponoids and specifically α-hydroxytropolones are promising lead candidates for development as anti-HBV drugs, providing that toxicity can be minimized. Potential anti-RNaseH drugs are envisioned to be employed in combination with the existing nucleos(t)ide analogs to suppress HBV replication far enough to block genomic maintenance, with the goal of eradicating infection.

Mitochondrial localization of p53 during adenovirus infection and regulation of its activity by E1B-19K.

Recent results have revealed that the p53 tumor suppressor protein possesses a direct transcription-independent apoptotic activity. During apoptosis induced by genotoxic stress, a small fraction of p53 is targeted to mitochondria where it initiates apoptosis by causing mitochondrial dysfunction. In adenovirus-infected cells, the expression of E1A protein enhances the accumulation of p53 during early phases of infection and during late times after infection, it is targeted for degradation by the combined action of E1B-55K and E4-orf6 proteins. The functional significance of E1A-mediated accumulation of p53 during early phases of viral replication is not known. Our studies with isogenic epithelial cell lines that differ only on the status of p53 indicate that Ad infection induces apoptosis by p53-dependent and -independent pathways and both pathways are suppressed by E1B-19K. We show that during early phase of Ad infection, a fraction of p53 is targeted to the mitochondria. In virus infected cells, a large fraction of the viral antiapoptosis protein E1B-19K is also localized in mitochondria during early and late phases of infection. Coimmunoprecipitation analysis has revealed that p53 and E1B-19K form a complex in mitochondria. The interaction of 19K involves two noncontiguous regions located around amino-acid residues 14–15 and 123–124. On p53, the mutations within the DNA-binding domain reduce interaction with E1B-19K. Our studies also suggest that 19K may additionally complex with the multidomain mitochondrial proapoptotic protein BAK, thereby reducing the level of p53 interaction with BAK. We suggest that p53-induced apoptosis may be important for efficient cell lysis and viral spread and that E1B-19K may neutralize the apoptotic activity of p53 at multiple levels.

Hepatitis B virus replication is blocked by a 2-hydroxyisoquinoline-1,3(2H,4H)-dione (HID) inhibitor of the viral ribonuclease H activity.

Nucleos(t)ide analog drugs profoundly suppress Hepatitis B virus (HBV) replication but rarely cure the infection, so therapy is usually life-long. The nucleos(t)ide analogs inhibit the viral DNA polymerase and often push HBV to the brink of extinction, so it may be possible to eradicate HBV by suppressing HBV replication further. The HBV ribonuclease H (RNaseH) is a logical new drug target because it is the second of only two viral enzymes essential for viral replication. We recently developed a low throughput screening pipeline for inhibitors of the HBV RNaseH and viral replication. Here, we screened a series of twenty-three nitrogen-based polyoxygenated heterocycles including sixteen 2-hydroxyisoquinoline-1,3(2H,4H)-dione derivatives for anti-HBV RNaseH activity. Nine compounds inhibited the HBV RNaseH, but activity was marginal for eight of them. Compound #1 [2-hydroxyisoquinoline-1,3(2H,4H)-dione, HID] was the best hit with an IC50 of 28.1μM and an EC50 of 4.2μM. It preferentially suppressed accumulation of the viral plus-polarity DNA strand in replication inhibition assays, indicating that replication was blocked due to suppression of HBV RNaseH activity. It had a CC50 of 75μM, yielding a therapeutic index of ∼18. The EC50 value was 7-fold lower than the IC50, possibly due to cellular retention or metabolism of the compound, or higher affinity for the full-length enzyme than the recombinant form used for screening. These data indicate that the 2-hydroxyisoquinoline-1,3(2H,4H)-diones will have different structure-activity relationships for the HBV and HIV RNaseHs. Therefore, HID compounds may provide a foundation for development of more effective RNaseH inhibitors of HBV replication.

Adenovirus type 5 E1A and E6 proteins of low-risk cutaneous beta-human papillomaviruses suppress cell transformation through interaction with FOXK1/K2 transcription factors.

