Janet V. Cross

Keap1 Is a Redox-Regulated Substrate Adaptor Protein for a Cul3-Dependent Ubiquitin Ligase Complex

ABSTRACT The bZIP transcription factor Nrf2 controls a genetic program that protects cells from oxidative damage and maintains cellular redox homeostasis. Keap1, a BTB-Kelch protein, is the major upstream regulator of Nrf2 and controls both the subcellular localization and steady-state levels of Nrf2. In this report, we demonstrate that Keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Keap1 assembles into a functional E3 ubiquitin ligase complex with Cul3 and Rbx1 that targets multiple lysine residues located in the N-terminal Neh2 domain of Nrf2 for ubiquitin conjugation both in vivo and in vitro. Keap1-dependent ubiquitination of Nrf2 is inhibited following exposure of cells to quinone-induced oxidative stress and sulforaphane, a cancer-preventive isothiocyanate. A mutant Keap1 protein containing a single cysteine-to-serine substitution at residue 151 within the BTB domain of Keap1 is markedly resistant to inhibition by either quinone-induced oxidative stress or sulforaphane. Inhibition of Keap1-dependent ubiquitination of Nrf2 correlates with decreased association of Keap1 with Cul3. Neither quinone-induced oxidative stress nor sulforaphane disrupts association between Keap1 and Nrf2. Our results suggest that the ability of Keap1 to assemble into a functional E3 ubiquitin ligase complex is the critical determinant that controls steady-state levels of Nrf2 in response to cancer-preventive compounds and oxidative stress.

Oxidative stress inhibits MEKK1 by site-specific glutathionylation in the ATP-binding domain

Many intracellular signalling events are accompanied by generation of reactive oxygen species in cells. Oxidation of protein thiol groups is an emerging theme in signal-transduction research. We have found that MEKK1 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase kinase 1], an upstream activator of the SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase) pathway, is directly inhibited by cysteine alkylation using NEM (N-ethylmaleimide). The related kinase, ASK1 (apoptosis signal-regulating kinase 1), was not inhibited, but was instead activated by NEM. Inhibition of MEKK1 requires a single unique cysteine residue (Cys1238) in the ATP-binding domain of MEKK1. Oxidative stress induced by menadione (2-methyl-1,4-naphthoquinone) also inhibited MEKK1, but activated ASK1, in cells. MEKK1 inhibition by menadione also required Cys1238. Oxidant-inhibited MEKK1 was re-activated by dithiothreitol and glutathione, supporting reversible cysteine oxidation as a mechanism. Using various chemical probes, we excluded modification by S-nitrosylation or oxidation of cysteine to sulphenic acid. Oxidant-inhibited MEKK1 migrated normally on non-reducing gels, excluding the possibility of intra- or inter-molecular disulphide bond formation. MEKK1 was inhibited by glutathionylation in vitro, and MEKK1 isolated from menadione-treated cells was shown by MS to be modified by glutathione on Cys1238. Our results support a model whereby the redox environment within the cell selectively regulates stress signalling through MEKK1 versus ASK1, and may thereby participate in the induction of apoptosis by oxidative stress.

Macrophage Migration Inhibitory Factor Promotes Tumor Growth and Metastasis by Inducing Myeloid-Derived Suppressor Cells in the Tumor Microenvironment

The macrophage migration inhibitory factor (MIF), an inflammatory cytokine, is overexpressed in many solid tumors and is associated with poor prognosis. We previously identified inhibitors of MIF within a class of natural products with demonstrated anti-cancer activities. We therefore sought to determine how MIF contributes to tumor growth and progression. We show in this study that in murine tumors including the 4T1 model of aggressive, spontaneously metastatic breast cancer in immunologically intact mice, tumor-derived MIF promotes tumor growth and pulmonary metastasis through control of inflammatory cells within the tumor. Specifically, MIF increases the prevalence of a highly immune suppressive subpopulation of myeloid-derived suppressor cells (MDSCs) within the tumor. In vitro, MIF promotes differentiation of myeloid cells into the same population of MDSCs. Pharmacologic inhibition of MIF reduces MDSC accumulation in the tumor similar to MIF depletion and blocks the MIF-dependent in vitro differentiation of MDSCs. Our results demonstrate that MIF is a therapeutically targetable mechanism for control of tumor growth and metastasis through regulation of the host immune response and support the potential utility of MIF inhibitors, either alone or in combination with standard tumor-targeting therapeutic or immunotherapy approaches.

