Role of TLR-2 in the activation of nuclear factor kappaB by oxidative stress in cardiac myocytes.
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Growing evidence from patients with heart failure and from experimental animal models implicates effectors of innate immunity in the pathogenesis of this syndrome. The expression of the innate immunity signaling protein, Toll-like receptor 4 (TLR4), is increased in cardiac myocytes in situ and in failing myocardium, but the mechanism by which TLRs may be activated in the failing heart remains unclear. We report that TLR2, which is expressed in cardiac myocytes, participates in the response of these cells to oxidative stress, a major contributor to the pathogenesis of cardiac dysfunction. Hydrogen peroxide increased nuclear factor kappaB (NF-kappaB) activation in Chinese hamster ovary fibroblasts that overexpress TLR2 but not in normal or TLR4-overexpressing Chinese hamster ovary cells, an effect that was abrogated by an alpha-TLR2 antibody. In neonatal rat ventricular myocytes, the alpha-TLR2 antibody inhibited hydrogen peroxide-induced nuclear translocation of NF-kappaB and activator protein-1 (AP-1). Inhibition of TLR2 had no effect on tumor necrosis factor alpha-induced NF-kappaB or AP-1 activation, on the DNA binding of the basal transcription factor Oct-1, or on hydrogen peroxide-induced phosphorylation of p38 MAP kinase. Importantly, oxidative stress-induced cytotoxicity was enhanced by blocking TLR2. Given the importance of cytotoxicity and apoptosis to the pathology of the ischemic heart, an anti-apoptotic effect of TLR2 in cardiac myocytes exposed to elevated levels of ROS may limit further cardiac dysfunction. (+info)
The beta-amyloid precursor protein functions as a cytosolic anchoring site that prevents Fe65 nuclear translocation.
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In this study we addressed the question of the intracellular localization of Fe65, an adaptor protein interacting with the beta-amyloid precursor protein (APP) and with the transcription factor CP2/LSF/LBP1. By using tagged Fe65 expression vectors, we observed that a significant fraction of Fe65 is localized in the nucleus of transfected COS7 cells. Furthermore, the isolation of nuclei from untransfected PC12 cells allowed us to observe that a part of the endogenous Fe65 is present in the nuclear extract. The analysis of Fe65 mutant constructs demonstrated that the region of the protein required for its nuclear translocation includes the WW domain, and that, on the other hand, a small fragment of 100 residues, including this WW domain, contains enough structural information to target a reporter protein (green fluorescent protein (GFP)-GFP) to the nucleus. To evaluate whether the Fe65-APP interaction could affect Fe65 intracellular trafficking, COS7 cells were cotransfected with APP(695) or APP(751) and with GFP-Fe65 expression vectors. These experiments demonstrated that Fe65 is no longer translocated to the nucleus when the cells overexpress APP, whereas the nuclear targeting of GFP-Fe65 mutants, unable to interact with APP, is unaffected by the coexpression of APP, thus suggesting that the interaction with APP anchors Fe65 in the cytosol. (+info)
Caspases disrupt the nuclear-cytoplasmic barrier.
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During apoptosis, caspases, a family of proteases, disassemble a cell by cleaving a set of proteins. Caspase-3 plays a major role in the dissassembly of the nucleus by processing several nuclear substrates. The question is how caspase-3 which is usually cytoplasmic, gains access to its nuclear targets. It was suggested that caspase-3 is actively transported to the nucleus through the nuclear pores. We found that caspase-9, which is activated earlier than caspase-3, directly or indirectly inactivates nuclear transport and increases the diffusion limit of the nuclear pores. This increase allows caspase-3 and other molecules that could not pass through the nuclear pores in living cells to enter or leave the nucleus during apoptosis by diffusion. Hence, caspase-9 contributes to cell disassembly by disrupting the nuclear cytoplasmic barrier. (+info)
Dissection of autophagosome biogenesis into distinct nucleation and expansion steps.
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Rapamycin, an antifungal macrolide antibiotic, mimics starvation conditions in Saccharomyces cerevisiae through activation of a general G(0) program that includes widespread effects on translation and transcription. Macroautophagy, a catabolic membrane trafficking phenomenon, is a prominent part of this response. Two views of the induction of autophagy may be considered. In one, up-regulation of proteins involved in autophagy causes its induction, implying that autophagy is the result of a signal transduction mechanism leading from Tor to the transcriptional and translational machinery. An alternative hypothesis postulates the existence of a dedicated signal transduction mechanism that induces autophagy directly. We tested these possibilities by assaying the effects of cycloheximide and specific mutations on the induction of autophagy. We find that induction of autophagy takes place in the absence of de novo protein synthesis, including that of specific autophagy-related proteins that are up-regulated in response to rapamycin. We also find that dephosphorylation of Apg13p, a signal transduction event that correlates with the onset of autophagy, is also independent of new protein synthesis. Finally, our data indicate that autophagosomes that form in the absence of protein synthesis are significantly smaller than normal, indicating a role for de novo protein synthesis in the regulation of autophagosome expansion. Our results define the existence of a signal transduction-dependent nucleation step and a separate autophagosome expansion step that together coordinate autophagosome biogenesis. (+info)
Nmd3p is a Crm1p-dependent adapter protein for nuclear export of the large ribosomal subunit.
