Overexpression of a Novel Noxo1 Mutant Increases Ros Production and Noxo1 Relocalisation.

Noxo1, the organizing element of the Nox1-dependent NADPH oxidase complex responsible for producing reactive oxygen species, has been described to be degraded by the proteasome. We mutated a D-box in Noxo1 to express a protein with limited degradation and capable of maintaining Nox1 activation. Wild-type (wt) and mutated Noxo1 (mut1) proteins were expressed in different cell lines to characterize their phenotype, functionality, and regulation. Mut1 increases ROS production through Nox1 activity affects mitochondrial organization and increases cytotoxicity in colorectal cancer cell lines. Unexpectedly the increased activity of Noxo1 is not related to a blockade of its proteasomal degradation since we were unable in our conditions to see any proteasomal degradation either for wt or mut1 Noxo1. Instead, D-box mutation mut1 leads to an increased translocation from the membrane soluble fraction to a cytoskeletal insoluble fraction compared to wt Noxo1. This mut1 localization is associated in cells with a filamentous phenotype of Noxo1, which is not observed with wt Noxo1. We found that mut1 Noxo1 associates with intermediate filaments such as keratin 18 and vimentin. In addition, Noxo1 D-Box mutation increases Nox1-dependent NADPH oxidase activity. Altogether, Nox1 D-box does not seem to be involved in Noxo1 degradation but rather related to the maintenance of the Noxo1 membrane/cytoskeleton balance.

Myotoxin-3 from the Pacific Rattlesnake Crotalus oreganus oreganus Venom Is a New Microtubule-Targeting Agent.

Microtubule targeting agents (MTA) are anti-cancer molecules that bind tubulin and interfere with the microtubule functions, eventually leading to cell death. In the present study, we used an in vitro microtubule polymerization assay to screen several venom families for the presence of anti-microtubule activity. We isolated myotoxin-3, a peptide of the crotamine family, and three isoforms from the venom of the Northern Pacific rattlesnake Crotalus oreganus oreganus, which was able to increase tubulin polymerization. Myotoxin-3 turned out to be a cell-penetrating peptide that slightly diminished the viability of U87 glioblastoma and MCF7 breast carcinoma cells. Myotoxin 3 also induced remodeling of the U87 microtubule network and decreased MCF-7 microtubule dynamic instability. These effects are likely due to direct interaction with tubulin. Indeed, we showed that myotoxin-3 binds to tubulin heterodimer with a Kd of 5.3 µM and stoichiometry of two molecules of peptide per tubulin dimer. Our results demonstrate that exogenous peptides are good candidates for developing new MTA and highlight the richness of venoms as a source of pharmacologically active molecules.

Patient-derived antibodies reveal the subcellular distribution and heterogeneous interactome of LGI1.

Autoantibodies against leucine-rich glioma-inactivated 1 (LGI1) occur in patients with encephalitis who present with frequent focal seizures and a pattern of amnesia consistent with focal hippocampal damage. To investigate whether the cellular and subcellular distribution of LGI1 may explain the localization of these features, and hence gain broader insights into LGI1's neurobiology, we analysed the detailed localization of LGI1 and the diversity of its protein interactome, in mouse brains using patient-derived recombinant monoclonal LGI1 antibodies. Combined immunofluorescence and mass spectrometry analyses showed that LGI1 is enriched in excitatory and inhibitory synaptic contact sites, most densely within CA3 regions of the hippocampus. LGI1 is secreted in both neuronal somatodendritic and axonal compartments, and occurs in oligodendrocytic, neuro-oligodendrocytic and astro-microglial protein complexes. Proteomic data support the presence of LGI1-Kv1-MAGUK complexes, but did not reveal LGI1 complexes with postsynaptic glutamate receptors. Our results extend our understanding of regional, cellular and subcellular LGI1 expression profiles and reveal novel LGI1-associated complexes, thus providing insights into the complex biology of LGI1 and its relationship to seizures and memory loss.

Mechanism of Zn2+ and Ca2+ Binding to Human S100A1.

