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1.
Cell ; 149(3): 578-89, 2012 Apr 27.
Artículo en Inglés | MEDLINE | ID: mdl-22541429

RESUMEN

During heat shock stress, importin ß family-mediated nucleocytoplasmic trafficking is downregulated, whereas nuclear import of the molecular chaperone Hsp70s is upregulated. Here, we identify a nuclear import pathway that operates during heat shock stress and is mediated by an evolutionarily conserved protein named "Hikeshi," which does not belong to the importin ß family. Hikeshi binds to FG-Nups and translocates through nuclear pores on its own, showing characteristic features of nuclear transport carriers. In reconstituted transport, Hikeshi supports the nuclear import of the ATP form of Hsp70s, but not the ADP form, indicating the importance of the Hsp70 ATPase cycle in the import cycle. In living cells, depletion of Hikeshi inhibits heat shock-induced nuclear import of Hsp70s, reduces cell viability after heat shock stress, and significantly delays the attenuation and reversion of multiple heat shock-induced nuclear phenotypes. Nuclear Hsp70s rescue the effect of Hikeshi depletion at least in part. Thus, Hsp70s counteract heat shock-induced damage by acting inside of the nucleus.


Asunto(s)
Transporte Activo de Núcleo Celular , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Respuesta al Choque Térmico , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Núcleo Celular/metabolismo , Células HeLa , Humanos , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Poro Nuclear/metabolismo , Alineación de Secuencia
2.
Genes Cells ; 29(9): 782-791, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-38987995

RESUMEN

Heat stress strongly triggers the nuclear localization of the molecular chaperone HSP70. Hikeshi functions as a unique nuclear import carrier of HSP70. However, how the nuclear import of HSP70 is activated in response to heat stress remains unclear. Here, we investigated the effects of heat on the nuclear import of HSP70. In vitro transport assays revealed that pretreatment of the test samples with heat facilitated the nuclear import of HSP70. Furthermore, binding of Hikeshi to HSP70 increased when temperatures rose. These results indicated that heat is one of the factors that activates the nuclear import of HSP70. Previous studies showed that the F97A mutation in Hikeshi in an extended loop induced an opening in the hydrophobic pocket and facilitated the translocation of Hikeshi through the nuclear pore complex. We found that nuclear accumulation of HSP70 occurred at a lower temperature in cells expressing the Hikeshi-F97A mutant than in cells expressing wild-type Hikeshi. Collectively, our results show that the movement of the extended loop may play an important role in the interaction of Hikeshi with both FG (phenylalanine-glycine)-nucleoporins and HSP70 in a temperature-dependent manner, resulting in the activation of nuclear import of HSP70 in response to heat stress.


Asunto(s)
Transporte Activo de Núcleo Celular , Proteínas HSP70 de Choque Térmico , Respuesta al Choque Térmico , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Humanos , Núcleo Celular/metabolismo , Unión Proteica , Calor , Células HeLa , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/genética , Mutación , Proteínas Portadoras
3.
Cell Struct Funct ; 49(2): 31-46, 2024 Jul 03.
Artículo en Inglés | MEDLINE | ID: mdl-38839376

RESUMEN

In metazoans, the nuclear envelope (NE) disassembles during the prophase and reassembles around segregated chromatids during the telophase. The process of NE formation has been extensively studied using live-cell imaging. At the early step of NE reassembly in human cells, specific pattern-like localization of inner nuclear membrane (INM) proteins, connected to the nuclear pore complex (NPC), was observed in the so-called "core" region and "noncore" region on telophase chromosomes, which corresponded to the "pore-free" region and the "pore-rich" region, respectively, in the early G1 interphase nucleus. We refer to these phenomena as NE subdomain formation. To biochemically investigate this process, we aimed to develop an in vitro NE reconstitution system using digitonin-permeabilized semi-intact mitotic human cells coexpressing two INM proteins, emerin and lamin B receptor, which were labeled with fluorescent proteins. The targeting and accumulation of INM proteins to chromosomes before and after anaphase onset in semi-intact cells were observed using time-lapse imaging. Our in vitro NE reconstitution system recapitulated the formation of the NE subdomain, as in living cells, although chromosome segregation and cytokinesis were not observed. This in vitro NE reconstitution required the addition of a mitotic cytosolic fraction supplemented with a cyclin-dependent kinase inhibitor and energy sources. The cytoplasmic soluble factor(s) dependency of INM protein targeting differed among the segregation states of chromosomes. Furthermore, the NE reconstituted on segregated chromosomes exhibited active nucleocytoplasmic transport competency. These results indicate that the chromosome status changes after anaphase onset for recruiting NPC components.