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In eukaryotic cells, nuclear export of nascent ribosomal subunits through the nuclear pore complex depends on the small GTPase Ran. However, neither the nuclear export signals (NESs) for the ribosomal subunits nor the receptor proteins, which recognize the NESs and mediate export of the subunits, have been identified. We showed previously that Nmd3p is an essential protein from yeast that is required for a late step in biogenesis of the large (60S) ribosomal subunit. Here, we show that Nmd3p shuttles and that deletion of the NES from Nmd3p leads to nuclear accumulation of the mutant protein, inhibition of the 60S subunit biogenesis, and inhibition of the nuclear export of 60S subunits. Moreover, the 60S subunits that accumulate in the nucleus can be coimmunoprecipitated with the NES-deficient Nmd3p. 60S subunit biogenesis and export of truncated Nmd3p were restored by the addition of an exogenous NES. To identify the export receptor for Nmd3p we show that Nmd3p shuttling and 60S export is blocked by the Crm1p-specific inhibitor leptomycin B. These results identify Crm1p as the receptor for Nmd3p export. Thus, export of the 60S subunit is mediated by the adapter protein Nmd3p in a Crm1p-dependent pathway. (+info)
The fission yeast ran GTPase is required for microtubule integrity.
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The microtubule cytoskeleton plays a pivotal role in cytoplasmic organization, cell division, and the correct transmission of genetic information. In a screen designed to identify fission yeast genes required for chromosome segregation, we identified a strain that carries a point mutation in the SpRan GTPase. Ran is an evolutionarily conserved eukaryotic GTPase that directly participates in nucleocytoplasmic transport and whose loss affects many biological processes. Recently a transport-independent effect of Ran on spindle formation in vitro was demonstrated, but the in vivo relevance of these findings was unclear. Here, we report the characterization of a Schizosaccharomyces pombe Ran GTPase partial loss of function mutant in which nucleocytoplasmic protein transport is normal, but the microtubule cytoskeleton is defective, resulting in chromosome missegregation and abnormal cell shape. These abnormalities are exacerbated by microtubule destabilizing drugs, by loss of the spindle checkpoint protein Mph1p, and by mutations in the spindle pole body component Cut11p, indicating that SpRan influences microtubule integrity. As the SpRan mutant phenotype can be partially suppressed by the presence of extra Mal3p, we suggest that SpRan plays a role in microtubule stability. (+info)
Primate foamy virus Pol proteins are imported into the nucleus.
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Mouse monoclonal antibodies (MAbs) that specifically detect the 127 kDa Pol precursor and the 85 kDa reverse transcriptase/RNase H (RT/RN) or pr127 and the 40 kDa integrase (IN) in immunoblot and immunofluorescence assays (IFA) were used to investigate the subcellular localization of primate foamy virus (PFV) proteins. IFA of cells infected with PFV using the anti-Pol MAbs and rabbit anti-capsid (Gag) serum revealed that both the Gag and Pol proteins are transported into the nucleus. Transfection of cells with eukaryotic expression constructs for pr127(Pol), p85(RT/RN) and p40(IN) served to show Gag-independent subcellular localization of Pol proteins. Interestingly, not only the Pol precursor and IN molecules were found to be localized to the nucleus, but also the RT/RN subdomain. It is therefore suggested that PFV cores bear at least three separate nuclear localization signals, one in Gag and two in Pol. The latter appear to be localized to the two Pol subdomains. (+info)
Interaction of the herpes simplex virus type 1 packaging protein UL15 with full-length and deleted forms of the UL28 protein.
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The UL15 and UL28 proteins of herpes simplex virus type 1 are both required for the packaging of replicated viral DNA into the viral capsid. We have expressed UL28 and a functional epitope-tagged form of UL15 in mammalian and insect cells. Immunoprecipitation experiments confirmed that the two proteins can interact. In agreement with previous results, UL15, when expressed alone, entered the nucleus but UL28 remained cytoplasmic. When co-expressed the two proteins co-localized in the nucleus. Six UL28 deletion mutants were constructed and similarly analysed. The results obtained by immunoprecipitation and immunofluorescence were consistent and demonstrate that at least two separate regions of the UL28 polypeptide chain have the ability to interact with UL15. Surprisingly, three of the mutants prevented the UL15 protein from localizing to the cell nucleus, and these were not functional in a transient DNA packaging assay. Of the three UL28 mutant proteins that entered the nucleus with UL15, one containing an internal deletion of 13 amino acids was able to complement a UL28 null mutant in both DNA packaging and virus yield assays, demonstrating that this region of the protein is not essential for function. In addition to interacting with the UL28 protein we also demonstrated that UL15 molecules can interact with each other, and that sequences within the second exon contribute to this interaction. (+info)