RESUMEN
Self-renewing somatic tissues rely on progenitors to support the continuous tissue regeneration. The gene regulatory network maintaining progenitor function remains incompletely understood. Here we show that NUP98 and RAE1 are highly expressed in epidermal progenitors, forming a separate complex in the nucleoplasm. Reduction of NUP98 or RAE1 abolishes progenitors' regenerative capacity, inhibiting proliferation and inducing premature terminal differentiation. Mechanistically, NUP98 binds on chromatin near the transcription start sites of key epigenetic regulators (such as DNMT1, UHRF1 and EZH2) and sustains their expression in progenitors. NUP98's chromatin binding sites are co-occupied by HDAC1. HDAC inhibition diminishes NUP98's chromatin binding and dysregulates NUP98 and RAE1's target gene expression. Interestingly, HDAC inhibition further induces NUP98 and RAE1 to localize interdependently to the nucleolus. These findings identified a pathway in progenitor maintenance, where HDAC activity directs the high levels of NUP98 and RAE1 to directly control key epigenetic regulators, escaping from nucleolar aggregation.
Asunto(s)
Cromatina , Proteínas de Transporte Nucleocitoplasmático , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Cromatina/genética , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/metabolismo , Sitios de UniónRESUMEN
RNA-binding proteins (RBPs) are essential factors required for the physiological function of neurons, muscle, and other tissue types. In keeping with this, a growing body of genetic, clinical, and pathological evidence indicates that RBP dysfunction and/or gene mutation leads to neurodegeneration and myopathy. Here, we summarize the current understanding of matrin 3 (MATR3), a poorly understood RBP implicated not only in ALS and frontotemporal dementia but also in distal myopathy. We begin by reviewing MATR3's functions, its regulation, and how it may be involved in both sporadic and familial neuromuscular disease. We also discuss insights gleaned from cellular and animal models of MATR3 pathogenesis, the links between MATR3 and other disease-associated RBPs, and the mechanisms underlying RBP-mediated disorders.
Asunto(s)
Enfermedades Neuromusculares/genética , Enfermedades Neuromusculares/fisiopatología , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/fisiología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/fisiología , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Modelos Animales de Enfermedad , Miopatías Distales/genética , Miopatías Distales/fisiopatología , Demencia Frontotemporal/genética , Demencia Frontotemporal/fisiopatología , Regulación de la Expresión Génica , Humanos , Ratones Noqueados , Mutación , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/deficiencia , Proteínas de Unión al ARN/químicaRESUMEN
Shedding, loss of expression, or internalization of natural killer group 2, member D (NKG2D) ligands from the tumor cell surface leads to immune evasion, which is associated with poor prognosis in patients with cancer. In many cancers, matrix metalloproteinases cause the proteolytic shedding of NKG2D ligands. However, it remained unclear how to protect NKG2D ligands from shedding. Here, we showed that the shedding of the mouse NKG2D ligand Rae-1 can be prevented by two critical acetyltransferases, GCN5 and PCAF, which acetylate the lysine residues of Rae-1 to avoid shedding both in vitro and in vivo. In contrast, mutations at lysines 80 and 87 of Rae-1 abrogated this acetylation and thereby desensitized tumor cells to NKG2D-dependent immune surveillance. Notably, the protein levels of GCN5 correlated with the expression levels of the human NKG2D ligand ULPB1 in a human tumor tissue microarray and, more importantly, with prolonged overall survival in many cancers. Our results suggest that the acetylation of Rae-1 protein at lysines 80 and 87 by GCN5 and PCAF protects Rae-1 from shedding so as to activate NKG2D-dependent immune surveillance. This discovery may shed light on new targets for NKG2D immunotherapy in cancer treatment.
