Sorcin Links Calcium Signaling to Vesicle Trafficking,Regulates Polo-Like Kinase 1 and Is Necessary for Mitosis

Sorcin, a protein overexpressed in many multi-drug resistant cancers, dynamically localizes to distinct subcellular sites in 3T3-L1 fibroblasts during cell-cycle progression. During interphase sorcin is in the nucleus, in the plasma membrane, in endoplasmic reticulum (ER) cisternae, and in ER-derived vesicles localized along the microtubules. These vesicles are positive to RyR, SERCA, calreticulin and Rab10. At the beginning of mitosis, sorcin-containing vesicles associate with the mitotic spindle, and during telophase are concentrated in the cleavage furrow and, subsequently, in the midbody. Sorcin regulates dimensions and calcium load of the ER vesicles by inhibiting RYR and activating SERCA. Analysis of sorcin interactome reveals calcium-dependent interactions with many proteins, including Polo-like kinase 1 (PLK1), Aurora A and Aurora B kinases. Sorcin interacts physically with PLK1, is phosphorylated by PLK1 and induces PLK1 autophosphorylation, thereby regulating kinase activity. Knockdown of sorcin results in major defects in mitosis and cytokinesis, increase in the number of rounded polynucleated cells, blockage of cell progression in G2/M, apoptosis and cell death. Sorcin regulates calcium homeostasis and is necessary for the activation of mitosis and cytokinesis.     

Modulation of Activity of Known Cytotoxic Ruthenium(III) Compound (KP418) with Hampered Transmembrane Transport in Electrochemotherapy In Vitro and In Vivo

To increase electrochemotherapy (ECT) applicability, the effectiveness of new drugs is being tested in combination with electroporation. Among them two ruthenium(III) compounds, (imH)[trans-RuCl₄(im)(DMSO-S)] (NAMI-A) and Na[trans-RuCl₄(ind)₂] (KP1339), proved to possess increased antitumor effectiveness when combined with electroporation. The objective of our experimental work was to determine influence of electroporation on the cytotoxic and antitumor effect of a ruthenium(III) compound with hampered transmembrane transport, (imH)[trans-RuCl₄(im)₂] (KP418) in vitro and in vivo and to determine changes in metastatic potential of cells after ECT with KP418 in vitro. In addition, platinum compound cisplatin (CDDP) and ruthenium(III) compound NAMI-A were included in the experiments as reference compounds. Our results show that electroporation leads to increased cellular accumulation and cytotoxicity of KP418 in murine melanoma cell lines with low and high metastatic potential, B16-F1 and B16-F10, but not in murine fibrosarcoma cell line SA-1 in vitro which is probably due to variable effectiveness of ECT in different cell lines and tumors. Electroporation does not potentiate the cytotoxicity of KP418 as prominently as the cytotoxicity of CDDP. We also showed that the metastatic potential of cells which survived ECT with KP418 or NAMI-A does not change in vitro: resistance to detachment, invasiveness, and re-adhesion of cells after ECT is not affected. Experiments in murine tumor models B16-F1 and SA-1 showed that ECT with KP418 does not have any antitumor effect while ECT with CDDP induces significant dose-dependent tumor growth delay in the two tumor models used in vivo.     

Interaction of leptospires with murine microglial cells

Leptospires, the agents of leptospirosis, exert tropism for the central nervous system, in the course of mammal infection. We evaluated the interaction between murine microglial cells and strains of pathogenic L. interrogans leptospires and non-pathogenic L. biflexa leptospires, mainly by flow cytometric assays. In the absence of opsonic conditions microglia are capable of ingesting--even quite slowly--the spirochetes and killing the non-pathogenic strain. The adhesion to microglia, which is quick and relevant for all the strains, does not involve the CR3 integrin receptor. These findings suggest that the murine microglia--in non opsonic conditions in vitro--do not effectively clear the pathogenic leptospires.     

