RESUMEN
Repair of DNA double-strand breaks (DSBs) elicits three-dimensional (3D) chromatin topological changes. A recent finding reveals that 53BP1 assembles into a 3D chromatin topology pattern around DSBs. How this formation of a higher-order structure is configured and regulated remains enigmatic. Here, we report that SLFN5 is a critical factor for 53BP1 topological arrangement at DSBs. Using super-resolution imaging, we find that SLFN5 binds to 53BP1 chromatin domains to assemble a higher-order microdomain architecture by driving damaged chromatin dynamics at both DSBs and deprotected telomeres. Mechanistically, we propose that 53BP1 topology is shaped by two processes: (1) chromatin mobility driven by the SLFN5-LINC-microtubule axis and (2) the assembly of 53BP1 oligomers mediated by SLFN5. In mammals, SLFN5 deficiency disrupts the DSB repair topology and impairs non-homologous end joining, telomere fusions, class switch recombination, and sensitivity to poly (ADP-ribose) polymerase inhibitor. We establish a molecular mechanism that shapes higher-order chromatin topologies to safeguard genomic stability.
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Cromatina , Reparación del ADN , Animales , Cromatina/genética , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Mamíferos/metabolismo , Proteínas de Unión a Telómeros/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteínas de Ciclo Celular/metabolismoRESUMEN
The understanding of schwannoma tumorigenesis has been reshaped by the recent identification of SH3PXD2A::HTRA1 fusion in 10% of intracranial/spinal schwannomas. Nonetheless, pathologic features of schwannomas harboring this fusion, as well as its prevalence outside intracranial/spinal locations, have not been characterized. We screened 215 consecutive schwannomas for their clinicopathologic characteristics and fusion status using reverse-transcriptase polymerase chain reaction (RT-PCR). Among 29 (13.5%) fusion-positive schwannomas, the most prevalent location was peripheral somatic tissue (30.7%, 19/62), followed by spinal/paraspinal (18.4%, 7/38), body cavity/deep structures (10%, 2/20), intracranial (1.3%, 1/75), and viscera (0/13). All 8 cellular, 4 microcystic/reticular, and 3 epithelioid schwannomas were fusion-negative, as were 41/42 nonschwannomatous peripheral nerve sheath tumors. Remarkably, a distinct 'serpentine' palisading pattern, comprising ovoid/plump cells shorter than usual schwannian cells in a hyalinized stroma, was identified in most fusion-positive cases and the schwannomatous component of the only fusion-positive malignant peripheral nerve sheath tumor. To validate this finding, 60 additional cases were collected, including 36 with (≥10% arbitrarily) and 24 without appreciable serpentine histology, of which 29 (80.6%) and 2 (8.3%) harbored the fusion, respectively. With percentages of 'serpentine' areas scored, 10% was determined as the optimal practical cut-off to predict the fusion status (sensitivity, 0.950; specificity, 0.943). Fusion positivity was significantly associated with serpentine histology, smaller tumors, younger patients, and peripheral somatic tissue, while multivariate logistic linear regression analysis only identified serpentine histology and location as independent fusion-predicting factors. RNA in situ hybridization successfully detected the fusion junction, highly concordant with RT-PCR results. Gene expression profiling on 18 schwannomas demonstrated segregation largely consistent with fusion status. Fusion-positive cases expressed significantly higher HTRA1 mRNA abundance, perhaps exploitable as a biomarker. In summary, we systematically characterize a series of 60 SH3PXD2A::HTRA1 fusion-positive schwannomas, showing their distinctive morphology and location-specific prevalence for the first time.