The impact of environmental metals in young urbanites' brains

Air pollution exposures are linked to cognitive and olfaction deficits, oxidative stress, neuroinflammation and neurodegeneration including frontal hyperphosphorylated tau and diffuse amyloid plaques in Mexico City children and young adults. Mexico City residents are chronically exposed to fine particulate matter (PM(2.5)) concentrations (containing toxic combustion and industrial metals) above the annual standard (15 μg/m(3)) and to contaminated water and soil. Here, we sought to address the brain-region-specific effects of metals and key neuroinflammatory and DNA repair responses in two air pollution targets: frontal lobe and olfactory bulb from 12 controls vs. 47 Mexico City children and young adults average age 33.06±4.8 SE years. Inductively coupled plasma mass spectrometry (metal analysis) and real time PCR (for COX2, IL1β and DNA repair genes) in target tissues. Mexico City residents had higher concentrations of metals associated with PM: manganese (p=0.003), nickel and chromium (p=0.02) along with higher frontal COX2 mRNA (p=0.008) and IL1β (p=0.0002) and COX2 (p=0.005) olfactory bulb indicating neuroinflammation. Frontal metals correlated with olfactory bulb DNA repair genes and with frontal and hippocampal inflammatory genes. Frontal manganese, cobalt and selenium increased with age in exposed subjects. Together, these findings suggest PM-metal neurotoxicity causes brain damage in young urbanites, the olfactory bulb is a target of air pollution and participates in the neuroinflammatory response and since metal concentrations vary significantly in Mexico City urban sub-areas, place of residency has to be integrated with the risk for CNS detrimental effects particularly in children.

JNK-mediated Phosphorylation of Cdc25C Regulates Cell Cycle Entry and G2/M DNA Damage Checkpoint

c-Jun NH2-terminal Kinases (JNKs) play a central role in the cellular response to a wide variety of stress signals. After their activation, JNKs induce phosphorylation of substrates, which control proliferation, migration, survival, and differentiation. Recent studies suggest that JNKs may also play a role in cell cycle control, although the underlying mechanisms are largely unexplored. Here we show that JNK directly phosphorylates Cdc25C at serine 168 during G2 phase of the cell cycle. Cdc25C phosphorylation by JNK negatively regulates its phosphatase activity and thereby Cdk1 activation, enabling a timely control of mitosis onset. Unrestrained phosphorylation by JNK, as obtained by a cell cycle-stabilized form of JNK or as seen in some human tumors, results in aberrant cell cycle progression. Additionally, UV irradiation-induced G2/M checkpoint requires inactivation of Cdc25C by JNK phosphorylation. JNK phosphorylation of Cdc25C as well as Cdc25A establishes a novel link between stress signaling and unperturbed cell cycle and checkpoint pathways.

Nutrient isothiocyanates covalently modify and inhibit the inflammatory cytokine macrophage migration inhibitory factor (MIF).

Dietary ITCs (isothiocyanates) prevent cancer and show other bioactivities in vivo. As electrophiles, ITCs may covalently modify cellular proteins. Using a novel proteomics screen, we identified MIF (macrophage migration inhibitory factor) as the principal target of nutrient ITCs in intact cells. ITCs covalently modify the N-terminal proline residue of MIF and extinguish its catalytic tautomerase activity. MIF deficiency does not prevent induction of Phase 2 gene expression, a hallmark of many cancer chemopreventives, including ITCs. Due to the emerging role of MIF in the control of malignant cell growth and its clear involvement in inflammation, inhibition of MIF by nutrient ITCs suggests therapeutic strategies for inflammatory diseases and cancer.

Thiol oxidation of cell signaling proteins: Controlling an apoptotic equilibrium.

Studies of cell signal transduction have predominantly focused on regulation of protein function by phosphorylation. However, recent efforts have begun to uncover another layer of regulation mediated by direct oxidation of cysteine residues in signaling proteins. Typically induced during signaling responses accompanied by generation of reactive oxygen species, these thiol modifications have a variety of functional consequences for target proteins. Using specific signaling protein targets as examples, we discuss how thiol oxidation generally activates pro‐apoptotic signaling pathways while inhibiting pathways that promote cell survival. We propose a model in which thiol oxidation acts to control the equilibrium between survival and apoptosis, fine tuning cellular responses that play a central role in the apoptotic decision‐making process. We identify areas of focus for future work, including a better understanding of specificity in thiol oxidation events, and a critical need for approaches to examine these modifications under physiologically relevant signaling conditions.

