RESUMO
Myopathies are a clinically and etiologically heterogeneous group of disorders that can range from limb girdle muscular dystrophy (LGMD) to syndromic forms with associated features including intellectual disability. Here, we report the identification of mutations in transport protein particle complex 11 (TRAPPC11) in three individuals of a consanguineous Syrian family presenting with LGMD and in five individuals of Hutterite descent presenting with myopathy, infantile hyperkinetic movements, ataxia, and intellectual disability. By using a combination of whole-exome or genome sequencing with homozygosity mapping, we identified the homozygous c.2938G>A (p.Gly980Arg) missense mutation within the gryzun domain of TRAPPC11 in the Syrian LGMD family and the homozygous c.1287+5G>A splice-site mutation resulting in a 58 amino acid in-frame deletion (p.Ala372_Ser429del) in the foie gras domain of TRAPPC11 in the Hutterite families. TRAPPC11 encodes a component of the multiprotein TRAPP complex involved in membrane trafficking. We demonstrate that both mutations impair the binding ability of TRAPPC11 to other TRAPP complex components and disrupt the Golgi apparatus architecture. Marker trafficking experiments for the p.Ala372_Ser429del deletion indicated normal ER-to-Golgi trafficking but dramatically delayed exit from the Golgi to the cell surface. Moreover, we observed alterations of the lysosomal membrane glycoproteins lysosome-associated membrane protein 1 (LAMP1) and LAMP2 as a consequence of TRAPPC11 dysfunction supporting a defect in the transport of secretory proteins as the underlying pathomechanism.
Assuntos
Deficiência Intelectual/genética , Transtornos dos Movimentos/genética , Doenças Musculares/genética , Distrofia Muscular do Cíngulo dos Membros/genética , Deleção de Sequência , Proteínas de Transporte Vesicular/metabolismo , Adolescente , Adulto , Ataxia/genética , Mapeamento Cromossômico , Consanguinidade , Creatina Quinase/sangue , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Exoma , Feminino , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Homozigoto , Humanos , Proteína 2 de Membrana Associada ao Lisossomo , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/metabolismo , Masculino , Transtornos dos Movimentos/patologia , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Doenças Musculares/patologia , Distrofia Muscular do Cíngulo dos Membros/patologia , Linhagem , Ligação Proteica , Transporte Proteico , Sítios de Splice de RNA , Síria , Proteínas de Transporte Vesicular/genética , Adulto JovemRESUMO
Acollinearity of annihilation photons (APA) introduces spatial blur in positron emission tomography (PET) imaging. This phenomenon increases proportionally with the scanner diameter and it has been shown to follow a Gaussian distribution. This last statement can be interpreted in two ways: the magnitude of the acollinearity angle, or the angular deviation of annihilation photons from perfect collinearity. As the former constitutes the partial integral of the latter, a misinterpretation could have significant consequences on the resulting spatial blurring. Previous research investigating the impact of APA in PET imaging has assumed the Gaussian nature of its angular deviation, which is consistent with experimental results. However, a comprehensive analysis of several simulation software packages for PET data acquisition revealed that the magnitude of APA was implemented as a Gaussian distribution. We quantified the impact of this misinterpretation of APA by comparing simulations obtained with GATE, which is one of these simulation programs, to an in-house modification of GATE that models APA deviation as following a Gaussian distribution. We show that the APA misinterpretation not only alters the spatial blurring profile in image space, but also considerably underestimates the impact of APA on spatial resolution. For an ideal PET scanner with a diameter of 81 cm, the APA point source response simulated under the first interpretation has a cusp shape with 0.4 mm FWHM. This is significantly different from the expected Gaussian point source response of 2.1 mm FWHM reproduced under the second interpretation. Although this misinterpretation has been found in several PET simulation tools, it has had a limited impact on the simulated spatial resolution of current PET scanners due to its small magnitude relative to the other factors. However, the inaccuracy it introduces in estimating the overall spatial resolution of PET scanners will increase as the performance of newer devices improves.
RESUMO
Self-assembled micelles of amphiphilic block copolymers (ABPs) with stimuli-responsive degradation (SRD) properties have a great promise as nanotherapeutics exhibiting enhanced release of encapsulated therapeutics into targeted cells. Here, thiol-responsive degradable micelles based on a new ABP consisting of a pendant disulfide-labeled methacrylate polymer block (PHMssEt) and a hydrophilic poly(ethylene oxide) (PEO) block were investigated as effective intracellular nanocarriers of anticancer drugs. In response to glutathione (GSH) as a cellular trigger, the cleavage of pendant disulfide linkages in hydrophobic PHMssEt blocks of micellar cores caused the destabilization of self-assembled micelles due to change in hydrophobic/hydrophilic balance. Such GSH-triggered micellar destabilization changed their size distribution with an appearance of large aggregates and led to enhanced release of encapsulated anticancer drugs. Cell culture results from flow cytometry and confocal laser scanning microscopy for cellular uptake as well as cell viability measurements for high anticancer efficacy suggest that new GSH-responsive degradable PEO-b-PHMssEt micelles offer versatility in multifunctional drug delivery applications.