Asunto(s)
Lisina/metabolismo , Mutación , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Neoplasias/metabolismo , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Acetilación , Línea Celular Tumoral , Proteínas Ligadas a GPI/metabolismo , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisina/genética , Trasplante de Neoplasias , Neoplasias/genética , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Estabilidad Proteica , Análisis de Supervivencia , Análisis de Matrices Tisulares , Escape del Tumor , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic that is a serious global health problem. Evasion of IFN-mediated antiviral signaling is a common defense strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to efficiently block STAT1 and STAT2 nuclear translocation in order to impair transcriptional induction of IFN-stimulated genes (ISGs). Our results demonstrate that the viral accessory protein Orf6 exerts this anti-IFN activity. We found that SARS-CoV-2 Orf6 localizes at the nuclear pore complex (NPC) and directly interacts with Nup98-Rae1 via its C-terminal domain to impair docking of cargo-receptor (karyopherin/importin) complex and disrupt nuclear import. In addition, we show that a methionine-to-arginine substitution at residue 58 impairs Orf6 binding to the Nup98-Rae1 complex and abolishes its IFN antagonistic function. All together our data unravel a mechanism of viral antagonism in which a virus hijacks the Nup98-Rae1 complex to overcome the antiviral action of IFN.
Asunto(s)
COVID-19/metabolismo , Interferones/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT2/metabolismo , Proteínas Virales/metabolismo , Transporte Activo de Núcleo Celular , Animales , Sitios de Unión , Chlorocebus aethiops , Células HEK293 , Humanos , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Unión Proteica , Transducción de Señal , Células VeroRESUMEN
Viruses employ multiple strategies to inhibit host mRNA nuclear export. Distinct to the generally nonselective inhibition mechanisms, ORF10 from gammaherpesviruses inhibits mRNA export in a transcript-selective manner by interacting with Rae1 (RNA export 1) and Nup98 (nucleoporin 98). We now report the structure of ORF10 from MHV-68 (murine gammaherpesvirus 68) bound to the Rae1-Nup98 heterodimer, thereby revealing detailed intermolecular interactions. Structural and functional assays highlight that two highly conserved residues of ORF10, L60 and M413, play critical roles in both complex assembly and mRNA export inhibition. Interestingly, although ORF10 occupies the RNA-binding groove of Rae1-Nup98, the ORF10-Rae1-Nup98 ternary complex still maintains a comparable RNA-binding ability due to the ORF10-RNA direct interaction. Moreover, mutations on the RNA-binding surface of ORF10 disrupt its function of mRNA export inhibition. Our work demonstrates the molecular mechanism of ORF10-mediated selective inhibition and provides insights into the functions of Rae1-Nup98 in regulating host mRNA export.
Asunto(s)
Transporte de ARN/fisiología , ARN Mensajero/metabolismo , Transactivadores/metabolismo , Animales , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Ratones , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/metabolismo , ARN Mensajero/química , Células Sf9 , Transactivadores/químicaRESUMEN
Excitatory Amino Acid Transporters (EAATs) are plasma membrane proteins responsible for maintenance of low extracellular concentrations of glutamate in the CNS. Dysfunction in their activity is implicated in various neurological disorders. Glutamate transport by EAATs occurs through the movement of the central transport domain relative to the scaffold domain in the EAAT membrane protein. Previous studies suggested that residues located within the interface of these two domains in EAAT2, the main subtype of glutamate transporter in the brain, are involved in regulating transport rates. We used mutagenesis, structure-function relationship, surface protein expression and electrophysiology studies, in transfected COS-7 cells and oocytes, to examine residue glycine at position 298, which is located within this interface. Mutation G298A results in increased transport rate without changes in surface expression, suggesting a more hydrophobic and larger alanine results in facilitated transport movement. The increased transport rate does not involve changes in sodium affinity. Electrophysiological currents show that G298A increase both transport and anion currents, suggesting faster transitions through the transport cycle. This work identifies a region critically involved in setting the glutamate transport rate.