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Neoplasias de la Vaina del Nervio , Neurilemoma , Humanos , Neurilemoma/patología , Neoplasias de la Vaina del Nervio/patología , Transformación Celular Neoplásica , Proteínas Adaptadoras del Transporte VesicularRESUMEN
Extraskeletal myxoid chondrosarcoma (EMC) is an ultrarare sarcoma typically exhibiting myxoid/reticular histology and NR4A3 translocation. However, morphologic variants and the relevance of non-EWSR1::NR4A3 fusions remain underexplored. Three challenging pan-Trk-expressing cases, featuring cellular to solid histology, were subjected to RNA exome sequencing (RES), unveiling different NR4A3-associated fusions. Alongside RES-analyzed cases, fluorescence in situ hybridization was performed to confirm 58 EMCs, with 48 available for pan-Trk immunostaining and KIT sequencing. Except for 1 (2%) NR4A3-rearranged EMC without identifiable partners, 46 (79%), 9 (16%), and 2 (3%) cases harbored EWSR1::NR4A3, TAF15::NR4A3, and TCF12::NR4A3 fusions, respectively. Five EWSR1::NR4A3-positive EMCs occurred in the subcutis (3) and bone (2). Besides 43 classical cases, there were 8 cellular, 4 rhabdoid/anaplastic, 2 solid, and 1 mixed tumor-like variants. Tumor cells were oval/spindle to pleomorphic and formed loose myxoid/reticular to compact sheet-like or fascicular patterns, imparting broad diagnostic considerations. RES showed upregulation of NTRK2/3, KIT, and INSM1. Moderate-to-strong immunoreactivities of pan-Trk, CD117, and INSM1 were present in 35.4%, 52.6%, and 54.6% of EMCs, respectively. KIT p. E554K mutation was detected in 2/48 cases. TAF15::NR4A3 was significantly associated with size >10 cm (78%, P = .025). Size >10 cm, moderate-to-severe nuclear pleomorphism, metastasis at presentation, TAF15::NR4A3 fusion, and the administration of chemotherapy portended shorter univariate disease-specific survival, whereas only size >10 cm (P = .004) and metastasis at presentation (P = .032) remained prognostically independent. Conclusively, EMC may manifest superficial or osseous lesions harboring EWSR1::NR4A3, underrecognized solid or anaplastic histology, and pan-Trk expression, posing tremendous challenges. Most TAF15::NR4A3-positive cases were >10 cm in size, ie, a crucial independent prognosticator, whereas pathogenic KIT mutation rarely occurred.
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Condrosarcoma , Receptores de Esteroides , Sarcoma , Factores Asociados con la Proteína de Unión a TATA , Humanos , Hibridación Fluorescente in Situ , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Condrosarcoma/genética , Condrosarcoma/diagnóstico , Sarcoma/genética , Factores Asociados con la Proteína de Unión a TATA/genética , Proteínas Represoras/genética , Proteínas de Unión al ADN/genética , Receptores de Esteroides/genética , Receptores de Hormona Tiroidea/genéticaRESUMEN
The human RecQ helicase BLM is involved in the DNA damage response, DNA metabolism, and genetic stability. Loss of function mutations in BLM cause the genetic instability/cancer predisposition syndrome Bloom syndrome. However, the molecular mechanism underlying the regulation of BLM in cancers remains largely elusive. Here, we demonstrate that the deubiquitinating enzyme USP37 interacts with BLM and that USP37 deubiquitinates and stabilizes BLM, thereby sustaining the DNA damage response (DDR). Mechanistically, DNA double-strand breaks (DSB) promotes ATM phosphorylation of USP37 and enhances the binding between USP37 and BLM. Moreover, knockdown of USP37 increases BLM polyubiquitination, accelerates its proteolysis, and impairs its function in DNA damage response. This leads to enhanced DNA damage and sensitizes breast cancer cells to DNA-damaging agents in both cell culture and in vivo mouse models. Collectively, our results establish a novel molecular mechanism for the USP37-BLM axis in regulating DSB repair with an important role in chemotherapy and radiotherapy response in human cancers.
