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Telomere capping and cellular checkpoints: clues from fruit flies
Full text linkIn most organisms, telomeres consist of repetitive G-rich sequences that are elongated by a specific reverse transcriptase, telomerase. A large number of proteins are recruited by these terminal repeats, forming specialized structures that regulate telomerase activity and protect telomeres from degradation and recombination. Drosophila lacks telomerase and telomere length is maintained by transposition of three specialized retrotransposons. In addition, unlike yeast and mammals, Drosophila telomeres are epigenetically determined, sequence-independent structures. However, several proteins required for Drosophila telomere behavior are evolutionarily conserved. These include the Mre11-Rad50-Nbs (MRN) complex and the Ataxia Telangiectasia Mutated (ATM) kinase, which are required to prevent telomeric fusions. In addition, recent studies have provided evidence that Drosophila uncapped telomeres elicit a DNA damage response (DDR) just as dysfunctional yeast and human telomeres. Uncapped Drosophila telomeres also activate the spindle assembly checkpoint (SAC) by recruiting the SAC kinase BubR1. Telomere-induced DDR and SAC both require the wild type function of the MRN complex. In addition, while DDR is mediated by ATR kinase, SAC activation requires both the ATM and ATR activities. These results indicate that the DNA repair systems play multiple roles at Drosophila telomeres, highlighting the importance of this model organism for investigations on the relationships between DNA repair and telomere maintenance
An essential function of AP-1 heterodimers in Drosophila.
No full textFos and Jun proteins homo- or heterodimerize to form functional AP-1 transcription factors. Drosophila mutants lacking either Jun or Fos display indistinguishable dorsal open phenotypes, indicating an essential function of both Jun and Fos for embryonic dorsal closure. Here we present experiments to determine the basis for this dual requirement. By combining mutant alleles and transgenes expressing Fos and Jun variants with altered dimerization preferences, fly lines were generated in which only specifically defined dimer variants can form. Phenotypic analysis of these mutants reveals that homodimers of Fos or of Jun cannot replace the function of the heterodimeric complex. This defect is not explained by the lower stability of homodimers as compared to heterodimers, because 'pseudo-homodimers' which are as stable as native Jun-Fos heterodimers cannot substitute for their function. We conclude that Jun and Fos play complementary roles that are both required for signal transduction and gene activation during dorsal closure
The Drosophila Nbs protein functions in multiple pathways for the maintenance of genome stability
No full textThe Mre11/Rad50/Nbs (MRN) complex and the two protein kinases ATM and ATR play critical roles in the response to DNA damage and telomere maintenance in mammalian systems. It has been previously shown that mutations in the Drosophila mre11 and rad50 genes cause both telomere fusion and chromosome breakage. Here, we have analyzed the role of the Drosophila nbs gene in telomere protection and the maintenance of chromosome integrity. Larval brain cells of nbs mutants display telomeric associations (TAs) but the frequency of these TAs is lower than in either mre11 or rad50 mutants. Consistently, Rad50 accumulates in the nuclei of wild-type cells but not in those of nbs cells, indicating that Nbs mediates transport of the Mre11/Rad50 complex in the nucleus. Moreover, epistasis analysis revealed that rad50 nbs, tefu (ATM) nbs, and mei-41 (ATR) nbs double mutants have significantly higher frequencies of TAs than either of the corresponding single mutants. This suggests that Nbs and the Mre11/Rad50 complex play partially independent roles in telomere protection and that Nbs functions in both ATR- and ATM-controlled telomere protection pathways. In contrast, analysis of chromosome breakage indicated that the three components of the MRN complex function in a single pathway for the repair of the DNA damage leading to chromosome aberrations
The mechanism of telomere protection: a comparison between Drosophila and humans
No full textDrosophila telomeres are maintained by transposition of specialized retrotransposons rather than by telomerase activity, and their stability is independent of the sequence of DNA termini. Recent studies have identified several proteins that protect Drosophila telomeres from fusion events. These proteins include the telomere capping factors HP1/ORC-associated protein (HOAP) and heterochromatin protein 1 (HP1), the Rad50 and Mre11 DNA repair proteins that are required for HOAP and HP1 localization at telomeres, and the ATM kinase. Another telomere- protecting factor identified in Drosophila is UbcD1, a polypeptide highly homologous to class I ubiquitin-conjugating E2 enzymes. In addition, it has been shown that HP1 and both components of the Drosophila Ku70/80 heterodimer act as negative regulators of telomere length. Except for HOAP, all these proteins are conserved in humans and are associated with human telomeres. Collectively, these results indicate that Drosophila is an excellent model system for the analysis of the mechanisms of telomere maintenance. In past and current studies, 15 Drosophila genes have been identified that prevent telomeric fusion, and it has been estimated that the Drosophila genome contains at least 40 genes required for telomere protection. We believe that the molecular characterization of these genes will lead to identification of many novel human genes with roles in telomere maintenance
Drosophila Fos mediates ERK and JNK signals via distinct phosphorylation sites.
