Analysis of the eukaryotic topoisomerase II DNA gate: a single-molecule FRET and structural perspective

Type II DNA topoisomerases (topos) are essential and ubiquitous enzymes that perform important intracellular roles in chromosome condensation and segregation, and in regulating DNA supercoiling. Eukaryotic topo II, a type II topoisomerase, is a homodimeric enzyme that solves topological entanglement problems by using the energy from ATP hydrolysis to pass one segment of DNA through another by way of a reversible, enzyme-bridged double-stranded break. This DNA break is linked to the protein by a phosphodiester bond between the active site tyrosine of each subunit and backbone phosphate of DNA. The opening and closing of the DNA gate, a critical step for strand passage during the catalytic cycle, is coupled to this enzymatic cleavage/religation of the backbone. This reversible DNA cleavage reaction is the target of a number of anticancer drugs, which can elicit DNA damage by affecting the cleavage/religation equilibrium. Because of its clinical importance, many studies have sought to determine the manner in which topo II interacts with DNA. Here we highlight recent single-molecule fluorescence resonance energy transfer and crystallographic studies that have provided new insight into the dynamics and structure of the topo II DNA gate

Characterization of BTBD1 and BTBD2, two similar BTB-domain-containing Kelch-like proteins that interact with Topoisomerase I

BACKGROUND: Two-hybrid screening for proteins that interact with the core domain of human topoisomerase I identified two novel proteins, BTBD1 and BTBD2, which share 80% amino acid identities. RESULTS: The interactions were confirmed by co-precipitation assays demonstrating the physical interaction of BTBD1 and BTBD2 with 100 kDa topoisomerase I from HeLa cells. Deletion mapping using two-hybrid and GST-pulldown assays demonstrated that less than the C-terminal half of BTBD1 is sufficient for binding topoisomerase I. The topoisomerase I sequences sufficient to bind BTBD2 were mapped to residues 215 to 329. BTBD2 with an epitope tag localized to cytoplasmic bodies. Using truncated versions that direct BTBD2 and TOP1 to the same cellular compartment, either the nucleus or the cytoplasm, co-localization was demonstrated in co-transfected Hela cells. The supercoil relaxation and DNA cleavage activities of topoisomerase I in vitro were affected little or none by co-incubation with BTBD2. Northern analysis revealed only a single sized mRNA for each BTBD1 and BTBD2 in all human tissues tested. Characterization of BTBD2 mRNA revealed a 255 nucleotide 90% GC-rich region predicted to encode the N-terminus. BTBD1 and BTBD2 are widely if not ubiquitously expressed in human tissues, and have two paralogs as well as putative orthologs in C. elegans and D. melanogaster. CONCLUSIONS: BTBD1 and BTBD2 belong to a small family of uncharacterized proteins that appear to be specific to animals. Epitope-tagged BTBD2 localized to cytoplasmic bodies. The characterization of BTBD1 and BTBD2 and their interaction with TOP1 is underway

DNA topoisomerases I from Pseudomonas aeruginosa and vaccinia virus and their use as drug targets

Pseudomonas aeruginosa encodes a putative topoisomerase with sequence similarity to the type IB topoisomerase enzyme from vaccinia virus. Residues in the active site are conserved, notably Tyr292 which would be predicted to form the transient covalent bond to DNA. The gene encoding the P. aeruginosa topoisomerase I (PAT) was cloned and expressed in E. coli. The enzyme relaxes supercoiled DNA, while a mutant of P. aeruginosa containing a Tyr292 to Phe substitution at the active site was found to be catalytically inert. This is consistent with the role of Tyr in forming the covalent intermediate. Like vaccinia topoisomerase (VT), PAT relaxes DNA in the absence of ATP. Unlike VT, PAT does not relax supercoiled DNA without MgCl2 (or MnCl2) present. In addition, high concentration of NaCl is not able to substitute for MgCl2 as seen for VT. A truncated derivative of the topoisomerase lacking residues 1-98 relaxes DNA in the absence of the N-terminal domain, with both full length and truncated enzyme exhibiting equivalent requirements for divalent cations. Data shows that P. aeruginosa encodes a functional topoisomerase with significant similarity to the type IB enzyme encoded by poxviruses. Fluoroquinolones are antibacterial agents in clinical use with activity against DNA gyrase and DNA topoisomerase IV. P. aeruginosa is an opportunistic pathogen causing life-threatening diseases. Sparfloxacin, enrofloxacin and norfloxacin fluoroquinolones, are able to inhibit the PAT at high concentrations but other drugs belonging to this family are unable to do so. VT is ¡Ö32 KDa and one-third the size of the human topoisomerase ¡Ö 100 KDa. It shares sequence and biochemical similarities with the human topoisomerase. The VT binds duplex DNA with stringent specificity for transesterification at 5`-(C/T) CCTT site, where the 3` phosphate of the incised strand is linked to the Tyr274 of the enzyme to form a covalent cleavage complex. The fluoroquinolone enrofloxacin inhibits relaxation of supercoiled DNA by VT in a Mg2+-dependent fashion. Further results indicate that the mechanism by which enrofloxacin inhibits VT is by preventing formation of the covalent complex which suggest that fluoroquinolones may be structurally optimized to target type IB topoisomerases

