Ubiquitin-nanoparticle interactions by solution NMR spectroscopy

The potential use of nanoparticles (NPs) in biomedical applications has attracted considerable interest inthe last years. NPs introduced in a biological environment interact with a collection of biomolecules,including proteins. NPs associating with proteins may determine changes in protein conformation, inter-fere with protein-protein interactions, and affect signal communication pathways [1]. Therefore, the study of NP-induced functional perturbations of proteins implicated in the regulation of key biochemical path-ways is particularly relevant. Ubiquitin (Ub) is a small cytosolic protein playing a central role in numerousbiological processes including protein degradation, cell signaling, and DNA repair [2]. It can be predicted that its interaction with NPs may affect cellular pathways. In this respect, we characterized, at atomic level,the interactions of Ub with two different size and chemical composition NPs. The first NP that was testedwas fullerenol, a polyhydroxylated [60]fullerene NP. These carbon based NPs have several potential bio-medical applications, including their use as drug carriers, antiviral drugs, enzyme inhibitors, contrastagents, antioxidants, and photosensitizers [3]. To characterize the fullerenol-Ub interactions, site-resolved chemical shift and intensity perturbations of Ub’s NMR signals, together with 15N spin relaxation ratechanges, were used [4]. The obtained data were consistent with interactions involving fullerenol clusters adsorbing reversibly to monomer ic Ub (and dimeric Ub), and targeting specific binding epitopes, coinci-dent with functional recognition sites of Ub. Furthermore, we observed that fullerenol almost completely abolished the formation of di-Ub and longer chains in vitro, suggesting that fullerenol NPs may effectively interfere with protein-mediated functional communication, eliciting cytotoxic effects. The second kind ofexamined NPs were SrF2 NPs. Considerable importance in biomedical lumin escence is the application ofthese NPs doped with rare lanthanide ions, due to their ability to produce up-conversion emission [5].NMR spectroscopy, up-conversion luminescence measurements and isothermal titration calorimetry wereused to probe the Ub SrF2 NP interactions. As in the case of fullerenol NPs, the analysis of NMR data indi-cated the occurrence of a reversible equilibrium between free and NP-bound protein forms. The identifica-tion of similar interaction epitopes suggests a similar impact on functional biomolecular communication.Our findings support the view that NPs may affect fundamental interaction patterns of Ub, with possiblenanotoxic consequences on cell homeostasis. On the other hand, the specific inhibition of critical Ub inter-actions through competitive binding of NPs to polyUb chains could represent a new potential opportunityfor pharmacologi cal intervention against cancer development.