Asunto(s)
Mitosis , Membrana Nuclear , Proteínas Nucleares , Humanos , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Células HeLa , Receptor de Lamina B , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Cromosomas Humanos/metabolismo , Poro Nuclear/metabolismo , Cromosomas/metabolismo
4.
Adv Exp Med Biol ; 1461: 61-78, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-39289274

RESUMEN

Nuclear transport is the basis for the biological reaction of eukaryotic cells, as it is essential to coordinate nuclear and cytoplasmic events separated by nuclear envelope. Although we currently understand the basic molecular mechanisms of nuclear transport in detail, many unexplored areas remain. For example, it is believed that the regulations and biological functions of the nuclear transport receptors (NTRs) highlights the significance of the transport pathways in physiological contexts. However, physiological significance of multiple parallel transport pathways consisting of more than 20 NTRs is still poorly understood, because our knowledge of each pathway, regarding their substrate information or how they are differently regulated, is still limited. In this report, we describe studies showing how nuclear transport systems in general are affected by temperature rises, namely, thermal stress or heat stress. We will then focus on Importin α family members and unique transport factor Hikeshi, because these two NTRs are affected in heat stress. Our present review will provide an additional view to point out the importance of diversity of the nuclear transport pathways in eukaryotic cells.


Asunto(s)
Transporte Activo de Núcleo Celular , Respuesta al Choque Térmico , Humanos , Respuesta al Choque Térmico/fisiología , Animales , Núcleo Celular/metabolismo , alfa Carioferinas/metabolismo , alfa Carioferinas/genética
5.
Proc Natl Acad Sci U S A ; 118(46)2021 11 16.
Artículo en Inglés | MEDLINE | ID: mdl-34750262

RESUMEN

DNA molecules are atomic-scale information storage molecules that promote reliable information transfer via fault-free repetitions of replications and transcriptions. Remarkable accuracy of compacting a few-meters-long DNA into a micrometer-scale object, and the reverse, makes the chromosome one of the most intriguing structures from both physical and biological viewpoints. However, its three-dimensional (3D) structure remains elusive with challenges in observing native structures of specimens at tens-of-nanometers resolution. Here, using cryogenic coherent X-ray diffraction imaging, we succeeded in obtaining nanoscale 3D structures of metaphase chromosomes that exhibited a random distribution of electron density without characteristics of high-order folding structures. Scaling analysis of the chromosomes, compared with a model structure having the same density profile as the experimental results, has discovered the fractal nature of density distributions. Quantitative 3D density maps, corroborated by molecular dynamics simulations, reveal that internal structures of chromosomes conform to diffusion-limited aggregation behavior, which indicates that 3D chromatin packing occurs via stochastic processes.


Asunto(s)
Cromatina/genética , Cromosomas/genética , Línea Celular Tumoral , ADN/genética , Células HCT116 , Humanos , Metafase/genética , Difracción de Rayos X/métodos , Rayos X
6.
Genes Cells ; 26(6): 360-380, 2021 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-33711210