S100A1 is a member of the S100 family of small ubiquitous Ca2+-binding proteins, which participates in the regulation of cell differentiation, motility, and survival. It exists as homo- or heterodimers. S100A1 has also been shown to bind Zn2+, but the molecular mechanisms of this binding are not yet known. In this work, using ESI-MS and ITC, we demonstrate that S100A1 can coordinate 4 zinc ions per monomer, with two high affinity (KD~4 and 770 nm) and two low affinity sites. Using competitive binding experiments between Ca2+ and Zn2+ and QM/MM molecular modeling we conclude that Zn2+ high affinity sites are located in the EF-hand motifs of S100A1. In addition, two lower affinity sites can bind Zn2+ even when the EF-hands are saturated by Ca2+, resulting in a 2Ca2+:S100A1:2Zn2+ conformer. Finally, we show that, in contrast to calcium, an excess of Zn2+ produces a destabilizing effect on S100A1 structure and leads to its aggregation. We also determined a higher affinity to Ca2+ (KD~0.16 and 24 µm) than was previously reported for S100A1, which would allow this protein to function as a Ca2+/Zn2+-sensor both inside and outside cells, participating in diverse signaling pathways under normal and pathological conditions.

Isolation of an Anti-Tumour Disintegrin: Dabmaurin-1, a Peptide Lebein-1-Like, from Daboia mauritanica Venom.

In the soft treatment of cancer tumours, consequent downregulation of the malignant tissue angiogenesis constitutes an efficient way to stifle tumour development and metastasis spreading. As angiogenesis requires integrin-promoting endothelial cell adhesion, migration, and vessel tube formation, integrins represent potential targets of new therapeutic anti-angiogenic agents. Our work is a contribution to the research of such therapeutic disintegrins in animal venoms. We report isolation of one peptide, named Dabmaurin-1, from the hemotoxic venom of snake Daboia mauritanica, and we evaluate its potential anti-tumour activity through in vitro inhibition of the human vascular endothelial cell HMECs functions involved in tumour angiogenesis. Dabmaurin-1 altered, in a dose-dependent manner, without any significant cytotoxicity, HMEC proliferation, adhesion, and their mesenchymal migration onto various extracellular matrix proteins, as well as formation of capillary-tube mimics on MatrigelTM. Via experiments involving HMEC or specific cancers cells integrins, we demonstrated that the above Dabmaurin-1 effects are possibly due to some anti-integrin properties. Dabmaurin-1 was demonstrated to recognize a broad panel of prooncogenic integrins (αvß6, αvß3 or αvß5) and/or particularly involved in control of angiogenesis α5ß1, α6ß4, αvß3 or αvß5). Furthermore, mass spectrometry and partial N-terminal sequencing of this peptide revealed, it is close to Lebein-1, a known anti-ß1 disintegrin from Macrovipera lebetina venom. Therefore, our results show that if Dabmaurin-1 exhibits in vitro apparent anti-angiogenic effects at concentrations lower than 30 nM, it is likely because it acts as an anti-tumour disintegrin.

AaHIV a sodium channel scorpion toxin inhibits the proliferation of DU145 prostate cancer cells.

Prostate cancer is the most highly diagnosed cancer in men worldwide. It is characterized by high proliferation, great invasion and metastatic potential. Sodium channel subtypes have been identified as highly expressed in different prostate cancer cell lines. In this study, we have screened the negatively charged fractions of Androctonus australis (Aa) scorpion venom to identify active peptides on DU145 prostate cancer cells proliferation. The most active compound was identified to be the sodium channel peptide AaHIV with an IC50 value of 15 µM. At this concentration, AaHIV had low effect on the adhesion of DU145 cells to fibronectin. When compared to other Na+ channel Aa toxins, AaHIV was found to be 2 times more active than AaHI and AaHII on DU145 cells proliferation and slightly less active than AaHII on their adhesion. The three peptides are inactive on DU145 cells migration. AaHIV was found to be 16 times more active than veratridine, asteroidal alkaloid from plants of the lily family widely used as a sodium channel activator. Electrophysiological experiments showed that the AaHIV toxin activates Nav1.6 channel, suggesting that this sodium channel subtype is implicated in the proliferation of DU145 prostate cancer cells.