Assuntos
Dissulfetos/química , Portadores de Fármacos , Glutationa/administração & dosagem , Metacrilatos/química , Nanoestruturas , Polietilenoglicóis/química , Doxorrubicina/administração & dosagem , Citometria de Fluxo , Glutationa/química , Células HEK293 , Humanos , Micelas , Microscopia Eletrônica de Transmissão , Espectrofotometria UltravioletaRESUMO
Transport of mRNA is an efficient mechanism to target proteins to specific regions of a cell. Although it is well documented that mRNAs are transported in ribonucleoprotein (RNP) complexes, several of the mechanisms involved in complex formation and localization are poorly understood. Staufen (Stau) 1, a double-stranded RNA-binding protein, is a well accepted marker of mRNA transport complexes. In this manuscript, we provide evidence that Stau1 self-associates in live cells using immunoprecipitation and bioluminescence resonance energy transfer (BRET) assays. The double-stranded RNA-binding domains dsRBD3 and dsRBD4 contributed about half of the signal, suggesting that Stau1 RNA-binding activity is involved in Stau1 self-association. Protein-protein interaction also occurred, via dsRBD5 and dsRBD2, as shown by in vitro pull-down, yeast two-hybrid, and BRET assays in live cells. Interestingly, Stau1 self-association contributes to the formation of oligomeric complexes as evidenced by the coexpression of split Renilla luciferase halves covalently linked to Stau1 in a protein complementation assay (PCA) combined with a BRET assay with Stau1-YFP. Moreover, we showed that these higher-order Stau1-containing complexes carry RNAs when the RNA stain SYTO 14 was used as the energy acceptor in the PCA/BRET assay. The oligomeric composition of Stau1-containing complexes and the presence of specific mRNAs have been confirmed by biochemical approaches involving two successive immunoprecipitations of Stau1-tagged molecules followed by qRT-PCR amplification. Altogether, these results indicate that Stau1 self-associates in mRNPs via its multiple functional domains that can select mRNAs to be transported and establish protein-protein interaction.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Transporte de RNA , Proteínas de Ligação a RNA/metabolismo , Linhagem Celular , Humanos , Imunoprecipitação , Medições Luminescentes , Multimerização Proteica , Ribonucleoproteínas/metabolismoRESUMO
Leukotrienes (LT) and platelet-activating factor (PAF) are important lipid mediators of inflammation. We and others reported previously that autacoids such as adenosine, histamine, prostaglandin E2, and beta-adrenergic agents inhibit LT biosynthesis in activated human polymorphonuclear leukocytes (PMN). In this study, we demonstrate that CGS-21680 (a selective agonist of the adenosine A2A receptor) and histamine also potently inhibit PAF biosynthesis in agonist [formyl Met-Leu-Phe (fMLP)]- and thapsigargin-activated human PMN. The observed inhibitions of PAF biosynthesis were reversed effectively by exogenous 1-O-alkyl-lyso-sn-glyceryl-3-phosphocholine (lyso-PAF), suggesting that these effects of CGS-21680 and histamine implicate the blockade of cytosolic phospholipase A2alpha (cPLA2alpha) activity and lyso-PAF release and that the acetyl-coenzyme A/lyso-PAF acetyl transferase is not inhibited by the autacoids. Accordingly, the cPLA2alpha inhibitor pyrrophenone completely blocked PAF formation, and lyso-PAF similarly prevented this effect of pyrrophenone. The inhibitory effects of CGS-21680 and histamine on PAF biosynthesis were prevented by the protein kinase A inhibitor H-89, supporting roles for the Gs -coupled receptors A2A and H2, respectively, and cyclic adenosine monophosphate in the inhibitory mechanism. The fMLP-induced phosphorylations of p38 and extracellular signal-regulated kinase 1/2 were not altered significantly by the CGS-21680, indicating that inhibition of these kinases is not involved in the inhibitory effect of the adenosine A2A receptor ligand on LT and PAF biosynthesis. These data further emphasize the multiple and potent inhibitory effects of adenosine and histamine on leukocyte functions, in particular, on the biosynthesis of two classes of important lipid mediators and their putative regulatory roles in immune processes in health and diseases.
