RESUMEN
BACKGROUND: Free labile hemin acts as a damage-associated molecular pattern during acute and chronic hemolysis and muscle injury, supporting platelet activation and thrombosis. OBJECTIVES: To investigate the anti-thrombotic potential of hydroxychloroquine on hemolysis-induced platelet activation and arterial thrombosis. METHODS: The effect of hydroxychloroquine on hemin-induced platelet activation and hemolysis-induced platelet recruitment and aggregation was measured in washed platelets and hemolyzed blood, respectively. Its effect on ferric-chloride (FeCl3)-induced arterial thrombosis and lung perfusion following hemin injection was assessed in wild-type mice. RESULTS: Erythrocyte lysis and endothelial cell activation cooperatively supported platelet aggregation and thrombosis at arterial shear stress. This thrombotic effect was reversed by hydroxychloroquine. In a purified system, hydroxychloroquine inhibited platelet build-up on immobilized von Willebrand factor in hemolyzed blood without altering initial platelet recruitment. Hydroxychloroquine inhibited hemin-induced platelet activation and phosphatidylserine exposure independently of reactive oxygen species generation. In the presence of hemin, hydroxychloroquine did not alter glycoprotein VI shedding but reduced C-type-lectin-like-2 expression on platelets. In vivo, hydroxychloroquine reversed pulmonary perfusion decline induced by exogenous administration of hemin. In arterial thrombosis models, hydroxychloroquine inhibited ferric-chloride-induced thrombosis in the carotid artery and reduced von Willebrand factor accumulation in the thrombi. CONCLUSION: Hydroxychloroquine inhibited hemolysis-induced arterial thrombosis ex vivo and improved pulmonary perfusion in hemin-treated mice, supporting a potential benefit of its use as an adjuvant therapy in hemolytic diseases to limit arterial thrombosis and to improve organ perfusion.
Asunto(s)
Hemina , Hemólisis , Hidroxicloroquina , Pulmón , Activación Plaquetaria , Trombosis , Animales , Hidroxicloroquina/farmacología , Hemólisis/efectos de los fármacos , Hemina/farmacología , Trombosis/tratamiento farmacológico , Trombosis/sangre , Pulmón/efectos de los fármacos , Pulmón/irrigación sanguínea , Activación Plaquetaria/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Agregación Plaquetaria/efectos de los fármacos , Compuestos Férricos , Humanos , Masculino , Cloruros , Modelos Animales de Enfermedad , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Factor de von Willebrand/metabolismoRESUMEN
Cytokinesis depends on a contractile actomyosin ring in many eukaryotes [1-3]. Myosin II is a key component of the actomyosin ring, although whether it functions as a motor or as an actin cross-linker to exert its essential role is disputed [1, 4, 5]. In Schizosaccharomyces pombe, the myo2-E1 mutation affects the upper 50 kDa sub-domain of the myosin II heavy chain, and cells carrying this lethal mutation are defective in actomyosin ring assembly at the non-permissive temperature [6, 7]. myo2-E1 also affects actomyosin ring contraction when rings isolated from permissive temperature-grown cells are incubated with ATP [8]. Here we report isolation of a compensatory suppressor mutation in the lower 50 kDa sub-domain (myo2-E1-Sup1) that reverses the inability of myo2-E1 to form colonies at the restrictive temperature. myo2-E1-Sup1 is capable of assembling normal actomyosin rings, although rings isolated from myo2-E1-Sup1 are defective in ATP-dependent contraction in vitro. Furthermore, the product of myo2-E1-Sup1 does not translocate actin filaments in motility assays in vitro. Superimposition of myo2-E1 and myo2-E1-Sup1 on available rigor and blebbistatin-bound myosin II structures suggests that myo2-E1-Sup1 may represent a novel actin translocation-defective allele. Actomyosin ring contraction and viability of myo2-E1-Sup1 cells depend on the late cytokinetic S. pombe myosin II isoform, Myp2p, a non-essential protein that is normally dispensable for actomyosin ring assembly and contraction. Our work reveals that Myo2p may function in two different and essential modes during cytokinesis: a motor activity-independent form that can promote actomyosin ring assembly and a motor activity-dependent form that supports ring contraction.
