Structure and Function of the Human Respiratory Syncytial Virus M2–1 Protein

Human respiratory syncytial virus (HRSV) is a non-segmented negative stranded RNA virus and is recognized as the most important viral agent of lower respiratory tract infection worldwide, responsible for up to 199,000 deaths each year. The only FDA-approved regime to prevent HRSV-mediated disease is pre-exposure administration of a humanized HRSV-specific monoclonal antibody, which although being effective, is not in widespread usage due to its cost. No HRSV vaccine exists and so there remains a strong need for alternative and complementary anti-HRSV therapies. The HRSV M2–1 protein is a transcription factor and represents an attractive target for the development of antiviral compounds, based on its essential role in the viral replication cycle. To this end, a detailed analysis of M2–1 structure and functions will aid in identifying rational targets for structure-based antiviral drug design that can be developed in future translational research. Here we present an overview of the current understanding of the structure and function of HRSV M2–1, drawing on additional information derived from its structural homologues from other related viruses

Inhibition of Bcl-2-dependent cell survival by a caspase inhibitor: a possible new pathway for Bcl-2 to regulate cell death

International audienceThe REtsAF cell line expresses a temperaturesensitive mutant of the SV40 large tumor antigen. At restrictive temperature (39.5³C), the cells undergo p53-mediated apoptosis, which can be inhibited by Bcl-2. Here, we show that Z-VADfmk, a caspase inhibitor, can suppress the Bcl-2-dependent cell survival at 39.5³C. This result suggests that a caspase-like activity can act as an inhibitor of apoptosis in this model, downstream of Bcl-2. Our results also suggest that this activity may be up-regulated by Bcl-2 and may be responsible for cleavage of the tumor suppressor Rb protein

Integration of a pAL2-1 homologous mitochondrial plasmid associated with life span extension in Podospora anserina

We isolated and characterized a novel spontaneous longevity mutant of Podospora anserina strain Wa32 carrying one of the pAL2-1 homologous mitochondrial plasmids. This mutant is at least ten fold longer-lived than the wild type, and is hence a formal suppressor of both the regular and the `plasmid-based¿ senescence process. We show that the longevity trait is maternally inherited and coincides with the presence of a copy of the plasmid integrated in the 5¿ UTR of the mitochondrial Complex I genes nd2 and nd3. This mutation is associated with complex alterations in the respiratory chain, including a dispensable induction of the alternative oxidase. It is also associated with a stabilization of the mitochondrial chromosome and a reduction of the overall cellular level of reactive oxygen species

p53 can promote mitochondria- and caspase-independent apoptosis

International audienceThe tumour suppressor p53 plays a pivotal role in suppressing tumorigenesis by inducing cell cycle arrest or apoptosis. Cell cycle arrest is mediated by transcriptional induction of genes whose products inhibit cell cycle progression. Conversely, the molecular events that lead to p53-dependent apoptosis are less clear. Transcriptional activation is commonly implicated but growing evidences show that transrepression and transcription-independent functions can also play a central role in p53-dependent apoptosis. 1 At the cellular level, all studies converge to the crucial role of both the mitochondrial pathway (cytochrome c release, 2 ROS production or/and DCm drop) 3 and caspase activation in p53-induced apopto-sis. 4 In this way, the Bcl-2 antiapoptotic protein as well as caspase inhibition were shown to protect cells from p53-induced apoptosis. We previously showed in rat embryo fibroblasts (e.g. the REtsAF cell line) expressing a temperature-sensitive mutant (tsA58) of the simian virus 40 large tumour antigen (LT) that LT inactivation leads to p53-mediated apoptosis. Moreover, we reported that while bcl-2 overexpression inhibits apopto-sis, caspase inhibition surprisingly accelerates apoptosis and moreover abolishes the protective effect of Bcl-2. 5 These data led us to postulate that caspase inhibition would unmask an alternative route for p53-induced cell death signal, which would lead to a caspase-independent and Bcl-2-insensitive cell death process. In order to ascertain that this new cell death process observed in the presence of ZVAD is dependent on p53 activity, REtsAF cells were transiently transfected with genes encoding temperature-sensitive dominant-negative mutants of p53 (p53 val135 and p53 ala143). 6,7 At restrictive temperature, these mutants are defective in their DNA binding domain and consequently loss their sequence-specific transactivation and transrepression properties. We observed that overexpression of either p53 val135 or p53 ala143 led to a decreased rate of apoptosis in the absence or in the presence of ZVAD (Figure 1a), demonstrating that active p53 is required for these two cell death pathways. Next, we controlled that this unexpected effect of ZVAD on p53-induced cell death could be reproduced in a more physiological model. For this purpose, we induced a p53-dependent apoptosis in primary rat embryo fibroblasts (RE) by addition of 100 mM etoposide, in the absence or in the presence of ZVAD. As observed in REtsAF cells, ZVAD treatment accelerated commitment to death of RE cells. As control, ZVAD reduced staurosporine-induced apoptosis of RE cells, showing its usual protector effect against this p53-independent cell death (Figure 1b). These results argue for a physiological relevance of the proapoptotic effect of ZVAD on p53-mediated apoptosis. In order to characterise the pattern of the p53-induced death of REtsAF cells in the presence of ZVAD, we examined the associated nuclear and cytological alterations. In the absence of ZVAD, the earliest morphological changes observed are the rounding up, the brightening phase, the shrinkage of the cells as well as the blebbing of the plasma membrane (Figure 1c). These events are associated with typical apoptotic chromatin compaction and fragmentation in globular, crescent-shaped figures (stage II chromatin condensation), and they preceded the breaking up of the cells into fragments as well as the fragmentation of DNA, as judged by the flow cytometric analysis of TUNEL-stained cells. In the presence of ZVAD, light microscopy observation showed that some typical morphological features of apoptosis such as loss of adherence and condensed cytoplasm were evident in dying REtsAF cells. Further fluorescence microscopy examination revealed chromatin condensed in lumps (stage I chromatin condensation), and rounded cells without microvilli or protu-sions on the surface (Figure 1c). However, specific events of the final degradation phase such as the nucleus and cell fragmentation do not occur whereas cells completely detached from the substratum. These observations indicate that p53 can promote two cell death pathways showing apoptotic features, one that is caspase-dependent and another that is independent of caspases. Recent data suggest that noncaspase proteases including calpains, cathepsins or serine proteases can also mediate cell death and bring about many of the morphological changes characteristic of apoptosis in a caspase-independent manner. 8 We investigated the possible involvement of these proteases in the novel route by which p53 signal cell death through the use of specific inhibitors: serine protease inhibitors (TPCK, TLCK), calpain protease inhibitors (ALLN, MDL), cysteine protease inhibitors (Lactacystine, Z-FA-fmk). Microscopic studies of cell morphology did not show notable differences in the amount of Z-VAD-dependent cellular death whether the cells were cultured in the presence or in the absence of the drugs. These results suggest that none of the tested proteases are involved in p53-induced apoptosis of REtsAF cells in the presence of ZVAD. Inasmuch as the mitochondrial pathway plays a central role in many models of p53-dependent apoptosis, we investigate

