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1.
Anticancer Res ; 41(9): 4563-4570, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34475084

RESUMO

BACKGROUND/AIM: Maspin has tumor-suppressor functions; however, its prognostic value in patients with oral squamous cell carcinoma (OSCC) remains unknown. We aimed to assess the prognostic importance of the subcellular localization of maspin in patients with OSCC. PATIENTS AND METHODS: Eighty resected specimens were analyzed by immunohistochemistry. Cytoplasmic-only expression observed in >10% of the tumor was defined as maspin-positive. RESULTS: The maspin-positive status (25%) was significantly associated with a higher recurrence rate and shorter disease-free survival (DFS). Cox's multivariate analysis showed that maspin-positive status was an independent factor for shorter DFS. All OSCC cell lines (HSC2, HSC3, HSC4, Ca9-22 and SAS) showed maspin protein localization to both the cytoplasm and nucleus using western blot analysis. In HSC4 cells, cell invasion was significantly increased in response to maspin knockdown. CONCLUSION: Cytoplasmic-only expression of maspin could be an independent poor prognostic factor for patients with OSCC.


Assuntos
Carcinoma de Células Escamosas/cirurgia , Citoplasma/metabolismo , Neoplasias Bucais/cirurgia , Serpinas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/metabolismo , Análise Multivariada , Prognóstico , Análise de Sobrevida
2.
Nat Commun ; 12(1): 4855, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381044

RESUMO

The vertebrate brain consists of diverse neuronal types, classified by distinct anatomy and function, along with divergent transcriptomes and proteomes. Defining the cell-type specific neuroproteomes is important for understanding the development and functional organization of neural circuits. This task remains challenging in complex tissue, due to suboptimal protein isolation techniques that often result in loss of cell-type specific information and incomplete capture of subcellular compartments. Here, we develop a genetically targeted proximity labeling approach to identify cell-type specific subcellular proteomes in the mouse brain, confirmed by imaging, electron microscopy, and mass spectrometry. We virally express subcellular-localized APEX2 to map the proteome of direct and indirect pathway spiny projection neurons in the striatum. The workflow provides sufficient depth to uncover changes in the proteome of striatal neurons following chemogenetic activation of Gαq-coupled signaling cascades. This method enables flexible, cell-type specific quantitative profiling of subcellular proteome snapshots in the mouse brain.


Assuntos
Ascorbato Peroxidases/metabolismo , Núcleo Celular/metabolismo , Corpo Estriado/metabolismo , Proteoma/metabolismo , Animais , Ascorbato Peroxidases/genética , Corpo Estriado/citologia , Citosol/metabolismo , Espectrometria de Massas , Camundongos , Vias Neurais , Neurônios/citologia , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Coloração e Rotulagem , Fluxo de Trabalho
3.
Int J Mol Sci ; 22(15)2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34361059

RESUMO

In vertebrates, nucleostemin (NS) is an important marker of proliferation in several types of stem and cancer cells, and it can also interact with the tumor-suppressing transcription factor p53. In the present study, the intra-nuclear diffusional dynamics of native NS tagged with GFP and two GFP-tagged NS mutants with deleted guanosine triphosphate (GTP)-binding domains were analyzed by fluorescence correlation spectroscopy. Free and slow binding diffusion coefficients were evaluated, either under normal culture conditions or under treatment with specific cellular proliferation inhibitors actinomycin D (ActD), 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), or trichostatin A (TSA). When treated with ActD, the fractional ratio of the slow diffusion was significantly decreased in the nucleoplasm. The decrease was proportional to ActD treatment duration. In contrast, DRB or TSA treatment did not affect NS diffusion. Interestingly, it was also found that the rate of diffusion of two NS mutants increased significantly even under normal conditions. These results suggest that the mobility of NS in the nucleoplasm is related to the initiation of DNA or RNA replication, and that the GTP-binding motif is also related to the large change of mobility.