Asunto(s)
Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/metabolismo , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Chlorocebus aethiops , Transportador 2 de Aminoácidos Excitadores/química , Femenino , Proteínas Asociadas a Matriz Nuclear/química , Estructura Secundaria de Proteína , Transporte de Proteínas/fisiología , Especificidad por Sustrato/fisiología , XenopusRESUMEN
Phosphatase and actin regulator 3/nuclear scaffold-associated protein phosphatase 1-inhibiting protein (Phactr3/Scapinin) is an actin- and protein phosphatase 1 (PP1)-binding protein known to negatively regulate axon elongation. In this study, we examined the expression pattern of Phactr3/Scapinin in several tissues and investigated the effect of Phactr3/Scapinin on dendritic morphology of cortical neurons. Results showed that Phactr3/Scapinin expression was up-regulated in the developing brain and enriched in neurons and in the postsynaptic density fraction, but not in astrocytes. Overexpression of wild type or mutant Phactr3/Scapinin, which lacked actin-binding activity, resulted in increased dendritic complexity and percentage of spines with a mushroom or stubby shape, as well as a decrease in spine density. However, overexpression of mutant Phactr3/Scapinin that lacked PP1-binding activity did not. Taken together, these findings suggest that Phactr3/Scapinin expression is neuronal and might contribute to synaptic formation via distinct actin- and PP1-binding domains involved in dendritic and axonal morphology, respectively.
Asunto(s)
Dendritas/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/metabolismo , Matriz Nuclear/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteína Fosfatasa 1/metabolismo , Animales , Corteza Cerebral/citología , Femenino , Masculino , Ratones Endogámicos C57BL , Mutación/genética , Unión Proteica , Dominios Proteicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Fracciones Subcelulares/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Calcium biominerals occur in all major animal phyla, and through biomolecular control, exhibit such diverse structures as exoskeletons, shells, bones, teeth and earstones (otoliths). Determining the three-dimensional expression of key biomineral proteins, however, has proven challenging as typical protein identification methods either lose spatial resolution during dissolution of the mineral phase or are costly and limited to two-dimensional expression of high abundance proteins. Here we present a modification of the CLARITY and ACT-PRESTO protocols to visualize and confirm, for the first time, the timing of expression and function of two key regulators of biomineralization.
Asunto(s)
Biomineralización , Minerales/química , Proteínas Asociadas a Matriz Nuclear/ultraestructura , Proteínas/ultraestructura , Exoesqueleto/química , Exoesqueleto/ultraestructura , Animales , Calcio/química , Carbonato de Calcio , Imagenología Tridimensional/métodos , Proteínas Asociadas a Matriz Nuclear/química , Diente/química , Diente/ultraestructuraRESUMEN
Chromosome translocation is a major cause of the onset and progression of diverse types of cancers. However, the mechanisms underlying this process remain poorly understood. Here, we identified a non-homologous end-joining protein, IFFO1, which structurally forms a heterotetramer with XRCC4. IFFO1 is recruited to the sites of DNA damage by XRCC4 and promotes the repair of DNA double-strand breaks in a parallel pathway with XLF. Interestingly, IFFO1 interacts with lamin A/C, forming an interior nucleoskeleton. Inactivating IFFO1 or its interaction with XRCC4 or lamin A/C leads to increases in both the mobility of broken ends and the frequency of chromosome translocation. Importantly, the destruction of this nucleoskeleton accounts for the elevated frequency of chromosome translocation in many types of cancer cells. Our results reveal that the lamin A/C-IFFO1-constituted nucleoskeleton prevents chromosome translocation by immobilizing broken DNA ends during tumorigenesis.
Asunto(s)
Carcinogénesis/genética , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Lamina Tipo A/metabolismo , Translocación Genética , Animales , Carcinoma/genética , Cromosomas Humanos , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Células HEK293 , Humanos , Proteínas de Filamentos Intermediarios/genética , Ratones , Matriz Nuclear/metabolismo , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas Asociadas a Matriz Nuclear/fisiologíaRESUMEN
Cortical force generators connect epithelial polarity sites with astral microtubules, allowing dynein movement to orient the mitotic spindle as astral microtubules depolymerize. Complexes of the LGN and NuMA proteins, fundamental components of force generators, are recruited to the cortex by Gαi-subunits of heterotrimeric G-proteins. They associate with dynein/dynactin and activate the motor activity pulling on astral microtubules. The architecture of cortical force generators is unknown. Here we report the crystal structure of NuMA:LGN hetero-hexamers, and unveil their role in promoting the assembly of active cortical dynein/dynactin motors that are required in orchestrating oriented divisions in polarized cells. Our work elucidates the basis for the structural organization of essential spindle orientation motors.
