The unusual properties of lactoferrin during its nascent phase

Lactoferrin, a multifunctional iron-binding protein containing 16 disulfides, is actively studied for its antibacterial and anti-carcinogenic properties. However, scarce information is nowadays available about its oxidative folding starting from the reduced and unfolded status. This study discovers unusual properties when this protein is examined in its reduced molten globule-like conformation. Using kinetic, CD and fluorescence analyses together with mass spectrometry, we found that a few cysteines display astonishing hyper-reactivity toward different thiol reagents. In details, four cysteines (i.e. 668, 64, 512 and 424) display thousands of times higher reactivity toward GSSG but normal against other natural disulfides. The formation of these four mixed-disulfides with glutathione probably represents the first step of its folding in vivo. A widespread low pKa decreases the reactivity of other 14 cysteines toward GSSG limiting their involvement in the early phase of the oxidative folding. The origin of this hyper-reactivity was due to transient lactoferrin-GSSG complex, as supported by fluorescence experiments. Lactoferrin represents another disulfide containing protein in addition to albumin, lysozyme, ribonuclease, chymotrypsinogen, and trypsinogen which shows cysteines with an extraordinary and specific hyper-reactivity toward GSSG confirming the discovery of a fascinating new feature of proteins in their nascent phase

The role of autophagy in resistance to targeted therapies

Autophagy is a self-degradative cellular process, involved in stress response such as starvation, hypoxia, and oxidative stress. This mechanism balances macro-molecule recycling to regulate cell homeostasis. In cancer, autophagy play a role in the development and progression, while several studies describe it as one of the key processes in drug resistance. In the last years, in addition to standard anti-cancer treatments such as chemotherapies and irradiation, targeted therapy became one of the most adopted strategies in clinical practices, mainly due to high specificity and reduced side effects. However, similar to standard treatments, drug resistance is the main challenge in most patients. Here, we summarize recent studies that investigated the role of autophagy in drug resistance after targeted therapy in different types of cancers. We highlight positive results and limitations of pre-clinical and clinical studies in which autophagy inhibitors are used in combination with targeted therapies. Refereed/Peer-reviewe

Lipoaspirate fluid proteome: A preliminary investigation by LC-MS top-down/bottom-up integrated platform of a high potential biofluid in regenerative medicine.

The lipoaspirate fluid (LAF) has recently emerged as a potentially valuable source in regenerative medicine. In particular, our group recently demonstrated that it is able to exert osteoinductive properties in vitro. This original observation stimulated the investigation of the proteomic component of LAF, by means of LC-ESI-LTQ-Orbitrap-MS top-down/bottom-up integrated approach, which represents the object of the present study. Top-down analyses required the optimization of sample pretreatment procedures to enable the correct investigation of the intact proteome. Bottom-up analyses have been directly applied to untreated samples after monodimensional SDS-PAGE separation. The analysis of the acid-soluble fraction of LAF by top-down approach allowed demonstrating the presence of albumin and haemoglobin fragments (i.e. VV- and LVV-hemorphin-7), thymosins β4 and β10 peptides, ubiquitin and acyl-CoA binding protein; adipogenesis regulatory factor, perilipin-1 fragments, and S100A6, along with their PTMs. Part of the bottom-up proteomic profile was reproducibly found in both tested samples. The bottom-up approach allowed demonstrating the presence of proteins, listed among the components of adipose tissue, and/or are comprised within the ASCs intracellular content and secreted proteome. Our data provide a first glance on the LAF molecular profile, which is consistent with its tissue environment. LAF appeared to contain bioactive proteins, peptides and paracrine factors, suggesting its potential translational exploitation

NATIVE CHARACTERIZATION AND QC PROFILING OF HUMAN AMNIOTIC MESENCHYMAL STROMAL CELL VESICULAR FRACTIONS FOR SECRETOME-BASED THERAPY

Human amniotic mesenchymal stromal cells (hAMSCs) have unique immunomodulatory properties making them attractive candidates for regenerative applications in inflammatory diseases. Most of their beneficial properties are mediated through their secretome. The bioactive factors concurring to its therapeutic activity are still unknown. Evidence suggests synergy between the two main components of the secretome, soluble factors and vesicular fractions, pivotal in shifting inflammation and promoting self-healing. Biological variability and the absence of quality control (QC) protocols hinder secretome-based therapy translation to clinical applications. Moreover, vesicular secretome contains a multitude of particles with varying size, cargos and functions whose complexity hinders full characterization and comprehension. This study achieved a significant advancement in secretome characterization by utilizing native, FFF-based separation and characterizing extracellular vesicles derived from hAMSCs. This was accomplished by obtaining dimensionally homogeneous fractions then characterized based on their protein content, potentially enabling the identification of subpopulations with diverse functionalities. This method proved to be successful as an independent technique for secretome profiling, with the potential to contribute to the standardization of a qualitative method. Additionally, it served as a preparative separation tool, streamlining populations before ELISA and LC-MS characterization. This approach facilitated the categorization of distinctive and recurring proteins, along with the identification of clusters associated with vesicle activity and functions. However, the presence of proteins unique to each fraction obtained through the FFF separation tool presents a challenge for further analysis of the protein content within these cargoes

Simultaneous stereoselective analysis by capillary electrophoresis of tramadol enantiomers and their main phase I metabolites in urine