A nanny model for intrinsically disordered proteins

Proteins without a well-defined tertiary structure are intrinsically unstable and are prone to degradation by the 20S proteasome. In my thesis, I investigate the protection mechanisms of intrinsically disordered (ID) protein regions via protein interactions using the AP-1 complex as a model system. AP-1 is composed of c-Fos and c-Jun proteins, out of which c-Fos has a shorter half-life than c-Jun. Interactions by c-Jun were shown to prolong the lifetime of c-Fos, leading to the proposal of the nanny model. This mechanism, where weak protein interactions protect unstructured regions without an induced folding, however, has never been probed directly. Here I investigate the nature of the interactions of c-Fos with c-Jun and how changes in disordered regions contribute to changes in half-life. I use mutational analysis to provide insight into changes in degradation rate as a function of the binding affinity in the bound form with c-Jun. I designed five mutants at the structured regions of c-Fos affecting specific contact sites (L165V, L172V) or charge separation (E175D, E189D, K190R) with c-Jun of which both modulate c-Fos turnover, proportionally to their impact on binding affinity. Interestingly, removal of the disordered region in the complex beyond the structured domain is observed to decrease c-Fos half-life indicating their role in the stability of the complex. The finding suggests that the protein turnover by the 20S proteasome can be fine-tuned by both structured and unstructured regions between c-Fos and c-Jun, consistent with the proposed 'nanny' model. These results highlight a novel aspect of disordered regions present in the bound form (fuzziness) in regulating protein half-life via fine-tuning the association rates between the two proteins. First, it demonstrates that the protection of disordered regions from degradation could be achieved without inducing a stable structure as confirmed by ECD spectroscopy. Binding to a partner generates a fuzzy complex, where fuzzy regions in protein complexes can serve as a nonspecific transient anchor. Second, the protection of disordered regions can be achieved with many binding configurations in the bound state without decreasing the conformational entropy. Thus, the protective role of fuzzy interactions from the 20S proteasome could also provide a possible explanation for how low-complexity sequence motifs involved in higher-order protein structures might serve as selective inhibitors of proteolysis.d

Synthesis of new polycyclic systems with potential antitumor activity and Angiogenic biological studies

