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1.
Mol Cell ; 84(10): 1980-1994.e8, 2024 May 16.
Artículo en Inglés | MEDLINE | ID: mdl-38759629

RESUMEN

Aggregation of proteins containing expanded polyglutamine (polyQ) repeats is the cytopathologic hallmark of a group of dominantly inherited neurodegenerative diseases, including Huntington's disease (HD). Huntingtin (Htt), the disease protein of HD, forms amyloid-like fibrils by liquid-to-solid phase transition. Macroautophagy has been proposed to clear polyQ aggregates, but the efficiency of aggrephagy is limited. Here, we used cryo-electron tomography to visualize the interactions of autophagosomes with polyQ aggregates in cultured cells in situ. We found that an amorphous aggregate phase exists next to the radially organized polyQ fibrils. Autophagosomes preferentially engulfed this amorphous material, mediated by interactions between the autophagy receptor p62/SQSTM1 and the non-fibrillar aggregate surface. In contrast, amyloid fibrils excluded p62 and evaded clearance, resulting in trapping of autophagic structures. These results suggest that the limited efficiency of autophagy in clearing polyQ aggregates is due to the inability of autophagosomes to interact productively with the non-deformable, fibrillar disease aggregates.


Asunto(s)
Amiloide , Autofagosomas , Autofagia , Proteína Huntingtina , Enfermedad de Huntington , Péptidos , Agregado de Proteínas , Proteína Sequestosoma-1 , Péptidos/metabolismo , Péptidos/química , Péptidos/genética , Humanos , Proteína Huntingtina/metabolismo , Proteína Huntingtina/genética , Proteína Huntingtina/química , Autofagosomas/metabolismo , Autofagosomas/ultraestructura , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genética , Amiloide/metabolismo , Amiloide/química , Amiloide/genética , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Microscopía por Crioelectrón , Animales , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/genética
2.
Nature ; 585(7824): 298-302, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32669707

RESUMEN

Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli1,2. Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as 'red laser'). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease.


Asunto(s)
Nucléolo Celular/enzimología , Nucléolo Celular/genética , ADN Ribosómico/genética , ARN Polimerasa II/metabolismo , ARN no Traducido/biosíntesis , ARN no Traducido/genética , Ribosomas/metabolismo , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Línea Celular Tumoral , Nucléolo Celular/fisiología , ADN Helicasas/metabolismo , ADN Intergénico/genética , Humanos , Enzimas Multifuncionales/metabolismo , Biosíntesis de Proteínas , Estructuras R-Loop , ARN Helicasas/metabolismo , ARN Polimerasa I/antagonistas & inhibidores , ARN Polimerasa I/metabolismo , Ribonucleasa H/metabolismo , Ribosomas/química , Ribosomas/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/patología
3.
Proc Natl Acad Sci U S A ; 107(43): 18398-403, 2010 Oct 26.
Artículo en Inglés | MEDLINE | ID: mdl-20937909

RESUMEN

Arginine methylation modulates diverse cellular processes and represents a molecular signature of germ-line-specific Piwi family proteins. A subset of Tudor domains recognize arginine methylation modifications, but the binding mechanism has been lacking. Here we establish that, like other germ-line Tudor proteins, the ancestral staphylococcal nuclease domain-containing 1 (SND1) polypeptide is expressed and associates with PIWIL1/Miwi in germ cells. We find that human SND1 binds PIWIL1 in an arginine methylation-dependent manner with a preference for symmetrically dimethylated arginine. The entire Tudor domain and a bifurcated SN domain are required for this binding activity, whereas the canonical Tudor domain alone is insufficient for methylarginine ligand binding. Crystal structures show that the intact SND1 extended Tudor domain forms a wide and negatively charged binding groove, which can accommodate distinct symmetrically dimethylated arginine peptides from PIWIL1 in different orientations. This analysis explains how SND1 preferentially recognizes symmetrical dimethylarginine via an aromatic cage and conserved hydrogen bonds, and provides a general paradigm for the binding mechanisms of methylarginine-containing peptides by extended Tudor domains.


Asunto(s)
Proteínas/química , Secuencia de Aminoácidos , Animales , Arginina/química , Proteínas Argonautas , Cristalografía por Rayos X , Endonucleasas , Humanos , Técnicas In Vitro , Masculino , Metilación , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas/genética , Proteínas/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Testículo/metabolismo
4.
J Immunol ; 179(6): 3780-91, 2007 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-17785815

RESUMEN

Activation of macrophages causes increased cell spreading, increased secretion of cytokines and matrix metalloproteinases, and enhanced phagocytosis. The intracellular mechanisms driving the up-regulation of these activities have not been completely clarified. We observe that classical activation of murine resident peritoneal or RAW 264.7 macrophages with a combination of IFN-gamma and LPS induces an increase in stabilized cytoplasmic microtubules (MTs), measured with an anti-acetylated alpha-tubulin Ab. We examined the mechanism of this MT stabilization and find that macrophage activation causes redistribution of the MT plus-end tracking protein, cytoplasmic linker protein-170 (CLIP-170). CLIP-170 is localized at the distal plus-ends of MTs in resting macrophages, but accumulates along the length of MTs in IFN-gamma/LPS-activated cells. A direct involvement of CLIP-170 in MT stabilization has not been thoroughly established. In this study, we show that expression of a mutant CLIP-170 chimeric protein (dominant-negative CLIP-170-GFP), lacking the MT-binding domain, prevents MT stabilization in activated RAW 264.7 macrophages. Furthermore, we find enhanced CLIP-170 association with MTs and MT stabilization by treating resting macrophages with okadaic acid, implicating the protein phosphatase 2A in CLIP-170 binding and MT stabilization in RAW 264.7 cells. Finally, we observed enhanced cell spreading and phagocytosis in both IFN-gamma/LPS-activated and okadaic acid-treated resting RAW 264.7 cells, which are markedly reduced in activated cells expressing dominant-negative CLIP-170-GFP. These results identify CLIP-170 as a key regulator of MT stabilization and establish a prominent role for stabilized MTs in cell spreading and phagocytosis in activated macrophages.


Asunto(s)
Forma de la Célula/inmunología , Activación de Macrófagos/inmunología , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/inmunología , Proteínas Asociadas a Microtúbulos/fisiología , Microtúbulos/metabolismo , Proteínas de Neoplasias/fisiología , Fagocitosis/inmunología , Animales , Proteínas Portadoras , Línea Celular , Forma de la Célula/efectos de los fármacos , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ácido Ocadaico/farmacología , Fagocitosis/efectos de los fármacos , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Fase de Descanso del Ciclo Celular/inmunología
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