Capillary zone electrophoresis was successfully applied to the enantiomeric resolution of racemic tramadol and its six phase I metabolites using carboxymethylated beta-cyclodextrin (CMB) added to the background electrolyte (BGE). Baseline resolution of tramadol and its metabolites was obtained in less than 30 min using a 50 mM phosphate buffer (pH 2.5) containing 5 mM of CMB. Chiral determinations of tramadol and its main three metabolites, O-demethyltramadol (M1), N-demethyltramadol (M2) and O-demethyl-N-demethyltramadol (M5), were performed in urine after a simple double liquid-liquid extraction of 200 microliters of biological material. In the tested concentration range (0.5-20 micrograms/ml, except for M2: 0.5-10 micrograms/ml) coefficients of correlation superior than 0.994 were obtained. Within-day variation determined on three different concentrations for each enantiomers showed accuracies ranging from 95.4% to 103.2%. The relative standard deviation (RSD) of these assays was determined to be less than 10.0%. Day-to-day variation presented accuracies ranging from 96.3% to 106.5% with a RSD less than 9.0%. After oral administration of 100 mg of tramadol hydrochloride to an healthy volunteer, the urinary excretion was monitored during 30 h. About 15% of the dose was excreted as unchanged tramadol. The enantiomeric ratios of all the excreted analytes, T, M1, M2 and M5, were found to be very different to 1.0, showing that a stereoselective metabolism of tramadol clearly occurred

Citrullination Post-Translational Modification: State of the Art of Brain Tumor Investigations and Future Perspectives

The present review aims to describe the state of the art of research studies investigating the citrullination post-translational modification in adult and pediatric brain tumors. After an introduction to the deimination reaction and its occurrence in proteins and polypeptide chains, the role of the citrullination post-translational modification in physiological as well as pathological states, including cancer, is summarized, and the recent literature and review papers on the topic are examined. A separate section deals with the specific focus of investigation of the citrullination post-translational modification in relation to brain tumors, examining the state of the art of the literature that mainly concerns adult and pediatric glioblastoma and posterior fossa pediatric tumors. We examined the literature on this emerging field of research, and we apologize in advance for any possible omission. Although only a few studies inspecting citrullination in brain tumors are currently available, the results interestingly highlighted different profiles of the citrullinome associated with different histotypes. The data outlined the importance of this post-translational modification in modulating cancer invasion and chemoresistance, influencing key factors involved in apoptosis, cancer cell communication through extracellular vesicle release, autophagy, and gene expression processes, which suggests the prospect of taking citrullination as a target of cancer treatment or as a source of potential diagnostic and prognostic biomarkers for potential clinical applications in the future

Analysis of the GFP-labelled β-dystroglycan interactome in HEK-293 transfected cells reveals novel intracellular networks

Highlights•HEK-293 cells were transfected with β-dystroglycan fused to GFP and the protein predominantly localized at the nuclear envelope.•Protein complexes bound to β-dystroglycan were investigated using immunoprecipitation by anti-GFP antibodies followed by shotgun proteomic analysis.•Novel potential interaction networks involving β-dystroglycan were identified in cytoskeleton, endoplasmatic reticulum, mitochondria and nucleus.•Lamin-Associated Polypeptide-1B (LAP1B) was identified as a novel protein that interacts with β-dystroglycan.AbstractDystroglycan (DG) is a cell adhesion complex that is widely expressed in tissues. It is composed by two subunits, α-DG, a highly glycosylated protein that interacts with several extracellular matrix proteins, and transmembrane β-DG whose cytodomain binds to the actin cytoskeleton. Glycosylation of α-DG is crucial for functioning as a receptor for its multiple extracellular binding partners. Perturbation of α-DG glycosylation is the central event in the pathogenesis of severe pathologies such as muscular dystrophy and cancer. β-DG acts as a scaffold for several cytoskeletal and nuclear proteins and very little is known about the fine regulation of some of these intracellular interactions and how they are perturbed in diseases.To start filling this gap by identifying uncharacterized intracellular networks preferentially associated with β-DG, HEK-293 cells were transiently transfected with a plasmid carrying the β-DG subunit with GFP fused at its C-terminus. With this strategy, we aimed at forcing β-DG to occupy multiple intracellular locations instead of sitting tightly at its canonical plasma membrane milieu, where it is commonly found in association with α-DG.Immunoprecipitation by anti-GFP antibodies followed by shotgun proteomic analysis, led to the identification of an interactome formed by 313 exclusive protein matches for β-DG binding. A series of already known β-DG interactors have been found, including ezrin and emerin, whilst significant new matches, which include potential novel β-DG interactors and their related networks, were identified in diverse subcellular compartments such as cytoskeleton, endoplasmic reticulum/Golgi, mitochondria, nuclear membrane and the nucleus itself. Of particular interest amongst the novel identified matches, Lamin-Associated Polypeptide-1B (LAP1B), an inner nuclear membrane protein whose mutations are known to cause nuclear envelopathies characterized by muscular dystrophy, was found to interact with β-DG in HEK-293 cells. This evidence was confirmed by immunoprecipitation, Western blotting and immunofluorescence experiments. We also found by immunofluorescence experiments that LAP1B looses its nuclear envelope localization in C2C12 DG-knock-out cells, suggesting that LAP1B requires β-DG for a proper nuclear localization.These results expand the role of β-DG as a nuclear scaffolding protein and provide novel evidence of a possible link between dystroglycanopathies and nuclear envelopathies displaying with muscular dystrophy