RESUMO
Huntington's disease (HD) is a neurodegenerative, age-onset disorder caused by a CAG DNA expansion in exon 1 of the HTT gene, resulting in a polyglutamine expansion in the huntingtin protein. Nuclear accumulation of mutant huntingtin is a hallmark of HD, resulting in elevated mutant huntingtin levels in cell nuclei. Huntingtin is normally retained at the endoplasmic reticulum via its N17 amphipathic α-helix domain but is released by oxidation of Met-8 during reactive oxygen species (ROS) stress. Huntingtin enters the nucleus via an importin ß1- and 2-dependent proline-tyrosine nuclear localization signal (PY-NLS), which has a unique intervening sequence in huntingtin. Here, we have identified the high-mobility group box 1 (HMGB1) protein as an interactor of the intervening sequence within the PY-NLS. Nuclear levels of HMGB1 positively correlated with varying levels of nuclear huntingtin in both HD and normal human fibroblasts. We also found that HMGB1 interacts with the huntingtin N17 region and that this interaction is enhanced by the presence of ROS and phosphorylation of critical serine residues in the N17 region. We conclude that HMGB1 is a huntingtin N17/PY-NLS ROS-dependent interactor, and this protein bridging is essential for relaying ROS sensing by huntingtin to its nuclear entry during ROS stress. ROS may therefore be a critical age-onset stress that triggers nuclear accumulation of mutant huntington in Huntington's disease.
Assuntos
Transporte Ativo do Núcleo Celular , Proteína HMGB1/fisiologia , Proteína Huntingtina/metabolismo , Espécies Reativas de Oxigênio/farmacologia , Sítios de Ligação , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Proteína Huntingtina/efeitos dos fármacos , Proteína Huntingtina/fisiologia , Sinais de Localização Nuclear , Proteínas Nucleares/metabolismo , Fosforilação , Ligação ProteicaRESUMO
Cofilin protein is involved in regulating the actin cytoskeleton during typical steady state conditions, as well as during cell stress conditions where cofilin saturates F-actin, forming cofilin-actin rods. Cofilin can enter the nucleus through an active nuclear localization signal (NLS), accumulating in nuclear actin rods during stress. Here, we characterize the active nuclear export of cofilin through a leptomycin-B-sensitive, CRM1-dependent, nuclear export signal (NES). We also redefine the NLS of cofilin as a bipartite NLS, with an additional basic epitope required for nuclear localization. Using fluorescence lifetime imaging microscopy (FLIM) and Förster resonant energy transfer (FRET) between cofilin moieties and actin, as well as automated image analysis in live cells, we have defined subtle mutations in the cofilin NLS that allow cofilin to bind actin in vivo and affect cofilin dynamics during stress. We further define the requirement of cofilin-actin rod formation in a system of cell stress by temporal live-cell imaging. We propose that cofilin nuclear shuttling is critical for the cofilin-actin rod stress response with cofilin dynamically communicating between the nucleus and cytoplasm during cell stress.
Assuntos
Actinas/metabolismo , Núcleo Celular/metabolismo , Cofilina 1/metabolismo , Estresse Fisiológico , Sequência de Aminoácidos , Animais , Linhagem Celular , Cofilina 1/química , Sequência Conservada , Técnicas de Silenciamento de Genes , Humanos , Carioferinas/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Mutação , Sinais de Exportação Nuclear , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/metabolismo , Fenótipo , Ligação Proteica , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína Exportina 1RESUMO
Among the known pathways of protein nuclear import, the karyopherin ß2/transportin pathway is only the second to have a defined nuclear localization signal (NLS) consensus. Huntingtin, a 350-kDa protein, has defined roles in the nucleus, as well as a CRM1/exportin-dependent nuclear export signal; however, the NLS and exact pathway of import have remained elusive. Here, using a live cell assay and affinity chromatography, we show that huntingtin has a karyopherin ß2-dependent proline-tyrosine (PY)-NLS in the amino terminus of the protein. This NLS comprises three consensus components: a basic charged sequence, a downstream conserved arginine, and a PY sequence. Unlike the classic PY-NLS, which has an unstructured intervening sequence between the consensus components, we show that a ß sheet structured region separating the consensus elements is critical for huntingtin NLS function. The huntingtin PY-NLS is also capable of import through the importin/karyopherin ß1 pathway but was not functional in all cell types tested. We propose that this huntingtin PY-NLS may comprise a new class of multiple import factor-dependent NLSs with an internal structural component that may regulate NLS activity.