Assuntos
Núcleo Celular/metabolismo , Dactinomicina/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Nucleares/metabolismo , Inibidores da Síntese de Ácido Nucleico/farmacologia , Transcrição Genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/genética , Células HeLa , Humanos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética
4.
Planta ; 254(3): 48, 2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34379202

RESUMO

MAIN CONCLUSION: During antipodal cells PCD, polytene chromosomes rearrangement, segregation of nucleoli components and extrusion of nuclear components occur, cytochrome c is released from the mitochondria and DNA breaks appear. We studied in detail the nuclei of cells of the antipodal complex of wheat embryo sac (Triticum aestivum L.) during programmed cell death (PCD). The antipodal complex has been reported to be formed before double fertilisation of the embryo sac. Polyploidisation leads to the formation of giant polytene chromosomes in the nuclei of antipodal cells. These chromosomes are involved in secretory functions and are important for the development of cellular endosperm. Terminal deoxynucleotidyl transferase dUTP nick end labelling assay and immunodetection revealed DNA breaks in the nuclei and release of cytochrome c from mitochondria into the cytoplasm of antipodal cells during PCD. We used transmission electron microscopy, immunodetection and histochemistry to analyse the characteristic structural changes in the nuclei of antipodal cells during PCD. These included sequential structural changes in the nuclei containing polytene chromosomes, segregation of some components of the nucleolus into the bodies of polytene chromosomes, extrusion of nucleolar components and parts of chromosomes into the cytoplasm of antipodal cells and then into the endosperm coenocyte. The obtained results expand the understanding of the structural changes of plant cells with giant polytene chromosomes during PCD.


Assuntos
Núcleo Celular , Triticum , Apoptose , Núcleo Celular/metabolismo , Endosperma , Mitocôndrias , Triticum/genética
5.
Nat Commun ; 12(1): 4657, 2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34341359

RESUMO

Correlative imaging and quantification of intracellular nanoparticles with the underlying ultrastructure is crucial for understanding cell-nanoparticle interactions in biological research. However, correlative nanoscale imaging of whole cells still remains a daunting challenge. Here, we report a straightforward nanoscopic approach for whole-cell correlative imaging, by simultaneous ionoluminescence and ultrastructure mapping implemented with a highly focused beam of alpha particles. We demonstrate that fluorescent nanodiamonds exhibit fast, ultrabright and stable emission upon excitation by alpha particles. Thus, by using fluorescent nanodiamonds as imaging probes, our approach enables quantification and correlative localization of single nanodiamonds within a whole cell at sub-30 nm resolution. As an application example, we show that our approach, together with Monte Carlo simulations and radiobiological experiments, can be employed to provide unique insights into the mechanisms of nanodiamond radiosensitization at the single whole-cell level. These findings may benefit clinical studies of radio-enhancement effects by nanoparticles in charged-particle cancer therapy.


Assuntos
Partículas alfa , Núcleo Celular/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Histonas/metabolismo , Nanodiamantes/efeitos da radiação , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células HeLa , Células Hep G2 , Humanos , Microscopia Confocal/métodos , Microscopia Eletrônica de Varredura/métodos , Nanodiamantes/química , Nanodiamantes/ultraestrutura , Fosforilação/efeitos da radiação
6.
Nat Commun ; 12(1): 4696, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34349113

RESUMO

Productive ribosomal RNA (rRNA) compaction during ribosome assembly necessitates establishing correct tertiary contacts between distant secondary structure elements. Here, we quantify the response of the yeast proteome to low temperature (LT), a condition where aberrant mis-paired RNA folding intermediates accumulate. We show that, at LT, yeast cells globally boost production of their ribosome assembly machinery. We find that the LT-induced assembly factor, Puf6, binds to the nascent catalytic RNA-rich subunit interface within the 60S pre-ribosome, at a site that eventually loads the nuclear export apparatus. Ensemble Förster resonance energy transfer studies show that Puf6 mimics the role of Mg2+ to usher a unique long-range tertiary contact to compact rRNA. At LT, puf6 mutants accumulate 60S pre-ribosomes in the nucleus, thus unveiling Puf6-mediated rRNA compaction as a critical temperature-regulated rescue mechanism that counters rRNA misfolding to prime export competence.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a RNA/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Temperatura Baixa , GTP Fosfo-Hidrolases/metabolismo , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteoma/metabolismo , Dobramento de RNA , Precursores de RNA/química , Precursores de RNA/metabolismo , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Subunidades Ribossômicas Maiores de Eucariotos/química , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
7.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360671