Asunto(s)
Antígenos Nucleares/metabolismo , Polaridad Celular , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Huso Acromático/metabolismo , Antígenos Nucleares/química , Antígenos Nucleares/genética , Antígenos Nucleares/aislamiento & purificación , Células CACO-2 , Proteínas de Ciclo Celular , Cristalografía por Rayos X , Complejo Dinactina/metabolismo , Dineínas/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/aislamiento & purificación , Microtúbulos/metabolismo , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/aislamiento & purificación , Unión Proteica/fisiología , Multimerización de Proteína/fisiología , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Many protein and peptide sequences are self-assembled into ß-sheet-rich fibrous structures called amyloids. Their atomic details provide insights into fundamental knowledge related to amyloid diseases. To study the detailed structure of the amyloid, we have developed a model system that mimics the self-assembling process of the amyloid within a water-soluble protein, termed peptide self-assembly mimic (PSAM). PSAM enables capturing of a peptide sequence within a water-soluble protein, thus making structural and energetics-related studies possible. In this work, we extend our PSAM approach to a naturally occurring chameleon sequence from αB crystallin. We chose "Val-Leu-Gly-Asp-Val (VLGDV)", a five amino-acid sequence, which forms a ß-turn in the native structure and a ß-barrel in the amyloid oligomer cylindrin, as a grafting sequence to the PSAM scaffold. The crystal structure revealed that the sequence grafting induced ß-sheet bending at the grafted site. We further investigated the role of the central glycine residue and found that its role in the ß-sheet bending is dependent on the neighboring residues. The ability of PSAM to observe the structural alterations induced by the grafted sequence provides an opportunity to evaluate the structural impact of a sequence from the peptide self-assembly.
Asunto(s)
Péptidos beta-Amiloides/química , Amiloide/química , Conformación Proteica en Lámina beta/genética , Cadena B de alfa-Cristalina/química , Secuencia de Aminoácidos/genética , Amiloide/genética , Péptidos beta-Amiloides/genética , Cristalografía por Rayos X , Modelos Moleculares , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/genética , Conformación Proteica , Estructura Secundaria de Proteína , Cadena B de alfa-Cristalina/genéticaRESUMEN
RNF8 plays a critical role in DNA damage response (DDR) to initiate ubiquitination-dependent signaling. To better characterize the role of RNF8 in UV-induced DDR, we searched for novel substrates of RNF8 and identified NONO as one intriguing substrate. We found that: (i) RNF8 ubiquitinates NONO and (ii) UV radiation triggers NONO ubiquitination and its subsequent degradation. Depletion of RNF8 inhibited UV-induced degradation of NONO, suggesting that RNF8 targets NONO for degradation in response to UV damage. In addition, we found that 3 NONO lysine residues (positions 279, 290 and 295) are important for conferring its instability in UV-DDR. Depletion of RNF8 or expression of NONO with lysine to arginine substitutions at positions 279, 290 and 295 prolonged CHK1 phosphorylation over an extended period of time. Furthermore, expression of the stable mutant, but not wild-type NONO, induced a prolonged S phase following UV exposure. Stable cell lines expressing the stable NONO mutant showed increased UV sensitivity in a clonogenic survival assay. Since RNF8 recruitment to the UV-damaged sites is dependent on ATR, we propose that RNF8-mediated NONO degradation and subsequent inhibition of NONO-dependent chromatin loading of TOPBP1, a key activator of ATR, function as a negative feedback loop critical for turning off ATR-CHK1 checkpoint signaling in UV-DDR.
