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Two aminobenzoic acid derivatives DAB-0 and DAB-1 showed distinct biological properties on murine bladder cancer (BCa) cell line MB49-I. In contrast to DAB-1, DAB-0 does not possess any anti-inflammatory activity and is less toxic. Furthermore, DAB-0 does not interfere with INFc-induced STAT1 activation and TNFa-induced IjB phosphorylation, while DAB-1 does. In order to rationalize these results, the binding efficacy of DAB-0 and DAB-1 with serum proteins such a human serum albumin (HSA), bovine serum albumin (BSA) and beta-lactoglobulin (b-LG) was investigated in aqueous solution at physiological pH. Multiple spectroscopic methods and thermodynamic analysis were used to determine the binding efficacy of DAB-0 and DAB-1 with serum proteins. Drug-protein conjugation was observed via through ionic contacts. DAB-1 forms stronger adducts than DAB-0, while b-LG shows more affinity with the order of stability b-LG>BSA>HSA. The stronger complexation of DAB-1 with serum proteins might account for its biological potential and transport in the blood. The binding efficacy ranged from 40 to 60%. Major alterations of protein secondary structures were detected upon drug complexation. Serum proteins are capable of delivering DAB-1 in vitro.Abbreviations: BSA: bovine serum albumin; DAB-0: N0-[4-(2,5-dioxo-pyrrolidin-1-yl)-benzoyl]-hydrazine carboxylic acid tert-butyl ester; DAB-1: N0-[4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-benzoyl]-hydrazine carboxylic acid tert-butyl est; FTIR: Fourier transform infrared; b-LG, beta-lactoglobulin; HAS: human serum albumin Fil: Chanphai, P.. Université du Québec a Montreal; Canadá Fil: Cloutier, F.. Université du Québec a Montreal; Canadá Fil: Oufqir, Y.. Université du Québec a Montreal; Canadá Fil: Leclerc, M.F.. Université du Québec a Montreal; Canadá Fil: Eijan, Ana Maria. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Reyes Moreno, C.. Université du Québec a Montreal; Canadá Fil: Bérubé, G.. Université du Québec a Montreal; Canadá Fil: Tajmir Riahi, H.A.. Université du Québec a Montreal; Canadá

Reelin is an extracellular glycoprotein involved in key cellular processes in developing and adult nervous system, including regulation of neuronal migration, synapse formation, and plasticity. Most of these roles are mediated by the intracellular phosphorylation of disabled-1 (Dab1), an intracellular adaptor molecule, in turn mediated by binding Reelin to its receptors. Altered expression and glycosylation patterns of Reelin in cerebrospinal and cortical extracts have been reported in Alzheimer’s disease. However, putative changes in Reelin are not described in natural prionopathies or experimental models of prion infection or toxicity. With this is mind, in the present study, we determined that Reelin protein and mRNA levels increased in CJD human samples and in mouse models of human prion disease in contrast to murine models of prion infection. However, changes in Reelin expression appeared only at late terminal stages of the disease, which prevent their use as an efficient diagnostic biomarker. In addition, increased Reelin in CJD and in in vitro models does not correlate with Dab1 phosphorylation, indicating failure in its intracellular signaling. Overall, these findings widen our understanding of the putative changes of Reelin in neurodegeneration. © 2016, Springer Science+Business Media New York.

Reelin is a large secreted extracellular glycoprotein that plays a critical role in the regulation of neuronal migration during brain development. Reelin is thought to guide migrating neurons by interacting with cell surface receptors, very low density lipoprotein receptor (VLDLR), apolipoprotein E receptor 2 (ApoER2), and a3 b1integrin, inducing tyrosine phosphorylation of the intracellular adapter protein Disabled-1 (Dab-1) that instructs neurons to reach their correct laminar position in the cortex. Recent evidence supports a role of Reelin in the control of cell proliferation through the activation of mitogen protein kinase (ERK) pathway. We have previously shown, by immunohistochemistry and stereological analysis, the expression of Reelin in both Glioblastoma (GBM) and in peritumor tissue. Moreover, we reported the expression of Reelin in cancer stem cells isolated from both tumor (GCSC) and peritumor tissue (PCSC), suggesting that this protein might contribute to neurosphere formation. Here we investigated by both Real-Time Polymerase Chain Reaction (RT-PCR) and Western Blotting (WB) analysis the gene and protein expression of Reelin and its intracellular adapter Dab-1 in GCSC and PCSC derived from four different patients. Analysis of gene and protein expression indicates higher level of Reelin and DAB-1 in PCSC compared to GCSC. These data suggest a possible role for Reelin and Dab-1 in the control of cell migration and GBM invasiveness., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