RESUMO

Regulated cell death (RCD) is a fundamental process common to nearly all living beings and essential for the development and tissue homeostasis in animals and humans. A wide range of molecules can induce RCD, including a number of viral proteolytic enzymes. To date, numerous data indicate that picornaviral 3C proteases can induce RCD. In most reported cases, these proteases induce classical caspase-dependent apoptosis. In contrast, the human hepatitis A virus 3C protease (3Cpro) has recently been shown to cause caspase-independent cell death accompanied by previously undescribed features. Here, we expressed 3Cpro in HEK293, HeLa, and A549 human cell lines to characterize 3Cpro-induced cell death morphologically and biochemically using flow cytometry and fluorescence microscopy. We found that dead cells demonstrated necrosis-like morphological changes including permeabilization of the plasma membrane, loss of mitochondrial potential, as well as mitochondria and nuclei swelling. Additionally, we showed that 3Cpro-induced cell death was efficiently blocked by ferroptosis inhibitors and was accompanied by intense lipid peroxidation. Taken together, these results indicate that 3Cpro induces ferroptosis upon its individual expression in human cells. This is the first demonstration that a proteolytic enzyme can induce ferroptosis, the recently discovered and actively studied type of RCD.


Assuntos
Proteases Virais 3C/metabolismo , Núcleo Celular/patologia , Ferroptose , Mitocôndrias/patologia , Proteases Virais 3C/genética , Células A549 , Núcleo Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Técnicas In Vitro , Peroxidação de Lipídeos , Mitocôndrias/metabolismo
8.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445470

RESUMO

In regular IVF, a portion of oocytes exhibit abnormal numbers of pronuclei (PN) that is considered as abnormal fertilization, and they are routinely discarded. However, it is known that abnormal ploidy still does not completely abandon embryo development and implantation. To explore the potential of cytoplasm from those abnormally fertilized oocytes, we developed a novel technique for the transfer of large cytoplasm between pronuclear-stage mouse embryos, and assessed its impact. A large volume of cytoplast could be efficiently transferred in the PN stage using a novel two-step method of pronuclear-stage cytoplasmic transfer (PNCT). PNCT revealed the difference in the cytoplasmic function among abnormally fertilized embryos where the cytoplasm of 3PN was developmentally more competent than 1PN, and the supplementing of fresh 3PN cytoplasm restored the impaired developmental potential of postovulatory "aged" oocytes. PNCT-derived embryos harbored significantly higher mitochondrial DNA copies, ATP content, oxygen consumption rate, and total cells. The difference in cytoplasmic function between 3PN and 1PN mouse oocytes probably attributed to the proper activation via sperm and may impact subsequent epigenetic events. These results imply that PNCT may serve as a potential alternative treatment to whole egg donation for patients with age-related recurrent IVF failure.


Assuntos
Núcleo Celular/patologia , Citoplasma/patologia , Embrião de Mamíferos/patologia , Desenvolvimento Embrionário , Fertilização In Vitro/métodos , Zigoto/patologia , Animais , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Embrião de Mamíferos/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos ICR , Zigoto/metabolismo
9.
Nucleic Acids Res ; 49(15): 8900-8922, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34370034

