Enforcing the positive charge of N-termini enhances membrane interaction and antitumor activity of bovine seminal ribonuclease

AbstractBinding to cell membrane, followed by translocation into the cytosol and RNA degradation, is a necessary requirement to convert a ribonuclease into a cytotoxin for malignant tumor cells. In this paper, we investigate the membrane binding attitude of bovine seminal ribonuclease (BS-RNase) and its variant G38K-BS-RNase, bearing an enforced cluster of positive charges at the N-termini surface. By using a combination of biophysical techniques, including CD, SPR and ESR, we find for the two proteins a common, two-step mechanism of interaction with synthetic liposomes, an initial binding to the bilayer surface, driven by electrostatic interactions, followed by a shallow penetration in the lipid core. Protein binding effectively perturbs lipid packing and dynamics. Remarkably, the higher G38K-BS-RNase membrane interacting capability well correlates with its increased cytotoxicity for tumor cells. Overall, these studies shed light on the mechanism of membrane binding and perturbation, proving definitely the importance of electrostatic interactions in the cytotoxic activity of BS-RNase, and provide a rational basis to design proteins with anticancer potential

Improving the quality of patient care in lung cancer: key factors for successful multidisciplinary team working

International Guidelines as well as Cancer Associations recommend a multidisciplinary approach to lung cancer care. A multidisciplinary team (MDT) can significantly improve treatment decision-making and patient coordination by putting different physicians and other health professionals “in the same room”, who collectively decide upon the best possible treatment. However, this is not a panacea for cancer treatment. The impact of multidisciplinary care (MDC) on patient outcomes is not univocal, while the effective functioning of the MDT depends on many factors. This review presents the available MDT literature with an emphasis on the key factors that characterize high-quality patient care in lung cancer. The study was conducted with a bibliographic search using different electronic databases (PubMed Central, Scopus, Google Scholar, and Google) referring to multidisciplinary cancer care settings. Many key elements appear consolidated, while others emerge as prevalent and actual, especially those related to visible barriers which work across geographic, organizational, and disciplinary boundaries. MDTs must be sustained by strategic management, structured within the entity, and cannot be managed as a separate care process. Furthermore, they need to coordinate with other teams (within and outside the organization) and join with the broad range of services delivered by multiple providers at various points of the cancer journey or within the system, with the vision of integrated care

NMR Studies on Structure and Dynamics of the Monomeric Derivative of BS-RNase: New Insights for 3D Domain Swapping

Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the aggregated state, these proteins acquire new biological functions, including selective cytotoxicity against tumour cells. RNase A is able to dislocate both N- and C-termini, but usually this process requires denaturing conditions. In contrast, bovine seminal ribonuclease (BS-RNase), which is a homo-dimeric protein sharing 80% of sequence identity with RNase A, occurs natively as a mixture of swapped and unswapped isoforms. The presence of two disulfides bridging the subunits, indeed, ensures a dimeric structure also to the unswapped molecule. In vitro, the two BS-RNase isoforms interconvert under physiological conditions. Since the tendency to swap is often related to the instability of the monomeric proteins, in these paper we have analysed in detail the stability in solution of the monomeric derivative of BS-RNase (mBS) by a combination of NMR studies and Molecular Dynamics Simulations. The refinement of NMR structure and relaxation data indicate a close similarity with RNase A, without any evidence of aggregation or partial opening. The high compactness of mBS structure is confirmed also by H/D exchange, urea denaturation, and TEMPOL mapping of the protein surface. The present extensive structural and dynamic investigation of (monomeric) mBS did not show any experimental evidence that could explain the known differences in swapping between BS-RNase and RNase A. Hence, we conclude that the swapping in BS-RNase must be influenced by the distinct features of the dimers, suggesting a prominent role for the interchain disulfide bridges

Design of more potent antitumor ribonucleases: a cluster of positive charges at N-termini enhances the cytotoxicity of bovine seminal ribonuclease via a membrane-mediated mechanism

The possible use of ribonucleases in the treatment of diseases, including cancer, has received a considerable interest in the last years. The necessary requirements that convert a ribonuclease into a cytotoxin for malignant tumour cells include binding to cell membrane, translocation into the cytosol and RNA degradation. Among the mammalian ribonucleases, only bovine seminal ribonuclease (BS-RNase) fulfils all these requirements (1). We have designed a BS_RNase variant with an enforced cluster of positive charges at the N-termini surface to improve its cytotoxic potential according to literature data indicating that the protein approaches the membrane with a preferred orientation (2). We found that the G38K-BS-RNase variant displays an increased citotoxicity for tumour cells and we hypothesized the higher cytotoxicity to be connected to its enhanced interaction with membranes. A detailed investigation of the membrane interaction mechanism of BS-RNase and G38K-BS-RNase, using a combination of biophysical techniques, including Surface Plasmon Resonance and Electron Spin Resonance, supports this idea, and indicates for both of them a common two-step mechanism, based on an initial binding to the bilayer surface followed by a shallow penetration in the lipid core. Protein binding , driven by electrostatic interactions, effectively perturbs lipid packing and dynamics. Our results, while ruling out the hypothesis that the protein crosses the membrane interior, support the hypothesis of a BS-RNase entry mediated by an endocytosis mechanism. Overall, these studies shed light on the mechanism of ribonuclease membrane binding and perturbation, and provide a rational basis to design proteins with higher antitumor potential