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Neoplasias de la Mama/genética , Reparación del ADN , Endopeptidasas/genética , Regulación Neoplásica de la Expresión Génica , RecQ Helicasas/genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Línea Celular Tumoral , ADN/genética , ADN/metabolismo , Roturas del ADN de Doble Cadena , Replicación del ADN , Endopeptidasas/metabolismo , Femenino , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Ratones , Fosforilación , Unión Proteica , Estabilidad Proteica , Proteolisis , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , RecQ Helicasas/metabolismo , Análisis de Supervivencia , Ubiquitinación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Transient receptor potential (TRP) cation channels, which are conserved across mammals, flies, fish, sea squirts, worms, and fungi, essentially contribute to cellular Ca2+ signaling. The activity of the unique TRP channel in yeast, TRP yeast channel 1 (TRPY1), relies on the vacuolar and cytoplasmic Ca2+ concentration. However, the mechanism(s) of Ca2+-dependent regulation of TRPY1 and possible contribution(s) of Ca2+-binding proteins are yet not well understood. Our results demonstrate a Ca2+-dependent binding of yeast calmodulin (CaM) to TRPY1. TRPY1 activity was increased in the cmd1-6 yeast strain, carrying a non-Ca2+-binding CaM mutant, compared with the parent strain expressing wt CaM (Cmd1). Expression of Cmd1 in cmd1-6 yeast rescued the wt phenotype. In addition, in human embryonic kidney 293 cells, hypertonic shock-induced TRPY1-dependent Ca2+ influx and Ca2+ release were increased by the CaM antagonist ophiobolin A. We found that coexpression of mammalian CaM impeded the activity of TRPY1 by reinforcing effects of endogenous CaM. Finally, inhibition of TRPY1 by Ca2+-CaM required the cytoplasmic amino acid stretch E33-Y92. In summary, our results show that TRPY1 is under inhibitory control of Ca2+-CaM and that mammalian CaM can replace yeast CaM for this inhibition. These findings add TRPY1 to the innumerable cellular proteins, which include a variety of ion channels, that use CaM as a constitutive or dissociable Ca2+-sensing subunit, and contribute to a better understanding of the modulatory mechanisms of Ca2+-CaM.
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Señalización del Calcio , Calcio/metabolismo , Calmodulina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Canales Catiónicos TRPC/metabolismo , Vacuolas/metabolismo , Calcio/química , Calmodulina/antagonistas & inhibidores , Calmodulina/química , Calmodulina/genética , Células HEK293 , Humanos , Dominios Proteicos , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Sesterterpenos/farmacología , Canales Catiónicos TRPC/química , Canales Catiónicos TRPC/genética , Vacuolas/química , Vacuolas/genéticaRESUMEN
PURPOSE: Sirtuin7 (SIRT7), as a member of the sirtuin and NAD+-dependent protein-modifying enzyme family, plays an important role in regulating cellular metabolism, stress responses, tumorigenesis, and aging. Ubiquitination and deubiquitination are reversible post-translational modifications that regulate protein stability, enzyme activity, protein-protein interactions, and cellular signaling transduction. However, whether SIRT7 is regulated by deubiquitination signaling is unclear. This study aims to elucidate the molecular mechanism of SIRT7 via deubiquitination signaling. METHODS: USP17L2 or SIRT7-targeting shRNAs were used to deplete USP17L2 or SIRT7. Western blot was applied to assess the effects of USP17L2 or SIRT7 depletion. A co-immunoprecipitation assay was used to detect the interaction relationship. Cell Counting Kit-8 assays were applied to assess the viability of breast cancer cells. An immunohistochemistry assay was employed to detect the protein level in samples from breast cancer patients, and the TCGA database was applied to analyze the survival rate of breast cancer patients. Statistical analyses were performed with the Student's t test (two-tailed unpaired) and χ2 test. RESULTS: We find that the deubiquitinase USP17L2 interacts with and deubiquitinates SIRT7, thereby increasing SIRT7 protein stability. In addition, USP17L2 regulates DNA damage repair through SIRT7. Furthermore, SIRT7 polyubiquitination is increased by knocking down of USP17L2, which leads to cancer cells sensitizing to chemotherapy. In breast cancer patient samples, high expression of USP17L2 is correlated with increased levels of SIRT7 protein. In conclusion, our study demonstrates that the USP17L2-SIRT7 axis is the new regulator in DNA damage response and chemo-response, suggesting that USP17L2 may be a prognostic factor and a potential therapeutic target in breast cancer. CONCLUSION: Our results highlighted that USP17L2 regulates the chemoresistance of breast cancer cells in a SIRT7-dependent manner. Moreover, the role of USP17L2 as a potential therapeutic target in breast cancer and a prognostic factor for patients was elucidated.
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Neoplasias de la Mama , Sirtuinas , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Línea Celular Tumoral , Daño del ADN , Enzimas Desubicuitinizantes/genética , Resistencia a Antineoplásicos/genética , Endopeptidasas/genética , Femenino , Humanos , NAD/genética , Sirtuinas/genética , Sirtuinas/metabolismoRESUMEN
We propose and demonstrate a received-signal-strength (RSS) based visible light positioning (VLP) system using a low-cost organic photovoltaic cell (OPVC) receiver (Rx). The OPVC is a passive device without the need of external power supply. It could detect VLC signal and harvest energy. Our developed OPVC has a high power conversion efficiency (PCE) of 9.8%. The VLP system can be operated at a low illumination of 130 lux. The regression machine learning (ML) algorithm is used to enhance the positioning accuracy.