RESUMEN

Mouse telomerase and the DNA polymerase alpha-primase complex elongate the leading and lagging strands of telomeres, respectively. To elucidate the molecular mechanism of lagging strand synthesis, we investigated the interaction between DNA polymerase alpha and two paralogs of the mouse POT1 telomere-binding protein (POT1a and POT1b). Yeast two-hybrid analysis and a glutathione S-transferase pull-down assay indicated that the C-terminal region of POT1a/b binds to the intrinsically disordered N-terminal region of p180, the catalytic subunit of mouse DNA polymerase alpha. Subcellular distribution analyses showed that although POT1a, POT1b, and TPP1 were localized to the cytoplasm, POT1a-TPP1 and POT1b-TPP1 coexpressed with TIN2 localized to the nucleus in a TIN2 dose-dependent manner. Coimmunoprecipitation and cell cycle synchronization experiments indicated that POT1b-TPP1-TIN2 was more strongly associated with p180 than POT1a-TPP1-TIN2, and this complex accumulated during the S phase. Fluorescence in situ hybridization and proximity ligation assays showed that POT1a and POT1b interacted with p180 and TIN2 on telomeric chromatin. Based on the present study and a previous study, we propose a model in which POT1a/b-TPP1-TIN2 translocates into the nucleus in a TIN2 dose-dependent manner to target the telomere, where POT1a/b interacts with DNA polymerase alpha for recruitment at the telomere for lagging strand synthesis.


Asunto(s)
ADN Polimerasa I/química , ADN Polimerasa I/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Secuencia de Aminoácidos , Aminopeptidasas/metabolismo , Animales , Especificidad de Anticuerpos/inmunología , Ciclo Celular , Bases de Datos Genéticas , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Genoma , Humanos , Ratones , Modelos Biológicos , Células 3T3 NIH , Unión Proteica , Homología de Secuencia de Aminoácido , Serina Proteasas/metabolismo , Complejo Shelterina , Relación Estructura-Actividad , Fracciones Subcelulares/metabolismo
7.
Exp Cell Res ; 386(1): 111686, 2020 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-31669744

RESUMEN

IER5 gene encodes an activator of HSF1 and is a p53 target gene. The IER5 protein forms a ternary complex with HSF1 and PP2A, and promotes PP2A-dependent dephosphorylation of HSF1 at a number of serine and threonine residues. This hypo-phosphorylated form of HSF1 is transcriptionally active and has been suggested to be responsible for the HSF1 activation observed in cancers. Here we report that IER5 possess a classical bipartite nuclear localization signal (NLS) at amino acids 217-244 that is highly conserved among species and that mediates complex formation with importin-α and importin-ß. We also demonstrate that the intact NLS is essential for HSF1 dephosphorylation and full activation by IER5. Thus, nuclear import of IER5 via importin-α and importin-ß may be essential for IER5 function.


Asunto(s)
Núcleo Celular/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Señales de Localización Nuclear , Proteínas Nucleares/metabolismo , Transporte Activo de Núcleo Celular , Línea Celular Tumoral , Secuencia Conservada , Células HEK293 , Factores de Transcripción del Choque Térmico/metabolismo , Humanos , Proteínas Inmediatas-Precoces/química , Carioferinas/metabolismo , Proteínas Nucleares/química , Fosforilación
8.
J Cell Sci ; 131(6)2018 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-29487178

RESUMEN

Although condensins play essential roles in mitotic chromosome assembly, Ki-67 (also known as MKI67), a protein localizing to the periphery of mitotic chromosomes, had also been shown to make a contribution to the process. To examine their respective roles, we generated a set of HCT116-based cell lines expressing Ki-67 and/or condensin subunits that were fused with an auxin-inducible degron for their conditional degradation. Both the localization and the dynamic behavior of Ki-67 on mitotic chromosomes were not largely affected upon depletion of condensin subunits, and vice versa. When both Ki-67 and SMC2 (a core subunit of condensins) were depleted, ball-like chromosome clusters with no sign of discernible thread-like structures were observed. This severe defective phenotype was distinct from that observed in cells depleted of either Ki-67 or SMC2 alone. Our results show that Ki-67 and condensins, which localize to the external surface and the central axis of mitotic chromosomes, respectively, have independent yet cooperative functions in supporting the structural integrity of mitotic chromosomes.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Cromosomas Humanos/metabolismo , Proteínas de Unión al ADN/metabolismo , Antígeno Ki-67/metabolismo , Mitosis , Complejos Multiproteicos/metabolismo , Adenosina Trifosfatasas/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Cromosomas Humanos/genética , Proteínas de Unión al ADN/genética , Humanos , Ácidos Indolacéticos/metabolismo , Antígeno Ki-67/genética , Complejos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transporte de Proteínas
9.
J Biol Chem ; 292(31): 13008-13021, 2017 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-28646110