A new role for FcgammaRIIA in the potentiation of human platelet activation induced by weak stimulation

The low affinity receptor for immunoglobulin G, FcgammaRIIA, is expressed in human platelets, mediates heparin-induced thrombocytopenia and participates to platelet activation induced by von Willebrand factor. In this work, we found that stimulation of platelets with agonists acting on G-protein-coupled receptors resulted in the tyrosine phosphorylation of FcgammaRIIA, through a mechanism involving a Src kinase. Treatment of platelets with the blocking monoclonal antibody IV.3 against FcgammaRIIA, but not with control IgG, inhibited platelet aggregation induced by TRAP1, TRAP4, the thromboxane analogue U46619, and low concentrations of thrombin. By contrast, platelet aggregation induced by high doses of thrombin was unaffected by blockade of FcgammaRIIA. We also found that the anti-FcgammaRIIA monoclonal antibody IV.3 inhibited pleckstrin phosphorylation and calcium mobilization induced by low, but not high, concentrations of thrombin. In addition, thrombin- or U46619-induced tyrosine phosphorylation of several substrates typically involved in FcgammaRIIA-mediated signalling, such as Syk and PLCgamma2, was clearly reduced by incubation with anti-FcgammaRIIA antibody IV.3. Upon stimulation with thrombin, FcgammaRIIA relocated in lipid rafts, and thrombin-induced tyrosine phosphorylation of FcgammaRIIA occurred within these membrane domains. Controlled disruption of lipid rafts by depleting membrane cholesterol prevented tyrosine phosphorylation of FcgammaRIIA and impaired platelet aggregation induced by U46619 or by low, but not high, concentrations of thrombin. These results indicate that FcgammaRIIA can be activated in human platelets downstream G-protein-coupled receptors and suggest a novel general mechanism for the reinforcement of platelet activation induced by low concentrations of agonists.     

c-Src-mediated phosphorylation of NoxA1 and Tks4 induces the reactive oxygen species (ROS)-dependent formation of functional invadopodia in human colon cancer cells

The NADPH oxidase family, consisting of Nox1-5 and Duox1-2, catalyzes the regulated formation of reactive oxygen species (ROS). Highly expressed in the colon, Nox1 needs the organizer subunit NoxO1 and the activator subunit NoxA1 for its activity. The tyrosine kinase c-Src is necessary for the formation of invadopodia, phosphotyrosine-rich structures which degrade the extracellular matrix (ECM). Many Src substrates are invadopodia components, including the novel Nox1 organizer Tks4 and Tks5 proteins. Nox1-dependent ROS generation is necessary for the maintenance of functional invadopodia in human colon cancer cells. However, the signals and the molecular machinery involved in the redox-dependent regulation of invadopodia formation remain unclear. Here, we show that the interaction of NoxA1 and Tks proteins is dependent on Src activity. Interestingly, the abolishment of Src-mediated phosphorylation of Tyr110 on NoxA1 and of Tyr508 on Tks4 blocks their binding and decreases Nox1-dependent ROS generation. The contemporary presence of Tks4 and NoxA1 unphosphorylable mutants blocks SrcYF-induced invadopodia formation and ECM degradation, while the overexpression of Tks4 and NoxA1 phosphomimetic mutants rescues this phenotype. Taken together, these results elucidate the role of c-Src activity on the formation of invadopodia and may provide insight into the mechanisms of tumor formation in colon cancers.     

Enriching the viral–host interactomes with interactions mediated by SH3 domains

Protein–protein interactions play an essential role in the regulation of most cellular processes. The process of viral infection is no exception and many viral pathogenic strategies involve targeting and perturbing host–protein interactions. The characterization of the host protein subnetworks disturbed by invading viruses is a major goal of viral research and may contribute to reveal fundamental biological mechanisms and to identify new therapeutic strategies. To assist in this approach, we have developed a database, VirusMINT, which stores in a structured format most of the published interactions between viral and host proteome. Although SH3 are the most ubiquitous and abundant class of protein binding modules, VirusMINT contains only a few interactions mediated by this domain class. To overcome this limitation, we have applied the whole interactome scanning experiment approach to identify interactions between 15 human SH3 domains and viral proline-rich peptides of two oncogenic viruses, human papillomavirus type 16 and human adenovirus A type 12. This approach identifies 114 new potential interactions between the human SH3 domains and proline-rich regions of the two viral proteomes.     

Can the IUCN criteria be effectively applied to peripheral isolated plant populations?