RESUMEN
AIMS: Spindle cell/sclerosing rhabdomyosarcomas (SC/SRMS) feature spindled and/or rounded rhabdomyosarcomatous cells within variably hyalinised stroma. Only 30-67% of SC/SRMSs harbour neomorphic MYOD1 p.L122R mutations, indicating heterogeneity in this RMS type. We compared MYOD1-mutant and non-mutant cases to characterise the histological and genetic spectrum of mutated SC/SRMS. METHODS AND RESULTS: Seventeen RMSs with spindled, sclerosing or hybrid histology were sequenced to identify MYOD1 and PIK3CA mutations and reappraised to assess histological features and myogenic immunophenotypes. Twelve SC/SRMSs harboured MYOD1 mutations, including homozygous p.L122R (n = 8), heterozygous p.L122R (n = 3) and heterozygous p.E118K (n = 1). MYOD1-mutant tumours affected nine females and three males aged 8-64 years (median = 22.5), had a median size of 4.2 cm (range = 2-22) and involved the head and neck (n = 7), extremities (n = 4) and mediastinum (n = 1). Fascicular/spindle histology was predominant in four cases, including one with heterologous lipoblasts in focally myxoid stroma. Four sclerosing cases mainly comprised rounded cells, including one with multinucleated tumour cells. Four cases were histologically hybrid. The only PIK3CA (p.H1047R) mutation was detected in a predominantly spindled MYOD1-p.L122R-mutated case, but not in its laser-microdissected lipoblast-containing area. All MYOD1-mutant cases exhibited diffuse MYOD1 expression but patchy myogenin reactivity. At final follow-up (median = 13.5 months), recurrences (n = 4), metastases (n = 2) or both (n = 1) occurred in seven MYOD1-mutant cases; one had died of disease. Five non-mutated cases were reclassified as spindle embryonal (n = 3), dense embryonal (n = 1) and unclassifiable (n = 1) RMSs. CONCLUSION: MYOD1-mutant RMSs are uncommonly mutated with PIK3CA and behave aggressively with an expanded morphological and genetic spectrum, including lipoblastic differentiation, multinucleated cells and the alternative p.E118K mutation.
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Proteína MioD/genética , Rabdomiosarcoma/genética , Rabdomiosarcoma/patología , Adolescente , Adulto , Niño , Fosfatidilinositol 3-Quinasa Clase I/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Estudios Retrospectivos , Sarcoma/genética , Sarcoma/patología , Adulto JovenRESUMEN
Cooperative binding of transcription factors is known to be important in the regulation of gene expression programs conferring cellular identities. However, current methods to measure cooperativity parameters have been laborious and therefore limited to studying only a few sequence variants at a time. We developed Coop-seq (cooperativity by sequencing) that is capable of efficiently and accurately determining the cooperativity parameters for hundreds of different DNA sequences in a single experiment. We apply Coop-seq to 12 dimer pairs from the Sox and POU families of transcription factors using 324 unique sequences with changed half-site orientation, altered spacing and discrete randomization within the binding elements. The study reveals specific dimerization profiles of different Sox factors with Oct4. By contrast, Oct4 and the three neural class III POU factors Brn2, Brn4 and Oct6 assemble with Sox2 in a surprisingly indistinguishable manner. Two novel half-site configurations can support functional Sox/Oct dimerization in addition to known composite motifs. Moreover, Coop-seq uncovers a nucleotide switch within the POU half-site when spacing is altered, which is mirrored in genomic loci bound by Sox2/Oct4 complexes.
