RESUMO
The toxic proline:arginine (PRn) poly-dipeptide encoded by the (GGGGCC)n repeat expansion in the C9orf72 form of heritable amyotrophic lateral sclerosis (ALS) binds to the central channel of the nuclear pore and inhibits the movement of macromolecules into and out of the nucleus. The PRn poly-dipeptide binds to polymeric forms of the phenylalanine:glycine (FG) repeat domain, which is shared by several proteins of the nuclear pore complex, including those in the central channel. A method of chemical footprinting was used to characterize labile, cross-ß polymers formed from the FG domain of the Nup54 protein. Mutations within the footprinted region of Nup54 polymers blocked both polymerization and binding by the PRn poly-dipeptide. The aliphatic alcohol 1,6-hexanediol melted FG domain polymers in vitro and reversed PRn-mediated enhancement of the nuclear pore permeability barrier. These data suggest that toxicity of the PRn poly-dipeptide results in part from its ability to lock the FG repeats of nuclear pore proteins in the polymerized state. Our study offers a mechanistic interpretation of PRn poly-dipeptide toxicity in the context of a prominent form of ALS.
Assuntos
Transporte Ativo do Núcleo Celular , Proteína C9orf72/farmacologia , Expansão das Repetições de DNA/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Biopolímeros , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Dipeptídeos/genética , Dipeptídeos/metabolismo , Dipeptídeos/farmacologia , Feminino , Glicóis/farmacologia , Humanos , Microscopia Confocal , Poro Nuclear/química , Poro Nuclear/efeitos dos fármacos , Poro Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , Oócitos/ultraestrutura , Permeabilidade/efeitos dos fármacos , Ligação Proteica , Domínios Proteicos , Aglutininas do Germe de Trigo/metabolismo , Aglutininas do Germe de Trigo/farmacologia , Xenopus laevisRESUMO
Two complementary approaches were used in search of the intracellular targets of the toxic PR poly-dipeptide encoded by the repeat sequences expanded in the C9orf72 form of amyotrophic lateral sclerosis. The top categories of PRn-bound proteins include constituents of non-membrane invested cellular organelles and intermediate filaments. PRn targets are enriched for the inclusion of low complexity (LC) sequences. Evidence is presented indicating that LC sequences represent the direct target of PRn binding and that interaction between the PRn poly-dipeptide and LC domains is polymer-dependent. These studies indicate that PRn-mediated toxicity may result from broad impediments to the dynamics of cell structure and information flow from gene to message to protein.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Dipeptídeos/metabolismo , Demência Frontotemporal/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Esclerose Lateral Amiotrófica/genética , Proteína C9orf72 , Expansão das Repetições de DNA , Dipeptídeos/química , Dipeptídeos/genética , Demência Frontotemporal/genética , Células HeLa , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Peptídeos/química , Peptídeos/genética , Domínios Proteicos , Proteínas/genéticaRESUMO
Many DNA and RNA regulatory proteins contain polypeptide domains that are unstructured when analyzed in cell lysates. These domains are typified by an over-representation of a limited number of amino acids and have been termed prion-like, intrinsically disordered or low-complexity (LC) domains. When incubated at high concentration, certain of these LC domains polymerize into labile, amyloid-like fibers. Here, we report methods allowing the generation of a molecular footprint of the polymeric state of the LC domain of hnRNPA2. By deploying this footprinting technique to probe the structure of the native hnRNPA2 protein present in isolated nuclei, we offer evidence that its LC domain exists in a similar conformation as that described for recombinant polymers of the protein. These observations favor biologic utility to the polymerization of LC domains in the pathway of information transfer from gene to message to protein.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/química , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Schistosoma japonicum/enzimologia , Tirosina/análiseRESUMO
Many RNA regulatory proteins controlling pre-messenger RNA splicing contain serine:arginine (SR) repeats. Here, we found that these SR domains bound hydrogel droplets composed of fibrous polymers of the low-complexity domain of heterogeneous ribonucleoprotein A2 (hnRNPA2). Hydrogel binding was reversed upon phosphorylation of the SR domain by CDC2-like kinases 1 and 2 (CLK1/2). Mutated variants of the SR domains changing serine to glycine (SR-to-GR variants) also bound to hnRNPA2 hydrogels but were not affected by CLK1/2. When expressed in mammalian cells, these variants bound nucleoli. The translation products of the sense and antisense transcripts of the expansion repeats associated with the C9orf72 gene altered in neurodegenerative disease encode GRn and PRn repeat polypeptides. Both peptides bound to hnRNPA2 hydrogels independent of CLK1/2 activity. When applied to cultured cells, both peptides entered cells, migrated to the nucleus, bound nucleoli, and poisoned RNA biogenesis, which caused cell death.