Asunto(s)
Miosina Tipo II/fisiología , Proteínas de Schizosaccharomyces pombe/fisiología , Schizosaccharomyces/fisiología , Citoesqueleto de Actina/metabolismo , Actomiosina/fisiología , CitocinesisRESUMEN
Removal of intron regions from pre-messenger RNA (pre-mRNA) requires spliceosome assembly with pre-mRNA, then subsequent spliceosome remodeling to allow activation for the two steps of intron removal. Spliceosome remodeling is carried out through the action of DExD/H-box ATPases that modulate RNA-RNA and protein-RNA interactions. The ATPase Prp16 remodels the spliceosome between the first and second steps of splicing by catalyzing release of first step factors Yju2 and Cwc25 as well as destabilizing U2-U6 snRNA helix I. How Prp16 destabilizes U2-U6 helix I is not clear. We show that the NineTeen Complex (NTC) protein Cwc2 displays genetic interactions with the U6 ACAGAGA, the U6 internal stem loop (ISL) and the U2-U6 helix I, all RNA elements that form the spliceosome active site. We find that one function of Cwc2 is to stabilize U2-U6 snRNA helix I during splicing. Cwc2 also functionally cooperates with the NTC protein Isy1/NTC30. Mutation in Cwc2 can suppress the cold sensitive phenotype of the prp16-302 mutation indicating a functional link between Cwc2 and Prp16. Specifically the prp16-302 mutation in Prp16 stabilizes Cwc2 interactions with U6 snRNA and destabilizes Cwc2 interactions with pre-mRNA, indicating antagonistic functions of Cwc2 and Prp16. We propose that Cwc2 is a target for Prp16-mediated spliceosome remodeling during pre-mRNA splicing.
Asunto(s)
Adenosina Trifosfatasas/genética , ARN Helicasas/genética , Precursores del ARN/metabolismo , Empalme del ARN , ARN Mensajero/metabolismo , ARN Nuclear Pequeño/química , Proteínas de Unión al ARN/genética , Proteínas de Saccharomyces cerevisiae/genética , Mutación , Conformación de Ácido Nucleico , Factores de Empalme de ARN , ARN Nuclear Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The NineTeen Complex (NTC) of proteins associates with the spliceosome during pre-mRNA splicing and is essential for both steps of intron removal. The NTC and other NTC-associated proteins are recruited to the spliceosome where they participate in regulating the formation and progression of essential spliceosome conformations required for the two steps of splicing. It is now clear that the NTC is an integral component of active spliceosomes from yeast to humans and provides essential support for the spliceosomal snRNPs (small nuclear ribonucleoproteins). In the present article, we discuss the identification and characterization of the yeast NTC and review recent work in yeast that supports the essential role for this complex in the regulation and fidelity of splicing.
Asunto(s)
Precursores del ARN/metabolismo , Empalme del ARN/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Empalmosomas/química , Empalmosomas/fisiología , Humanos , Modelos Biológicos , Conformación Molecular , Factores de Empalme de ARN , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Empalmosomas/metabolismoRESUMEN
Efficient and general procedures have been developed for the solution-phase preparation of substituted morpholine derivatives, and a library has been produced around generic structure 1. This library was designed with proprietary modeling software for use as a general screening library. The 30 R1 reagents were phenols, and the 275 R2 reagents were taken from five different reagent classes, giving a variety of product classes in the final library of 8250 potential products. All of the library members were generated from a common intermediate, mesylate (5), which was synthesized efficiently, in bulk, in three steps from N-benzylethanolamine (2). High-throughput chemistry using robotics was carried out to produce the 7907 library members, which were individually characterized by reversed-phase LC/MS analysis.