ABSTRACT The adenovirus (Adv) oncoprotein E1A stimulates cell proliferation and inhibits differentiation. These activities are primarily linked to the N-terminal region (exon 1) of E1A, which interacts with multiple cellular protein complexes. The C terminus (exon 2) of E1A antagonizes these processes, mediated in part through interaction with C-terminal binding proteins 1 and 2 (CtBP1/2). To identify additional cellular E1A targets that are involved in the modulation of E1A C-terminus-mediated activities, we undertook tandem affinity purification of E1A-associated proteins. Through mass spectrometric analysis, we identified several known E1A-interacting proteins as well as novel E1A targets, such as the forkhead transcription factors, FOXK1/K2. We identified a Ser/Thr-containing sequence motif in E1A that mediated interaction with FOXK1/K2. We demonstrated that the E6 proteins of two beta-human papillomaviruses (HPV14 and HPV21) associated with epidermodysplasia verruciformis also interacted with FOXK1/K2 through a motif similar to that of E1A. The E1A mutants deficient in interaction with FOXK1/K2 induced enhanced cell proliferation and oncogenic transformation. The hypertransforming activity of the mutant E1A was suppressed by HPV21 E6. An E1A-E6 chimeric protein containing the Ser/Thr domain of the E6 protein in E1A interacted efficiently with FOXK1/K2 and inhibited cell transformation. Our results suggest that targeting FOXK1/K2 may be a common mechanism for certain beta-HPVs and Adv5. E1A exon 2 mutants deficient in interaction with the dual-specificity kinases DYRK1A/1B and their cofactor HAN11 also induced increased cell proliferation and transformation. Our results suggest that the E1A C-terminal region may suppress cell proliferation and oncogenic transformation through interaction with three different cellular protein complexes: FOXK1/K2, DYRK(1A/1B)/HAN11, and CtBP1/2.

BAX/BAK–Independent Mitoptosis during Cell Death Induced by Proteasome Inhibition?

Proteasome inhibitors induce rapid death of cancer cells. We show that in epithelial cancer cells, such death is associated with dramatic and simultaneous up-regulation of several BH3-only proteins, including BIK, BIM, MCL-1S, NOXA, and PUMA, as well as p53. Elevated levels of these proteins seem to be the result of direct inhibition of their proteasomal degradation, induction of transcription, and active translation. Subsequent cell death is independent of BAX, and probably BAK, and proceeds through the intrinsic mitochondrial apoptosis pathway. We identify the cascade of molecular events responsible for cell death induced by a prototypical proteasome inhibitor, MG132, starting with rapid accumulation of BH3-only proteins in the mitochondria, proceeding through mitochondrial membrane permeabilization and subsequent loss of ΔΨm, and leading to irreversible changes of mitochondrial ultrastructure, degradation of mitochondrial network, and detrimental impairment of crucial mitochondrial functions. Our results also establish a rationale for the broader use of proteasome inhibitors to kill apoptosis-resistant tumor cells that lack functional BAX/BAK proteins. (Mol Cancer Res 2009;7(8):1268–84)

Evidence for Involvement of BH3-Only Proapoptotic Members in Adenovirus-Induced Apoptosis

ABSTRACT Mammalian cells infected with human adenoviruses (Ads) undergo an apoptotic response as a result of expression of the viral E1A proteins, and this process is suppressed by the viral E1B-19K protein. The intermediary steps in the Ad-induced apoptosis pathway are not fully resolved. The apical step in the canonical mammalian apoptosis pathway involves functional activation of one or more of the BH3-only BCL-2 family proapoptotic proteins. Previous reports have suggested that Ad-induced apoptosis may be initiated at checkpoints downstream of the BH3-only proteins. Here, we undertook genetic and biochemical studies to determine the roles of BH3-only proteins in Ad-induced apoptosis. We examined the activities of the cellular antiapoptosis protein BCL-xL and its mutants expressed from the E1B region of the Ad5 genome. Our results showed efficient suppression of Ad-induced apoptosis by a BCL-xL mutant (mt1) deficient in interaction with multidomain proapoptotic proteins BAX and BAK but proficient in interaction with BH3-only proteins, suggesting a role for BH3-only proteins in the initiation of Ad-induced apoptosis. Further, the antiapoptotic activity of BCL-xL mt1 in Ad-infected cells was observed in spite of BAK activation as a consequence of MCL-1 degradation. Analysis of the mRNA levels of various BH3-only members by reverse transcription-PCR revealed prominent activation of the Bik gene. Further, the BIK protein was also modified into an apoptotically enhanced phosphorylated form during the viral infection. In addition to BIK, enhanced level of BIM was observed in Ad-infected cells. Between the two major E1A proteins coded by the 12S and 13S mRNAs, the 13S product appeared to contribute to the activation of these BH3-only members and apoptosis during viral infection. Depletion of BIK by the use of small interfering RNA reduced the level of Ad-induced apoptosis. Our results are consistent with a model that activation of the BH3-only members may initiate Ad-induced apoptosis.