The IUCN criteria 2001 are considered one of the best methods to evaluate species extinction risk at the global and regional levels. The aim of this work is to test the applicability of the IUCN criteria to peripheral isolated plant populations (PIPPs). PIPPs have been a topic of scientific debate in Conservation Biology for about 15 years and international conventions such as ESPC address the issue of their conservation. Conservation measures often rely on Red Lists based on IUCN criteria, but there is little evidence supporting their application to PIPPs. In this work, we tested the hypothesis that PIPPs’ intrinsic restricted range and rarity lead to the overestimation of their extinction risk. We compared and analyzed four IUCN criteria (A, B, C, D), considering 17 species with PIPPs in different Italian administrative regions. Special attention must be given to the spatial scale at which PIPPs are assessed, the evaluation of the threats affecting the populations, and their decline. PIPPs should not be assessed within political boundaries and we propose a new area designation that better corresponds to the characteristics of PIPPs. Criterion B is strongly biased by restricted range and overestimates the extinction risk of PIPPs, particularly when the population decline is only suspected and not observed. In this case, criterion D more accurately assesses the status of PIPPs. Criterion A is also suitable for assessing PIPPs, because it is not affected by their phytogeographic rarity. The proposed statements could also be valid for the global assessment of narrow endemic species.     

Partner-Mediated Polymorphism of an Intrinsically Disordered Protein

Intrinsically disordered proteins (IDPs) recognize their partners through molecular recognition elements (MoREs). The MoRE of the C-terminal intrinsically disordered domain of the measles virus nucleoprotein (N_TAIL) is partly pre-configured as an α-helix in the free form and undergoes α-helical folding upon binding to the X domain (XD) of the viral phosphoprotein. Beyond XD, N_TAIL also binds the major inducible heat shock protein 70 (hsp70). So far, no structural information is available for the N_TAIL/hsp70 complex. Using mutational studies combined with a protein complementation assay based on green fluorescent protein reconstitution, we have investigated both N_TAIL/XD and N_TAIL/hsp70 interactions. Although the same N_TAIL region binds the two partners, the binding mechanisms are different. Hsp70 binding is much more tolerant of MoRE substitutions than XD, and the majority of substitutions lead to an increased N_TAIL/hsp70 interaction strength. Furthermore, while an increased and a decreased α-helicity of the MoRE lead to enhanced and reduced interaction strength with XD, respectively, the impact on hsp70 binding is negligible, suggesting that the MoRE does not adopt an α-helical conformation once bound to hsp70. Here, by showing that the α-helical conformation sampled by the free form of the MoRE does not systematically commit it to adopt an α-helical conformation in the bound form, we provide an example of partner-mediated polymorphism of an IDP and of the relative insensitiveness of the bound structure to the pre-recognition state. The present results therefore contribute to shed light on the molecular mechanisms by which IDPs recognize different partners.     

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine induces apoptosis in mouse nigrostriatal glia. Relevance to nigral neuronal death and striatal neurochemical changes

Swiss mice were given 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 25 mg/kg/day, for 5 consecutive days and killed at different days after MPTP discontinuance. Decreases in striatal tyrosine hydroxylase activity and levels of dopamine and its metabolites were observed 1 day after MPTP discontinuance. Ascorbic acid and glutamate levels had increased, dehydroascorbic acid and GSH decreased, whereas catabolites of high-energy phosphates (inosine, hypoxanthine, xanthine, and uric acid) were unchanged. In addition, gliosis was observed in both striatum and substantia nigra compacta (SNc). Sections of SNc showed some terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL)-positive cells. Neurochemical parameters of dopaminergic activity showed a trend toward recovery 3 days after MPTP discontinuance. At this time point, TUNEL-positive cells were detected in SNc; some of them showed nuclei with neuronal morphology. A late (days 6-11) increase in striatal dopamine oxidative metabolism, ascorbic acid oxidative status, and catabolites of high-energy phosphates were observed concomitant with nigral neuron and nigrostriatal glial cell apoptotic death, as revealed by TUNEL, acridine orange, and Hoechst staining, and transmission electron microscopy. These data suggest that MPTP-induced activation/apoptotic death of glial cells plays a key role in the sequential linkage of neurochemical and cellular events leading to dopaminergic nigral neuron apoptotic death.