Asunto(s)
Daño del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Factores de Transcripción de Octámeros/metabolismo , Proteínas de Unión al ARN/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Humanos , Lisina/metabolismo , Proteínas Asociadas a Matriz Nuclear/química , Factores de Transcripción de Octámeros/química , Proteínas de Unión al ARN/química , Fase S , Transducción de Señal , Ubiquitinación , Rayos UltravioletaRESUMEN
Long mammalian introns make it challenging for the RNA processing machinery to identify exons accurately. We find that LINE-derived sequences (LINEs) contribute to this selection by recruiting dozens of RNA-binding proteins (RBPs) to introns. This includes MATR3, which promotes binding of PTBP1 to multivalent binding sites within LINEs. Both RBPs repress splicing and 3' end processing within and around LINEs. Notably, repressive RBPs preferentially bind to evolutionarily young LINEs, which are located far from exons. These RBPs insulate the LINEs and the surrounding intronic regions from RNA processing. Upon evolutionary divergence, changes in RNA motifs within LINEs lead to gradual loss of their insulation. Hence, older LINEs are located closer to exons, are a common source of tissue-specific exons, and increasingly bind to RBPs that enhance RNA processing. Thus, LINEs are hubs for the assembly of repressive RBPs and also contribute to the evolution of new, lineage-specific transcripts in mammals. VIDEO ABSTRACT.
Asunto(s)
Ribonucleoproteínas Nucleares Heterogéneas/química , Elementos de Nucleótido Esparcido Largo , Proteínas Asociadas a Matriz Nuclear/química , Poliadenilación , Proteína de Unión al Tracto de Polipirimidina/química , Proteínas de Unión al ARN/química , ARN/química , Empalme Alternativo , Animales , Sitios de Unión , Exones , Células HeLa , Humanos , Intrones , Ratones , Mutación , Motivos de Nucleótidos , Filogenia , Unión Proteica , Mapeo de Interacción de Proteínas , Empalme del ARNRESUMEN
Abnormalities in nucleic acid processing are associated with the development of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Mutations in Matrin 3 (MATR3), a poorly understood DNA- and RNA-binding protein, cause familial ALS/FTD, and MATR3 pathology is a feature of sporadic disease, suggesting that MATR3 dysfunction is integrally linked to ALS pathogenesis. Using a rat primary neuron model to assess MATR3-mediated toxicity, we noted that neurons were bidirectionally vulnerable to MATR3 levels, with pathogenic MATR3 mutants displaying enhanced toxicity. MATR3's zinc finger domains partially modulated toxicity, but elimination of its RNA recognition motifs had no effect on survival, instead facilitating its self-assembly into liquid-like droplets. In contrast to other RNA-binding proteins associated with ALS, cytoplasmic MATR3 redistribution mitigated neurodegeneration, suggesting that nuclear MATR3 mediates toxicity. Our findings offer a foundation for understanding MATR3-related neurodegeneration and how nucleic acid binding functions, localization, and pathogenic mutations drive sporadic and familial disease.
Asunto(s)
Núcleo Celular/metabolismo , Neurotoxinas/toxicidad , Proteínas Asociadas a Matriz Nuclear/metabolismo , Ácidos Nucleicos/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Núcleo Celular/efectos de los fármacos , Supervivencia Celular , Células HEK293 , Humanos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Mutación/genética , Degeneración Nerviosa/patología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/genética , Unión Proteica , Estabilidad Proteica , Motivo de Reconocimiento de ARN , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Ratas , Ratas Long-Evans , Solubilidad , Dedos de ZincRESUMEN
To position the mitotic spindle within the cell, dynamic plus ends of astral microtubules are pulled by membrane-associated cortical force-generating machinery. However, in contrast to the chromosome-bound kinetochore structure, how the diffusion-prone cortical machinery is organized to generate large spindle-pulling forces remains poorly understood. Here, we develop a light-induced reconstitution system in human cells. We find that induced cortical targeting of NuMA, but not dynein, is sufficient for spindle pulling. This spindle-pulling activity requires dynein-dynactin recruitment by NuMA's N-terminal long arm, dynein-based astral microtubule gliding, and NuMA's direct microtubule-binding activities. Importantly, we demonstrate that cortical NuMA assembles specialized focal structures that cluster multiple force-generating modules to generate cooperative spindle-pulling forces. This clustering activity of NuMA is required for spindle positioning, but not for spindle-pole focusing. We propose that cortical Dynein-Dynactin-NuMA (DDN) clusters act as the core force-generating machinery that organizes a multi-arm ensemble reminiscent of the kinetochore.