RESUMO

In eukaryotes, the major nuclear export pathway for mature mRNAs uses the dimeric receptor TAP/p15, which is recruited to mRNAs via the multisubunit TREX complex, comprising the THO core and different export adaptors. Viruses that replicate in the nucleus adopt different strategies to hijack cellular export factors and achieve cytoplasmic translation of their mRNAs. No export receptors are known in plants, but Arabidopsis TREX resembles the mammalian complex, with a conserved hexameric THO core associated with ALY and UIEF proteins, as well as UAP56 and MOS11. The latter protein is an orthologue of mammalian CIP29. The nuclear export mechanism for viral mRNAs has not been described in plants. To understand this process, we investigated the export of mRNAs of the pararetrovirus CaMV in Arabidopsis and demonstrated that it is inhibited in plants deficient in ALY, MOS11 and/or TEX1. Deficiency for these factors renders plants partially resistant to CaMV infection. Two CaMV proteins, the coat protein P4 and reverse transcriptase P5, are important for nuclear export. P4 and P5 interact and co-localise in the nucleus with the cellular export factor MOS11. The highly structured 5' leader region of 35S RNAs was identified as an export enhancing element that interacts with ALY1, ALY3 and MOS11 in vitro.


Assuntos
Regiões 5' não Traduzidas , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/virologia , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas Virais/metabolismo , Transporte Ativo do Núcleo Celular , Arabidopsis/virologia , Proteínas de Arabidopsis/fisiologia , Proteínas do Capsídeo/metabolismo , Caulimovirus/genética , Caulimovirus/metabolismo , Núcleo Celular/metabolismo , Doenças das Plantas/virologia , RNA Viral/química , DNA Polimerase Dirigida por RNA/metabolismo
10.
J Cell Sci ; 134(2)2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-34432038

RESUMO

Bidirectional transport of macromolecules across the nuclear envelope is a hallmark of eukaryotic cells, in which the genetic material is compartmentalized inside the nucleus. The nuclear pore complex (NPC) is the major gateway to the nucleus and it regulates nucleocytoplasmic transport, which is key to processes including transcriptional regulation and cell cycle control. Accordingly, components of the nuclear transport machinery are often found to be dysregulated or hijacked in diseases. In this Cell Science at a Glance article and accompanying poster, we provide an overview of our current understanding of cargo transport through the NPC, from the basic transport signals and machinery to more emerging aspects, all from a 'cargo perspective'. Among these, we discuss the transport of large cargoes (>15 nm), as well as the roles of different cargo properties to nuclear transport, from size and number of bound nuclear transport receptors (NTRs), to surface and mechanical properties.


Assuntos
Membrana Nuclear , Poro Nuclear , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Células Eucarióticas/metabolismo , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo
11.
Nat Commun ; 12(1): 4908, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389711

RESUMO

C9ORF72 hexanucleotide GGGGCC repeat expansion is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat-containing RNA mediates toxicity through nuclear granules and dipeptide repeat (DPR) proteins produced by repeat-associated non-AUG translation. However, it remains unclear how the intron-localized repeats are exported and translated in the cytoplasm. We use single molecule imaging approach to examine the molecular identity and spatiotemporal dynamics of the repeat RNA. We demonstrate that the spliced intron with G-rich repeats is stabilized in a circular form due to defective lariat debranching. The spliced circular intron, instead of pre-mRNA, serves as the translation template. The NXF1-NXT1 pathway plays an important role in the nuclear export of the circular intron and modulates toxic DPR production. This study reveals an uncharacterized disease-causing RNA species mediated by repeat expansion and demonstrates the importance of RNA spatial localization to understand disease etiology.


Assuntos
Proteína C9orf72/genética , Núcleo Celular/metabolismo , Íntrons/genética , Biossíntese de Proteínas/genética , RNA/genética , Transporte Ativo do Núcleo Celular/genética , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Proteína C9orf72/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/genética , Expansão das Repetições de DNA/genética , Dipeptídeos/genética , Dipeptídeos/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Predisposição Genética para Doença/genética , Células HEK293 , Humanos , Microscopia de Fluorescência , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética
12.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201896

