Advances in the detection at the new XMCD and RIXS beamline of the ESRF

LAUREA MAGISTRALEThe European Synchrotron Radiation Facility is a synchrotron radiation source offering forefront experimental instrumentation for the investigation of the magnetic properties and the electronic structure of materials. Within a general upgrade programme of the facility, the new ID32 soft X-ray beamline has been built, offering two experimental end-stations: one for RIXS and the other for XMCD measurements. ERIXS, the new spectrometer of the RIXS branch, is designed to achieve an unprecedented resolving power, requiring the optimization of all its components. In the present work we explore the possibility of enhancing the spatial resolution of the CCD detector of the spectrometer, which can be done by calculating the centre of mass of the charge clouds generated by each photon. This approach introduces some artefacts in the reconstructed images that we were able to remove by using the so-called eta-algorithm and one of its possible extensions. In the meantime we carried out the very first experiment of the XMCD branch of ID32, studying the temperature dependence of weak ferromagnetism in samples of doped high-Tc superconducting cuprates

Optimized reaction coordinates for analysis of enhanced sampling

Atomistic simulations of biological processes offer insights at a high level of spatial and temporal resolution, but accelerated sampling is often required for probing timescales of biologically relevant processes. The resulting data need to be statistically reweighted and condensed in a concise yet faithful manner to facilitate interpretation. Here, we provide evidence that a recently proposed approach for the unsupervised determination of optimized reaction coordinate (RC) can be used for both analysis and reweighting of such data. We first show that for a peptide interconverting between helical and collapsed configurations, the optimal RC permits efficient reconstruction of equilibrium properties from enhanced sampling trajectories. Upon RC-reweighting, kinetic rate constants and free energy profiles are in good agreement with values obtained from equilibrium simulations. In a more challenging test, we apply the method to enhanced sampling simulations of the unbinding of an acetylated lysine-containing tripeptide from the bromodomain of ATAD2. The complexity of this system allows us to investigate the strengths and limitations of these RCs. Overall, the findings presented here underline the potential of the unsupervised determination of reaction coordinates and the synergy with orthogonal analysis methods, such as Markov state models and SAPPHIRE analysis

Molecular dynamics analysis of the structural properties of the transglutaminases of Kutzneria albida and Streptomyces mobaraensis

The microbial transglutaminase (TGase) from Streptomyces mobaraensis (MTGase) is widely used for industrial applications. However, in the last decades, TGases from other bacteria have been described. We focused our attention on TGase, from Kutzneria albida (KalbTGase), recently characterized as more selective than MTGase and proposed for applications in drug delivery. By comparison of the crystallographic structures, the volume of the catalytic site results smaller in KalbTGase. We compared KalbTGase and MTGase structural flexibility by molecular dynamics (MD) simulations at different conditions. KalbTGase is more rigid than MTGase at 300 K, but the catalytic site has a preserved conformation in both structures. Preliminary studies at higher temperatures suggest that KalbTGase acquires enhanced conformational flexibility far from the active site region. The volume of the catalytic active site pocket of KalbTGase at room temperature is smaller than that of MTGase, and decreases at 335 K, remaining stable after further temperature increase. On the contrary, in MTGase the pocket volume continues to decrease as the temperature increases. Overall, the results of our study suggest that at room temperature the enhanced specificity of KalbTGase could be related to a more closed catalytic pocket and lower flexibility than MTGase. Moreover, by preliminary results at higher temperature, KalbTGase structural flexibility suggests an adaptability to different substrates not recognized at room temperature. Lower adaptability of MTGase at higher temperature with a reduction of the catalytic pocket, instead, suggests a reduction of its activity

Sputum Proteomics reveals unique signatures linked to key outcomes in cystic fibrosis trials

Rationale Lung function (FEV1) and quality of life (QoL) are key outcomes in most interventional clinical trials conducted in people living with cystic fibrosis. However, no robust pre-clinical surrogates for FEV1 and QoL exist. The precise physiological mechanisms leading to treatment-related improvements in these outcomes are incompletely understood. In this post-hoc analysis we explored the relationship between changes in the sputum proteome and these outcomes with the aim of identifying translational biomarkers.Methods Paired sputum samples collected during the AZTEC-CF study (NCT02894684) pre and post 14 days of antibiotic treatment for an acute pulmonary exacerbation were included. Samples were analysed using in vitro Mesoscale Discovery (MSD) assays and by nano LC-MS/MS. Peptide identification and quantification was performed and the log-fold change for individual proteins and relationships between protein change and changes in FEV1 and QoL were evaluated.Results Distinct patterns were found between proteins that correlated with FEV1 and those that correlated with QoL improvements. FEV1 improvement was characterised by increases in bacterially-derived proteins accompanied by decreases in proteins relating to neutrophil degranulation. Conversely, changes in QoL were associated with increases in antiprotease and antioxidant proteins. MSD analysis revealed changes in some neutrophil-associated markers significantly correlated with FEV1 improvements, but no markers significantly correlated with QoL improvements.Conclusions These results suggest changes in two key CF clinical trial outcomes (FEV1 and QoL) may be underpinned by different physiological mechanisms. Understanding these divergent mechanisms is vital to fortify optimal clinical trial design in CF and panels of biomarkers may be needed to improve translational confidence.<br/