DNA and the nucleus still represent the main target of the traditional “cytotoxic chemotherapy” for the treatment of cancer. A particular interest has been awarded to the DNA Topoisomerases (topos), essential enzymes that regulate the topological state of DNA during cellular processes such as replication, transcription, recombination, and chromatin remodeling. The ability to interfere with these enzymes or generate enzyme-mediated damage is an effective strategy for cancer therapy and DNA topos (I and II) proved to be excellent targets of clinically significant classes of anticancer drugs. In this connection, I performed the synthesis of a series of benzo- and pyrido-thiopyrano indoles, characterized by a planar mojety and pendant protonable dialkylaminoalkyl side chains, as new potential DNA intercalators and Topoisomerases inhibitors. The antiproliferative activity of the novel derivatives, as well as their ability to intercalate into the DNA and eventually inhibit the Topoisomerases were evaluated along the course of the thesis by a research group of the Faculty of Pharmacy of the University of Padua. A more recent and alternative strategy for the treatment of cancer is represented by the so called “biological therapy”, which is based on the disproportion of the signaling pathways controlling growth and differentiation, between normal and cancer cells. In particular, the molecular basis of tumour angiogenesis, the process of new vessel formation, has been extensively studied, and the Vascular Endothelial Growth Factor (VEGF) pathway has emerged as one of the most important positive modulators of this process. The founding that expression of VEGF and of its receptors correlates with the degree of vascularization of many experimental and clinical tumours led to the rational design and development of agents targeting this pathway, both selectively and with a multi-target action involving other tyrosine kinases responsible for tumour pathogenesis. In this regard I synthesized a series of benzo- and pyrido-thiopyrano-fused pyrimidines as new potential VEGFR inhibitors. Then the ability of the novel derivatives to inhibit the kinase activity of the VEGFR-2, as well as their cytotoxic effect were determined by the research group of the University of Padua. Moreover, as a part of the collaboration with the University of Valencia, I tested the anti-angiogenic activity of selected compounds in the ex-vivo “rat aortic ring assay”. My collaboration with the University of Valencia also regarded the partecipation to an extensive project on the study of the mechanism of action of Carvedilol and the Vasodilating Beta-Blockers. In particular I focused my studies on the evaluation of the relationship between adrenoceptors and angiogenic growth, through the “rat aortic ring assay”, on the analysis of the changes in the ARs gene expression induced by Carvedilol and/or by other α/β ARs agonists, by RT-PCR, and finally on the analysis of intracellular signals coupled to α/β ARs (ERK 1/2 activation), by western blotting

Regulation of botulinum toxin complex formation in <i>Clostridium botulinum</i> type A NCTC 2916

Genomic DNA fragments encoding the silent type B neurotoxin gene from Clostridium botulinum NCTC 2916 have been cloned and the complete nucleotide sequence determined. The translated sequence revealed that the gene encoded a neurotoxin which was closely related to type B neurotoxin genes from Group I Clostridium botulinum. However among the nucleotide sequence differences, aG to T transition has interrupted the coding sequence with the formation of a stop codon. In addition the deletion of an adenine residue has resulted in a frame-shift mutation. Analysis of the DNA sequence contiguous with the silent type B neurotoxin gene revealed the presence of a gene encoding a Nontoxic-Nonhaemagglutinin protein which appears to share a bicistronic mRNA transcript with the type B neurotoxin gene. In the reverse orientation, the partial sequence of a gene encoding a haemagglutinin protein was found, typical of type A and B botulinal neurotoxin complexes. Separating the genes encoding the 'components of the neurotoxin complex was a gene of 178 amino acids which possessed features commonly associated with transcriptional factors. To facilitate the in vivo study of botulinal neurotoxin complex regulation, a gene transfer system using clostridial components has been developed. The minimal replicon of the cryptic plasmid pCB 102 from Clostridium butyricum NCIB 7423 was located to 1.6 kb DNA fragment by deletion analysis, enabling the identification of hitherto undiscovered putative ORFs and secondary structures, consistent with a replicative function. The replicon has been incorporated in to a number of Escherichia coli vectors resulting in a versatile series of shuttle vectors which have demonstrated high structural and segregational stabilities in a heterologous host Clostridium beyerinckii NCI NIB 8052. Gene transfer of a Group I Clostridium botulinum type A strain was demonstrated with a representative pCB 102-derived shuttle vector, pMTL540E. In addition, a 5.9 kb plasmid indigenous to C. botulimun NCTC 2916 was cloned and the complete nucleotide sequence determined. Eight putative ORFs have been identified, including a putative replication protein and recombinase

Abstracts from the 19th FGC

Plenary and poster session abstracts from the 19th Fungal Genetics Conferenc

Cellular mechanisms that regulate the endogenous mono-ADP-ribosylation of the G protein βγ subunit.