RESUMO

miR-29b2 and miR-29c play a suppressive role in breast cancer progression. C1orf132 (also named MIR29B2CHG) is the host gene for generating both microRNAs. However, the region also expresses longer transcripts with unknown functions. We employed bioinformatics and experimental approaches to decipher C1orf132 expression and function in breast cancer tissues. We also used the CRISPR/Cas9 technique to excise a predicted C1orf132 distal promoter and followed the behavior of the edited cells by real-time PCR, flow cytometry, migration assay, and RNA-seq techniques. We observed that C1orf132 long transcript is significantly downregulated in triple-negative breast cancer. We also identified a promoter for the longer transcripts of C1orf132 whose functionality was demonstrated by transfecting MCF7 cells with a C1orf132 promoter-GFP construct. Knocking-out the promoter by means of CRISPR/Cas9 revealed no alterations in the expression of the neighboring genes CD46 and CD34, while the expression of miR-29c was reduced by half. Furthermore, the promoter knockout elevated the migration ability of the edited cells. RNA sequencing revealed many up- and downregulated genes involved in various cellular pathways, including epithelial to mesenchymal transition and mammary gland development pathways. Altogether, we are reporting here the existence of an additional/distal promoter with an enhancer effect on miR-29 generation and an inhibitory effect on cell migration.


Assuntos
RNA Longo não Codificante/genética , Neoplasias de Mama Triplo Negativas/genética , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Regulação para Baixo , Transição Epitelial-Mesenquimal/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Células MCF-7 , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Regiões Promotoras Genéticas , RNA Longo não Codificante/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo
13.
Anticancer Res ; 41(7): 3401-3407, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34230135

RESUMO

BACKGROUND/AIM: Plakophilin 1 (PKP1) expression is inversely related to cancer grade. This study aimed to evaluate whether PKP1 is a prognostic marker for esophageal cancer (EC). MATERIALS AND METHODS: We tested immunohistochemically for PKP1 in squamous cell carcinoma EC specimens from 99 patients, including cytoplasmic (C), membrane (M), and nuclear (N) cellular areas, and analyzed their relationships with clinicopathological factors. RESULTS: PKP1stains were stratified into strong and weak for all three cellular areas. Staining was inversely related to tumor depth (C: p=0.002, M: p=0.00007, N: p=0.02), lymph node metastasis (C: p=0.003, M: p=0.001, N: p=0.004) and pathological stage (C: p=0.0004, M: p=0.0001, N: p=0.006). Cytoplasmic and membrane staining were inversely related to vessel invasion. Patients with strong C stain had a better overall survival than those with weak C stains (p=0.01). Disease-free survival of patients with strong M stains was better than that of those with weak staining (p=0.01). CONCLUSION: Cytoplasmic and membrane PKP1 expression is a possible prognostic marker for EC.


Assuntos
Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Placofilinas/metabolismo , Idoso , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Citoplasma/metabolismo , Citoplasma/patologia , Intervalo Livre de Doença , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Metástase Linfática/patologia , Masculino , Prognóstico
14.
Biol Lett ; 17(7): 20210194, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34314641

RESUMO

Intrapopulation variation in behaviour, including activity, boldness and aggressiveness, is becoming more widely recognized and is hypothesized to substantially affect ecological and evolutionary dynamics. Although previous studies used candidate-gene approaches and genome-wide association analyses to identify genes correlated with variations in activity and aggressiveness, behavioural variation may not be fully captured in the nuclear genome, as it does not account for mitochondrial genomes. Mitochondrial genes encode products that are key regulators of the cellular energy-producing pathways in metabolic processes and are thought to play a significant role in life-history and reproductive traits. In this study, we considered many isofemale lines of Drosophila immigrans established from two wild populations to investigate whether intrapopulation variation in the mitochondrial genome affected activity level within this species. We identified two major haplogroups in these populations, and activity levels in both larvae and adults differed significantly between the two haplogroups. This result indicated that intrapopulation variation in activity level may be partially controlled by mitochondrial genes, along with the interaction between nuclear and mitochondrial genes and the age of individual organisms.