Structures of ferroportin in complex with its specific inhibitor vamifeport

A central regulatory mechanism of iron homeostasis in humans involves ferroportin (FPN), the sole cellular iron exporter, and the peptide hormone hepcidin, which inhibits Fe$^{2+}$ transport and induces internalization and degradation of FPN. Dysregulation of the FPN/hepcidin axis leads to diverse pathological conditions, and consequently, pharmacological compounds that inhibit FPN-mediated iron transport are of high clinical interest. Here, we describe the cryo-electron microscopy structures of human FPN in complex with synthetic nanobodies and vamifeport (VIT-2763), the first clinical-stage oral FPN inhibitor. Vamifeport competes with hepcidin for FPN binding and is currently in clinical development for β-thalassemia and sickle cell disease. The structures display two distinct conformations of FPN, representing outward-facing and occluded states of the transporter. The vamifeport site is located in the center of the protein, where the overlap with hepcidin interactions underlies the competitive relationship between the two molecules. The introduction of point mutations in the binding pocket of vamifeport reduces its affinity to FPN, emphasizing the relevance of the structural data. Together, our study reveals conformational rearrangements of FPN that are of potential relevance for transport, and it provides initial insight into the pharmacological targeting of this unique iron efflux transporter

Primary cilia contribute to the aggressiveness of atypical teratoid/rhabdoid tumors

Atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant brain tumor in infants that is characterized by loss of nuclear expression of SMARCB1 or SMARCA4 proteins. Recent studies show that AT/RTs comprise three molecular subgroups, namely AT/RT-TYR, AT/RT-MYC and AT/RT-SHH. The subgroups show distinct expression patterns of genes involved in ciliogenesis, however, little is known about the functional roles of primary cilia in the biology of AT/RT. Here, we show that primary cilia are present across all AT/RT subgroups with specific enrichment in AT/RT-TYR patient samples. Furthermore, we demonstrate that primary ciliogenesis contributes to AT/RT biology in vitro and in vivo. Specifically, we observed a significant decrease in proliferation and clonogenicity following disruption of primary ciliogenesis in AT/RT cell line models. Additionally, apoptosis was significantly increased via the induction of STAT1 and DR5 signaling, as detected by proteogenomic profiling. In a Drosophila model of SMARCB1 deficiency, concomitant knockdown of several cilia-associated genes resulted in a substantial shift of the lethal phenotype with more than 20% of flies reaching adulthood. We also found significantly extended survival in an orthotopic xenograft mouse model of AT/RT upon disruption of primary ciliogenesis. Taken together, our findings indicate that primary ciliogenesis or its downstream signaling contributes to the aggressiveness of AT/RT and, therefore, may constitute a novel therapeutic target

Allosteric Antagonist Modulation of TRPV2 by Piperlongumine Impairs Glioblastoma Progression.

The use of computational tools to identify biological targets of natural products with anticancer properties and unknown modes of action is gaining momentum. We employed self-organizing maps to deconvolute the phenotypic effects of piperlongumine (PL) and establish a link to modulation of the human transient receptor potential vanilloid 2 (hTRPV2) channel. The structure of the PL-bound full-length rat TRPV2 channel was determined by cryo-EM. PL binds to a transient allosteric pocket responsible for a new mode of anticancer activity against glioblastoma (GBM) in which hTRPV2 is overexpressed. Calcium imaging experiments revealed the importance of Arg539 and Thr522 residues on the antagonistic effect of PL and calcium influx modulation of the TRPV2 channel. Downregulation of hTRPV2 reduces sensitivity to PL and decreases ROS production. Analysis of GBM patient samples associates hTRPV2 overexpression with tumor grade, disease progression, and poor prognosis. Extensive tumor abrogation and long term survival was achieved in two murine models of orthotopic GBM by formulating PL in an implantable scaffold/hydrogel for sustained local therapy. Furthermore, in primary tumor samples derived from GBM patients, we observed a selective reduction of malignant cells in response to PL ex vivo. Our results establish a broadly applicable strategy, leveraging data-motivated research hypotheses for the discovery of novel means tackling cancer