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Factores del Dominio POU/metabolismo , Factores de Transcripción SOX/metabolismo , Animales , ADN/química , ADN/metabolismo , Ratones , Modelos Moleculares , Factor 3 de Transcripción de Unión a Octámeros/química , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores del Dominio POU/química , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Factores de Transcripción SOX/química , Factores de Transcripción SOXB1/química , Factores de Transcripción SOXB1/metabolismoRESUMEN
Ouabain, a cardiotonic steroid and specific Na+ /K+ -ATPase inhibitor, has a potential to induce cancer cell apoptosis but the mechanisms of apoptosis induced by ouabain are not fully understand. The aim of this study was to investigate the cytotoxic effects of ouabain on human prostate cancer DU 145 cells in vitro. Cell morphological changes were examined by phase contrast microscopy. Cell viability, cell cycle distribution, cell apoptosis, DNA damage, the production of ROS and Ca2+ , and mitochondrial membrane potential (ΔΨm ) were measured by flow cytometry assay. Results indicated that ouabain induced cell morphological changes, decreased total cell viability, induced G0/G1 phase arrest, DNA damage, and cell apoptosis, increased ROS and Ca2+ production, but decreased the levels of ΔΨm in DU 145 cells. Ouabain also increased the activities of caspase-3, -8, and -9. Western blotting was used for measuring the alterations of apoptosis-associated protein expressions in DU 145 cells and results indicated that ouabain increased the expression of DNA damage associated proteins (pATMSer1981 , p-H2A.XSer139 , and p-p53Ser15 ) and ER-stress-associated proteins (Grp78, ATF6ß, p-PERKThr981 , PERK, eIF2A, GADD153, CaMKIIß, and caspase-4) in time-dependently. Furthermore, ouabain increased apoptosis-associated proteins (DR4, DR5, Fas, Fas Ligand, and FADD), TRAIL pathway, which related to extrinsic pathway, promoted the pro-apoptotic protein Bax, increased apoptotic-associated proteins, such as cytochrome c, AIF, Endo G, caspase-3, -8, and -9, but reduced anti-apoptotic protein Bcl-2 and Bcl-x in DU 145 cells. In conclusion, we may suggest that ouabain decreased cell viability and induced apoptotic cell death may via caspase-dependent and mitochondria-dependent pathways in human prostate cancer DU 145 cells.
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Apoptosis/efectos de los fármacos , Cardiotónicos/farmacología , Daño del ADN/efectos de los fármacos , Ouabaína/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Caspasa 3/metabolismo , Línea Celular Tumoral , Chaperón BiP del Retículo Endoplásmico , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Humanos , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
BACKGROUND: BATF family transcription factors (BATF, BATF2 and BATF3) form hetero-trimers with JUNB and either IRF4 or IRF8 to regulate cell fate in T cells and dendritic cells in vivo. While each combination of the hetero-trimer has a distinct role, some degree of cross-compensation was observed. The basis for the differential actions of IRF4 and IRF8 with BATF factors and JUNB is still unknown. We propose that the differences in function between these hetero-trimers may be caused by differences in their DNA binding preferences. While all three BATF family transcription factors have similar binding preferences when binding as a hetero-dimer with JUNB, the cooperative binding of IRF4 or IRF8 to the hetero-dimer/DNA complex could change the preferences. We used Spec-seq, which allows for the efficient and accurate determination of relative affinity to a large collection of sequences in parallel, to find differences between cooperative DNA binding of IRF4, IRF8 and BATF family members. RESULTS: We found that without IRF binding, all three hetero-dimer pairs exhibit nearly the same binding preferences to both expected wildtype binding sites TRE (TGA(C/G)TCA) and CRE (TGACGTCA). IRF4 and IRF8 show the very similar DNA binding preferences when binding with any of the three hetero-dimers. No major change of binding preferences was found in the half-sites between different hetero-trimers. IRF proteins bind with substantially lower affinity with either a single nucleotide spacer between IRF and BATF binding site or with an alternative mode of binding in the opposite orientation. In addition, the preference to CRE binding site was reduced with either IRF binding in all BATF-JUNB combinations. CONCLUSIONS: The specificities of BATF, BATF2 and BATF3 are all very similar as are their interactions with IRF4 and IRF8. IRF proteins binding adjacent to BATF sites increases affinity substantially compared to sequences with spacings between the sites, indicating cooperative binding through protein-protein interactions. The preference for the type of BATF binding site, TRE or CRE, is also altered when IRF proteins bind. These in vitro preferences aid in the understanding of in vivo binding activities.