Developmental cell death during Xenopus metamorphosis involves BID cleavage and caspase 2 and 8 activation.

International audienceElimination of tadpole organs during Xenopus metamorphosis is largely achieved through apoptosis, and recent evidence suggest involvement of the mitochondrial death route and bax-initiated caspase-3 and -9 deployment. However, events upstream of the activation of Bax are unknown. In other models, proteins of the BH3-only group such as BID are known to assure this function. We show that Xenopus bid transcript levels increase at metamorphosis in larval cells destined to disappear. This increase correlates with an abrupt rise in Caspase-2 and -8 mRNA levels and an enhanced activity of Caspase-2 and -8. In BIDGFP transgenic animal's tail regression is accelerated. The cleavage of BIDGFP fusion protein during natural or T(3)-induced metamorphosis was specifically inhibited by caspase-8 inhibitors. Our results show that tail regression at metamorphosis implicates an apoptotic pathway inducible by T(3) hormone in an organ autonomous manner and involving the cell death executioners BID and Caspases-2 and -8

Triumfettosterol Id and Triumfettosaponin, a New (Fatty Acyl)-Substituted Steroid and a Triterpenoid Dimer Bis(-D-glucopyranosyl) Ester from the Leaves of Wild Triumfetta cordifolia A. Rich. (Tiliaceae)

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Evidence for a mitochondrial localization of Rb protein

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Effects of 17β-Estradiol on Preadipocyte Proliferation in Human Adipose Tissue: Involvement of IGF1-R Signaling

Estrogens are known to stimulate the proliferation of human preadipocytes. However, the molecular mechanisms underlying the increased cell growth by these steroids are poorly understood. In the present study, we have demonstrated that the proliferative effect of 17beta-estradiol involves the induction of both cell cycle gene expressions, c-myc and cyclin D1. Moreover, the mitogenic effects of 17beta-estradiol are suppressed by the pure antagonist ICI 182780 suggesting that estradiol action is mediated by estrogen receptor (ER). We have also shown that 17beta-estradiol is able to inhibit human preadipocyte apoptosis capacity as reflected by DNA fragmentation experiments and the mRNA expression of the pro- and antiapoptotic genes. Finally, 17beta-estradiol significantly induces both mRNA and protein expression of RIGF1 in human preadipose cells via ER and thus reinforces the signaling pathway of the proliferative factor, IGF1. Taken together, these data reinforce the concept of cross-talk between IGF1- and ER-signaling pathways in preadipocytes and indicate that IGFI may be a critical regulator of estrogen-mediated preadipose growth

Inactivation of p53 Is Sufficient to Induce Development of Pulmonary Hypertension in Rats.

Pulmonary artery smooth muscle cells (PA-SMCs) in pulmonary arterial hypertension (PAH) show similarities to cancer cells. Due to the growth-suppressive and pro-apoptotic effects of p53 and its inactivation in cancer, we hypothesized that the p53 pathway could be altered in PAH. We therefore explored the involvement of p53 in the monocrotaline (MCT) rat model of pulmonary hypertension (PH) and the pathophysiological consequences of p53 inactivation in response to animal treatment with pifithrin-α (PFT, an inhibitor of p53 activity).PH development was assessed by pulmonary arterial pressure, right ventricular hypertrophy and arterial wall thickness. The effect of MCT and PFT on lung p53 pathway expression was evaluated by western blot. Fourteen days of daily PFT treatment (2.2 mg/kg/day), similar to a single injection of MCT (60 mg/kg), induced PH and aggravated MCT-induced PH. In the first week after MCT administration and prior to PH development, p53, p21 and MDM2 protein levels were significantly reduced; whereas PFT administration effectively altered the protein level of p53 targets. Anti-apoptotic and pro-proliferative effects of PFT were revealed by TUNEL and MTT assays on cultured human PA-SMCs treated with 50 μM PFT.Pharmacological inactivation of p53 is sufficient to induce PH with a chronic treatment by PFT, an effect related to its anti-apoptotic and pro-proliferative properties. The p53 pathway was down-regulated during the first week in the rat MCT model. These in vivo experiments implicate the p53 pathway at the initiation stages of PH pathogenesis