Requirement of BAX for Efficient Adenovirus-Induced Apoptosis

ABSTRACT Infection of human epithelial cells with adenoviruses induces an apoptosis paradigm that is efficiently suppressed by the expression of viral E1B-19K protein, which is a functional homolog of the cellular antiapoptosis protein BCL-2. The mechanisms of adenovirus (Ad)-induced apoptosis appear to involve the cellular BCL-2 family proapoptotic proteins. Recent genetic studies with fibroblasts derived from mutant mouse embryos indicate that a class of the BCL-2 family proapoptotic proteins (designated BH-123 or multidomain proteins) such as BAX and BAK constitutes an essential component of the core apoptosis machinery in animal cells. We have examined the role of BAX in Ad-induced apoptosis in human epithelial cells using two colon cancer cell lines, HCT116Bax (Bax+/−) and HCT116BaxKO (Bax−/−) (L. Zhang, J. Yu, B. H. Park, K. W. Kinzler, and B. Vogelstein, Science 290:989-992, 2000). Infection of Bax+/− cells with an Ad type 2 mutant (dl250) defective in expression of the E1B-19K protein resulted in enhanced cytopathic effect, large plaques on cell monolayers, fragmentation of cellular DNA, and enhanced cell death. These mutant phenotypes were not efficiently expressed in Bax−/− cells, suggesting that BAX is essential for Ad-induced apoptosis. Infection of Bax+/− cells with dl250 induced increased levels of an N-terminally processed form of BAX. Cells infected with the 19K mutant also contained enhanced levels of truncated BAX in membrane-inserted form. Our results suggest that at least a part of the mechanism utilized by E1B-19K to suppress apoptosis during Ad infection may involve modulation of the activities of BAX.

Inhibition of hepatitis B virus replication by N-hydroxyisoquinolinediones and related polyoxygenated heterocycles

Abstract We previously reported low sensitivity of the hepatitis B virus (HBV) ribonuclease H (RNaseH) enzyme to inhibition by N‐hydroxyisoquinolinedione (HID) compounds. Subsequently, our biochemical RNaseH assay was found to have a high false negative rate for predicting HBV replication inhibition, leading to underestimation of the number of HIDs that inhibit HBV replication. Here, 39 HID compounds and structurally related polyoxygenated heterocycles (POH), N‐hydroxypyridinediones (HPD), and flutimides were screened for inhibition of HBV replication in vitro. Inhibiting the HBV RNaseH preferentially blocks synthesis of the positive‐polarity DNA strand and causes accumulation of RNA:DNA heteroduplexes. Eleven HIDs and one HPD preferentially inhibited HBV positive‐polarity DNA strand accumulation. EC50s ranged from 0.69 &mgr;M to 19 &mgr;M with therapeutic indices from 2.4 to 71. Neither the HIDs nor the HPD had an effect on the ability of the polymerase to elongate DNA strands in capsids. HBV RNaseH inhibition by the HIDs was confirmed with an improved RNaseH assay and by detecting accumulation RNA:DNA heteroduplexes in HBV capsids from cells treated with a representative HID. Therefore, the HID scaffold is more promising for anti‐HBV drug discovery than we originally reported, and the HPD scaffold may hold potential for antiviral development. The preliminary structure‐activity relationship will guide optimization of the HID/HPDs as HBV inhibitors. HighlightsHID and HPD compounds inhibit HBV replication with low micromolar EC50 values.HID compounds inhibit HBV replication by blocking the viral RNaseH and they do not inhibit the viral reverse transcriptase.The HID and HPD compounds may be promising scaffolds for anti‐HBV drug development.

Efficacy and cytotoxicity in cell culture of novel α-hydroxytropolone inhibitors of hepatitis B virus ribonuclease H

Abstract Chronic Hepatitis B virus (HBV) infection is a major worldwide public health problem. Current direct‐acting anti‐HBV drugs target the HBV DNA polymerase activity, but the equally essential viral ribonuclease H (RNaseH) activity is unexploited as a drug target. Previously, we reported that &agr;–hydroxytropolone compounds can inhibit the HBV RNaseH and block viral replication. Subsequently, we found that our biochemical RNaseH assay underreports efficacy of the &agr;‐hydroxytropolones against HBV replication. Therefore, we conducted a structure‐activity analysis of 59 troponoids against HBV replication in cell culture. These studies revealed that antiviral efficacy is diminished by larger substitutions on the tropolone ring, identified key components in the substitutions needed for high efficacy, and revealed that cytotoxicity correlates with increased lipophilicity of the &agr;‐hydroxytropolones. These data provide key guidance for further optimization of the &agr;‐hydroxytropolone scaffold as novel HBV RNaseH inhibitors. HighlightsEfficacy of &agr;‐hydroxytropolones against HBV is diminished by bulky substitutions on the troponoid ring.Carbonyl, lactone, or sulfone groups at R2 on the troponoid ring enhance activity against HBV.No cell‐specific cytotoxicity was found among cells of hepatic, kidney, or stellate‐cell origin.Cytotoxicity of the troponoids correlated with a hydroxyl in the &agr; position on the troponoid ring and overall lipophilicity.