RESUMEN

The protein mini-chromosome maintenance 10 (Mcm10) was originally identified as an essential yeast protein in the maintenance of mini-chromosome plasmids. Subsequently, Mcm10 has been shown to be required for both initiation and elongation during chromosomal DNA replication. However, it is not fully understood how the multiple functions of Mcm10 are coordinated or how Mcm10 interacts with other factors at replication forks. Here, we identified and characterized the Mcm2-7-interacting domain in human Mcm10. The interaction with Mcm2-7 required the Mcm10 domain that contained amino acids 530-655, which overlapped with the domain required for the stable retention of Mcm10 on chromatin. Expression of truncated Mcm10 in HeLa cells depleted of endogenous Mcm10 via siRNA revealed that the Mcm10 conserved domain (amino acids 200-482) is essential for DNA replication, whereas both the conserved and the Mcm2-7-binding domains were required for its full activity. Mcm10 depletion reduced the initiation frequency of DNA replication and interfered with chromatin loading of replication protein A, DNA polymerase (Pol) α, and proliferating cell nuclear antigen, whereas the chromatin loading of Cdc45 and Pol ϵ was unaffected. These results suggest that human Mcm10 is bound to chromatin through the interaction with Mcm2-7 and is primarily involved in the initiation of DNA replication after loading of Cdc45 and Pol ϵ.


Asunto(s)
Cromatina/metabolismo , Replicación del ADN , Componente 2 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Complejo de Reconocimiento del Origen/metabolismo , Origen de Réplica , Transporte Activo de Núcleo Celular , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Componente 2 del Complejo de Mantenimiento de Minicromosoma/química , Componente 7 del Complejo de Mantenimiento de Minicromosoma/química , Proteínas de Mantenimiento de Minicromosoma/antagonistas & inhibidores , Proteínas de Mantenimiento de Minicromosoma/química , Proteínas de Mantenimiento de Minicromosoma/genética , Mutagénesis Sitio-Dirigida , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Estabilidad Proteica , Interferencia de ARN , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Mutación Silenciosa , Homología Estructural de Proteína
10.
Am J Hum Genet ; 97(4): 555-66, 2015 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-26411495

RESUMEN

The nuclear pore complex (NPC) is a huge protein complex embedded in the nuclear envelope. It has central functions in nucleocytoplasmic transport, nuclear framework, and gene regulation. Nucleoporin 107 kDa (NUP107) is a component of the NPC central scaffold and is an essential protein in all eukaryotic cells. Here, we report on biallelic NUP107 mutations in nine affected individuals who are from five unrelated families and show early-onset steroid-resistant nephrotic syndrome (SRNS). These individuals have pathologically focal segmental glomerulosclerosis, a condition that leads to end-stage renal disease with high frequency. NUP107 is ubiquitously expressed, including in glomerular podocytes. Three of four NUP107 mutations detected in the affected individuals hamper NUP107 binding to NUP133 (nucleoporin 133 kDa) and NUP107 incorporation into NPCs in vitro. Zebrafish with nup107 knockdown generated by morpholino oligonucleotides displayed hypoplastic glomerulus structures and abnormal podocyte foot processes, thereby mimicking the pathological changes seen in the kidneys of the SRNS individuals with NUP107 mutations. Considering the unique properties of the podocyte (highly differentiated foot-process architecture and slit membrane and the inability to regenerate), we propose a "podocyte-injury model" as the pathomechanism for SRNS due to biallelic NUP107 mutations.