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Astrócitos/metabolismo , Nucléolo Celular/metabolismo , Dipeptídeos/metabolismo , Demência Frontotemporal/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Proteínas/genética , Processamento Alternativo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Astrócitos/patologia , Proteína C9orf72 , Morte Celular , Células Cultivadas , Dipeptídeos/genética , Dipeptídeos/farmacologia , Transportador 2 de Aminoácido Excitatório , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Proteínas de Transporte de Glutamato da Membrana Plasmática/genética , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato , Fosforilação , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/metabolismo , RNA Antissenso/antagonistas & inibidores , RNA Antissenso/biossíntese , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , RNA Ribossômico/antagonistas & inibidores , RNA Ribossômico/biossíntese , Sequências Repetitivas de Aminoácidos , Transcrição GênicaRESUMO
The low-complexity (LC) domains of the products of the fused in sarcoma (FUS), Ewings sarcoma (EWS), and TAF15 genes are translocated onto a variety of different DNA-binding domains and thereby assist in driving the formation of cancerous cells. In the context of the translocated fusion proteins, these LC sequences function as transcriptional activation domains. Here, we show that polymeric fibers formed from these LC domains directly bind the C-terminal domain (CTD) of RNA polymerase II in a manner reversible by phosphorylation of the iterated, heptad repeats of the CTD. Mutational analysis indicates that the degree of binding between the CTD and the LC domain polymers correlates with the strength of transcriptional activation. These studies offer a simple means of conceptualizing how RNA polymerase II is recruited to active genes in its unphosphorylated state and released for elongation following phosphorylation of the CTD.
Assuntos
RNA Polimerase II/química , RNA Polimerase II/metabolismo , Ativação Transcricional , Células HeLa , Humanos , Hidrogéis/química , Hidrogéis/metabolismo , Repetições de Microssatélites , Fosforilação , Polimerização , Estrutura Terciária de Proteína , Proteína EWS de Ligação a RNA/metabolismo , Proteína FUS de Ligação a RNA/química , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Fatores Associados à Proteína de Ligação a TATA/química , Fatores Associados à Proteína de Ligação a TATA/metabolismoRESUMO
Eukaryotic cells contain assemblies of RNAs and proteins termed RNA granules. Many proteins within these bodies contain KH or RRM RNA-binding domains as well as low complexity (LC) sequences of unknown function. We discovered that exposure of cell or tissue lysates to a biotinylated isoxazole (b-isox) chemical precipitated hundreds of RNA-binding proteins with significant overlap to the constituents of RNA granules. The LC sequences within these proteins are both necessary and sufficient for b-isox-mediated aggregation, and these domains can undergo a concentration-dependent phase transition to a hydrogel-like state in the absence of the chemical. X-ray diffraction and EM studies revealed the hydrogels to be composed of uniformly polymerized amyloid-like fibers. Unlike pathogenic fibers, the LC sequence-based polymers described here are dynamic and accommodate heterotypic polymerization. These observations offer a framework for understanding the function of LC sequences as well as an organizing principle for cellular structures that are not membrane bound.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Hidrogel de Polietilenoglicol-Dimetacrilato/metabolismo , Proteínas de Ligação a RNA/análise , RNA/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Sistema Livre de Células , Grânulos Citoplasmáticos/química , Células-Tronco Embrionárias/metabolismo , Masculino , Camundongos , Modelos Moleculares , Células NIH 3T3 , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Testículo/citologia , Testículo/metabolismo , Difração de Raios XRESUMO
Cellular granules lacking boundary membranes harbor RNAs and their associated proteins and play diverse roles controlling the timing and location of protein synthesis. Formation of such granules was emulated by treatment of mouse brain extracts and human cell lysates with a biotinylated isoxazole (b-isox) chemical. Deep sequencing of the associated RNAs revealed an enrichment for mRNAs known to be recruited to neuronal granules used for dendritic transport and localized translation at synapses. Precipitated mRNAs contain extended 3' UTR sequences and an enrichment in binding sites for known granule-associated proteins. Hydrogels composed of the low complexity (LC) sequence domain of FUS recruited and retained the same mRNAs as were selectively precipitated by the b-isox chemical. Phosphorylation of the LC domain of FUS prevented hydrogel retention, offering a conceptual means of dynamic, signal-dependent control of RNA granule assembly.