A Low Molecular Weight Protein from the Sea Anemone Anemonia viridis with an Anti-Angiogenic Activity.

Sea anemones are a remarkable source of active principles due to a decentralized venom system. New blood vessel growth or angiogenesis is a very promising target against cancer, but the few available antiangiogenic compounds have limited efficacy. In this study, a protein fraction, purified from tentacles of Anemonia viridis, was able to limit endothelial cells proliferation and angiogenesis at low concentration (14 nM). Protein sequences were determined with Edman degradation and mass spectrometry in source decay and revealed homologies with Blood Depressing Substance (BDS) sea anemones. The presence of a two-turn alpha helix observed with circular dichroism and a trypsin activity inhibition suggested that the active principle could be a Kunitz-type inhibitor, which may interact with an integrin due to an Arginine Glycin Aspartate (RGD) motif. Molecular modeling showed that this RGD motif was well exposed to solvent. This active principle could improve antiangiogenic therapy from existing antiangiogenic compounds binding on the Vascular Endothelial Growth Factor (VEGF).

Inactivation of the arn operon and loss of aminoarabinose on lipopolysaccharide as the cause of susceptibility to colistin in an atypical clinical isolate of proteus vulgaris.

Colistin has become a last-line antibiotic for the treatment of multidrug-resistant bacterial infections; however, resistance to colistin has emerged in recent years. Some bacteria, such as Proteus and Serratia spp., are intrinsically resistant to colistin although the exact mechanism of resistance is unknown. Here we identified the molecular support for intrinsic colistin resistance in Proteus spp. by comparative genomic, transcriptomic and proteomic analyses of colistin-susceptible (CSUR P1868_S) and colistin-resistant (CSUR P1867_R) strains of an atypical Proteus vulgaris. A significant difference in outer membrane glycoside structures in both strains that was corroborated by MALDI-TOF/MS analysis was found, which showed an absence of 4-amino-4-deoxy-l-arabinose (L-Ara4N) in the outer membrane lipid A moiety of the susceptible strain. Comparative genomic analysis with other resistant strains of P. vulgaris available in a local database found a mutation in the arnBCADTEF operon of the susceptible strain. Transcriptomic analysis of genes belonging to the arnBCADTEF operon showed a significant decrease in mRNA expression level of these genes in the susceptible strain, supporting addition of L-Ara4N in the outer membrane lipid A moiety as an explanation for colistin resistance. Insertion of the arnD gene that was suggested to be altered in the susceptible strain by in silico analysis led to a 16-fold increase of colistin MIC in the susceptible strain, confirming its role in colistin resistance in this species. Here we show that constitutive activation of the arn operon and addition of L-Ara4N is the main molecular mechanism of colistin resistance in P. vulgaris.

Omics analysis of mouse brain models of human diseases.

The identification of common gene/protein profiles related to brain alterations, if they exist, may indicate the convergence of the pathogenic mechanisms driving brain disorders. Six genetically engineered mouse lines modelling neurodegenerative diseases and neuropsychiatric disorders were considered. Omics approaches, including transcriptomic and proteomic methods, were used. The gene/protein lists were used for inter-disease comparisons and further functional and network investigations. When the inter-disease comparison was performed using the gene symbol identifiers, the number of genes/proteins involved in multiple diseases decreased rapidly. Thus, no genes/proteins were shared by all 6 mouse models. Only one gene/protein (Gfap) was shared among 4 disorders, providing strong evidence that a common molecular signature does not exist among brain diseases. The inter-disease comparison of functional processes showed the involvement of a few major biological processes indicating that brain diseases of diverse aetiologies might utilize common biological pathways in the nervous system, without necessarily involving similar molecules.

An ultrasensitive LC-MS/MS method with liquid phase extraction to determine paclitaxel in both cell culture medium and lysate promising quantification of drug nanocarriers release in vitro.

