Laura Zuccaro

Molecular mechanism of the camptothecin resistance of Glu710Gly topoisomerase IB mutant analyzed in vitro and in silico

BackgroundDNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase IB can be inhibited by several compounds that act through different mechanisms, including clinically used drugs, such as the derivatives of the natural compound camptothecin that reversibly bind the covalent topoisomerase-DNA complex, slowing down the religation of the cleaved DNA strand, thus inducing cell death. Three enzyme mutations, which confer resistance to irinotecan in an adenocarcinoma cell line, were recently identified but the molecular mechanism of resistance was unclear.MethodsThe three resistant mutants have been investigated in S. cerevisiae model system following their viability in presence of increasing amounts of camptothecin. A systematical analysis of the different catalytic steps has been made for one of these mutants (Glu710Gly) and has been correlated with its structural-dynamical properties studied by classical molecular dynamics simulation.ResultsThe three mutants display a different degree of camptothecin resistance in a yeast cell viability assay. Characterization of the different steps of the catalytic cycle of the Glu710Gly mutant indicated that its resistance is related to a high religation rate that is hardly affected by the presence of the drug. Analysis of the dynamic properties through simulation indicate that the mutant displays a much lower degree of correlation in the motion between the different protein domains and that the linker almost completely loses its correlation with the C-terminal domain, containing the active site tyrosine.ConclusionsThese results indicate that a fully functional linker is required to confer camptothecin sensitivity to topoisomerase I since the destabilization of its structural-dynamical properties is correlated to an increase of religation rate and drug resistance.

Real-Time Label-Free Direct Electronic Monitoring of Topoisomerase Enzyme Binding Kinetics on Graphene

Monolayer graphene field-effect sensors operating in liquid have been widely deployed for detecting a range of analyte species often under equilibrium conditions. Here we report on the real-time detection of the binding kinetics of the essential human enzyme, topoisomerase I interacting with substrate molecules (DNA probes) that are immobilized electrochemically on to monolayer graphene strips. By monitoring the field-effect characteristics of the graphene biosensor in real-time during the enzyme-substrate interactions, we are able to decipher the surface binding constant for the cleavage reaction step of topoisomerase I activity in a label-free manner. Moreover, an appropriate design of the capture probes allows us to distinctly follow the cleavage step of topoisomerase I functioning in real-time down to picomolar concentrations. The presented results are promising for future rapid screening of drugs that are being evaluated for regulating enzyme activity.

Tuning the isoelectric point of graphene by electrochemical functionalization

The ability to control the charge-potential landscape at solid-liquid interfaces is pivotal to engineer novel devices for applications in sensing, catalysis and energy conversion. The isoelectric point (pI)/point of zero charge (pzc) of graphene plays a key role in a number of physico-chemical phenomena occurring at the graphene-liquid interface. Supported by theory, we present here a methodology to identify the pI/pzc of (functionalized) graphene, which also allows for estimating the nature and extent of ion adsorption. The pI of bare graphene (as-prepared, chemical vapor deposition (CVD)-grown) is found to be less than 3.3, which we can continuously modify up to 7.5 by non-covalent electrochemical attachment of aromatic amino groups, preserving the favorable electronic properties of graphene throughout. Modelling all the observed results with detailed theory, we also show that specific adsorption of ions and the substrate play only an ancillary role in our capability to tune the pI of graphene.

The human topoisomerase 1B Arg634Ala mutation results in camptothecin resistance and loss of inter-domain motion correlation

Human topoisomerase 1B, the unique target of the natural anticancer compound camptothecin, catalyzes the unwinding of supercoiled DNA by introducing transient single strand nicks and providing covalent protein-DNA adducts. The functional properties and the drug reactivity of the single Arg634Ala mutant have been investigated in comparison to the wild type enzyme. The mutant is characterized by an identical relaxation and cleavage rate but it displays resistance to camptothecin as indicated by a viability assay of the yeast cells transformed with the mutated protein. The mutant also displays a very fast religation rate that is only partially reduced by the presence of the drug, suggesting that this is the main reason for its resistance. A comparative analysis of the structural-dynamical properties of the native and mutant proteins by molecular dynamics simulation indicates that mutation of Arg634 brings to a loss of motion correlation between the different domains and in particular between the linker and the C-terminal domain, containing the catalytic tyrosine residue. These results indicate that the loss of motion correlation and the drug resistance are two strongly correlated events.

Identifying Chemical Functionalization on Individual Carbon Nanotubes and Graphene by Local Vibrational Fingerprinting

Chemical functionalization of carbon nanotubes (CNTs) and graphene allows for fine-tuning their physical and chemical properties to realize fascinating new fundamental phenomena as well as exotic applications. A primary challenge in such endeavors is the need to identify the chemical nature of attached functionalities at a single-nano-object level in a spatially resolved manner. Here we report the vibrational fingerprinting of functional groups that are attached to individual CNTs and graphene flakes. In order to achieve this, we decorate noncovalently functionalized CNTs and graphene with nanoparticles, which leads to the appearance of Raman peaks that can be correlated with the vibrational modes characteristic of the functional groups with diffraction-limited spatial resolution. The presented strategy is generic enough to be extended to other chemical modification routes on a range of nanostructures and hence will allow for rapid characterization of chemical modification of individual (semi)conducting nanostructures.