Assuntos
Proteínas do Tecido Nervoso/genética , Sinais de Localização Nuclear/fisiologia , Proteínas Nucleares/genética , beta Carioferinas/genética , Animais , Humanos , Proteína Huntingtina , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Mapeamento de Peptídeos/métodos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , beta Carioferinas/metabolismoRESUMO
Two serine residues within the first 17 amino acid residues of huntingtin (N17) are crucial for modulation of mutant huntingtin toxicity in cell and mouse genetic models of Huntington's disease. Here we show that the stress-dependent phosphorylation of huntingtin at Ser13 and Ser16 affects N17 conformation and targets full-length huntingtin to chromatin-dependent subregions of the nucleus, the mitotic spindle and cleavage furrow during cell division. Polyglutamine-expanded mutant huntingtin is hypophosphorylated in N17 in both homozygous and heterozygous cell contexts. By high-content screening in live cells, we identified kinase inhibitors that modulated N17 phosphorylation and hence huntingtin subcellular localization. N17 phosphorylation was reduced by casein kinase-2 inhibitors. Paradoxically, IKKß kinase inhibition increased N17 phosphorylation, affecting huntingtin nuclear and subnuclear localization. These data indicate that huntingtin phosphorylation at Ser13 and Ser16 can be modulated by small-molecule drugs, which may have therapeutic potential in Huntington's disease.
Assuntos
Caseína Quinase II/antagonistas & inibidores , Doença de Huntington/metabolismo , Quinase I-kappa B/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Inibidores de Proteínas Quinases/farmacologia , Animais , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Imunofluorescência , Proteína Huntingtina , Doença de Huntington/enzimologia , Doença de Huntington/genética , Camundongos , Mutação , Fosforilação , Serina/genética , Fuso Acromático/metabolismo , TransfecçãoAssuntos
Actinas/análise , Actinas/química , Citofotometria/métodos , Peptídeos/metabolismo , Coloração e Rotulagem/métodos , Fatores de Despolimerização de Actina/genética , Fatores de Despolimerização de Actina/metabolismo , Actinas/genética , Animais , Linhagem Celular Transformada , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia de Fluorescência/métodos , Neurônios/metabolismo , Peptídeos/genética , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Nuclear accumulation of the polyglutamine-expanded mutant huntingtin protein remains one of the most predictive cell biological phenotypes of Huntington's disease (HD) progression in patient brain samples and mouse models of the disease. Yet, the relationship between huntingtin nuclear import, neuronal dysfunction and toxicity is not fully understood and it remains unclear whether nuclear accumulation is required for disease onset. Here, we discuss several studies that have guided current understanding of this subject, and highlight our recent data detailing the discovery of a karyopherin ß1/ß2-type nuclear localization signal near the N-terminus of huntingtin. This signal can function through multiple pathways of nuclear import, and may also be responsible for huntingtin import into the primary cilium. This work represents a significant step forward in our knowledge of the regulatory pathways that govern huntingtin nuclear accumulation and will allow direct examination of both normal and mutant huntingtin nuclear function. This work also suggests a re-examination of the cell biology of any protein that contains a multi-pathway nuclear localization signal. The possibility of targeting huntingtin nuclear import therapeutically and the potential impacts of such a strategy for the treatment of HD are also discussed.