The stability and activity of human neuroserpin are modulated by a salt bridge that stabilises the reactive centre loop

Neuroserpin (NS) is an inhibitory protein belonging to the serpin family and involved in several pathologies, including the dementia Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), a genetic neurodegenerative disease caused by accumulation of NS polymers. Our Molecular Dynamics simulations revealed the formation of a persistent salt bridge between Glu289 on strand s2C and Arg362 on the Reactive Centre Loop (RCL), a region important for the inhibitory activity of NS. Here, we validated this structural feature by simulating the Glu289Ala mutant, where the salt bridge is not present. Further, MD predictions were tested in vitro by purifying recombinant Glu289Ala NS from E. coli. The thermal and chemical stability along with the polymerisation propensity of both Wild Type and Glu289Ala NS were characterised by circular dichroism, emission spectroscopy and non-denaturant gel electrophoresis, respectively. The activity of both variants against the main target protease, tissue-type plasminogen activator (tPA), was assessed by SDS-PAGE and chromogenic kinetic assay. Our results showed that deletion of the salt bridge leads to a moderate but clear reduction of the overall protein stability and activity

Wildtype and A30P mutant alpha-synuclein form different fibril structures

Parkinson's Disease (PD) is a neurodegenerative movement disorder affecting millions of people worldwide. One of the key players in the development of the disease is the protein α-synuclein (aSN), which aggregates in the brain of PD patients. The aSN mutant A30P has been reported to cause early-onset familial PD and shows different aggregation behavior compared to wt aSN. Here we use a multidisciplinary approach to compare the aggregation process of wt and A30P aSN. In agreement with previous studies, we observe an initial lag phase followed by a continuous structural development of fibrils until reaching an apparent monomer-aggregate equilibrium state and a plateau in Thioflavin T (ThT) fluorescence intensity. However, at later timepoints A30P shows greater propensity than αSN wt to form dense bundled fibril networks. Combining small angle x-ray scattering, x-ray fibre diffraction and linear dichroism, we demonstrate that while the microscopic structure of the individual fibril essentially remains constant throughout the experiment, the formation of dense A30P fibril networks occur through a continuous assembly pathway while the formation of less dense wt fibril networks with fewer contact points follows a continuous path during the elongation phase and a second rearrangement phase after reaching the ThT fluorescence plateau. Our work thus highlights that structural rearrangements proceed beyond the plateau in ThT-based monitoring of the fibrillation process, and the density and morphology of the resulting fibril networks is highly dependent on the aSN form studied

Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation

Bone disease is the most frequent complication in multiple myeloma (MM) resulting in osteolytic lesions, bone pain, hypercalcemia and renal failure. In MM bone disease the perfect balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) activity is lost in favour of OCs, thus resulting in skeletal disorders. Since exosomes have been described for their functional role in cancer progression, we here investigate whether MM cell-derived exosomes may be involved in OCs differentiation. We show that MM cells produce exosomes which are actively internalized by Raw264.7 cell line, a cellular model of osteoclast formation. MM cell-derived exosomes positively modulate pre-osteoclast migration, through the increasing of CXCR4 expression and trigger a survival pathway. MM cell-derived exosomes play a significant pro-differentiative role in murine Raw264.7 cells and human primary osteoclasts, inducing the expression of osteoclast markers such as Cathepsin K (CTSK), Matrix Metalloproteinases 9 (MMP9) and Tartrate-resistant Acid Phosphatase (TRAP). Pre-osteoclast treated with MM cell-derived exosomes differentiate in multinuclear OCs able to excavate authentic resorption lacunae. Similar results were obtained with exosomes derived from MM patient's sera. Our data indicate that MM-exosomes modulate OCs function and differentiation. Further studies are needed to identify the OCs activating factors transported by MM cell-derived exosomes

Proof-of-Concept Study on the Use of Tangerine-Derived Nanovesicles as siRNA Delivery Vehicles toward Colorectal Cancer Cell Line SW480

In the last years, the field of nanomedicine and drug delivery has grown exponentially, providing new platforms to carry therapeutic agents into the target sites. Extracellular vesicles (EVs) are ready-to-use, biocompatible, and non-toxic nanoparticles that are revolutionizing the field of drug delivery. EVs are involved in cell-cell communication and mediate many physiological and pathological processes by transferring their bioactive cargo to target cells. Recently, nanovesicles from plants (PDNVs) are raising the interest of the scientific community due to their high yield and biocompatibility. This study aims to evaluate whether PDNVs may be used as drug delivery systems. We isolated and characterized nanovesicles from tangerine juice (TNVs) that were comparable to mammalian EVs in size and morphology. TNVs carry the traditional EV marker HSP70 and, as demonstrated by metabolomic analysis, contain flavonoids, organic acids, and limonoids. TNVs were loaded with DDHD1-siRNA through electroporation, obtaining a loading efficiency of 13%. We found that the DDHD1-siRNA complex TNVs were able to deliver DDHD1-siRNA to human colorectal cancer cells, inhibiting the target expression by about 60%. This study represents a proof of concept for the use of PDNVs as vehicles of RNA interference (RNAi) toward mammalian cells