Site specific replacements of a single loop nucleoside with a dibenzyl linker may switch the activity of TBA from anticoagulant to antiproliferative

Many antiproliferative G-quadruplexes (G4s) arise from the folding of GT-rich strands. Among these, the Thrombin Binding Aptamer (TBA), as a rare example, adopts a monomolecular well-defined G4 structure. Nevertheless, the potential anticancer properties of TBA are severely hampered by its anticoagulant action and, consequently, no related studies have appeared so far in the literature. We wish to report here that suitable chemical modifications in the TBA sequence can preserve its antiproliferative over anticoagulant activity. Particularly, we replaced one residue of the TT or TGT loops with a dibenzyl linker to develop seven new quadruplex-forming TBA based sequences (TBA-bs), which were studied for their structural (CD, CD melting, 1D NMR) and biological (fibrinogen, PT and MTT assays) properties. The three-dimensional structures of the TBA-bs modified at T13 (TBA-bs13) or T12 (TBA-bs12), the former endowed with selective antiproliferative activity, and the latter acting as potently as TBA in both coagulation and MTT assays, were further studied by 2D NMR restrained molecular mechanics. The comparative structural analyses indicated that neither the stability, nor the topology of the G4s, but the different localization of the two benzene rings of the linker was responsible for the loss of the antithrombin activity for TBA-bs13

Identification of the cellular targets and mechanism of action of the glycerophosphoinositols

The glycerophosphoinositols (GPIs) are ubiquitous, bioactive metabolites that are produced by the phospholipase A2 IVa activity on the membrane phosphoinositides. Glycerophosphoinositol (GroPIns) and glycerophosphoinositol 4-phosphate (GroPIns4P) are the most active and well studied of the GPIs. When added exogenously, the GPls can enter cells and have multiple effects, such as modulation of actin cytoskeleton organisation in fibroblasts and reduction of the invasive potential of metastatic cells. To gain insights into the molecular mechanisms of the effects of the GPIs, I set out to identify their protein targets. Therefore, a proteomic approach based on high- throughput differential-LC-MS/MS analysis was used with the modified GPIs: with a biotin moiety bound to their glycerol backbone. The targets identified include proteins involved in cell signalling, cytoskeleton organisation, protein folding and metabolic processes. Among these, I focussed my attention on Src-homology phosphatase-l (Shpl), a well-known regulator of Src activation, as it might be related to the reported signalling pathway leading to GroPlns4P-mediated modulation of the actin cytoskeleton, which involves Src. Based on biochemical data, I provide evidence of a direct interaction between Shp 1 and both GroPIns4P .and GroPIns. During my project, Shpl was studied in the context of GroPIns4P-induced membrane ruffle formation in NIH 3T3 fibroblasts, where inhibition of enzymatic activity of Shp 1 completely abolished GroPIns4P-mediated reorganisation of the actin cytoskeleton. In addition, I have also shown that GroPIns4P treatment results in the dephosphorylation of the inhibitory tyrosine residue of Src, as a consequence of increased binding between Shp 1 and Src. A role for Shp 1 is also demonstrated in GroPIns-mediated inhibition of tumour cell invasion. In A375MM melanoma cells, GroPIns treatment results in inhibition of extracellular matrix degradation, and this activity is suppressed when the inactive mutant form of Shp 1 is expressed, while it is essentially unaffected by expression of the native enzyme. In agreement with these results, a lack of effect of GroPIns was observed also in Shpl knock-down cells. In conclusion, my PhD project has led to the definition of Shpl as the first direct GPI target that has been identified to date, and it reveals a positive role for Shpl in the mechanisms of action of both GroPIns4 P and GroPIns.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Functional organization of the endoplasmic reticulum dictates the susceptibility of target cells to arsenite-induced mitochondrial superoxide formation, mitochondrial dysfunction and apoptosis

Arsenite induces many critical effects associated with the formation of reactive oxygen species (ROS) through different mechanisms. We focused on Ca2+-dependent mitochondrial superoxide (mitoO2-.) formation and addressed questions on the effects of low concentrations of arsenite on the mobilization of the cation from the endoplasmic reticulum and the resulting mitochondrial accumulation. Using various differentiated and undifferentiated cell types uniquely expressing the inositol-1, 4, 5-triphosphate receptor (IP3R), or both the IP3R and the ryanodine receptor (RyR), we determined that expression of this second Ca2+ channel is an absolute requirement for mitoO2-. formation and for the ensuing mitochondrial dysfunction and downstream apoptosis. In arsenite-treated cells, RyR was recruited after IP3R stimulation and agonist studies provided an indirect indication for a close apposition between RyR and mitochondria. It was also interesting to observe that arsenite fails to promote mitochondrial Ca2+ accumulation, mitoO2-. formation and mitochondrial toxicity in RyR-devoid cells, in which the IP3R is in close contact with the mitochondria. We therefore conclude that low dose arsenite-induced mitoO2- formation, and the resulting mitochondrial dysfunction and toxicity, are prerequisite of cell types expressing the RyR in close apposition with mitochondria