Assuntos
Drosophila , Genoma Mitocondrial , Animais , Núcleo Celular/metabolismo , DNA Mitocondrial/genética , Drosophila/genética , Variação Genética , Estudo de Associação Genômica Ampla , Mitocôndrias/genética
15.
Nat Commun ; 12(1): 4618, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34326347

RESUMO

The transcriptional co-activator and acetyltransferase p300 is required for fundamental cellular processes, including differentiation and growth. Here, we report that p300 forms phase separated condensates in the cell nucleus. The phase separation ability of p300 is regulated by autoacetylation and relies on its catalytic core components, including the histone acetyltransferase (HAT) domain, the autoinhibition loop, and bromodomain. p300 condensates sequester chromatin components, such as histone H3 tail and DNA, and are amplified through binding of p300 to the nucleosome. The catalytic HAT activity of p300 is decreased due to occlusion of the active site in the phase separated droplets, a large portion of which co-localizes with chromatin regions enriched in H3K27me3. Our findings suggest a model in which p300 condensates can act as a storage pool of the protein with reduced HAT activity, allowing p300 to be compartmentalized and concentrated at poised or repressed chromatin regions.


Assuntos
Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteína p300 Associada a E1A/metabolismo , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Células Cultivadas , Proteína p300 Associada a E1A/química , Humanos , Domínios Proteicos
16.
Nat Commun ; 12(1): 4502, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301937

RESUMO

Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Transporte Biológico , Fusão Celular , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Células Gigantes/metabolismo , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Camundongos , RNA-Seq/métodos , Transdução de Sinais/genética , Fatores de Transcrição/genética , Proteínas do Envelope Viral/genética
17.
Cell Prolif ; 54(8): e13088, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34240781

RESUMO

OBJECTIVES: Breast cancer-amplified sequence 3 (BCAS3) was initially found to be amplified in human breast cancer (BRCA); however, there has been little consensus on the functions of BCAS3 in breast tumours. MATERIALS AND METHODS: We analysed BCAS3 expression in BRCA using bio-information tools. Affinity purification and mass spectrometry were employed to identify BCAS3-associated proteins. GST pull-down and ubiquitination assays were performed to analyse the interaction mechanism between BCAS3/p53 and CUL4A-RING E3 ubiquitin ligase (CRL4A) complex. BCAS3 was knocked down individually or in combination with p53 in MCF-7 cells to further explore the biological functions of the BCAS3/p53 axis. The clinical values of BCAS3 for BRCA progression were evaluated via semiquantitative immunohistochemistry (IHC) analysis and Cox regression. RESULTS: We reported that the expression level of BCAS3 in BRCA was higher than that in adjacent normal tissues. High BCAS3 expression promoted growth, inhibited apoptosis and conferred chemoresistance in breast cancer cells. Mechanistically, BCAS3 overexpression fostered BRCA cell growth by interacting with the CRL4A complex and promoting ubiquitination and proteasomal degradation of p53. Furthermore, BCAS3 could regulate cell growth, apoptosis and chemoresistance through a p53-mediated mechanism. Clinically, BCAS3 overexpression was significantly correlated with a malignant phenotype. Moreover, higher expression of BCAS3 correlates with shorter overall survival (OS) in BRCA. CONCLUSIONS: The functional characterization of BCAS3 offers new insights into the oncogenic properties and chemotherapy resistance in breast cancer.


Assuntos
Neoplasias da Mama/patologia , Proteínas de Neoplasias/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Proteínas Culina/metabolismo , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Pessoa de Meia-Idade , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Prognóstico , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Taxa de Sobrevida , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
18.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299219

RESUMO

Infertility is a problem affecting an increasing number of couples worldwide. Currently, marker tests for male factor infertility are complex, highly technical and relatively subjective. Up to 40% of cases of male factor infertility are currently diagnosed as idiopathic therefore, there is a clear need for further research into better ways of diagnosing it. Changes in sperm telomere length have been associated with infertility and closely linked to DNA damage and fragmentation, which are also known to be related to infertility. However, telomere distribution is a parameter thus far underexplored as an infertility marker. Here, we assessed morphological parameters of sperm nuclei in fertile control and male factor infertile cohorts. In addition, we used 2D and 3D fluorescence in situ hybridization (FISH) to compare telomere distribution between these two groups. Our findings indicate that the infertile cohort sperm nuclei were, on average, 2.9% larger in area and showed subtle differences in sperm head height and width. Telomeres were mainly distributed towards the periphery of the nuclei in the control cohort, with diminishing telomere signals towards the center of the nuclei. Sperm nuclei of infertile males, however, had more telomere signals towards the center of the nuclei, a finding supported by 3D imaging. We conclude that, with further development, both morphology and telomere distribution may prove useful investigative tools in the fertility clinic.