Asunto(s)
Edad de Inicio , Mutación/genética , Síndrome Nefrótico/congénito , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Alelos , Animales , Células Cultivadas , Niño , Preescolar , Citoplasma/metabolismo , Femenino , Haplotipos , Humanos , Immunoblotting , Inmunoprecipitación , Lactante , Riñón/metabolismo , Riñón/patología , Masculino , Microscopía Fluorescente , Síndrome Nefrótico/etiología , Síndrome Nefrótico/patología , Poro Nuclear , Proteínas de Complejo Poro Nuclear/antagonistas & inhibidores , Oligorribonucleótidos Antisentido/farmacología , Linaje , Podocitos/metabolismo , Podocitos/patología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/antagonistas & inhibidores
11.
J Cell Sci ; 129(22): 4200-4212, 2016 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-27802161

RESUMEN

Lamin B receptor (LBR), an inner nuclear membrane (INM) protein, contributes to the functional integrity of the nucleus by tethering heterochromatin to the nuclear envelope. We have previously reported that the depletion of embryonic large molecule derived from yolk sac (ELYS; also known as AHCTF1), a component of the nuclear pore complex, from cells perturbs the localization of LBR to the INM, but little is known about the underlying molecular mechanism. In this study, we found that the depletion of ELYS promoted LBR phosphorylation at the residues known to be phosphorylated by cyclin-dependent kinase (CDK) and serine/arginine protein kinases 1 and 2 (SRPK1 and SRPK2, respectively). These phosphorylation events were most likely to be counter-balanced by protein phosphatase 1 (PP1), and the depletion of PP1 from cells consistently caused the mislocalization of LBR. These observations point to a new mechanism regulating the localization of LBR, which is governed by an ELYS-mediated phosphorylation network. This phosphorylation-dependent coordination between INM proteins and the nuclear pore complex might be important for the integrity of the nucleus.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Proteína Quinasa CDC2/metabolismo , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/metabolismo , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Lamina Tipo B/metabolismo , Mutación/genética , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Dominios Proteicos , Isoformas de Proteínas/metabolismo , Proteína Fosfatasa 1/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Receptores Citoplasmáticos y Nucleares/química , Receptor de Lamina B
12.
Genes Cells ; 22(11): 968-976, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-28980748

RESUMEN

Hikeshi mediates the heat stress-induced nuclear import of heat-shock protein 70 (HSP70s: HSP70/HSC70). Dysfunction of Hikeshi causes some serious effects in humans; however, the cellular function of Hikeshi is largely unknown. Here, we investigated the effects of Hikeshi depletion on the survival of human cells after proteotoxic stress and found opposite effects in HeLa and hTERT-RPE1 (RPE) cells; depletion of Hikeshi reduced the survival of HeLa cells, but increased the survival of RPE cells in response to proteotoxic stress. Hikeshi depletion sustained heat-shock transcription factor 1 (HSF1) activation in HeLa cells after recovery from stress, but introduction of a nuclear localization signal-tagged HSC70 in Hikeshi-depleted HeLa cells down-regulated HSF1 activity. In RPE cells, the HSF1 was efficiently activated, but the activated HSF1 was not sustained after recovery from stress, as in HeLa cells. Additionally, we found that p53 and subsequent up-regulation of p21 were higher in the Hikeshi-depleted RPE cells than in the wild-type cells. Our results indicate that depletion of Hikeshi renders HeLa cells proteotoxic stress-sensitive through the abrogation of the nuclear function of HSP70s required for HSF1 regulation. Moreover, Hikeshi depletion up-regulates p21 in RPE cells, which could be a cause of its proteotoxic stress resistant.


Asunto(s)
Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Respuesta al Choque Térmico , Epitelio Pigmentado de la Retina/metabolismo , Estrés Fisiológico , Transporte Activo de Núcleo Celular , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Supervivencia Celular , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Células HeLa , Factores de Transcripción del Choque Térmico/genética , Factores de Transcripción del Choque Térmico/metabolismo , Humanos , Epitelio Pigmentado de la Retina/citología , Telomerasa/genética
13.
Genes Cells ; 21(10): 1113-1124, 2016 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-27610954