Assuntos
Encéfalo/citologia , RNA/análise , RNA/metabolismo , Ribonucleoproteínas/química , Animais , Biotinilação , Encéfalo/metabolismo , Linhagem Celular , Sistema Livre de Células , Humanos , Isoxazóis/metabolismo , Camundongos , Transporte de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Measurements of the abundance of common metabolites in cultured embryonic stem (ES) cells revealed an unusual state with respect to one-carbon metabolism. These findings led to the discovery of copious expression of the gene encoding threonine dehydrogenase (TDH) in ES cells. TDH-mediated catabolism of threonine takes place in mitochondria to generate glycine and acetyl-coenzyme A (CoA), with glycine facilitating one-carbon metabolism via the glycine cleavage system and acetyl-CoA feeding the tricarboxylic acid cycle. Culture media individually deprived of each of the 20 amino acids were applied to ES cells, leading to the discovery that ES cells are critically dependent on one amino acid--threonine. These observations show that ES cells exist in a high-flux backbone metabolic state comparable to that of rapidly growing bacterial cells.
Assuntos
Células-Tronco Embrionárias/metabolismo , Treonina/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Células Cultivadas , Meios de Cultura , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Técnicas de Cultura de TecidosRESUMO
The neuronal PAS domain protein 3 (NPAS3) gene encoding a brain-enriched transcription factor was recently found to be disrupted in a family suffering from schizophrenia. Mice harboring compound disruptions in the NPAS3 and related NPAS1 genes manifest behavioral and neuroanatomical abnormalities reminiscent of schizophrenia. Herein we demonstrate that Npas3-/- mice are deficient in expression of hippocampal FGF receptor subtype 1 mRNA, most notably in the dentate gyrus. In vivo BrdUrd-labeling shows that basal neural precursor cell proliferation in the dentate gyrus of Npas3-/- mice is reduced by 84% relative to wild-type littermates. We propose that a deficiency in adult neurogenesis may cause the behavioral and neuroanatomical abnormalities seen in Npas3-/- mice, and we speculate that impaired neurogenesis may be involved in the pathophysiology of schizophrenia.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipocampo/citologia , Neurônios/citologia , Esquizofrenia/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Comportamento Animal , Proliferação de Células , Giro Denteado/anatomia & histologia , Giro Denteado/química , Giro Denteado/citologia , Giro Denteado/crescimento & desenvolvimento , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Hipocampo/química , Masculino , Camundongos , Camundongos Mutantes Neurológicos , Neurônios/metabolismo , Neurônios/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Esquizofrenia/metabolismo , Células-Tronco/metabolismoRESUMO
The hypoxia inducible factor (HIF) plays an important role in the progression of a number of pathophysiological processes including tumorigenesis. In addition to several well characterized oxygen-dependent modes of regulation, the function of the HIF transcription factor can also be influenced through the action of other regulatory pathways. Misregulation of these factors resulting in inappropriate HIF expression or activity can contribute to the progression of human cancers through the induction of genes promoting angiogenesis, glycolysis, cell survival, and metastasis, among other processes. The candidate tumor suppressor protein inhibitor of growth family member 4 (ING4) has recently been implicated as a repressor of angiogenesis and tumor growth through association with NF-kappaB. Here we demonstrate that suppression of ING4 further induces HIF transcriptional activity as well. ING4 directly associates with the HIF prolyl hydroxylase, an Fe(II)-dependent oxygenase previously shown to mediate HIF stability as a function of oxygen availability. However, rather than affecting HIF's stability, ING4 mediates HIF's activity. These data support a model in which, in addition to regulating HIF stability, HIF prolyl hydroxylases can modulate HIF function through the recruitment of ING4, a likely component of a chromatin-remodeling complex.