Glycerophosphoinositol Promotes Apoptosis of Chronic Lymphocytic Leukemia Cells by Enhancing Bax Expression and Activation

An imbalance in the expression of pro- and anti-apoptotic members of the Bcl-2 family of apoptosis-regulating proteins is one of the main biological features of CLL, highlighting these proteins as therapeutic targets for treatment of this malignancy. Indeed, the Bcl-2 inhibitor Venetoclax is currently used for both first-line treatment and treatment of relapsed or refractory CLL. An alternative avenue is the transcriptional modulation of Bcl-2 family members to tilt their balance towards apoptosis. Glycerophosphoinositol (GroPIns) is a biomolecule generated from membrane phosphoinositides by the enzymes phospholipase A(2) and lysolipase that pleiotropically affects key cellular functions. Mass-spectrometry analysis of GroPIns interactors recently highlighted the ability of GroPIns to bind to the non-receptor tyrosine phosphatase SHP-1, a known promoter of Bax expression, suggesting that GroPIns might correct the Bax expression defect in CLL cells, thereby promoting their apoptotic demise. To test this hypothesis, we cultured CLL cells in the presence of GroPIns, alone or in combination with drugs commonly used for treatment of CLL. We found that GroPIns alone increases Bax expression and apoptosis in CLL cells and enhances the pro-apoptotic activity of drugs used for CLL treatment in a SHP-1 dependent manner. Interestingly, among GroPIns interactors we found Bax itself. Short-term treatments of CLL cells with GroPIns induce Bax activation and translocation to the mitochondria. Moreover, GroPIns enhances the pro-apoptotic activity of Venetoclax and Fludarabine in CLL cells. These data provide evidence that GroPIns exploits two different pathways converging on Bax to promote apoptosis of leukemic cells and pave the way to new studies aimed at testing GroPIns in combination therapies for the treatment of CLL

The natural phosphoinositide derivative glycerophosphoinositol inhibits the lipopolysaccharide-induced inflammatory and thrombotic responses

Inflammatory responses are elicited through lipid products of phospholipase A(2) activity that acts on the membrane phospholipids, including the phosphoinositides, to form the proinflammatory arachidonic acid and, in parallel, the glycerophosphoinositols. Here, we investigate the role of the glycerophosphoinositol in the inflammatory response. We show that it is part of a negative feedback loop that limits proinflammatory and prothrombotic responses in human monocytes stimulated with lipopolysaccharide. This inhibition is exerted both on the signaling cascade initiated by the lipopolysaccharide with the glycerophosphoinositol-dependent decrease in IB kinase /, p38, JNK, and Erk1/2 kinase phosphorylation and at the nuclear level with decreased NF-B translocation and binding to inflammatory gene promoters. In a model of endotoxemia in the mouse, treatment with glycerophosphoinositol reduced TNF- synthesis, which supports the concept that glycerophosphoinositol inhibits the de novo synthesis of proinflammatory and prothrombotic compounds and might thus have a role as an endogenous mediator in the resolution of inflammation. As indicated, this effect of glycerophosphoinositol can also be exploited in the treatment of manifestations of severe inflammation by exogenous administration of the compound

Clinical, Genetic, and Histological Characterization of Patients with Rare Neuromuscular and Mitochondrial Diseases Presenting with Different Cardiomyopathy Phenotypes

: Cardiomyopathies are mostly determined by genetic mutations affecting either cardiac muscle cell structure or function. Nevertheless, cardiomyopathies may also be part of complex clinical phenotypes in the spectrum of neuromuscular (NMD) or mitochondrial diseases (MD). The aim of this study is to describe the clinical, molecular, and histological characteristics of a consecutive cohort of patients with cardiomyopathy associated with NMDs or MDs referred to a tertiary cardiomyopathy clinic. Consecutive patients with a definitive diagnosis of NMDs and MDs presenting with a cardiomyopathy phenotype were described. Seven patients were identified: two patients with ACAD9 deficiency (Patient 1 carried the c.1240C>T (p.Arg414Cys) homozygous variant in ACAD9; Patient 2 carried the c.1240C>T (p.Arg414Cys) and the c.1646G>A (p.Ar549Gln) variants in ACAD9); two patients with MYH7-related myopathy (Patient 3 carried the c.1325G>A (p.Arg442His) variant in MYH7; Patient 4 carried the c.1357C>T (p.Arg453Cys) variant in MYH7); one patient with desminopathy (Patient 5 carried the c.46C>T (p.Arg16Cys) variant in DES); two patients with mitochondrial myopathy (Patient 6 carried the m.3243A>G variant in MT-TL1; Patient 7 carried the c.253G>A (p.Gly85Arg) and the c.1055C>T (p.Thr352Met) variants in MTO1). All patients underwent a comprehensive cardiovascular and neuromuscular evaluation, including muscle biopsy and genetic testing. This study described the clinical phenotype of rare NMDs and MDs presenting as cardiomyopathies. A multidisciplinary evaluation, combined with genetic testing, plays a main role in the diagnosis of these rare diseases, and provides information about clinical expectations, and guides management

