ABSTRACT
Venoms comprise of complex mixtures of peptides evolved for predation and defensive purposes. Remarkably, some carnivorous cone snails can inject two distinct venoms in response to predatory or defensive stimuli, providing a unique opportunity to study separately how different ecological pressures contribute to toxin diversification. Here, we report the extraordinary defensive strategy of the Rhizoconus subgenus of cone snails. The defensive venom from this worm-hunting subgenus is unusually simple, almost exclusively composed of αD-conotoxins instead of the ubiquitous αA-conotoxins found in the more complex defensive venom of mollusc- and fish-hunting cone snails. A similarly compartmentalized venom gland as those observed in the other dietary groups facilitates the deployment of this defensive venom. Transcriptomic analysis of a Conus vexillum venom gland revealed the αD-conotoxins as the major transcripts, with lower amounts of 15 known and four new conotoxin superfamilies also detected with likely roles in prey capture. Our phylogenetic and molecular evolution analysis of the αD-conotoxins from five subgenera of cone snails suggests they evolved episodically as part of a defensive strategy in the Rhizoconus subgenus. Thus, our results demonstrate an important role for defence in the evolution of conotoxins.
Subject(s)
Conotoxins/chemistry , Conus Snail/genetics , Evolution, Molecular , Phylogeny , Transcriptome , Amino Acid Sequence , Animals , Australia , Cell Line , Conotoxins/genetics , Humans , Molecular Sequence Data , Sequence Analysis, RNA , Tandem Mass SpectrometryABSTRACT
Poly(ADP-ribose) polymerase 3 (PARP-3) is a novel member of the PARP family of enzymes that synthesize poly(ADP-ribose) on themselves and other acceptor proteins. Very little is known about this PARP, which is closely related to PARP-1 and PARP-2. By sequence analysis, we find that PARP-3 may be expressed in two isoforms which we studied in more detail to gain insight into their possible functions. We find that both PARP-3 isoforms, transiently expressed as GFP or FLAG fusions, are nuclear. Detection of endogenous PARP-3 with a specific antibody also shows a widespread nuclear distribution, appearing in numerous small foci and a small number of larger foci. Through co-localization experiments and immunoprecipitations, the larger nuclear foci were identified as Polycomb group bodies (PcG bodies) and we found that PARP-3 is part of Polycomb group protein complexes. Furthermore, using a proteomics approach, we determined that both PARP-3 isoforms are part of complexes comprising DNA-PKcs, PARP-1, DNA ligase III, DNA ligase IV, Ku70, and Ku80. Our findings suggest that PARP-3 is a nuclear protein involved in transcriptional silencing and in the cellular response to DNA damage.
Subject(s)
Cell Cycle Proteins/metabolism , DNA Damage/genetics , DNA Repair/genetics , DNA/genetics , Poly(ADP-ribose) Polymerases/metabolism , Repressor Proteins/metabolism , Amino Acid Sequence , Animals , Antigens, Nuclear/metabolism , Base Sequence , Cell Cycle Proteins/genetics , Cell Line , Chlorocebus aethiops , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Enzymologic , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Ku Autoantigen , Mass Spectrometry , Molecular Sequence Data , Poly(ADP-ribose) Polymerases/genetics , Polycomb-Group Proteins , Protein Binding , Repressor Proteins/geneticsABSTRACT
Bloom's syndrome (BS) is a rare human autosomal recessive disorder characterized by an increased risk to develop cancer of all types. BS cells are characterized by a generalized genetic instability including a high level of sister chromatid exchanges. BS arises through mutations in both alleles of the BLM gene which encodes a 3' - 5' DNA helicase identified as a member of the RecQ family. We developed polyclonal antibodies specific for the NH2- and COOH-terminal region of BLM. Using these antibodies, we analysed BLM expression during the cell cycle and showed that the BLM protein accumulates to high levels in S phase, persists in G2/M and sharply declines in G1, strongly suggestive of degradation during mitosis. The BLM protein is subject to post-translational modifications in mitosis, as revealed by slow migrating forms of BLM found in both demecolcine-treated cells and in mitotic cells isolated from non-treated asynchronous populations. Phosphatase treatment indicated that phosphorylation events were solely responsible for the appearance of the retarded moieties, a possible signal for subsequent degradation. Together, these results are consistent with a role of BLM in a replicative (S phase) and/or post-replicative (G2 phase) process. Oncogene (2000).
