Nadir Mechti

Intracellular distribution of microinjected antisense oligonucleotides.

Antisense oligomers constitute an attractive class of specific tools for genetic analysis and for potential therapeutic applications. Targets with different cellular locations have been described, such as mRNA translation initiation sites, pre-mRNA splicing sites, or the genes themselves. However the mechanism(s) of action and the intracellular distribution of antisense oligomers remain poorly understood. Antisense oligomers conjugated with various fluorochromes or with BrdUrd were microinjected into the cytoplasm of somatic cells, and their cellular distribution was monitored by fluorescence microscopy in fixed and nonfixed cells. A fast translocation in the nuclei and a concentration on nuclear structures were observed whatever probe was used. Nuclear transport occurs by diffusion since it is not affected by depletion of the intracellular ATP pool, temperature, or excess unlabeled oligomer. Accumulation of the oligomers in the nuclei essentially takes place on a set of proteins preferentially extracted between 0.2 M and 0.4 M NaCl as revealed by crosslinking of photosensitive oligomers. The relationship between nuclear location of antisense oligomers and their mechanism of action remains to be ascertained and could be of major interest in the design of more efficient antisense molecules.

Altered Memory Capacities and Response to Stress in p300/CBP-Associated Factor (PCAF) Histone Acetylase Knockout Mice

Chromatin remodeling by posttranslational modification of histones plays an important role in brain plasticity, including memory, response to stress and depression. The importance of H3/4 histones acetylation by CREB-binding protein (CBP) or related histone acetyltransferase, including p300, was specifically demonstrated using knockout (KO) mouse models. The physiological role of a related protein that also acts as a transcriptional coactivator with intrinsic histone acetylase activity, the p300/CBP-associated factor (PCAF), is poorly documented. We analyzed the behavioral phenotype of homozygous male and female PCAF KO mice and report a marked impact of PCAF deletion on memory processes and stress response. PCAF KO animals showed short-term memory deficits at 2 months of age, measured using spontaneous alternation, object recognition, or acquisition of a daily changing platform position in the water maze. Acquisition of a fixed platform location was delayed, but preserved, and no passive avoidance deficit was noted. No gender-related difference was observed. These deficits were associated with hippocampal alterations in pyramidal cell layer organization, basal levels of Fos immunoreactivity, and MAP kinase activation. PCAF KO mice also showed an exaggerated response to acute stress, forced swimming, and conditioned fear, associated with increased plasma corticosterone levels. Moreover, learning and memory impairments worsened at 6 and 12 months of age, when animals failed to acquire the fixed platform location in the water maze and showed passive avoidance deficits. These observations demonstrate that PCAF histone acetylase is involved lifelong in the chromatin remodeling necessary for memory formation and response to stress.

TRIM22 E3 ubiquitin ligase activity is required to mediate antiviral activity against encephalomyocarditis virus.

The interferon (IFN) system is a major effector of the innate immunity that allows time for the subsequent establishment of an adaptive immune response against a wide-range of pathogens. Their diverse biological actions are thought to be mediated by the products of specific but usually overlapping sets of cellular genes induced in the target cells. Ubiquitin ligase members of the tripartite motif (TRIM) protein family have emerged as IFN-induced proteins involved in both innate and adaptive immunity. In this report, we provide evidence that TRIM22 is a functional E3 ubiquitin ligase that is also ubiquitinated itself. We demonstrate that TRIM22 expression leads to a viral protection of HeLa cells against encephalomyocarditis virus infections. This effect is dependent upon its E3 ubiquitinating activity, since no antiviral effect was observed in cells expressing a TRIM22-deletion mutant defective in ubiquitinating activity. Consistent with this, TRIM22 interacts with the viral 3C protease (3C(PRO)) and mediates its ubiquitination. Altogether, our findings demonstrate that TRIM22 E3 ubiquitin ligase activity represents a new antiviral pathway induced by IFN against picornaviruses.