Assuntos
Adenosina/metabolismo , Histamina/metabolismo , Neutrófilos/metabolismo , Fosfolipases A/metabolismo , Fator de Ativação de Plaquetas/análogos & derivados , Fator de Ativação de Plaquetas/antagonistas & inibidores , Adenosina/análogos & derivados , Adenosina/farmacologia , Agonistas do Receptor A2 de Adenosina , Anti-Hipertensivos/farmacologia , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Fosfolipases A2 do Grupo IV , Histamina/farmacologia , Humanos , Leucotrienos/biossíntese , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Lipídeos de Membrana/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Fenetilaminas/farmacologia , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/imunologia , Fosfolipídeos/metabolismo , Fosforilação/efeitos dos fármacos , Fator de Ativação de Plaquetas/biossíntese , Fator de Ativação de Plaquetas/metabolismo , Pirrolidinas/farmacologia , Receptor A2A de Adenosina/metabolismo , Receptores de Formil Peptídeo/efeitos dos fármacos , Receptores de Formil Peptídeo/metabolismo , Receptores Histamínicos H2/efeitos dos fármacos , Receptores Histamínicos H2/metabolismoRESUMO
Variations of dopamine (DA) levels induced by drugs of abuse or in the context of Parkinson's disease modulate the number of dendritic spines in medium spiny neurons (MSNs) of the striatum, showing that DA plays a major role in the structural plasticity of MSNs. However, little is presently known regarding early spine development in MSNs occurring before the arrival of cortical inputs and in particular about the role of DA and D1 (D1R) and D2 (D2R) DA receptors. A cell culture model reconstituting early cellular interactions between MSNs, intrinsic cholinergic interneurons and DA neurons was used to study the role of DA in spine formation. After 5 or 10 days in vitro, the presence of DA neurons increased the number of immature spine-like protrusions. In MSN monocultures, chronic activation of D1R or D2R also increased the number of spines and spinophilin expression in MSNs, suggesting a direct role for these receptors. In DA-MSN cocultures, chronic blockade of D1R or D2R reduced the number of dendritic spines. Interestingly, the combined activation or blockade of both D1R and D2R failed to elicit more extensive spine formation, suggesting that both receptors act through a mechanism that is not additive. Finally, we found increased ionotropic glutamate receptor responsiveness and miniature excitatory postsynaptic current (EPSC) frequency in DA-MSN co-cultures, in parallel with a higher number of spines containing PSD-95, suggesting that the newly formed spines present functional post-synaptic machinery preparing the MSNs to receive additional glutamatergic contacts. These results represent a first step in the understanding of how dopamine neurons promote the structural plasticity of MSNs during the development of basal ganglia circuits.
Assuntos
Corpo Estriado/fisiologia , Espinhas Dendríticas/fisiologia , Dopamina/fisiologia , Neurônios Dopaminérgicos/fisiologia , Receptores de Dopamina D1/fisiologia , Receptores de Dopamina D2/fisiologia , Animais , Células Cultivadas , Técnicas de Cocultura , Corpo Estriado/citologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Camundongos , Camundongos Transgênicos , Neurônios/fisiologiaRESUMO
Messenger ribonucleoprotein particles (mRNPs) are used to transport mRNAs along neuronal dendrites to their site of translation. Numerous mRNA-binding and regulatory proteins within mRNPs finely regulate the fate of bound-mRNAs. Their specific combination defines different types of mRNPs that in turn are related to specific synaptic functions. One of these mRNA-binding proteins, Staufen2 (Stau2), was shown to transport dendritic mRNAs along microtubules. Its knockdown expression in neurons was shown to change spine morphology and synaptic functions. To further understand the molecular mechanisms by which Stau2 modulates synaptic function in neurons, it is important to identify and characterize protein co-factors that regulate the fate of Stau2-containing mRNPs. To this end, a proteomic approach was used to identify co-immunoprecipitated proteins in Staufen2-containing mRNPs isolated from embryonic rat brains. The proteomic approach identified mRNA-binding proteins (PABPC1, hnRNP H1, YB1 and hsc70), proteins of the cytoskeleton (alpha- and beta-tubulin) and RUFY3 a poorly characterized protein. While PABPC1 and YB1 associate with Stau2-containing mRNPs through RNAs, hsc70 is directly bound to Stau2 and this interaction is regulated by ATP. PABPC1 and YB1 proteins formed puncta in dendrites of embryonic rat hippocampal neurons. However, they poorly co-localized with Stau2 in the large dendritic complexes suggesting that they are rather components of Stau2-containing mRNA particles. All together, these results represent a further step in the characterization of Stau2-containing mRNPs in neurons and provide new tools to study and understand how Stau2-containing mRNPs are transported, translationally silenced during transport and/or locally expressed according to cell needs.