Replacement of the human topoisomerase linker domain with the plasmodial counterpart renders the enzyme camptothecin resistant.

A human/plasmodial hybrid enzyme, generated by swapping the human topoisomerase IB linker domain with the corresponding domain of the Plasmodium falciparum enzyme, has been produced and characterized. The hybrid enzyme displays a relaxation activity comparable to the human enzyme, but it is characterized by a much faster religation rate. The hybrid enzyme is also camptothecin resistant. A 3D structure of the hybrid enzyme has been built and its structural-dynamical properties have been analyzed by molecular dynamics simulation. The analysis indicates that the swapped plasmodial linker samples a conformational space much larger than the corresponding domain in the human enzyme. The large linker conformational variability is then linked to important functional properties such as an increased religation rate and a low drug reactivity, demonstrating that the linker domain has a crucial role in the modulation of the topoisomerase IB activity.

Rolling circle amplification-based detection of human topoisomerase I activity on magnetic beads.

A high-sensitivity assay has been developed for the detection of human topoisomerase I with single molecule resolution. The method uses magnetic sepharose beads to concentrate rolling circle products, produced by the amplification of DNA molecules circularized by topoisomerase I and detectable with a confocal microscope as single and discrete dots, once reacted with fluorescent probes. Each dot, corresponding to a single cleavage-religation event mediated by the enzyme, can be counted due to its high signal/noise ratio, allowing detection of 0.3pM enzyme and representing a valid method to detect the enzyme activity in highly diluted samples.

Mutations of human DNA topoisomerase I at poly (ADP-ribose) binding sites: modulation of camptothecin activity by ADP-ribose polymers

BackgroundDNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase I belongs to the family of poly(ADP-ribose)-binding proteins and is the target of camptothecin derived anticancer drugs. Poly(ADP-ribosyl)ation occurs at specific sites of the enzyme inhibiting the cleavage and enhancing the religation steps during the catalytic cycle. Thus, ADP-ribose polymers antagonize the activity of topoisomerase I poisons, whereas PARP inhibitors increase their antitumor effects.MethodsUsing site-directed mutagenesis we have analyzed the interaction of human topoisomerase I and poly(ADP-ribose) through enzymatic activity and binding procedures.ResultsMutations of the human topoisomerase I hydrophobic or charged residues, located on the putative polymer binding sites, are not sufficient to abolish or reduce the binding of the poly(ADP-ribose) to the protein. These results suggest either the presence of additional binding sites or that the mutations are not enough perturbative to destroy the poly(ADP-ribose) interaction, although in one mutant they fully abolish the enzyme activity.ConclusionsIt can be concluded that mutations at the hydrophobic or charged residues of the putative polymer binding sites do not interfere with the ability of poly(ADP-ribose) to antagonize the antitumor activity of topoisomerase I poisons.

A small organic compound enhances the religation reaction of human topoisomerase I and identifies crucial elements for the religation mechanism

The different steps of the human Top1 (topoisomerase I) catalytic cycle have been analysed in the presence of a pentacyclic-diquinoid synthetic compound. The experiments indicate that it efficiently inhibits the cleavage step of the enzyme reaction, fitting well into the catalytic site. Surprisingly the compound, when incubated with the binary topoisomerase–DNA cleaved complex, helps the enzyme to remove itself from the cleaved DNA and close the DNA gap, increasing the religation rate. The compound also induces the religation of the stalled enzyme–CPT (camptothecin)–DNA ternary complex. Analysis of the molecule docked over the binary complex, together with its chemical properties, suggests that the religation enhancement is due to the presence on the compound of two oxygen atoms that act as hydrogen acceptors. This property facilitates the deprotonation of the 5′ DNA end, suggesting that this is the limiting step in the topoisomerase religation mechanism.

Binding Kinetics of Methylene Blue on Monolayer Graphene Investigated by Multiparameter Surface Plasmon Resonance

In this paper, we study the interaction of a small dye molecule, namely, methylene blue (MB) with graphene surfaces using surface plasmon resonance (SPR). We show that by utilizing all of the parameters of the SPR angular dip and exploiting the fact that MB absorbs light at the operating wavelength, it is possible to detect the binding of small molecules that would otherwise not give a significant signal. The binding of MB to unmodified graphene is found to be stronger than that for gold. By studying the interaction at modified surfaces, we demonstrate that electrostatic effects play a dominant role in the binding of MB on to graphene. Furthermore, following the binding kinetics at various concentrations allows us to estimate apparent equilibrium binding and rate constants for the interaction of MB with graphene.