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Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Factores Reguladores del Interferón/genética , Análisis de Secuencia de ADN/métodos , Factores de Transcripción/genética , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Sitios de Unión , Humanos , Factores Reguladores del Interferón/química , Factores Reguladores del Interferón/metabolismo , Ratones , Multimerización de Proteína , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The nuclear import receptor Kap114 carries transcription factors and other cargos across nuclear pores into the nucleus. Here we show that yeast Kap114 is modified by SUMO (small ubiquitin-related modifier) and that sumoylation is required for Kap114-mediated nuclear import. Among the four known SUMO-specific E3 ligases in yeast, Mms21 is the preferred E3 enzyme responsible for the covalent attachment of SUMO to the Kap114 protein. Kap114 is sumoylated on lysine residue 909, which is part of a ΨKxD/E sumoylation consensus motif. Kap114 containing a lysine-to-arginine point mutation at position 909 mislocalizes to the nucleus and is defective in promoting nuclear import. Similarly, mutants defective in sumoylation or desumoylation specifically accumulate Kap114 in the nucleus and are blocked in import of Kap114 cargos. Ran-GTP is not sufficient to disassemble Kap114/cargo complexes, which necessitates additional cargo release mechanisms in the nucleus. Remarkably, sumoylation of Kap114 greatly stimulates cargo dissociation in vitro. We propose that sumoylation occurs at the site of Kap114 cargo function and that SUMO is a cargo release factor involved in intranuclear targeting.
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Transporte Activo de Núcleo Celular , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sumoilación , beta Carioferinas/metabolismo , Lisina/metabolismo , Mutación Puntual , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligasas/metabolismo , beta Carioferinas/genéticaRESUMEN
Multiple export receptors passage bound pre-ribosomes through nuclear pore complexes (NPCs) by transiently interacting with the Phe-Gly (FG) meshwork of their transport channels. Here, we reveal how the non-FG interacting yeast mRNA export factor Gly-Leu-FG lethal 2 (Gle2) functions in the export of the large pre-ribosomal subunit (pre-60S). Structure-guided studies uncovered conserved platforms used by Gle2 to export pre-60S: an uncharacterized basic patch required to bind pre-60S, and a second surface that makes non-FG contacts with the nucleoporin Nup116. A basic patch mutant of Gle2 is able to function in mRNA export, but not pre-60S export. Thus, Gle2 provides a distinct interaction platform to transport pre-60S to the cytoplasm. Notably, Gle2's interaction platforms become crucial for pre-60S export when FG-interacting receptors are either not recruited to pre-60S or are impaired. We propose that large complex cargos rely on non-FG as well as FG-interactions for their efficient translocation through the nuclear pore complex channel.
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Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Sitios de Unión , Datos de Secuencia Molecular , Mutación , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/genética , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alineación de SecuenciaRESUMEN
Nuclear export of mRNAs and pre-ribosomal subunits (pre40S and pre60S) is fundamental to all eukaryotes. While genetic approaches in budding yeast have identified bona fide export factors for mRNAs and pre60S subunits, little is known regarding nuclear export of pre40S subunits. The yeast heterodimeric transport receptor Mex67-Mtr2 (TAP-p15 in humans) binds mRNAs and pre60S subunits in the nucleus and facilitates their passage through the nuclear pore complex (NPC) into the cytoplasm by interacting with Phe-Gly (FG)-rich nucleoporins that line its transport channel. By exploiting a combination of genetic, cell-biological, and biochemical approaches, we uncovered an unanticipated role of Mex67-Mtr2 in the nuclear export of 40S pre-ribosomes. We show that recruitment of Mex67-Mtr2 to pre40S subunits requires loops emanating from its NTF2-like domains and that the C-terminal FG-rich nucleoporin interacting UBA-like domain within Mex67 contributes to the transport of pre40S subunits to the cytoplasm. Remarkably, the same loops also recruit Mex67-Mtr2 to pre60S subunits and to the Nup84 complex, the respective interactions crucial for nuclear export of pre60S subunits and mRNAs. Thus Mex67-Mtr2 is a unique transport receptor that employs a common interaction surface to participate in the nuclear export of both pre-ribosomal subunits and mRNAs. Mex67-Mtr2 could engage a regulatory crosstalk among the three major export pathways for optimal cellular growth and proliferation.