Mono-ADP-ribosylation is a reversible, post-translational modification of cellular proteins that has been implicated in regulation of signal transduction, muscle cell differentiation, and protein trafficking and secretion. The reaction is catalysed by mono-ADP-ribosyltransferases that transfer a single ADP-ribose moiety from p-NAD+ to a specific amino-acid of acceptor proteins. An ADP-ribosylation reaction occurs in intact cells on the p subunit of heterotrimeric G proteins that is carried out by an arginine- specific, plasma-membrane-associated, mono-ADP-ribosyltransferase. This modification is reversed by a cytosolic ADP-ribosylhydrolase that regenerates native Py dimer by releasing the bound ADP-ribose. Once ADP-ribosylated, the py dimer is inactive towards its effector enzymes, such as adenylyl cyclase, phosphoinositide 3-kinase and phospholipase C. It thus appears that endogenous P subunit mono-ADP-ribosylation might represent a novel cellular mechanism for the modulation of the G-protein-mediated signal transduction machinery through a direct regulation of the py dimer. In this study, the mechanisms that regulate endogenous mono-ADP-ribosylation of the p subunit have been investigated. The reaction appears to be under hormonal control both in vitro and in vivo, since the levels of ADP-ribosylated p are increased upon activation of certain G-protein-coupled receptors (GPCRs), such as thrombin, serotonin and cholecystokinin receptors. Conversely, hormonal stimulation by additional GPCRs, such as the GnRH receptor, can lead to a decrease in p subunit mono-ADP-ribosylation. Thus, ADP-ribosylation of the py dimer can be differentially regulated by different GPCRs in a receptor-type-dependent manner. In addition, the involvement of the ADP-ribosylating factor ARF6 in GnRH-mediated regulation of p subunit mono-ADP-ribosylation is demonstrated. Indeed, removal of ARF6 from plasma membranes results in loss of GnRH-mediated inhibition of p subunit mono-ADP-ribosylation, which can be fully restored by re-addition of purified ARF6. In conclusion, the results reported in this thesis allow the definition of the mechanisms that regulated endogenous ADP-ribosylation of the p subunit, and demonstrate a novel role for ARF6 in hormonal regulation of p subunit mono-ADP-ribosylation

Experimental and Computational Analysis of the Synucleins

The synuclein proteins α, β and γ which are located in the brain, have been a subject of intense research. Of particular interest is α-synuclein, which is found in misfolded forms in Lewy bodies that are associated with Parkinson\u27s disease. Despite the efforts of researchers across the world, the physiological structure and function of the synucleins remains elusive. In recent years, highly controversial reports by some investigators indicate that in its natural form, α-synuclein exists as a tetramer instead of as an intrinsically unstructured monomer. This dissertation presents results of the experimental and computational analysis of the synucleins. First, we investigated methods of destroying protein fibrils from α-synuclein. We report that low temperature plasma can disrupt synuclein fibrils and proposed that neuronal macrophages can eliminate the resulting structures via phagocytosis. We also conducted inhibition studies to investigate the mechanism by which β-synuclein inhibits α-synuclein fibrillation. Using our experimental conditions, β-synuclein readily formed fibrils while α-synuclein inhibited β-synuclein fibrillation. We show, for the first time, that two fibril forming proteins when incubated together have an inhibitory effect on each other. This important information can be employed in the future development of inhibitors of formation. In order to determine the native structure of the synucleins, we expressed and isolated multimeric forms of the synuclein proteins. We found that expression of β-synuclein in which we did not boil the bacterial lysate. yielded a high molecular weight multimeric β-synuclein form. This study has established the basis for further research with the ultimate aim of forming a well-diffracting crystal that will lead to solving a high resolution X-ray crystallographic structure of the native state. Finally, computational approaches involving bioinformatics and molecular modeling were employed to establish a superfamily for the synucleins. The hypothesis is based on the fact that the structure and function of the synucleins could be inferred from that of their related proteins, whose structure and function have been resolved. Our computational results indicated that the synucleins seem to be orphans in the animal kingdom but share sequence similarity with an endoglucanase enzyme from Acetobacter pomorum bacterium, a CRE-DUR-1 protein from a nematode, a cytochrome c protein from a spiral bacterium and a protein from the Tasmanian Devil. This study led to the development of a proposed evolutionary model for the synucleins, which hypothesized that β-synuclein, encoded by seven exons, is the oldest of the synucleins. α-Synuclein, encoded by 6 exons, evolved to contain amino acid sequences to prevent fibril formation such as the change from threonine 53 to alanine. Together, these results further our understanding of the synuclein proteins from a myriad of experimental and computational vantage points