Alexandre Philips

Comparative genomics: Insecticide resistance in mosquito vectors

Resistance to insecticides among mosquitoes that act as vectors for malaria (Anopheles gambiae) and West Nile virus (Culex pipiens) emerged more than 25 years ago in Africa, America and Europe; this resistance is frequently due to a loss of sensitivity of the insects acetylcholinesterase enzyme to organophosphates and carbamates. Here we show that this insensitivity results from a single amino-acid substitution in the enzyme, which we found in ten highly resistant strains of C. pipiens from tropical (Africa and Caribbean) and temperate (Europe) areas, as well as in one resistant African strain of A. gambiae. Our identification of this mutation may pave the way for designing new insecticides.

Timing of cyclin E gene expression depends on the regulated association of a bipartite repressor element with a novel E2F complex.

Transient induction of the cyclin E gene in late G1 gates progression into S. We show that this event is controlled via a cyclin E repressor module (CERM), a novel bipartite repressor element located near the cyclin E transcription start site. CERM consists of a variant E2F‐binding site and a contiguous upstream AT‐rich sequence which cooperate during G0/G1 to delay cyclin E expression until late G1. CERM binds the protein complex CERC, which disappears upon progression through G0–G1 and reappears upon entry into the following G1. CERC disappearance correlates kinetically with the liberation of the CERM module in vivo and cyclin E transcriptional induction. CERC contains E2F4/DP1 and a pocket protein, and sediments faster than classical E2F complexes in a glycerol gradient, suggesting the presence of additional components in a novel high molecular weight complex. Affinity purified CERC binds to CERM but not to canonical E2F sites, thus displaying behavior different from known E2F complexes. In cells nullizygous for members of the Rb family, CERC is still detectable and CERM‐dependent repression is functional. Thus p130, p107 and pRb function interchangeably in CERC. Notably, the CERC–CERM complex dissociates prematurely in pRb−/− cells in correspondence with the premature expression of cyclin E. Thus, we identify a new regulatory module that controls repression of G1‐specific genes in G0/G1.

Activation of cyclin D1 expression by the ERK5 cascade

Transcriptional activation of the cyclin D1 gene is a key step in cell proliferation. Accordingly, cyclin D1 overexpression is frequently an early step in neoplastic transformation, particularly in mammary epithelium. Numerous studies have linked elevated cyclin D1 promoter activity to a sustained activation of the ERK1/2 cascade. Here we show that the ERK5 cascade, a distinct mitogen-induced MAPK pathway, can also drive cyclin D1 expression. In CCL39 cells, serum induces a strong, prolonged peak of ERK1/2 and ERK5 phosphorylation, and subsequently elevates cyclin D1 mRNA and protein levels. Overexpression of constitutively active MEK5 and wt ERK5 induces a cyclin D1 reporter gene (D1 −973-luciferase) at least as well as constitutively active MEK1. Activation is blocked by kinase-dead mutants of ERK5 and ERK2, respectively. Mutation of the CRE at −50 in the cyclin D1 promoter decreases activation by the ERK5 but not the ERK1/2 cascade. Importantly, expression of kinase-dead ERK5 diminishes endogenous cyclin D1 protein induction by serum in CCL39 cells and the breast cancer cell lines MCF-7 and HS579. These data identify the cyclin D1 gene as a novel target of the ERK5 cascade, an observation with important implications in cancers involving cyclin D1 deregulation.

Oct-1 Potentiates CREB-Driven Cyclin D1 Promoter Activation via a Phospho-CREB- and CREB Binding Protein-Independent Mechanism

ABSTRACT Cyclin D1, the regulatory subunit for mid-G1 cyclin-dependent kinases, controls the expression of numerous cell cycle genes. A cyclic AMP-responsive element (CRE), located upstream of the cyclin D1 mRNA start site, integrates mitogenic signals that target the CRE-binding factor CREB, which can recruit the transcriptional coactivator CREB-binding protein (CBP). We describe an alternative mechanism for CREB-driven cyclin D1 induction that involves the ubiquitous POU domain protein Oct-1. In the breast cancer cell line MCF-7, overexpression of Oct-1 or its POU domain strongly increases transcriptional activation of cyclin D1 and GAL4 reporter genes that is specifically dependent upon CREB but independent of Oct-1 DNA binding. Gel retardation and chromatin immunoprecipitation assays confirm that POU forms a complex with CREB bound to the cyclin D1 CRE. In solution, CREB interaction with POU requires the CREB Q2 domain and, notably, occurs with CREB that is not phosphorylated on Ser 133. Accordingly, Oct-1 also potently enhances transcriptional activation mediated by a Ser133Ala CREB mutant. Oct-1/CREB synergy is not diminished by the adenovirus E1A 12S protein, a repressor of CBP coactivator function. In contrast, E1A strongly represses CBP-enhanced transactivation by CREB phosphorylated on Ser 133. Our observation that Oct-1 potentiates CREB-dependent cyclin D1 transcriptional activity independently of Ser 133 phosphorylation and E1A-sensitive coactivator function offers a new paradigm for the regulation of cyclin D1 induction by proliferative signals.

