A homology model of restriction endonuclease SfiI in complex with DNA

BACKGROUND: Restriction enzymes (REases) are commercial reagents commonly used in recombinant DNA technologies. They are attractive models for studying protein-DNA interactions and valuable targets for protein engineering. They are, however, extremely divergent: the amino acid sequence of a typical REase usually shows no detectable similarities to any other proteins, with rare exceptions of other REases that recognize identical or very similar sequences. From structural analyses and bioinformatics studies it has been learned that some REases belong to at least four unrelated and structurally distinct superfamilies of nucleases, PD-DxK, PLD, HNH, and GIY-YIG. Hence, they are extremely hard targets for structure prediction and homology-based inference of sequence-function relationships and the great majority of REases remain structurally and evolutionarily unclassified. RESULTS: SfiI is a REase which recognizes the interrupted palindromic sequence 5'GGCCNNNN^NGGCC3' and generates 3 nt long 3' overhangs upon cleavage. SfiI is an archetypal Type IIF enzyme, which functions as a tetramer and cleaves two copies of the recognition site in a concerted manner. Its sequence shows no similarity to other proteins and nothing is known about the localization of its active site or residues important for oligomerization. Using the threading approach for protein fold-recognition, we identified a remote relationship between SfiI and BglI, a dimeric Type IIP restriction enzyme from the PD-DxK superfamily of nucleases, which recognizes the 5'GCCNNNN^NGGC3' sequence and whose structure in complex with the substrate DNA is available. We constructed a homology model of SfiI in complex with its target sequence and used it to predict residues important for dimerization, tetramerization, DNA binding and catalysis. CONCLUSIONS: The bioinformatics analysis suggest that SfiI, a Type IIF enzyme, is more closely related to BglI, an "orthodox" Type IIP restriction enzyme, than to any other REase, including other Type IIF REases with known structures, such as NgoMIV. NgoMIV and BglI belong to two different, very remotely related branches of the PD-DxK superfamily: the α-class (EcoRI-like), and the β-class (EcoRV-like), respectively. Thus, our analysis provides evidence that the ability to tetramerize and cut the two DNA sequences in a concerted manner was developed independently at least two times in the evolution of the PD-DxK superfamily of REases. The model of SfiI will also serve as a convenient platform for further experimental analyses

Tau inhibits tubulin oligomerization induced by prion protein

AbstractIn previous studies we have demonstrated that prion protein (PrP) interacts with tubulin and disrupts microtubular cytoskeleton by inducing tubulin oligomerization. These observations may explain the molecular mechanism of toxicity of cytoplasmic PrP in transmissible spongiform encephalopathies (TSEs). Here, we check whether microtubule associated proteins (MAPs) that regulate microtubule stability, influence the PrP-induced oligomerization of tubulin. We show that tubulin preparations depleted of MAPs are more prone to oligomerization by PrP than those containing traces of MAPs. Tau protein, a major neuronal member of the MAPs family, reduces the effect of PrP. Importantly, phosphorylation of Tau abolishes its ability to affect the PrP-induced oligomerization of tubulin. We propose that the binding of Tau stabilizes tubulin in a conformation less susceptible to oligomerization by PrP. Since elevated phosphorylation of Tau leading to a loss of its function is observed in Alzheimer disease and related tauopathies, our results point at a possible molecular link between these neurodegenerative disorders and TSEs

Increased acetylation of lysine 317/320 of p53 caused by BCR-ABL protects from cytoplasmic translocation of p53 and mitochondria-dependent apoptosis in response to DNA damage

Chronic myeloid leukemia (CML) is a disorder of hematopoietic stem cells caused by the expression of BCR-ABL. Loss of p53 has not been implicated as important for the development of CML. Mutations in p53 protein are infrequent, however they correlate with the disease progression. The absence of p53 mutations does not exclude the possibility that other dysfunctions play an important role in CML pathology. Acetylation represents a very potent posttranslational mechanism regulating p53 stability, transcriptional activity and localization. In this study we have investigated whether the expression of BCR-ABL could influence the acetylation of p53, specifically at lysine 317/320 (K317/K320), which has been shown to regulate nuclear export and transcription-independent apoptotic activity of p53. We found that BCR-ABL expression increases K317 acetylation of p53 and is able to prevent a drop in acetylation observed upon DNA damage, followed by translocation of p53 to the cytoplasm and by Bax activation. We have shown that this site plays a crucial role in the regulation of p53 localization and p53-dependent, Bax-mediated apoptosis. Our study presents a novel BCR-ABL-dependent mechanism protecting from DNA-damage-induced cell death. It can, in addition to already known mechanisms, explain the resistance to p53-dependent apoptosis observed in CML cells expressing wt p53. We propose that the acetyltransferases regulating the p53 acetylation could be an interesting and potent target for therapeutic intervention

Increased acetylation of lysine 317/320 of p53 caused by BCR-ABL protects from cytoplasmic translocation of p53 and mitochondria-dependent apoptosis in response to DNA damage

Chronic myeloid leukemia (CML) is a disorder of hematopoietic stem cells caused by the expression of BCR-ABL. Loss of p53 has not been implicated as important for the development of CML. Mutations in p53 protein are infrequent, however they correlate with the disease progression. The absence of p53 mutations does not exclude the possibility that other dysfunctions play an important role in CML pathology. Acetylation represents a very potent posttranslational mechanism regulating p53 stability, transcriptional activity and localization. In this study we have investigated whether the expression of BCR-ABL could influence the acetylation of p53, specifically at lysine 317/320 (K317/K320), which has been shown to regulate nuclear export and transcription-independent apoptotic activity of p53. We found that BCR-ABL expression increases K317 acetylation of p53 and is able to prevent a drop in acetylation observed upon DNA damage, followed by translocation of p53 to the cytoplasm and by Bax activation. We have shown that this site plays a crucial role in the regulation of p53 localization and p53-dependent, Bax-mediated apoptosis. Our study presents a novel BCR-ABL-dependent mechanism protecting from DNA-damage-induced cell death. It can, in addition to already known mechanisms, explain the resistance to p53-dependent apoptosis observed in CML cells expressing wt p53. We propose that the acetyltransferases regulating the p53 acetylation could be an interesting and potent target for therapeutic intervention