Clinical, Genetic, and Histological Characterization of Patients with Rare Neuromuscular and Mitochondrial Diseases Presenting with Different Cardiomyopathy Phenotypes

: Cardiomyopathies are mostly determined by genetic mutations affecting either cardiac muscle cell structure or function. Nevertheless, cardiomyopathies may also be part of complex clinical phenotypes in the spectrum of neuromuscular (NMD) or mitochondrial diseases (MD). The aim of this study is to describe the clinical, molecular, and histological characteristics of a consecutive cohort of patients with cardiomyopathy associated with NMDs or MDs referred to a tertiary cardiomyopathy clinic. Consecutive patients with a definitive diagnosis of NMDs and MDs presenting with a cardiomyopathy phenotype were described. Seven patients were identified: two patients with ACAD9 deficiency (Patient 1 carried the c.1240C>T (p.Arg414Cys) homozygous variant in ACAD9; Patient 2 carried the c.1240C>T (p.Arg414Cys) and the c.1646G>A (p.Ar549Gln) variants in ACAD9); two patients with MYH7-related myopathy (Patient 3 carried the c.1325G>A (p.Arg442His) variant in MYH7; Patient 4 carried the c.1357C>T (p.Arg453Cys) variant in MYH7); one patient with desminopathy (Patient 5 carried the c.46C>T (p.Arg16Cys) variant in DES); two patients with mitochondrial myopathy (Patient 6 carried the m.3243A>G variant in MT-TL1; Patient 7 carried the c.253G>A (p.Gly85Arg) and the c.1055C>T (p.Thr352Met) variants in MTO1). All patients underwent a comprehensive cardiovascular and neuromuscular evaluation, including muscle biopsy and genetic testing. This study described the clinical phenotype of rare NMDs and MDs presenting as cardiomyopathies. A multidisciplinary evaluation, combined with genetic testing, plays a main role in the diagnosis of these rare diseases, and provides information about clinical expectations, and guides management

A signalling cascade involving receptor-activated phospholipase A2, glycerophosphoinositol 4-phosphate, Shp1 and Src in the activation of cell motility

Background: Shp1, a tyrosine-phosphatase-1 containing the Src-homology 2 (SH2) domain, is involved in inflammatory and immune reactions, where it regulates diverse signalling pathways, usually by limiting cell responses through dephosphorylation of target molecules. Moreover, Shp1 regulates actin dynamics. One Shp1 target is Src, which controls many cellular functions including actin dynamics. Src has been previously shown to be activated by a signalling cascade initiated by the cytosolic-phospholipase A2 (cPLA2) metabolite glycerophosphoinositol 4-phosphate (GroPIns4P), which enhances actin polymerisation and motility. While the signalling cascade downstream Src has been fully defined, the mechanism by which GroPIns4P activates Src remains unknown. Methods: Affinity chromatography, mass spectrometry and co-immunoprecipitation studies were employed to identify the GroPIns4P-interactors; among these Shp1 was selected for further analysis. The specific Shp1 residues interacting with GroPIns4P were revealed by NMR and validated by site-directed mutagenesis and biophysical methods such as circular dichroism, isothermal calorimetry, fluorescence spectroscopy, surface plasmon resonance and computational modelling. Morphological and motility assays were performed in NIH3T3 fibroblasts. Results: We find that Shp1 is the direct cellular target of GroPIns4P. GroPIns4P directly binds to the Shp1-SH2 domain region (with the crucial residues being Ser 118, Arg 138 and Ser 140) and thereby promotes the association between Shp1 and Src, and the dephosphorylation of the Src-inhibitory phosphotyrosine in position 530, resulting in Src activation. As a consequence, fibroblast cells exposed to GroPIns4P show significantly enhanced wound healing capability, indicating that GroPIns4P has a stimulatory role to activate fibroblast migration. GroPIns4P is produced by cPLA2 upon stimulation by diverse receptors, including the EGF receptor. Indeed, endogenously-produced GroPIns4P was shown to mediate the EGF-induced cell motility. Conclusions: This study identifies a so-far undescribed mechanism of Shp1/Src modulation that promotes cell motility and that is dependent on the cPLA2 metabolite GroPIns4P. We show that GroPIns4P is required for EGF-induced fibroblast migration and that it is part of a cPLA2/GroPIns4P/Shp1/Src cascade that might have broad implications for studies of immune-inflammatory response and cancer. [Figure not available: see fulltext.

Asthma in patients admitted to emergency department for COVID-19: prevalence and risk of hospitalization

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