Insecticide resistance: a silent base prediction

Response to a challenging environment proceeds through adaptation, the result of stochastic processes (chance) and of the influence of history (constraint) [1]. Adaptations, such as pesticide resistance, provide an opportunity to study historical constraints. Insecticides, widely used since the mid 1950s, have elicited numerous cases of resistance. Specific amino acid changes at unique or few critical positions of the target protein explain resistance to the major classes of insecticides, such as cyclodienes, organochlorines, pyrethroids, carbamates and organophosphates (OPs) [2–6] and sometimes lead to extremely high resistance levels (>1000 fold). In mosquitoes, a single glycine (Gly) to serine (Ser) substitution at position 119 (Torpedo nomenclature) in the acetylcholinesterase (AChE1) gene confers high levels of resistance to carbamates and OPs [3]. This G119S substitution was selected at least twice independently in Culex pipiens, once in Anopheles albimanus and once in Anopheles gambiae, suggesting that there are only few possibilities to generate high AChE1 insensitivity [7]. Although heavily controlled with carbamates and OPs, the mosquito vector of dengue and yellow fever, Aedes aegypti, never developed high levels of resistance. We first checked whether the G119S mutation in AChE1 is ineffective in this species. We cloned the complete AChE1 cDNA, encoding a protein 96.4% similar to C. pipiens AChE1, (supplemental data) and produced wild-type and G119S mutant recombinant proteins. Ae. aegypti AChE1 behaved exactly like C. pipiens AChE1: wild-type proteins were inhibited at identical doses of the carbamate insecticide propoxur (IC50 = 5 x 10–7 M), while G119S proteins remained insensitive up to 10–2 M propoxur (Figure 1). It is, therefore, unlikely that the low resistance in Ae. aegypti resulted from particular biochemical properties of its AChE1. Alternatively, Ae. aegypti AChE1 may not be able to evolve to the G119S substitution. Notably, in Ae. aegypti glycine 119 of AChE1 is encoded by a GGA codon, whereas GGC was found in all the other species analysed so far [3,7]. This silent third base change represents an extraordinarily heavy constraint. It decreases the probability of a spontaneous G119S substitution by several orders of magnitude . As Ser can be encoded by AGY or TCN, substitution to Ser requires only one mutation when Gly is encoded by GGY, whereas two are required when Gly is encoded by GGR. We thus hypothesized that high levels of resistance cannot emerge if the G119S substitution requires more than a single step mutation, a situation that could be described as a ‘codon constraint’. Accordingly, knowing the sequence of codon 119 should allow the prediction of the ability of a given mosquito species to develop high OP-resistance. We first checked all known acetylcholinesterase amino acid sequences (79 animal species), and found that a glycine is present at position 119 in all species, except in ascidians and Schistosoma, which show a serine. This suggests that presence of a glycine is critical, and that no other amino acids are allowed in this position, except serine. We next analysed the sequence of codon 119 in 26 natural populations of Ae. aegypti collected in 12 countries. In all samples, the glycine was encoded by GGA (serineimmutable), which fits with the

Resveratrol protects against protease inhibitor-induced reactive oxygen species production, reticulum stress and lipid raft perturbation.

Objective:HIV protease inhibitors have been successfully used in highly active antiretroviral therapy of HIV-1 infection, but their benefits are compromised by a number of clinically important adverse side-effects. Several studies showed that protease inhibitors induce sarco/endoplasmic reticulum stress and overproduction of reactive oxygen species (ROS), but the hierarchy of these events was never established in protease inhibitor-treated cells. Our objective was to determine whether ROS production and sarco/endoplasmic reticulum stress were co-induced by protease inhibitors in human primary skeletal myotubes and whether antioxidant treatment with resveratrol could protect against protease inhibitor-induced cellular damages. Design and methods:We analyzed in cultures of primary human skeletal myotubes the effects of four protease inhibitors (atazanavir, lopinavir, ritonavir and saquinavir) on ROS production (by measuring the reduction of nitro blue tetrazolium and by fluorescence microscopy using CM-H2DCFDA), on sarco/endoplasmic reticulum stress induction (by immunofluorescence and electron microscopy) and on the expression and localization at lipid rafts of Caveolin 3 and Flotillin 1, two major components of lipid rafts (by immunoblotting and biochemical experiments). Cells were co-incubated with resveratrol to assess its protective effects. Results:In myotubes, protease inhibitors increased ROS production, altered sarco/endoplasmic reticulum morphology, increased expression of C/EBP homologous protein, a sarco/endoplasmic reticulum stress marker, and decreased expression and localization at lipid rafts of Caveolin 3 and Flotillin 1. Importantly, these deleterious protease inhibitor effects were inhibited by the antioxidant resveratrol. Conclusion:Our findings demonstrate a protective effect of resveratrol against protease inhibitor-induced sarco/endoplasmic reticulum stress in human myotubes.