Assuntos
Dano ao DNA , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Cabeça do Espermatozoide/patologia , Telômero/metabolismo , Biomarcadores/metabolismo , Estudos de Casos e Controles , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Estudos de Coortes , Humanos , Infertilidade Masculina/genética , Masculino , Cabeça do Espermatozoide/metabolismo , Telômero/genética
19.
J Cell Sci ; 134(13)2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34313312

RESUMO

The shuttling of transcription factors and transcriptional regulators into and out of the nucleus is central to the regulation of many biological processes. Here we describe a new method for studying the rates of nuclear entry and exit of transcriptional regulators. A photo-responsive LOV (light-oxygen-voltage) domain from Avena sativa is used to sequester fluorescently labelled transcriptional regulators YAP1 and TAZ (also known as WWTR1) on the surface of mitochondria and to reversibly release them upon blue light illumination. After dissociation, fluorescent signals from the mitochondria, cytoplasm and nucleus are extracted by a bespoke app and used to generate rates of nuclear entry and exit. Using this method, we demonstrate that phosphorylation of YAP1 on canonical sites enhances its rate of nuclear export. Moreover, we provide evidence that, despite high intercellular variability, YAP1 import and export rates correlate within the same cell. By simultaneously releasing YAP1 and TAZ from sequestration, we show that their rates of entry and exit are correlated. Furthermore, combining the optogenetic release of YAP1 with lattice light-sheet microscopy reveals high heterogeneity of YAP1 dynamics within different cytoplasmic regions, demonstrating the utility and versatility of our tool to study protein dynamics. This article has an associated First Person interview with Anna M. Dowbaj, joint first author of the paper.


Assuntos
Transporte Ativo do Núcleo Celular , Optogenética , Proteínas de Plantas , Avena , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
Int J Mol Sci ; 22(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34298951

RESUMO

The chemokines CCL5 and CXCL4 are deposited by platelets onto endothelial cells, inducing monocyte arrest. Here, the fate of CCL5 and CXCL4 after endothelial deposition was investigated. Human umbilical vein endothelial cells (HUVECs) and EA.hy926 cells were incubated with CCL5 or CXCL4 for up to 120 min, and chemokine uptake was analyzed by microscopy and by ELISA. Intracellular calcium signaling was visualized upon chemokine treatment, and monocyte arrest was evaluated under laminar flow. Whereas CXCL4 remained partly on the cell surface, all of the CCL5 was internalized into endothelial cells. Endocytosis of CCL5 and CXCL4 was shown as a rapid and active process that primarily depended on dynamin, clathrin, and G protein-coupled receptors (GPCRs), but not on surface proteoglycans. Intracellular calcium signals were increased after chemokine treatment. Confocal microscopy and ELISA measurements in cell organelle fractions indicated that both chemokines accumulated in the nucleus. Internalization did not affect leukocyte arrest, as pretreatment of chemokines and subsequent washing did not alter monocyte adhesion to endothelial cells. Endothelial cells rapidly and actively internalize CCL5 and CXCL4 by clathrin and dynamin-dependent endocytosis, where the chemokines appear to be directed to the nucleus. These findings expand our knowledge of how chemokines attract leukocytes to sites of inflammation.


Assuntos
Núcleo Celular/metabolismo , Quimiocina CCL5/metabolismo , Células Endoteliais/metabolismo , Fator Plaquetário 4/metabolismo , Transporte Ativo do Núcleo Celular , Linhagem Celular , Humanos
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