RESUMO
This work identifies the combination of enzymatic transfer and click labeling as an efficient method for the site-specific tagging of RNA molecules for biophysical studies. A double-activated analog of the ubiquitous co-substrate S-adenosyl-l-methionine was employed to enzymatically transfer a five carbon chain containing a terminal alkynyl moiety onto RNA. The tRNA:methyltransferase Trm1 transferred the extended alkynyl moiety to its natural target, the N2 of guanosine 26 in tRNA(Phe). LC/MS and LC/MS/MS techniques were used to detect and characterize the modified nucleoside as well as its cycloaddition product with a fluorescent azide. The latter resulted from a labeling reaction via Cu(I)-catalyzed azide-alkyne 1,3-cycloaddition click chemistry, producing site-specifically labeled RNA whose suitability for single molecule fluorescence experiments was verified in fluorescence correlation spectroscopy experiments.
Assuntos
Química Click , RNA de Transferência de Fenilalanina/química , tRNA Metiltransferases/metabolismo , Alcinos/metabolismo , Sequência de Bases , Corantes Fluorescentes , Dados de Sequência Molecular , Compostos Orgânicos , RNA de Transferência de Fenilalanina/metabolismo , S-Adenosilmetionina/análogos & derivados , S-Adenosilmetionina/química , Espectrometria de FluorescênciaRESUMO
Common dietary components including vitamins A and D, omega-3 and probiotics are now widely accepted to be essential to protect against many diseases with an inflammatory nature. On the other hand, high-fat diets are documented to exert multiple deleterious effects, including fatty liver diseases. Here we discuss the effect of dietary components on regulatory T cell (Treg) homeostasis, a central element of the immune system to prevent chronic tissue inflammation. Accordingly, evidence on the impact of dietary components on diseases in which Tregs play an influential role will be discussed. We will review chronic tissue-specific autoimmune and inflammatory conditions such as inflammatory bowel disease, type 1 diabetes mellitus, multiple sclerosis, rheumatoid arthritis and allergies among chronic diseases where dietary factors could have a direct influence via modulation of Tregs homeostasis and functions.
Assuntos
Suplementos Nutricionais , Linfócitos T Reguladores/imunologia , Animais , Dieta Hiperlipídica/efeitos adversos , Ácidos Graxos Ômega-3/administração & dosagem , Ácidos Graxos Ômega-3/uso terapêutico , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Vitaminas/administração & dosagem , Vitaminas/uso terapêuticoRESUMO
Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs), which are formed from multiple copies of ~30 different nucleoporins (Nups) and inserted into the double nuclear membrane. Many of these Nups are organized into subcomplexes, of which the Y-shaped Nup84 complex is the major constituent of the nuclear and cytoplasmic rings. The Nup82-Nup159-Nsp1 complex is another module that, however, is only assembled into the cytoplasmic ring. By means of crosslinking mass spectrometry, biochemical reconstitution, and molecular modeling, we identified a short linear motif in the unstructured N-terminal region of Chaetomium thermophilum Nup145C, a subunit of the Y-complex, that is sufficient to recruit the Nup82 complex, but only in its assembled state. This finding points to a more general mechanism that short linear motifs in structural Nups can act as sensors to cooperatively connect pre-assembled NPC modules, thereby facilitating the formation and regulation of the higher-order NPC assembly.
Assuntos
Chaetomium/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Poro Nuclear/metabolismo , Motivos de Aminoácidos , Chaetomium/química , Chaetomium/genética , Proteínas Fúngicas/genética , Modelos Moleculares , Poro Nuclear/química , Poro Nuclear/genética , Ligação ProteicaRESUMO
Nuclear pore complexes (NPCs) are 110-megadalton assemblies that mediate nucleocytoplasmic transport. NPCs are built from multiple copies of ~30 different nucleoporins, and understanding how these nucleoporins assemble into the NPC scaffold imposes a formidable challenge. Recently, it has been shown how the Y complex, a prominent NPC module, forms the outer rings of the nuclear pore. However, the organization of the inner ring has remained unknown until now. We used molecular modeling combined with cross-linking mass spectrometry and cryo-electron tomography to obtain a composite structure of the inner ring. This architectural map explains the vast majority of the electron density of the scaffold. We conclude that despite obvious differences in morphology and composition, the higher-order structure of the inner and outer rings is unexpectedly similar.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Poro Nuclear/ultraestrutura , Transporte Ativo do Núcleo Celular , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Células HeLa , Humanos , Espectrometria de Massas , Modelos Moleculares , Matriz Nuclear/metabolismo , Matriz Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genéticaRESUMO
Nuclear pore complexes (NPCs) mediate transport between the nucleus and cytoplasm. NPCs are composed of â¼30 nucleoporins (Nups), most of which are organized in stable subcomplexes. How these modules are interconnected within the large NPC framework has been unknown. Here we show a mechanism of how supercomplexes can form between NPC modules. The Nup192 inner-pore-ring complex serves as a seed to which the Nup82 outer-ring complex and Nsp1 channel complex are tethered. The linkage between these subcomplexes is generated by short sequences within linker Nups. The conserved Nup145N is the most versatile connector of NPC modules because it has three discrete binding sites for Nup192, Nup170 and Nup82. We assembled a large part of a Chaetomium thermophilum NPC protomer in vitro, providing a step forward toward the reconstitution of the entire NPC.