The antiphospholipid syndrome (APS) is a non-inflammatory autoimmune disease clinically characterized by the occurrence of either venous or arterial thrombosis or the presence of specific pregnancy complications. Serological criteria are the persistent presence of antibodies directed against cardiolipin or β2-glycoprotein I (β2-GPI) or a prolonged clotting time due to the presence of lupus anticoagulant in plasma. The precise mechanism behind the pathology of APS is largely unknown. Previous work demonstrated that blood platelets incubated with β2-GPI, dimerized by auto-antibodies or monoclonal antibodies become pro-thrombotic. A recombinant dimeric β2-GPI in which the dimerization domain of clotting factor XI (apple4) was used for dimerization, mimics the pro-thrombotic properties of β2-GPI-anti-β2-GPI complexes. In perfusion studies we found that upon dimerization, β2-GPI binds to the platelet receptors Apolipoprotein receptor E2' and Glycoprotein Ibα (ApoER2' and GPIbα). Platelets express 3 different splice variants of ApoER2 which can all participate in signaling events. The splice variant of ApoER2 on platelets expressed the most abundant and participating the strongest in ApoER2 signaling lacks LDL-binding domains 3, 4, 5 and 6 (ApoER2-750 or ApoER2'). Domain deletion studies showed that the binding site for dimeric β2-GPI resides in the LDL-binding domain I of ApoER2'. LDL-binding domain I was expressed in all three ApoER2 splice variants present on platelets. Solid phase binding assays showed the binding site for dimeric β2-GPI on GPIbα in close proximity of the thrombin biding site. The interaction between dimeric β2-GPI and GPIbα was depending on the presence of Zn2+. Dimeric β2-GPI bound to both ApoER2' and GPIbα via its domain V and the binding site for negatively charged phospholipids and ApoER2' did not overlap. The binding site on dimeric β2-GPI for GPIbα and ApoER2 did not overlap either. We furthermore showed that ApoER2' and GPIbα are present in a receptor complex on platelets independent of the presence of dimeric β2-GPI or cytoskeletal association. To test physiological relevance of the interactions of dimeric β2-GPI with ApoER2' and GPIbα, perfusion studies were performed. The interaction of dimeric β2-GPI with both ApoER2' and GPIbα and subsequent downstream receptor specific signaling events were required for platelet activation and adhesion to dimeric β2-GPI and fibronectin under conditions of flow. The transient dissociation of ApoER2 from its adaptor protein Dab-1 however, was not inhibited when dimeric β2-GPI binding to GPIbα binding was blocked. Also, GPIbα translocation towards the cytoskeleton via its adaptor protein 14-3-3-ζ could not be inhibited by blocking dimeric β2-GPI binding to ApoER2'. These observations suggest the presence of a common pathway downstream of the initial signaling events. This common pathway is currently under investigation. The work presented in this thesis opens new therapeutic windows in the treatment of APS. Molecular understanding of protein-protein interactions and subsequent downstream events will help understanding the mechanisms involved in the pathology of APS. In-vitro, blocking the binding of dimerized β2-GPI to either ApoER2' or GPIbα was sufficient to block the pro-thrombotic phenotype of blood platelets induced by binding of dimeric β2-GPI. Animal studies are currently being used to test efficacy of blocking the interaction of dimeric β2-GPI with either GPIbα or ApoER2'.

The antiphospholipid syndrome (APS) is a non-inflammatory autoimmune disease clinically characterized by the occurrence of either venous or arterial thrombosis or the presence of specific pregnancy complications. Serological criteria are the persistent presence of antibodies directed against cardiolipin or β2-glycoprotein I (β2-GPI) or a prolonged clotting time due to the presence of lupus anticoagulant in plasma. The precise mechanism behind the pathology of APS is largely unknown. Previous work demonstrated that blood platelets incubated with β2-GPI, dimerized by auto-antibodies or monoclonal antibodies become pro-thrombotic. A recombinant dimeric β2-GPI in which the dimerization domain of clotting factor XI (apple4) was used for dimerization, mimics the pro-thrombotic properties of β2-GPI-anti-β2-GPI complexes. In perfusion studies we found that upon dimerization, β2-GPI binds to the platelet receptors Apolipoprotein receptor E2' and Glycoprotein Ibα (ApoER2' and GPIbα). Platelets express 3 different splice variants of ApoER2 which can all participate in signaling events. The splice variant of ApoER2 on platelets expressed the most abundant and participating the strongest in ApoER2 signaling lacks LDL-binding domains 3, 4, 5 and 6 (ApoER2-750 or ApoER2'). Domain deletion studies showed that the binding site for dimeric β2-GPI resides in the LDL-binding domain I of ApoER2'. LDL-binding domain I was expressed in all three ApoER2 splice variants present on platelets. Solid phase binding assays showed the binding site for dimeric β2-GPI on GPIbα in close proximity of the thrombin biding site. The interaction between dimeric β2-GPI and GPIbα was depending on the presence of Zn2+. Dimeric β2-GPI bound to both ApoER2' and GPIbα via its domain V and the binding site for negatively charged phospholipids and ApoER2' did not overlap. The binding site on dimeric β2-GPI for GPIbα and ApoER2 did not overlap either. We furthermore showed that ApoER2' and GPIbα are present in a receptor complex on platelets independent of the presence of dimeric β2-GPI or cytoskeletal association. To test physiological relevance of the interactions of dimeric β2-GPI with ApoER2' and GPIbα, perfusion studies were performed. The interaction of dimeric β2-GPI with both ApoER2' and GPIbα and subsequent downstream receptor specific signaling events were required for platelet activation and adhesion to dimeric β2-GPI and fibronectin under conditions of flow. The transient dissociation of ApoER2 from its adaptor protein Dab-1 however, was not inhibited when dimeric β2-GPI binding to GPIbα binding was blocked. Also, GPIbα translocation towards the cytoskeleton via its adaptor protein 14-3-3-ζ could not be inhibited by blocking dimeric β2-GPI binding to ApoER2'. These observations suggest the presence of a common pathway downstream of the initial signaling events. This common pathway is currently under investigation. The work presented in this thesis opens new therapeutic windows in the treatment of APS. Molecular understanding of protein-protein interactions and subsequent downstream events will help understanding the mechanisms involved in the pathology of APS. In-vitro, blocking the binding of dimerized β2-GPI to either ApoER2' or GPIbα was sufficient to block the pro-thrombotic phenotype of blood platelets induced by binding of dimeric β2-GPI. Animal studies are currently being used to test efficacy of blocking the interaction of dimeric β2-GPI with either GPIbα or ApoER2'.