The quantification of paclitaxel, a chemotherapy drug used to treat different types of cancers, has been performed from complete cell culture medium and cell lysate samples using a simple liquid-liquid extraction procedure in conjunction with liquid chromatography tandem mass spectrometry (LC-MS/MS). A simple sample preparation using methanol and acetic acid as a weaker acid was applied to avoid paclitaxel destruction and to achieve recovery exceeding 80 % from both matrices spiked with paclitaxel and docetaxel used as internal standard. This rapid, simple, selective and sensitive method enabled the quantification of paclitaxel within the linear range of 1-250nM in culture medium and 5-250nM in cell lysate. The lower limit of quantification was achieved in cell culture medium and cell lysates at 0.2 and 1pmol, respectively. This method was successfully applied to human non-small cell lung carcinoma cells (A549 cells) in order to quantify the amount of paclitaxel in both cell culture medium and lysate after incubation with 5, 50 and 100nM of paclitaxel. This ultra-sensitive method promises the quantification of ultra-low concentrations of paclitaxel released from any nanocarriers, allowing the determination of the kinetic profile of drug release, which is an essential parameter to validate the use of nanocarriers for drug delivery in cancer therapy.

An Early Postnatal Oxytocin Treatment Prevents Social and Learning Deficits in Adult Mice Deficient for Magel2, a Gene Involved in Prader-Willi Syndrome and Autism.

BACKGROUND: Mutations of MAGEL2 have been reported in patients presenting with autism, and loss of MAGEL2 is also associated with Prader-Willi syndrome, a neurodevelopmental genetic disorder. This study aimed to determine the behavioral phenotype of Magel2-deficient adult mice, to characterize the central oxytocin (OT) system of these mutant mice, and to test the curative effect of a peripheral OT treatment just after birth. METHODS: We assessed the social and cognitive behavior of Magel2-deficient mice, analyzed the OT system of mutant mice treated or not by a postnatal administration of OT, and determined the effect of this treatment on the brain. RESULTS: Magel2 inactivation induces a deficit in social recognition and social interaction and a reduced learning ability in adult male mice. In these mice, we reveal anatomical and functional modifications of the OT system and show that these defects change from birth to adulthood. Daily administration of OT in the first postnatal week was sufficient to prevent deficits in social behavior and learning abilities in adult mutant male mice. We show that this OT treatment partly restores a normal OT system. Thus, we report that an alteration of the OT system around birth has long-term consequences on behavior and on cognition. Importantly, an acute OT treatment of Magel2-deficient pups has a curative effect. CONCLUSIONS: Our study reveals that OT plays a crucial role in setting social behaviors during a period just after birth. An early OT treatment in this critical period could be a novel therapeutic approach for the treatment of neurodevelopmental disorders such as Prader-Willi syndrome and autism.

Gold nanoparticles prepared by laser ablation in aqueous biocompatible solutions: assessment of safety and biological identity for nanomedicine applications.

Due to excellent biocompatibility, chemical stability, and promising optical properties, gold nanoparticles (Au-NPs) are the focus of research and applications in nanomedicine. Au-NPs prepared by laser ablation in aqueous biocompatible solutions present an essentially novel object that is unique in avoiding any residual toxic contaminant. This paper is conceived as the next step in development of laser-ablated Au-NPs for future in vivo applications. The aim of the study was to assess the safety, uptake, and biological behavior of laser-synthesized Au-NPs prepared in water or polymer solutions in human cell lines. Our results showed that laser ablation allows the obtaining of stable and monodisperse Au-NPs in water, polyethylene glycol, and dextran solutions. The three types of Au-NPs were internalized in human cell lines, as shown by transmission electron microscopy. Biocompatibility and safety of Au-NPs were demonstrated by analyzing cell survival and cell morphology. Furthermore, incubation of the three Au-NPs in serum-containing culture medium modified their physicochemical characteristics, such as the size and the charge. The composition of the protein corona adsorbed on Au-NPs was investigated by mass spectrometry. Regarding composition of complement C3 proteins and apolipoproteins, Au-NPs prepared in dextran solution appeared as a promising drug carrier. Altogether, our results revealed the safety of laser-ablated Au-NPs in human cell lines and support their use for theranostic applications.

Monitoring therapeutic monoclonal antibodies in brain tumor.