RESUMEN

Although the condensin complexes and topoisomerase IIα (TopoIIα) are the central players in mitotic chromosome formation, they are insufficient for its completion, and additional factors involved in the process have been extensively sought. In this study, we examined the possibility that Ki67, a perichromosomal protein widely used as a cell proliferation marker, is one such factor. Using a combination of auxin-inducible degron and CRISPR-Cas9-based gene editing technologies, we generated a human HCT116 cell line in which Ki67 is rapidly depleted in a few hours. The removal of Ki67 before mitotic entry did not impact the early mitotic chromosome assembly observed in prophase but subsequently resulted in the formation of misshapen mitotic chromosomes. When Ki67 was removed after mitotic entry, preassembled rod-shaped mitotic chromosomes became disorganized. In addition, we show that Ki67 and TopoIIα are reciprocally coimmunoprecipitated from mitotic cell extracts. These observations indicate that Ki67 aids the finalization of mitotic chromosome formation and helps maintain rod-shaped chromosome architecture, likely in collaboration with TopoIIα. Together, these findings represent a new model in which mitotic chromosome architecture is supported both internally and externally.


Asunto(s)
Cromosomas Humanos/fisiología , Antígeno Ki-67/fisiología , Mitosis/fisiología , Antígenos de Neoplasias/fisiología , ADN-Topoisomerasas de Tipo II/fisiología , Proteínas de Unión al ADN/fisiología , Células HCT116 , Células HeLa , Humanos , Antígeno Ki-67/genética , Modelos Biológicos
14.
J Med Genet ; 53(2): 132-7, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26545878

RESUMEN

BACKGROUND: Leukodystrophies are genetic white matter disorders affecting the formation or maintenance of myelin. Among the recently discovered genetic defects associated with leukodystrophies, several genes converge on a common mechanism involving protein transcription/translation and ER stress response. METHODS: The genetic basis of a novel congenital leukodystrophy, associated with early onset spastic paraparesis, acquired microcephaly and optic atrophy was studied in six patients from three unrelated Ashkenazi-Jewish families. To this end we used homozygosity mapping, exome analysis, western blot (Hikeshi, HSF1-pS326 and b-actin) in patient fibroblasts, indirect immunofluorescence (HSP70 and HSF1) in patient fibroblasts undergoing heat shock stress, nuclear injection of plasmids expressing Hikeshi or EGFP in patient fibroblasts, in situ hybridization and Immunoblot analysis of Hikeshi in newborn and adult mouse brain. RESULTS: All the patients were homozygous for a missense mutation, p.Val54Leu, in C11ORF73 encoding HSP70 nuclear transporter protein, Hikeshi. The mutation segregated with the disease in the families and was carried by 1:200 Ashkenazi-Jewish individuals. The mutation was associated with undetectable level of Hikeshi in the patients' fibroblasts and with lack of nuclear HSP70 during heat shock stress, a phenomenon which was reversed upon the introduction of normal human Hikeshi to the patients cells. Hikeshi was found to be expressed in central white matter of mouse brain. CONCLUSIONS: These data underscore the importance of Hikeshi for HSP70 relocation into the nucleus. It is likely that in the absence of Hikeshi, HSP70 cannot attenuate the multiple heat shock induced nuclear phenotypes, leaving the cells unprotected during heat shock stress. We speculate that the sudden death of three of the six patients following a short febrile illness and the life-threatening myo-pericarditis in the fourth are the result of excess extra-nuclear HSP70 level which initiates cytokine release or provide target for natural killer cells. Alternatively, nuclear HSP70 might play an active role in stressed cells protection.


Asunto(s)
Proteínas Portadoras/genética , Efecto Fundador , Judíos/genética , Leucoencefalopatías/genética , Mutación , Adolescente , Animales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Preescolar , Femenino , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Masculino , Ratones , Atrofias Ópticas Hereditarias/genética , Linaje
15.
Traffic ; 15(7): 727-48, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24766099

RESUMEN

Importin-ß family proteins (Imp-ßs) are nucleocytoplasmic transport receptors (NTRs) that import and export proteins and RNAs through the nuclear pores. The family consists of 14-20 members depending on the biological species, and each member transports a specific group of cargoes. Thus, the Imp-ßs mediate multiple, parallel transport pathways that can be regulated separately. In fact, the spatiotemporally differential expressions and the functional regulations of Imp-ßs have been reported. Additionally, the biological significance of each pathway has been characterized by linking the function of a member of Imp-ßs to a cellular consequence. Connecting these concepts, the regulation of the transport pathways conceivably induces alterations in the cellular physiological states. However, few studies have linked the regulation of an importin-ß family NTR to an induced cellular response and the corresponding cargoes, despite the significance of this linkage in comprehending the biological relevance of the transport pathways. This review of recent reports on the regulation and biological functions of the Imp-ßs highlights the significance of the transport pathways in physiological contexts and points out the possibility that the identification of yet unknown specific cargoes will reinforce the importance of transport regulation.