TRIM22 Inhibits Influenza A Virus Infection by Targeting the Viral Nucleoprotein for Degradation

ABSTRACT Tripartite motif (TRIM) protein superfamily members are emerging as important effectors of the innate immune response against viral infections. In particular, TRIM22 was reported to exert antiviral activity against RNA viruses, such as hepatitis B virus (HBV), encephalomyocarditis virus (ECMV), and human immunodeficiency virus type 1 (HIV-1). We demonstrate here, for the first time, that TRIM22 is upregulated by influenza A virus (IAV) infection at both mRNA and protein levels in human alveolar epithelial A549 cells. Conversely, TRIM22 potently restricted IAV replication, in that prevention of TRIM22 expression by means of short hairpin RNA led to a 10-fold enhancement of IAV replication in these cells. Depletion of TRIM22 also reduced the anti-IAV activity of alpha interferon (IFN-α), suggesting that TRIM22 is an important IFN-stimulated gene that is required for maximal suppression of IAV by type I IFN. Furthermore, the IAV infectious titer decreased up to 100-fold in MDCK cells expressing exogenous human TRIM22. Restriction of IAV replication was accounted for by the interaction between TRIM22 and the viral nucleoprotein (NP), resulting in its polyubiquitination and degradation in a proteasome-dependent manner. Thus, TRIM22 represents a novel restriction factor upregulated upon IAV infection that curtails its replicative capacity in epithelial cells.

A20/TNFAIP3, a new estrogen-regulated gene that confers tamoxifen resistance in breast cancer cells.

The zinc-finger protein A20/TNFAIP3, an inhibitor of nuclear factor-κB (NF-κB) activation, has been shown to protect MCF-7 breast carcinoma cells from TNFα-induced apoptosis. As estrogen receptor (ER) status is an important parameter in the development and progression of breast cancer, we analysed the effect of 17β-estradiol (E2) treatment on the expression of A20. We found that A20 is a new E2-regulated gene, whose expression correlates with ER expression in both cell lines and tumor samples. With the aim of investigating the impact of A20 expression on MCF-7 cells in response to ER ligands, we established stably transfected-MCF-7 cells overexpressing A20 (MCF-7-A20). These cells exhibited a phenotype of resistance to the 4-hydroxy-tamoxifen cytostatic and pro-apoptotic actions and of hyper-response to E2. Dysregulations in bax, bcl2, bak, phospho-bad, cyclin D1, cyclin E2, cyclin D2 and cyclin A2 proteins expression were shown to be related to the resistant phenotype developed by the MCF-7-A20 cells. Interestingly, we found that A20 was also overexpressed in MVLN and VP tamoxifen-resistant cell lines. Furthermore, high A20 expression levels were observed in more aggressive breast tumors (ER-negative, progesterone receptor-negative and high histological grade). These overall findings strongly suggest that A20 is a key protein involved in tamoxifen resistance, and thus represents both a new breast cancer marker and a promising target for developing new strategies to prevent the emergence of acquired mechanisms of drug resistance in breast cancer.

TRIM22 Inhibits HIV-1 Transcription Independently of Its E3 Ubiquitin Ligase Activity, Tat, and NF-κB-Responsive Long Terminal Repeat Elements

ABSTRACT Previous studies identified clones of the U937 promonocytic cell line that were either permissive or nonpermissive for human immunodeficiency virus type 1 (HIV-1) replication. These clones were investigated further in the search for host restriction factors that could explain their differential capacity to support HIV-1 replication. Among known HIV-1 restriction factors screened, tripartite motif-containing protein 22 (TRIM22) was the only factor constitutively expressed in nonpermissive and absent in permissive U937 cells. Stable TRIM22 knockdown (KD) rescued HIV-1 long-terminal-repeat (LTR)-driven transcription in KD-nonpermissive cells to the levels observed in permissive cells. Conversely, transduction-mediated expression of TRIM22 in permissive cells reduced LTR-driven luciferase expression by ∼7-fold, supporting a negative role of TRIM22 in HIV-1 transcription. This finding was further confirmed in the human T cell line A3.01 expressing TRIM22. Moreover, overexpression of TRIM22 in 293T cells significantly impaired basal and phorbol myristate acetate-ionomycin-induced HIV-1 LTR-driven gene expression, whereas inhibition of tumor necrosis factor alpha-induced viral transcription was a consequence of lower basal expression. In agreement, TRIM22 equally inhibited an LTR construct lacking the tandem NF-κB binding sites. In addition, TRIM22 did not affect Tat-mediated LTR transactivation. Finally, these effects were independent of TRIM22 E3 ubiquitin-ligase activity. In the context of replication-competent virus, significantly higher levels of HIV-1 production were observed in KD-nonpermissive versus control nonpermissive U937 cells after infection. In contrast, lower peak levels of HIV-1 replication characterized U937 and A3.01 cells expressing TRIM22 versus their control transduced counterpart. Thus, nuclear TRIM22 significantly impairs HIV-1 replication, likely by interfering with Tat- and NF-κB-independent LTR-driven transcription.

