Nucleocytoplasmic transport
Traffic into and out of the nucleus in interphase eukaryotic cells occurs through huge protein assemblies called nuclear pore complexes (NPCs). Small molecules can pass through NPCs by passive diffusion. The passage of most proteins and protein-RNA complexes, however, depends on dedicated transport receptors that recognize their cargo through short peptide sequences that mark it for nuclear import or export. The interaction between the nuclear transport receptors and the import/export signals is controlled by the Ran system, which ensures that the cargo is loaded and released in the appropriate cellular compartment.
Our research focused on the structure of the NPC, its ability to discriminate between tagged and non-tagged macromolecular cargo, and its ability to the support passive diffusion of small molecules along with facilitated, receptor-mediated macromolecular transport. In addition, we constructed a model explaining how NPCs are able to maintain extensive bi-directional fluxes of macromolecules and macromolecular complexes without experiencing debilitating traffic jams.
Protein folding and binding
Understanding how proteins fold and interact with other macromolecules or small molecules are central objectives in structural biology.
Most of our studies were carried out using the single-molecule manipulation technique - dynamic force spectroscopy. We initially focused on the interactions of the prototypic nuclear transport receptor importin ß1 with the GDP- and GTP-bound forms of Ran and on the modulation of these interactions by Ran’s effector protein, RanBP1. We then used the importin ß1-Ran(GTP) complex as a model to measure, for the first time, the free energy landscape roughness of protein-related reactions. Roughness is a characteristic property of protein energy landscapes and has important effects on the folding and binding of proteins as well as on their behaviour at equilibrium.
In later studies we concentrated on protein folding/unfolding. Combining dynamic force spectroscopy with coarse-grained- and all-atom steered molecular dynamics simulations we were able to delineate the mechanical unfolding pathway of the small enzyme acylphosphatase (AcP) at single secondary structure resolution along with with the antagonistic effect ligand binding has on this process. Notably, we observed the opposite effect, i.e., facilitation of forced unfolding upon binding of ligand, for the interferon receptor subunit IFNAR1. This is the first experimental demonstration that ligand binding lends a protein more easily mechanically unfolded. Lastly, in a study combining conventional bulk experimental methods with molecular dynamics simulations we showed that the thermodynamic stability of a protein can be enhanced by modifications that increase its folded state entropy
Facilitated and selective nuclear import of DNA
Efficient nuclear import of DNA is a prerequisite for the success of therapeutic approaches such as gene therapy and DNA vaccination, which rely on gene transfer. The problem we addressed concerns the transfer efficacy of DNA molecules delivered into the cells by synthetic (non-viral) vectors. Such carriers provide attractive alternative to viral-based delivery systems because they are safer, have a larger DNA packing capacity, and are simpler to produce. However, their transfection efficiency is poor, largely because of the inability of the DNA to passively diffuse in the cytoplasm and across the NPC.
We have developed a method that facilitates the transport of the DNA through the cytoplasm and enables it to translocate through the NPC into the nucleus. It is based on the modification of the DNA by binding sites for endogenous DNA-binding proteins that, by virtue of their function, carry one or more nuclear localization signals. These proteins then bind the modified DNA inside the cytoplasm and carry it into the nucleus along existing nuclear protein import pathways. This method provides a safe and cost-effective way for the delivery of DNA into the nucleus of any cell population.
An important advantage of the above piggyback transfer approach is that it allows for therapeutic selectivity by choosing transport mediators that are tissue-specific or over-expressed in diseased states. We have constructed DNA vectors that contain multiple binding sites for members of the rapid-response transcription factor family nuclear factor ϰB (NFkappaB). Abnormal NFkappaB activity is implicated in a number of autoimmune and malignant disorders. In many of these disorders, NFkappaB malfunction is due to a failure in the regulation of their induction and, consequently, of their cytoplasmic retention. This results in constitutive shuttling of the proteins to the nucleus regardless of their activation state. Nuclear import of DNA modified with NFkappaB binding sites should thus be greatly facilitated in diseased cells compared to cells with normal NFkappaB activity. Exploiting this, we hope to achieve therapeutic specificity that can be subsequently utilized for suicide gene therapy of certain B-cell lymphomas and leukaemias as well as some breast cancers.