Asunto(s)
Transporte Activo de Núcleo Celular , beta Carioferinas/metabolismo , Animales , Humanos
16.
EMBO J ; 31(7): 1644-53, 2012 Apr 04.
Artículo en Inglés | MEDLINE | ID: mdl-22343941

RESUMEN

How a long strand of genomic DNA is compacted into a mitotic chromosome remains one of the basic questions in biology. The nucleosome fibre, in which DNA is wrapped around core histones, has long been assumed to be folded into a 30-nm chromatin fibre and further hierarchical regular structures to form mitotic chromosomes, although the actual existence of these regular structures is controversial. Here, we show that human mitotic HeLa chromosomes are mainly composed of irregularly folded nucleosome fibres rather than 30-nm chromatin fibres. Our comprehensive and quantitative study using cryo-electron microscopy and synchrotron X-ray scattering resolved the long-standing contradictions regarding the existence of 30-nm chromatin structures and detected no regular structure >11 nm. Our finding suggests that the mitotic chromosome consists of irregularly arranged nucleosome fibres, with a fractal nature, which permits a more dynamic and flexible genome organization than would be allowed by static regular structures.


Asunto(s)
Cromosomas Humanos/química , Mitosis , Nucleosomas/química , Cromatina/química , Cromatina/ultraestructura , Cromosomas Humanos/ultraestructura , Microscopía por Crioelectrón , Células HeLa , Humanos , Nucleosomas/ultraestructura , Pliegue de Proteína , Dispersión del Ángulo Pequeño , Difracción de Rayos X
17.
J Biol Chem ; 289(33): 22877-22887, 2014 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-25012651

RESUMEN

Ki67 is a protein widely used as cell-proliferation marker, with its cellular functions being hardly unveiled. In this paper, we present the direct interaction between Ki67 and PP1γ, a protein phosphatase showing characteristic accumulation on anaphase chromosomes via the canonical PP1-binding motif within Ki67. In cells depleted of Ki67, PP1γ is targeted to anaphase chromosomes less efficiently. Additionally, overexpression of Ki67, but not a mutant form without the ability to bind PP1γ, induced ectopic localization of PP1γ οn metaphase chromosomes. These observations demonstrate that Ki67 is one factor that defines the cellular behavior of PP1γ in anaphase. To explore the specific roles of the subset of PP1γ recruited on chromosome via its interaction with Ki67 (PP1γ-Ki67), endogenous Ki67 was replaced with a Ki67 mutant deficient in its ability to interact with PP1γ. Although no obvious defects in the progression of mitosis were observed, the timing of dephosphorylation of the mutant Ki67 in anaphase was delayed, indicating that Ki67 itself is one of the substrates of PP1γ-Ki67.


Asunto(s)
Anafase/fisiología , Cromosomas Humanos/metabolismo , Antígeno Ki-67/metabolismo , Proteína Fosfatasa 1/metabolismo , Cromosomas Humanos/genética , Células HeLa , Humanos , Antígeno Ki-67/genética , Mutación , Fosforilación/fisiología , Proteína Fosfatasa 1/genética
18.
Biochim Biophys Acta ; 1840(9): 2953-60, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24797038

RESUMEN

BACKGROUND: In eukaryotic cells, molecular trafficking between the nucleus and cytoplasm is a highly regulated process related to cellular homeostasis and cellular signaling. However, various cellular stresses induce the perturbation of conventional nucleocytoplasmic transport pathways, resulting in the nucleocytoplasmic redistribution of many functional proteins. SCOPE OF REVIEW: We describe the recent insights into the mechanism and functions of nuclear import of cytosolic chaperone HSP70 under stress conditions and the cellular distribution and functions of its co-chaperones. MAJOR CONCLUSIONS: Hikeshi mediates the nuclear import of the molecular chaperone HSP70. A few of the regulators of the HSP70 chaperone system also accumulate in the nucleus under heat stress conditions. These proteins function collaboratively to protect cells from stress-induced damage and aid in the recovery of cells from stress. GENERAL SIGNIFICANCE: Studies on the regulation of nucleocytoplasmic transport under several cellular stresses should provide new insights into the fundamental principles of protein homeostasis (proteostasis) in both compartments, the nucleus and cytoplasm.


Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Animales , Humanos
19.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 3): 473-83, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25760597

RESUMEN

Hikeshi is a nuclear transport receptor required for cell survival after stress. It mediates heat-shock-induced nuclear import of 70 kDa heat-shock proteins (Hsp70s) through interactions with FG-nucleoporins (FG-Nups), which are proteins in nuclear pore complexes (NPCs). Here, the crystal structure of human Hikeshi is presented at 1.8 Šresolution. Hikeshi forms an asymmetric homodimer that is responsible for the interaction with Hsp70s. The asymmetry of Hikeshi arises from the distinct conformation of the C-terminal domain (CTD) and the flexibility of the linker regions of each monomer. Structure-guided mutational analyses showed that both the flexible linker region and the CTD are important for nuclear import of Hsp70. Pull-down assays revealed that only full-length Hsp70s can interact with Hikeshi. The N-terminal domain (NTD) consists of a jelly-roll/ß-sandwich fold structure which contains hydrophobic pockets involved in FG-Nup recognition. A unique extended loop (E-loop) in the NTD is likely to regulate the interactions of Hikeshi with FG-Nups. The crystal structure of Hikeshi explains how Hikeshi participates in the regulation of nuclear import through the recognition of FG-Nups and which part of Hikeshi affects its binding to Hsp70. This study is the first to yield structural insight into this highly unique import receptor.


Asunto(s)
Proteínas Portadoras/química , Proteínas Nucleares/química , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología
20.
Mol Cell Proteomics ; 12(1): 145-57, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23087160

RESUMEN

The human importin-ß family consists of 21 nucleocytoplasmic transport carrier proteins that carry proteins and RNAs across the nuclear envelope through nuclear pores in specific directions. These transport carriers are responsible for the nucleocytoplasmic transport of thousands of proteins, but the cargo allocation of each carrier, which is necessary information if one wishes to understand the physiological context of transport, is poorly characterized. To address this issue, we developed a high-throughput method to identify the cargoes of transport carriers by applying stable isotope labeling by amino acids in cell culture to construct an in vitro transport system. Our method can be outlined in three steps. (1) Cells are cultured in a medium containing a stable isotope. (2) The cell membranes of the labeled cells are permeabilized, and proteins extracted from unlabeled cells are transported into the nuclei of the permeabilized cells. In this step, the reaction system is first depleted of all importin-ß family carriers and then supplemented with a particular importin-ß family carrier of interest. (3) Proteins in the nuclei are extracted and analyzed quantitatively via LC-MS/MS. As an important test case, we used this method to identify cargo proteins of transportin, a representative member of the importin-ß family. As expected, the identified candidate cargo proteins included previously reported transportin cargoes as well as new potential cargoes, which we corroborated via in vitro binding assays. The identified cargoes are predominately RNA-interacting proteins, affirming that cargoes allotted to the same carrier share functional characteristics. Finally, we found that the transportin cargoes possessed at least two classes of signal sequences: the well characterized PY-nuclear localization signals specific for transportin, and Lys/Arg-rich segments capable of binding to both transportin and importin-ß. Thus, our method will be useful for linking a carrier to features shared among its cargoes and to specific nuclear localization signals.


Asunto(s)
Transporte Activo de Núcleo Celular , Señales de Localización Nuclear/metabolismo , beta Carioferinas/análisis , Secuencia de Aminoácidos , Aminoácidos , Membrana Celular , Cromatografía Liquida , Humanos , Marcaje Isotópico , Membrana Nuclear/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteómica , Espectrometría de Masas en Tándem , beta Carioferinas/metabolismo
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