Asunto(s)
Antígenos Nucleares/metabolismo , Complejo Dinactina/metabolismo , Dineínas/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Huso Acromático/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Antígenos Nucleares/química , Proteínas de Ciclo Celular , Línea Celular , Humanos , Ácidos Indolacéticos/farmacología , Luz , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Mutación/genética , Proteínas Asociadas a Matriz Nuclear/química , Optogenética , Paclitaxel/farmacología , Fenotipo , Dominios ProteicosRESUMEN
Connexins are integral membrane building blocks that form gap junctions, enabling direct cytoplasmic exchange of ions and low-molecular-mass metabolites between adjacent cells. In the heart, gap junctions mediate the propagation of cardiac action potentials and the maintenance of a regular beating rhythm. A number of connexin interacting proteins have been described and are known gap junction regulators either through direct effects (e.g., kinases) or the formation of larger multifunctional complexes (e.g., cytoskeleton scaffold proteins). Most connexin partners can be categorized as either proteins promoting coupling by stimulating forward trafficking and channel opening or inhibiting coupling by inducing channel closure, internalization, and degradation. While some interactions have only been implied through co-localization using immunohistochemistry, others have been confirmed by biophysical methods that allow detection of a direct interaction. Our understanding of these interactions is, by far, most well developed for connexin 43 (Cx43) and the scope of this review is to summarize our current knowledge of their functional and regulatory roles. The significance of these interactions is further exemplified by demonstrating their importance at the intercalated disc, a major hub for Cx43 regulation and Cx43 mediated effects.
Asunto(s)
Conexina 43/genética , Citoesqueleto/genética , Uniones Comunicantes/genética , Mapas de Interacción de Proteínas/genética , Fenómenos Biofísicos , Comunicación Celular/genética , Conexina 43/química , Citoesqueleto/química , Uniones Comunicantes/química , Humanos , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/genéticaRESUMEN
Ran-guanosine triphosphatase orchestrates mitotic spindle assembly by modulation of the interaction between Importin-α/-ß and spindle assembly factors (SAFs). The inhibition of SAFs performed by importins needs to be done without much sequestration from abundant nuclear localization signal (NLS) -containing proteins. However, the molecular mechanisms that determine NLS-binding selectivity and that inhibit activity of Importin-ß-regulated SAFs (e.g., nuclear mitotic apparatus protein [NuMA]) remain undefined. Here, we present a crystal structure of the Importin-α-NuMA C terminus complex showing a novel binding pattern that accounts for selective NLS recognition. We demonstrate that, in the presence of Importin-α, Importin-ß inhibits the microtubule-binding function of NuMA. Further, we have identified a high-affinity microtubule-binding region that lies carboxyl-terminal to the NLS, which is sterically masked by Importin-ß on being bound by Importin-α. Our study provides mechanistic evidence of how Importin-α/-ß regulates the NuMA functioning required for assembly of higher-order microtubule structures, further illuminating how Ran-governed transport factors regulate diverse SAFs and accommodate various cell demands.