Subject(s)
Adenosine Triphosphatases/genetics , Bloom Syndrome/genetics , DNA Helicases/genetics , Gene Expression Regulation, Enzymologic , Proteasome Endopeptidase Complex , Adenosine Triphosphatases/metabolism , Bloom Syndrome/enzymology , Bloom Syndrome/metabolism , DNA Helicases/metabolism , Demecolcine/pharmacology , G2 Phase , HeLa Cells , Humans , Mitosis , Peptide Hydrolases/metabolism , Phosphorylation , Protease Inhibitors/pharmacology , Protein Processing, Post-Translational , RecQ Helicases , S Phase , Tumor Cells, CulturedABSTRACT
Bloom's syndrome (BS), a rare genetic disease, arises through mutations in both alleles of the BLM gene which encodes a 3'-5' DNA helicase identified as a member of the RecQ family. BS patients exhibit a high predisposition to development of all types of cancer affecting the general population and BLM-deficient cells display a strong genetic instability. We recently showed that BLM protein expression is regulated during the cell cycle, accumulating to high levels in S phase, persisting in G2/M and sharply declining in G1, suggesting a possible implication of BLM in a replication (S phase) and/or post-replication (G2 phase) process. Here we show that, in response to ionizing radiation, BLM-deficient cells exhibit a normal p53 response as well as an intact G1/S cell cycle checkpoint, which indicates that ATM and p53 pathways are functional in BS cells. We also show that the BLM defect is associated with a partial escape of cells from the gamma-irradiation-induced G2/M cell cycle checkpoint. Finally, we present data demonstrating that, in response to ionizing radiation, BLM protein is phosphorylated and accumulates through an ATM-dependent pathway. Altogether, our data indicate that BLM participates in the cellular response to ionizing radiation by acting as an ATM kinase downstream effector.
Subject(s)
Adenosine Triphosphatases/metabolism , Cell Cycle/radiation effects , DNA Helicases/metabolism , Gamma Rays , Protein Serine-Threonine Kinases/metabolism , Adenosine Triphosphatases/genetics , Ataxia Telangiectasia Mutated Proteins , Bloom Syndrome/enzymology , Bloom Syndrome/metabolism , Bloom Syndrome/pathology , Blotting, Western , Cell Cycle Proteins , Cell Line, Transformed , DNA Helicases/genetics , DNA-Binding Proteins , Flow Cytometry , G1 Phase/drug effects , G2 Phase/drug effects , Gene Deletion , Humans , Kinetics , Mitosis/drug effects , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation/radiation effects , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , RecQ Helicases , S Phase/drug effects , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor ProteinsABSTRACT
Human copper-zinc superoxide dismutase (Cu,Zn-SOD) participates in the control of reactive oxygen intermediate intracellular concentration. In this study, we show that phorbol 12-myristate 13-acetate (PMA) increases Cu,Zn-SOD mRNA expression within 30 min. The sequence between nucleotides -71 and -29 is essential for both basal and PMA-induced gene expression. This region includes an Sp1-binding site that is also recognized by a possible Sp1-like protein and by Egr-1 in a PMA-inducible manner. Egr-1 and two splicing variants of the Egr-related protein WT1 were able to transactivate the SOD1 promoter in co-transfection experiments. Sp1 and the possible Sp1-like proteins bind to two overlapping, but distinct sequences. However, Egr-1 and Sp1 seem to interact with two sites that are either identical or very close to each other. None of these sites fit the consensus sequences previously reported for these proteins. Analysis of various mutants of the SOD1 proximal promoter revealed that the region that binds Sp1 and Egr-1 is required for both basal and Egr-1-driven expression. Interplay between different members of the Sp1 family, Egr-1, and different splicing variants of WT1 in the SOD1 proximal promoter may provide clues about the physiological function of Cu,Zn-SOD.