No full textDuring Drosophila development Fos acts downstream from the JNK pathway. Here we show that it can also mediate ERK signaling in wing vein formation and photoreceptor differentiation. Drosophila JNK and ERK phosphorylate D-Fos with overlapping, but distinct, patterns. Analysis of flies expressing phosphorylation site point mutants of D-Fos revealed that the transcription factor responds differentially to JNK and ERK signals. Mutations in the phosphorylation sites for JNK interfere specifically with the biological effects of JNK activation, whereas mutations in ERK phosphorylation sites affect responses to the EGF receptor-Ras-ERK pathway. These results indicate that the distinction between ERK and JNK signals can be made at the level of D-Fos, and that different pathway-specific phosphorylated forms of the protein can elicit different responses
Adenovirus-mediated gene transfer of an IL-6 antagonist
No full textIL-6 is a pleiotropic cytokine and plays a major role in inflammation and in the immune response. Altered serum levels of IL-6 have been described in several pathologies such as myeloma, EBV-lymphoma and chronic autoimmune disease. Here we report data on the utilization of a hIL-6 receptor superantagonist with a gene therapy approach. The superantagonist used in this work possesses very high affinity for the hIL-6 receptor, and is therefore an excellent candidate for the treatment of IL-6-dependent diseases. To obtain an efficient in vivo delivery method, we constructed a recombinant adenovirus expressing the IL-6 receptor superantagonist by inserting the cDNA, controlled by the RSV promoter, into a first generation replication-incompetent adenoviral vector. Recombinant virus allowed correct expression of the transgene in vitro. Supernatants of infected cells specifically inhibited IL-6-induced transcriptional activation in hepatoma cells and blocked the IL-6-dependent proliferation of human myeloma cells. After intravenous injection of the recombinant virus into mice, nanomolar amounts of antagonist were produced in the serum, and these were able completely to inhibit IL-6 bioactivity. Gene transfer of such an antagonist offers a practical means of imposing long-term blockade of IL-6 activity in vivo for investigational and therapeutic purposes
Cytokine gene expression in intestine of rat during the postnatal developmental period: increased IL-1 expression at weaning.