Mechanisms Involved in Stimulation of Human Immunodeficiency Virus Type 1 Replication by Aminooxypentane RANTES

ABSTRACT Aminooxypentane (AOP)-RANTES is a potent inhibitor of nonsyncytium-inducing (NSI), CCR5-tropic (R5) human immunodeficiency virus type 1 (HIV-1) isolates. Although classical chemotactic responses are not induced in primary leukocytes by AOP-RANTES, recent studies suggest that a remnant of cell signaling occurs upon binding of receptor to this compound. We have detected a breakthrough of NSI/R5 replication from the inhibitory effects of high AOP-RANTES concentrations (<100 nM). A stimulation of different primary syncytium-inducing (SI), CXCR4-tropic (X4) HIV-1 isolates was also observed in the presence of AOP-RANTES. This stimulation was also observed after 110 h in PCR and RT-PCR for minus-strand strong-stop DNA and unspliced and multiply spliced RNA, respectively. However, there was significant variability between different SI/X4 or NSI/R5 HIV-1 isolates with regard to this AOP-RANTES-mediated stimulation or breakthrough, respectively. To further define the mechanism(s) responsible for this AOP-RANTES effect, we performed detailed retroviral replication studies with an NSI/R5 (B-92BR021) and SI/X4 (D-92UG021) HIV-1 isolate in the presence of the drug. Treatment of peripheral blood mononuclear cells with 125 nM AOP-RANTES and virus did not alter coreceptor expression, HIV-1 entry, reverse transcription, or mRNA transcription from the long terminal repeat, but it did result in increased HIV-1 integration. This AOP-RANTES-mediated increase in HIV-1 integration was diminished by treatment with pertussis toxin. Phosphorylation of the mitogen-activated protein kinase (MAPK) isoforms, extracellular signal-regulated kinase 1 (ERK1) and ERK2, was increased in a CD4+ CCR5+ U87 cell line treated with AOP-RANTES or with an NSI/R5 HIV-1 isolate. These findings suggest that AOP-RANTES may induce a MAPK/ERK signal transduction pathway upon binding to a G-protein-coupled receptor. MAPK/ERK1 and -2 appear to phosphorylate the HIV-1 preintegration complex, a step necessary for nuclear translocation and successful integration.

Acetylation of apurinic/apyrimidinic endonuclease-1 regulates Helicobacter pylori-mediated gastric epithelial cell apoptosis.

BACKGROUND & AIMS Helicobacter pylori-induced gastric epithelial cell (GEC) apoptosis is a complex process that includes activation of the tumor suppressor p53. p53-mediated apoptosis involves p53 activation, bax transcription, and cytochrome c release from mitochondria. Apurinic/apyrimidinic endonuclease-1 (APE-1) regulates transcriptional activity of p53, and H pylori induce APE-1 expression in human GECs. H pylori infection increases intracellular calcium ion concentration [Ca2+]i of GECs, which induces APE-1 acetylation. We investigated the effects of H pylori infection and APE-1 acetylation on GEC apoptosis. METHODS AGS cells (wild-type or with suppressed APE-1), KATO III cells, and cells isolated from gastric biopsy specimens were infected with H pylori. Effects were examined by immunoblotting, real-time reverse-transcription polymerase chain reaction, immunoprecipitation, immunofluorescence microscopy, chromatin immunoprecipitation, mobility shift, DNA binding, and luciferase assays. RESULTS H pylori infection increased [Ca2+]i and acetylation of APE-1 in GECs, but the acetylation status of APE-1 did not affect the transcriptional activity of p53. In GECs, expression of a form of APE-1 that could not be acetylated increased total and mitochondrial levels of Bax and induced release of cytochrome c and fragmentation of DNA; expression of wild-type APE-1 reduced these apoptotic events. We identified a negative calcium response element in the human bax promoter and found that poly (adenosine diphosphate-ribose) polymerase 1 recruited the acetylated APE-1/histone deacetylase-1 repressor complex to bax nCaRE. CONCLUSIONS H pylori-mediated acetylation of APE-1 suppresses Bax expression; this prevents p53-mediated apoptosis when H pylori infect GECs.

SAPK/JNK regulates cdc2/cyclin B kinase through phosphorylation and inhibition of cdc25c.

Cells undergo M phase arrest in response to stresses like UV irradiation or DNA damage. Stress-activated protein kinase (SAPK, also known as c-Jun N-terminal kinase, JNK) is activated by such stress stimuli. We addressed the potential effects of SAPK activation on cell cycle regulatory proteins. Activation of SAPK strongly correlated with inhibition of cdc2/cyclin B kinase, an important regulator of G2/M phase. SAPK directly phosphorylated the cdc2 regulator, cdc25c, in vitro on serine 168 (S168). This residue was highly phosphorylated in vivo in response to stress stimuli. cdc25c phosphorylated on S168 in cells lacks phosphatase activity, and expression of a S168A mutant of cdc25c reversed the inhibition of cdc2/cyclin B kinase activity by cell stress. Antibodies directed against phosphorylated S168 detect increased phosphorylation of S168 after cell stress. We conclude that SAPK regulates cdc2/cyclin B kinase following stress events by a novel mechanism involving inhibitory phosphorylation of the cdc2-activating phosphatase cdc25c on S168.