Cloning and expression of an alternatively spliced mRNA encoding a soluble form of the human interleukin-6 signal transducer gp130.

Both gp130 and alternatively spliced sgp130 were also transcribed by the myeloma cell lines XG‐1, XG‐2, XG‐4, XG‐4CNTF XG‐6, XG‐7, XG‐9, XG‐10, U266 and RPMI 8226. However, XG‐4A cells derived from XG‐4 cells, but growing independently of exogenous IL‐6, did not transcribe sgp130 mRNA. A possible interference with intracrine stimulatory factors by alternatively spliced sgp130 needs to be further investigated.

Drug specific resistance to oxaliplatin is associated with apoptosis defect in a cellular model of colon carcinoma.

To investigate acquired resistance to oxaliplatin, we selected two resistant clones from the HCT116 cell line. We found that the resistant phenotype was associated with resistance to oxaliplatin‐induced apoptosis as demonstrated by FACS analysis and by Western blotting of caspase 3 activation. In addition, the resistant phenotype showed a concomitant resistance to lonidamine and arsenic trioxide which are inducers of mitochondrial apoptosis. Furthermore, a complete loss of Bax expression due to a frameshift mutation was observed in the most resistant clone. Taken together, these findings suggest that altered mitochondrial‐mediated apoptosis could play a role in oxaliplatin resistance.

The viral RNA recognition sensor RIG-I is degraded during encephalomyocarditis virus (EMCV) infection.

RNA helicase-like receptors MDA-5 but not RIG-I has been shown to be essential for triggering innate immune responses against picornaviruses. However, virus-host co-evolution has selected for viruses capable of replicating despite host cells antiviral defences. In this report, we demonstrate that RIG-I is degraded during encephalomyocarditis virus (EMCV) infection. This effect is mediated by both the viral-encoded 3C protease and caspase proteinase. In addition, we show that RIG-I overexpression confers IFN-beta promoter activation during EMCV infection, in MDA-5 knockout (MDA-5(-/-)) mouse embryo fibroblasts. This induction is followed by a strong inhibition reflecting the ability of EMCV to disrupt RIG-I signalling. Taken together, our data strongly suggest that during evolution RIG-I has been involved for triggering innate immune response to picornavirus infections.

TRIM22 Inhibits HIV-1 Transcription Independently of Its E3-Ubiquitin Ligase Activity, Tat and NF-κB Responsive LTR Elements

ABSTRACT Previous studies identified clones of the U937 promonocytic cell line that were either permissive or nonpermissive for human immunodeficiency virus type 1 (HIV-1) replication. These clones were investigated further in the search for host restriction factors that could explain their differential capacity to support HIV-1 replication. Among known HIV-1 restriction factors screened, tripartite motif-containing protein 22 (TRIM22) was the only factor constitutively expressed in nonpermissive and absent in permissive U937 cells. Stable TRIM22 knockdown (KD) rescued HIV-1 long-terminal-repeat (LTR)-driven transcription in KD-nonpermissive cells to the levels observed in permissive cells. Conversely, transduction-mediated expression of TRIM22 in permissive cells reduced LTR-driven luciferase expression by ∼7-fold, supporting a negative role of TRIM22 in HIV-1 transcription. This finding was further confirmed in the human T cell line A3.01 expressing TRIM22. Moreover, overexpression of TRIM22 in 293T cells significantly impaired basal and phorbol myristate acetate-ionomycin-induced HIV-1 LTR-driven gene expression, whereas inhibition of tumor necrosis factor alpha-induced viral transcription was a consequence of lower basal expression. In agreement, TRIM22 equally inhibited an LTR construct lacking the tandem NF-κB binding sites. In addition, TRIM22 did not affect Tat-mediated LTR transactivation. Finally, these effects were independent of TRIM22 E3 ubiquitin-ligase activity. In the context of replication-competent virus, significantly higher levels of HIV-1 production were observed in KD-nonpermissive versus control nonpermissive U937 cells after infection. In contrast, lower peak levels of HIV-1 replication characterized U937 and A3.01 cells expressing TRIM22 versus their control transduced counterpart. Thus, nuclear TRIM22 significantly impairs HIV-1 replication, likely by interfering with Tat- and NF-κB-independent LTR-driven transcription.