No full textIn the present study we have investigate whether cytokines are constitutively and differently expressed in intestine during the differentiative processes that take place at weaning. We have analyzed the expression of IL-1 beta, IL-2, IL-4 and IFN gamma by polymerase chain reaction in Peyer's patches (PP) and in intestine deprived of PP (I-PP) of rats from 16 to 30 days of age. The results showed a constitutive and marked expression of the cytokines already before weaning, with the exception of IL-2 in PP and IFN gamma in I-PP. IL-beta was the only cytokine to show a different expression at various ages with an initial increase at 19 days and a further elevation at 21 days when intestinal epithelium passes through major differentiative stages, suggesting an involvement of this cytokine in intestinal development. We have also tested whether treatment of rats with the immunosuppressor cyclosporin A (CsA) could affect intestinal differentiation. The results showed that only some markers of differentiation were affected (proliferation of staminal crypt cells and length of crypts). This was probably due to a direct effect rather than an immunomediated effect of CsA, since treatment of three intestinal cell lines (Caco-2, HT-29, FRIC) with CsA indicated that this drug can exert a cytostatic activity on intestinal cells
Unprotected Drosophila melanogaster telomeres activate the spindle assembly checkpoint
No full textIn both yeast and mammals, uncapped telomeres activate the DNA damage response (DDR) and undergo end-to-end fusion. Previous work has shown that the Drosophila HOAP protein, encoded by the caravaggio (cav) gene, is required to prevent telomeric fusions. Here we show that HOAP-depleted telomeres activate both the DDR and the spindle assembly checkpoint (SAC). The cell cycle arrest elicited by the DDR was alleviated by mutations in mei-41 (encoding ATR), mus304 (ATRIP), grp (Chk1) and rad50 but not by mutations in tefu (ATM). The SAC was partially overridden by mutations in zw10 (also known as mit(1)15) and bubR1, and also by mutations in mei-41, mus304, rad50, grp and tefu. As expected from SAC activation, the SAC proteins Zw10, Zwilch, BubR1 and Cenp-meta (Cenp-E) accumulated at the kinetochores of cav mutant cells. Notably, BubR1 also accumulated at cav mutant telomeres in a mei-41-, mus304-, rad50-, grp- and tefu-dependent manner. Our results collectively suggest that recruitment of BubR1 by dysfunctional telomeres inhibits Cdc20-APC function, preventing the metaphase-to-anaphase transition
The Drosophila modigliani (moi) gene encodes a HOAP-interacting protein required for telomere protection
Full text linkSeveral proteins have been identified that protect Drosophila telomeres from fusion events. They include UbcD1, HP1, HOAP, the components of the Mre11-Rad50-Nbs (MRN) complex, the ATM kinase, and the putative transcription factor Woc. Of these proteins, only HOAP has been shown to localize specifically at telomeres. Here we show that the modigliani gene encodes a protein (Moi) that is enriched only at telomeres, colocalizes and physically interacts with HOAP, and is required to prevent telomeric fusions. Moi is encoded by the bicistronic CG31241 locus. This locus produces a single transcript that contains 2 ORFs that specify different essential functions. One of these ORFs encodes the 20-kDa Moi protein. The other encodes a 60-kDa protein homologous to RNA methyltransferases that is not required for telomere protection (Drosophila Tat-like). Moi and HOAP share several properties with the components of shelterin, the protein complex that protects human telomeres. HOAP and Moi are not evolutionarily conserved unlike the other proteins implicated in Drosophila telomere protection. Similarly, none of the shelterin subunits is conserved in Drosophila, while most human nonshelterin proteins have Drosophila homologues. This suggests that the HOAP-Moi complex, we name "terminin," plays a specific role in the DNA sequence-independent assembly of Drosophila telomeres. We speculate that this complex is functionally analogous to shelterin, which binds chromosome ends in a sequence-dependent manner
Definition of a complex binding site for gp130 in human interleukin-6
No full textThe helical cytokine interleukin-6 (IL-6) assembles a multiprotein receptor complex. The starting event in the activation of intracellular signaling is the binding of the IL-6/IL-6R alpha subcomplex to two gp130 chains. The homodimerization of gp130 is triggered by two distinct and independent regions of IL-6 called sites 2 and 3. Several IL-6 antagonists have been obtained that affect signaling, but not IL-6 IL-6R alpha subcomplex formation. In this paper, we analyze in detail the impact of these antagonists on gp130 binding and dimerization and show that each signaling variant affects gp130 dimerization in vitro and that biological activity on cells decreases in precise parallel to the decrease in gp130 dimerization in vitro. All IL-6 antagonists can be classified into two groups, mapping at either site 2 or 3 in correspondence to their mode of interaction with gp130. We found that site 3 is a large region, which includes residues at the beginning of helix D spatially flanked by residues in the putative AB loop and located at one extremity of the cytokine 4-helix bundle. Interestingly, in leukemia inhibitory factor, another cytokine that signals through gp130, site 3, is topologically conserved but has evolved to bind leukemia inhibitory factor receptor
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