Bevacizumab induces normalization of abnormal blood vessels, making them less leaky. By binding to vascular endothelial growth factor, it indirectly attacks the vascular tumor mass. The optimal delivery of targeted therapies including monoclonal antibodies or anti-angiogenesis drugs to the target tissue highly depends on the blood-brain barrier permeability. It is therefore critical to investigate how drugs effectively reach the tumor. In situ investigation of drug distribution could provide a better understanding of pharmacological agent action and optimize chemotherapies for solid tumors. We developed an imaging method coupled to protein identification using matrix-assisted laser desorption/ionization mass spectrometry. This approach monitored bevacizumab distribution within the brain structures, and especially within the tumor, without any labeling.

Peptide ligation from alkoxyamine based radical addition.

Intermolecular radical 1,2-addition (IRA) of N-tert-butyl-N-(1-diethylphosphono-2,2-dimethylpropyl)aminoxyl (SG1) based alkoxyamines onto activated olefins is used as a tool for peptide ligation. This strategy relies on simple peptide pre-derivatization to obtain (i) a SG1 nitroxide functionalized resin peptide at its N-terminus (SG1-peptide alkoxyamine), (ii) a vinyl functionalized peptide (either at its C-terminus or N-terminus), and does not require any coupling agents.

MALDI imaging and in-source decay for top-down characterization of glioblastoma.

Glioblastoma multiforme is one of the most common intracranial tumors encountered in adults. This tumor of very poor prognosis is associated with a median survival rate of approximately 14 months. One of the major issues to better understand the biology of these tumors and to optimize the therapy is to obtain the molecular structure of glioblastoma. MALDI molecular imaging enables location of molecules in tissues without labeling. However, molecular identification in situ is not an easy task. In this paper, we used MALDI imaging coupled to in-source decay to characterize markers of this pathology. We provided MALDI molecular images up to 30 µm spatial resolution of mouse brain tissue sections. MALDI images showed the heterogeneity of the glioblastoma. In the various zones and at various development stages of the tumor, using our top-down strategy, we identified several proteins. These proteins play key roles in tumorigenesis. Particular attention was given to the necrotic area with characterization of hemorrhage, one of the most important poor prognosis factors in glioblastoma.

Tubacin prevents neuronal migration defects and epileptic activity caused by rat Srpx2 silencing in utero.

Altered development of the human cerebral cortex can cause severe malformations with often intractable focal epileptic seizures and may participate in common pathologies, notably epilepsy. This raises important conceptual and therapeutic issues. Two missense mutations in the sushi repeat-containing protein SRPX2 had been previously identified in epileptic disorders with or without structural developmental alteration of the speech cortex. In the present study, we aimed to decipher the precise developmental role of SRPX2, to have a better knowledge on the consequences of its mutations, and to start addressing therapeutic issues through the design of an appropriate animal model. Using an in utero Srpx2 silencing approach, we show that SRPX2 influences neuronal migration in the developing rat cerebral cortex. Wild-type, but not the mutant human SRPX2 proteins, rescued the neuronal migration phenotype caused by Srpx2 silencing in utero, and increased alpha-tubulin acetylation. Following in utero Srpx2 silencing, spontaneous epileptiform activity was recorded post-natally. The neuronal migration defects and the post-natal epileptic consequences were prevented early in embryos by maternal administration of tubulin deacetylase inhibitor tubacin. Hence epileptiform manifestations of developmental origin could be prevented in utero, using a transient and drug-based therapeutic protocol.

Tubulin isoforms identified in the brain by MALDI in-source decay.

Identification of biomarkers is a major issue for enhancement of chemotherapies. The molecular characterization of tissues necessitates the identification of thousands of biomolecules each participating in physiopathological processes. MALDI in-source decay (ISD) fragmentation has already been proven to be effective for protein characterization. However, the difficulty to identify proteins from complex mixtures such as tissue sections can limit the applications of this technique. In this study, we evidenced that tubulin has an unusual fragmentation pathway in the MALDI source. This striking property allowed the detecting of several mouse brain tubulin isotypes simultaneously by simply using laser fragmentation. Tubulin isoforms are consistent markers of a bad prognosis of solid tumors and could be the target of targeted chemotherapies. Such a direct molecular printout of tubulin in tissues is a milestone that should be useful either at preclinical or clinical stage.