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Proteínas de Transporte de Membrana , Proteínas Nucleares , Proteínas de Transporte Nucleocitoplasmático , ARN Mensajero/metabolismo , Proteínas de Unión al ARN , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transporte Activo de Núcleo Celular/genética , Dimerización , Regulación Fúngica de la Expresión Génica , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Estructura Terciaria de Proteína , Transporte de ARN/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
A host of observations demonstrating the relationship between nuclear architecture and processes such as gene expression have led to a number of new technologies for interrogating chromosome positioning. Whereas some of these technologies reconstruct intermolecular interactions, others have enhanced our ability to visualize chromosomes in situ. Here, we describe an oligonucleotide- and PCR-based strategy for fluorescence in situ hybridization (FISH) and a bioinformatic platform that enables this technology to be extended to any organism whose genome has been sequenced. The oligonucleotide probes are renewable, highly efficient, and able to robustly label chromosomes in cell culture, fixed tissues, and metaphase spreads. Our method gives researchers precise control over the sequences they target and allows for single and multicolor imaging of regions ranging from tens of kilobases to megabases with the same basic protocol. We anticipate this technology will lead to an enhanced ability to visualize interphase and metaphase chromosomes.
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Pintura Cromosómica/métodos , Genoma/genética , Hibridación Fluorescente in Situ/métodos , Sondas de Oligonucleótidos/metabolismo , Animales , Caenorhabditis elegans/genética , Núcleo Celular/metabolismo , Cromosomas/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Femenino , Biblioteca de Genes , Humanos , Interfase/genética , Metafase/genética , Ratones , Ovario/citología , Ovario/metabolismo , Coloración y EtiquetadoRESUMEN
A simple method is demonstrated to improve the folding reliability and sheet resistance of silver nanowire (Ag NW)-based transparent conductive films (TCFs) without overcoated layers or complicated processes. As-coated Ag NW film is immersed in a silver-surrounded solution, in which the silver was reduced by Tollen's reagent. Therefore, the reduced silver can be deposited on the surface and crossing of Ag NWs to form a weld-like junction and bind the Ag NWs together, hence decreasing the contact resistance of Ag NW structure at wire-to-wire junctions by increasing the contact surface area. Compared with original Ag NW TCFs, this Ag NW junction structure not only shows compatibility with conventional wet processes, but also demonstrates outstanding flexibility, even when the entire film is folded. This could be attributed to the robustly weld-like wire-to-wire junction of Ag NWs, which enhances its mechanical characteristics. The results demonstrated the feasibility of this alternative approach for the development of an effective low-power-consumption route for fabricating foldable transparent electrodes. Moreover, the results also indicated the practicability of applying this foldable transparent electrode in the fabrication of highly flexible and wearable optoelectronic devices.
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BACKGROUND: Recurrent oral cancer incurred grave outcome. Tumor microenvironment features, like tumor-infiltrating lymphocytes (TILs) or tumor stromal ratio (TSR) had prognostic significance in various cancers. We aimed to evaluate the impact of stromal categorization which incorporated the stromal TILs and TSR on survival outcomes in recurrent oral cancer. METHODS: 162 patients who received surgery-based treatment between 2010 and 2020 were recruited. Outcomes were 5-year overall survival (OS) and disease-specific survival (DSS). The impact of stromal categorization of recurrent primary tumor or node on 5-year OS and DSS were assessed with the Kaplan-Meier method. Multivariate analysis was performed, incorporating variables at initial treatment and salvage surgery. Patients were further categorized using a survival decision tree. RESULTS: Mean age was 56.1 (SD, 11.3) years; 153 patients (94.4%) were male; 51 patients (31.5%) had stromal category III. Local recurrence occurred in 94 patients (58%), regional recurrence in 55 (34%), and loco-regional recurrence in 13 (8%). Patients with stromal category III had poorer 5-year OS and DSS. Prior radiotherapy, advanced recurrent stage, positive surgical margin, and stromal category III were independent prognosticators for 5-year OS and DSS. In survival decision tree analysis, patients with prior radiotherapy and stromal category III had the worst outcomes. CONCLUSION: Stromal categorization is associated with outcomes in recurrent oral cancer. Patients with poor prognosticators, such as stromal categorization III, prior radiation, and advanced stage may require closer follow-up and intensive treatment.