Asunto(s)
Antígenos Nucleares/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Huso Acromático/metabolismo , beta Carioferinas/metabolismo , Animales , Antígenos Nucleares/química , Antígenos Nucleares/genética , Proteínas de Ciclo Celular , Humanos , Microtúbulos/metabolismo , Modelos Moleculares , Complejos Multiproteicos , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Huso Acromático/química , Huso Acromático/genética , Relación Estructura-Actividad , Xenopus , alfa Carioferinas/metabolismo , beta Carioferinas/química , beta Carioferinas/genética , Proteína de Unión al GTP ran/metabolismoRESUMEN
Scaffold attachment factor B1 (SAFB1) is an integral component of the nuclear matrix of vertebrate cells. It binds to DNA on scaffold/matrix attachment region elements, as well as to RNA and a multitude of different proteins, affecting basic cellular activities such as transcription, splicing and DNA damage repair. In the present study, we show that enhancer of rudimentary homologue (ERH) is a new molecular partner of SAFB1 and its 70% homologous paralogue, scaffold attachment factor B2 (SAFB2). ERH interacts directly in the nucleus with the C-terminal Arg-Gly-rich region of SAFB1/2 and co-localizes with it in the insoluble nuclear fraction. ERH, a small ubiquitous protein with striking homology among species and a unique structure, has also been implicated in fundamental cellular mechanisms. Our functional analyses suggest that the SAFB/ERH interaction does not affect SAFB1/2 function in transcription (e.g. as oestrogen receptor α co-repressors), although it reverses the inhibition exerted by SAFB1/2 on the splicing kinase SR protein kinase 1 (SRPK1), which also binds on the C-terminus of SAFB1/2. Accordingly, ERH silencing decreases lamin B receptor and SR protein phosphorylation, which are major SRPK1 substrates, further substantiating the role of SAFB1 and SAFB2 in the co-ordination of nuclear function.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Estrógenos/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular , Animales , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Proteínas de Unión a la Región de Fijación a la Matriz/química , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Microscopía Fluorescente , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Ratas , Receptores de Estrógenos/química , Receptores de Estrógenos/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Factores de Empalme Serina-Arginina/química , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/química , Factores de Transcripción/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Protein interactions involving intrinsically disordered proteins (IDPs) comprise a variety of binding modes, from the well-characterized folding upon binding to dynamic fuzzy complexes. To date, most studies concern the binding of an IDP to a structured protein, while the interaction between two IDPs is poorly understood. In this study, NMR, smFRET, and molecular dynamics (MD) simulation are combined to characterize the interaction between two IDPs, the C-terminal domain (CTD) of protein 4.1G and the nuclear mitotic apparatus (NuMA) protein. It is revealed that CTD and NuMA form a fuzzy complex with remaining structural disorder. Multiple binding sites on both proteins were identified by molecular dynamics and mutagenesis studies. This study provides an atomic scenario in which two IDPs bearing multiple binding sites interact with each other in dynamic equilibrium. The combined approach employed here could be widely applicable for investigating IDPs and their dynamic interactions.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Sitios de Unión , Proteínas del Citoesqueleto/química , Transferencia Resonante de Energía de Fluorescencia , Lógica Difusa , Células HeLa , Humanos , Proteínas Intrínsecamente Desordenadas/química , Espectroscopía de Resonancia Magnética , Proteínas de la Membrana/química , Simulación de Dinámica Molecular , Mutagénesis , Proteínas Asociadas a Matriz Nuclear/química , Unión Proteica , Conformación ProteicaRESUMEN
Both cell-cell adhesion and oriented cell division play prominent roles in establishing tissue architecture, but it is unclear how they might be coordinated. Here, we demonstrate that the cell-cell adhesion protein E-cadherin functions as an instructive cue for cell division orientation. This is mediated by the evolutionarily conserved LGN/NuMA complex, which regulates cortical attachments of astral spindle microtubules. We show that LGN, which adopts a three-dimensional structure similar to cadherin-bound catenins, binds directly to the E-cadherin cytosolic tail and thereby localizes at cell-cell adhesions. On mitotic entry, NuMA is released from the nucleus and competes LGN from E-cadherin to locally form the LGN/NuMA complex. This mediates the stabilization of cortical associations of astral microtubules at cell-cell adhesions to orient the mitotic spindle. Our results show how E-cadherin instructs the assembly of the LGN/NuMA complex at cell-cell contacts, and define a mechanism that couples cell division orientation to intercellular adhesion.