Anopheles salivary gland proteomes from major malaria vectors.

BACKGROUND: Antibody responses against Anopheles salivary proteins can indicate individual exposure to bites of malaria vectors. The extent to which these salivary proteins are species-specific is not entirely resolved. Thus, a better knowledge of the diversity among salivary protein repertoires from various malaria vector species is necessary to select relevant genus-, subgenus- and/or species-specific salivary antigens. Such antigens could be used for quantitative (mosquito density) and qualitative (mosquito species) immunological evaluation of malaria vectors/host contact. In this study, salivary gland protein repertoires (sialomes) from several Anopheles species were compared using in silico analysis and proteomics. The antigenic diversity of salivary gland proteins among different Anopheles species was also examined. RESULTS: In silico analysis of secreted salivary gland protein sequences retrieved from an NCBInr database of six Anopheles species belonging to the Cellia subgenus (An. gambiae, An. arabiensis, An. stephensi and An. funestus) and Nyssorhynchus subgenus (An. albimanus and An. darlingi) displayed a higher degree of similarity compared to salivary proteins from closely related Anopheles species. Additionally, computational hierarchical clustering allowed identification of genus-, subgenus- and species-specific salivary proteins. Proteomic and immunoblot analyses performed on salivary gland extracts from four Anopheles species (An. gambiae, An. arabiensis, An. stephensi and An. albimanus) indicated that heterogeneity of the salivary proteome and antigenic proteins was lower among closely related anopheline species and increased with phylogenetic distance. CONCLUSION: This is the first report on the diversity of the salivary protein repertoire among species from the Anopheles genus at the protein level. This work demonstrates that a molecular diversity is exhibited among salivary proteins from closely related species despite their common pharmacological activities. The involvement of these proteins as antigenic candidates for genus-, subgenus- or species-specific immunological evaluation of individual exposure to Anopheles bites is discussed.

Mass spectrometry imaging is moving toward drug protein co-localization.

Mass spectrometry (MS)-based technology provides label-free localization of molecules in tissue samples. Drugs, proteins, lipids and metabolites can easily be monitored in their environment. Resolution can be achieved down to the cellular level (10-20 µm) for conventional matrix-assisted laser desorption/ionization (MALDI) imaging, or even to the subcellular level for more complex technologies such as secondary ionization mass spectrometry (SIMS) imaging. One question remains: are we going to be able to investigate functional relationships between drugs and proteins and compare with localized phenomena? This review describes the various spatial levels of investigation offered by mass spectrometry imaging (MSI), and the advantages and disadvantages compared with other labeling technologies.

In-source decay and pseudo tandem mass spectrometry fragmentation processes of entire high mass proteins on a hybrid vacuum matrix-assisted laser desorption ionization-quadrupole ion-trap time-of-flight mass spectrometer.

In-source decay (ISD), although a process known for decades in mass spectrometry, has a renewed interest due to increased theoretical knowledge in fragmentation processes of large biomolecules coupled with technological improvements. We report here an original method consisting of isolating matrix-assisted laser desorption ionization (MALDI)-generated in-source fragments of large proteins and subsequently performing selective fragmentation experiments (up to four cycles) using a hybrid MALDI quadrupole ion-trap time-of-flight mass spectrometer (MALDI-QIT-TOF). This technology takes advantage of keeping high resolution on the selection of precursors and detection of fragments. It allows exhaustive N- and C-terminal sequencing of proteins. In this work, human serum albumin (HSA), ß-casein, and recombinant Tau proteins were submitted to in source decay in the MALDI source. The fragments were stored in the ion-trap and submitted to sequential collision-induced dissociation (CID). Finally, ISD and pseudo MS(n) were performed on oxidized Tau protein and acetylated bovine serum albumin to identify amino acid modifications. This work highlights the potential of the MALDI-QIT-TOF instrument for pseudo MS(n) strategies and top down proteomics.