The retinoblastoma protein is essential for cyclin A repression in quiescent cells

Cyclin A is a positive regulatory component of kinases required for the progression through S phase and for the transition between the G2 and M phases of the cell division cycle. Previous studies have demonstrated that the promoter of its gene is under transcriptional repression in quiescent cells. Whereas the DNA sequences mediating this effect have been clearly delineated, the nature of the proteins acting in trans is still debated. Indirect observations suggest the involvement of proteins related to the retinoblastoma tumor suppressor protein (pRb). However, the precise role of these proteins has been difficult to assess, since most experiments designed to analyse their function have been carried out in transformed cell lines. Nevertheless, a current model has emerged whereby the role of the p130 protein would be restricted to resting and early G1 cells and p107, absent in quiescent cells, would be involved later in the control of the G1/S transition, whilst pRb would be effective throughout the cell cycle. We show here that cyclin A transcriptional inhibition is relieved in primary fibroblasts from pRb(−/−) embryos and not in fibroblasts from p130(−/−), p107(−/−) or even p130(−/−)/p107(−/−) double mutant embryos. This suggests a unique role for pRb in controlling the extinction of specific genes in G0, providing thus the first example of non-overlapping functions achieved by the different pocket proteins.

Extinction of rac1 and Cdc42Hs signalling defines a novel p53-dependent apoptotic pathway.

Apoptosis is a normal physiological process which eliminates cells that do not receive adequate extracellular signals. One of the pathways signalling apoptosis is controlled by the small GTPases of the Rho family, also involved in cell proliferation, differentiation and motility. Another major apoptosis signalling pathway involves the p53 tumour suppressor which is activated by a variety of stress and mediates growth arrest or apoptosis in normal cells. We show here that upon detachment from the extracellular matrix, fibroblasts undergo rapid apoptosis that can be rescued by constitutive activation of Rac1 and Cdc42Hs GTPases. Conversely, inhibition of Rac1 and Cdc42Hs efficiently triggers apoptosis in adherent cells. Interestingly, apoptosis is not observed in p53−/− cells either cultured in suspension or inhibited for Rac1 and Cdc42Hs activity. Moreover, Rac1 and Cdc42Hs extinction in normal cells activates endogenous p53. Using specific inhibitors of MAPK pathways, we demonstrate that, in our experimental system, p38 signals survival, while ERK activity is required for apoptosis. Our data constitute the first demonstration that Rac1 and Cdc42Hs control pathways that require simultaneous signalling through MAPK ERK and p53 to induce apoptosis.

CHF: a novel factor binding to cyclin A CHR corepressor element

Cell cycle modulation of cyclin A expression is due to the periodic relief of a transcriptional repression mediated by a bipartite negative DNA regulatory region. The 5′ element (Cell Cycle Responsive Element: CCRE; cell Cycle Dependent Element: CDE) is clearly occupied in a cyclic manner in vivo, whereas the 3′ element, whose sequence is shared by B-myb, cdc25C and cdc2 genes (cell Cycle gene Homology Region: CHR), is involved in more subtle interactions. Mutation of either element results in complete deregulation of cyclin A promoter activity. Whereas some reports claim that E2F/DP can bind to the CCRE/CDE, the nature of the protein(s) interacting with the CHR is unknown. In the present work we have characterized an activity present in quiescent cells and absent in cells blocked in S phase, which binds specifically to cyclin A CHR, but not to B-myb, or to cdc25C, or to cdc2 CHRs. A 90 kD protein, named CHF (cyclin A CHR binding factor), has been identified through preparative electrophoresis and UV crosslinking experiments. In order to address in more functional terms the binding of CHF to cyclin A CHR, we developed in vitro and in vivo oligonucleotide competition assays. Both in vitro transcription and in vivo microinjection experiments demonstrate that a functional difference exists between the composite CCRE/CDE-CHR repressor regions of cell cycle regulated genes such as cyclin A and cdc25C.

A CDE/CHR-like element mediates repression of transcription of the mouse RB2 (p130) gene.

The bipartite repressor elements, termed cell cycle‐dependent element (CDE)/cell cycle regulatory element (CCRE)‐cell cycle homology region (CHR) control the growth‐dependent transcription of the cyclin A, cdc25C, cdc2 genes. Here, we have identified a functional element displaying the signature of the CDE–CHR in the promoter of the mouse RB2 (p130) gene, encoding the retinoblastoma protein family (pRB)‐related protein p130. This element locates close to the major transcription start site where it makes major groove contacts with proteins that can be detected in a cellular context using in vivo genomic footprinting techniques. Inactivation of either the CDE or CHR sequence strongly up‐regulates the p130 promoter activity in exponentially growing cells, a situation where endogenous p130 gene expression is almost undetectable. Electrophoretic mobility shift assays suggest that two different protein complexes bind independently to the p130 CDE and CHR elements, and that the protein(s) bound to the CDE might be related to those bound on cyclin A and cdc2 promoters.