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Carcinoma de Células Escamosas , Neoplasias de la Boca , Humanos , Persona de Mediana Edad , Tasa de Supervivencia , Recurrencia Local de Neoplasia/patología , Carcinoma de Células Escamosas/patología , Neoplasias de la Boca/patología , Pronóstico , Terapia Recuperativa/métodos , Estudios Retrospectivos , Microambiente TumoralRESUMEN
We explored the impact of stromal tumor-infiltrating lymphocytes (sTILs) on the prognostic value of an early death model for advanced buccal cancer. We assessed 121 patients with advanced buccal cancer who underwent primary tumor resection at a medical center. Predictors of early death and 5-year overall survival (OS) were analyzed using Cox regression models. Performance of models was evaluated with the Harrell C and Akaike information criterion. The net reclassification improvement of the early death model was also calculated relative to the 5-year OS model for one-year all-cause mortality. A total of 121 patients with advanced buccal cancer were recruited. Mean age was 56.1 ± 9.8 years; 117 (96.7%) patients were male. sTILs ≤30%, clinical nodal disease, pathological nodal disease, poor differentiation, lymphovascular invasion, perineural invasion, WPOI 5, and no adjuvant radiotherapy were risk factors for early death in univariate analysis. In multivariate analysis, clinical TNM, sTILs, clinical nodal disease, poor differentiation, lymphovascular invasion, and no adjuvant RT were independent factors for early death. sTILs, pathological nodal disease, poor differentiation, lymphovascular invasion, and no adjuvant RT were independent factors for early death in the multivariate model with pathological TNM. The discriminatory ability was better for early death model for 1-year all-cause mortality. Finally, incorporation of sTILs into the early death model increased net reclassification by 21% for the clinical TNM model and 28% for the pathological TNM model. Addition of sTILs improved the early death model, which may help physicians to identify high-risk patients for more intensive treatment and follow-up.
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Linfocitos Infiltrantes de Tumor , Neoplasias de la Boca , Humanos , Masculino , Persona de Mediana Edad , Femenino , Linfocitos Infiltrantes de Tumor/patología , Neoplasias de la Boca/patología , Neoplasias de la Boca/mortalidad , Pronóstico , Estadificación de Neoplasias , Invasividad Neoplásica , Anciano , Adulto , Factores de Riesgo , Estudios Retrospectivos , Metástasis Linfática/patologíaRESUMEN
Homoneura picta belongs to the Homoneurinae subfamily of Lauxaniidae, and it is widely distributed and common in China. This study reports the newly sequenced mitochondrial genome of H. picta. The sequence is 15,469 bp long and contains 37 genes (13 protein-coding, 22 tRNA, and 2 rRNA genes) and a control region. The overall base composition is 38.4% for A, 37.7% for T, 14.1% for C, and 9.8% for G, with a bias toward A + T (76.1%). Phylogenetic analysis show that Homoneura is a sister genus of Cestrotus. We have successfully sequenced the mitochondrial genome of H. picta, which can be useful in investigating the phylogenetic status of Homoneurinae. Our results provide data for further studies of phylogeny in Diptera.
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The coexistence of pesticides and plastic film residues in agricultural soils poses a significant threat to soil organisms due to their potential long-term contamination and combined toxic effects. Specifically, earthworms are at risk of simultaneously ingesting residual pesticides and microplastics, yet the impact of this combined exposure on their intestinal health and function remains poorly understood. In this study, earthworm (Eisenia fetida) were single and combined exposed to three particle sizes (10 µm, 500 µm, and 2 mm) of polyethylene microplastics (PE MPs) and imidacloprid (IMI) for 28 days, respectively. Our findings underscore that compared to single exposures, the combined exposure inflicted more profound injuries on intestinal tissues and elicited a heightened activation of intestinal digestive enzymes. Furthermore, the combined exposure significantly perturbed the relative abundance of several pivotal metabolic-associated gut microbiota, fostering an enrichment of pathogenic species. Metabolomics analysis showed combined exposure increased differential metabolites, disrupting amino acid, fatty acid, and carbohydrate metabolism in earthworm intestines, potentially hindering nutrient absorption and causing toxic metabolite accumulation. An integrated omics analysis implies that combined exposures have the potential to disrupt the relative abundance of crucial gut microbiota in earthworms, thereby altering their intestinal metabolism and subsequently impacting intestinal health and functionality. Overall, the results reveal that combined exposure of IMI and PE MPs exacerbate the negative effects on earthworm gut health, and this study holds significant implications for the holistic understanding of the combined toxic effects of microplastics and pesticide on soil ecosystems.