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1.
Cell Prolif ; 54(5): e13034, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33931895

RESUMEN

OBJECTIVES: Dysfunction of autophagy results in accumulation of depolarized mitochondria and breakdown of self-renewal and pluripotency in ESCs. However, the regulators that control how mitochondria are degraded by autophagy for pluripotency regulation remains largely unknown. This study aims to dissect the molecular mechanisms that regulate mitochondrial homeostasis for pluripotency regulation in mouse ESCs. MATERIALS AND METHODS: Parkin+/+ and parkin-/- ESCs were established from E3.5 blastocysts of parkin+/- x parkin+/- mating mice. The pink1-/- , optn-/- and ndp52-/- ESCs were generated by CRISPR-Cas9. shRNAs were used for function loss assay of target genes. Mito-Keima, ROS and ATP detection were used to investigate the mitophagy and mitochondrial function. Western blot, Q-PCR, AP staining and teratoma formation assay were performed to evaluate the PSC stemness. RESULTS: PINK1 or OPTN depletion impairs the degradation of dysfunctional mitochondria during reprogramming, and reduces the reprogramming efficiency and quality. In ESCs, PINK1 or OPTN deficiency leads to accumulation of dysfunctional mitochondria and compromised pluripotency. The defective mitochondrial homeostasis and pluripotency in pink1-/- ESCs can be compensated by gain expression of phosphomimetic Ubiquitin (Ub-S65D) together with WT or a constitutively active phosphomimetic OPTN mutant (S187D, S476D, S517D), rather than constitutively inactive OPTN (S187A, S476A, S517A) or a Ub-binding dead OPTN mutant (D477N). CONCLUSIONS: The mitophagy receptor OPTN guards ESC mitochondrial homeostasis and pluripotency by scavenging damaged mitochondria through TBK1-activated OPTN binding of PINK1-phosphorylated Ubiquitin.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Mitofagia , Proteínas Quinasas/metabolismo , Animales , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/genética , Reprogramación Celular , Edición Génica , Proteínas de Transporte de Membrana/deficiencia , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ubiquitina/metabolismo
2.
Int J Mol Sci ; 22(6)2021 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-33802105

RESUMEN

Several lines of evidence suggest the existence in the eukaryotic cells of a tight, yet largely unexplored, connection between DNA replication and sister chromatid cohesion. Tethering of newly duplicated chromatids is mediated by cohesin, an evolutionarily conserved hetero-tetrameric protein complex that has a ring-like structure and is believed to encircle DNA. Cohesin is loaded onto chromatin in telophase/G1 and converted into a cohesive state during the subsequent S phase, a process known as cohesion establishment. Many studies have revealed that down-regulation of a number of DNA replication factors gives rise to chromosomal cohesion defects, suggesting that they play critical roles in cohesion establishment. Conversely, loss of cohesin subunits (and/or regulators) has been found to alter DNA replication fork dynamics. A critical step of the cohesion establishment process consists in cohesin acetylation, a modification accomplished by dedicated acetyltransferases that operate at the replication forks. Defects in cohesion establishment give rise to chromosome mis-segregation and aneuploidy, phenotypes frequently observed in pre-cancerous and cancerous cells. Herein, we will review our present knowledge of the molecular mechanisms underlying the functional link between DNA replication and cohesion establishment, a phenomenon that is unique to the eukaryotic organisms.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/fisiología , Replicación del ADN/fisiología , Fase G1/fisiología , Telofase/fisiología , Animales , Humanos
3.
Int J Mol Sci ; 22(6)2021 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-33809701

RESUMEN

Evidence suggests that augmented expression of a certain gene can influence the efficacy of targeted and conventional chemotherapies. Here, we tested whether the high expression of enhancer of the rudimentary homolog (ERH), which serves as a prognostic factor in some cancers, can influence the efficacy of anthocyanins isolated from fruits of Vitis coignetiae Pulliat, Meoru in Korea (AIMs) on human gastric cancer cells. The anticancer efficacy of AIMs was augmented in ERH-transfected MKN28 cells (E-MKN28 cells). Molecularly, ERH augmented AIM-induced caspase-dependent apoptosis by activating caspase-3 and -9. The ERH-augmented apoptotic effect was related to mitochondrial depolarization and inhibition of antiapoptotic proteins, XIAP, and Bcl-2. In addition, reactive oxygen species (ROS) generation was augmented in AIMs-treated E-MKN28 cells compared to AIMs-treated naïve MKN28 cells. In conclusion, ERH augmented AIM-induced caspase-dependent mitochondrial-related apoptosis in MKN28 cells. A decrease in expression of Bcl-2 and subsequent excessive ROS generation would be the mechanism for ERH-augmented mitochondrial-related apoptosis in AIMs-treated MKN28 cells. A decrease in expression of XIAP would be another mechanism for ERH-augmented caspase-dependent apoptosis in AIMs-treated MKN28 cells.


Asunto(s)
Antocianinas/farmacología , Apoptosis/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Neoplasias Gástricas/metabolismo , Factores de Transcripción/metabolismo , Vitis/química , Caspasas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Neoplasias Gástricas/patología , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo
4.
Nat Commun ; 12(1): 2028, 2021 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-33795686

RESUMEN

Germline mutations in BRAF and other components of the MAPK pathway are associated with the congenital syndromes collectively known as RASopathies. Here, we report the association of Septo-Optic Dysplasia (SOD) including hypopituitarism and Cardio-Facio-Cutaneous (CFC) syndrome in patients harbouring mutations in BRAF. Phosphoproteomic analyses demonstrate that these genetic variants are gain-of-function mutations leading to activation of the MAPK pathway. Activation of the MAPK pathway by conditional expression of the BrafV600E/+ allele, or the knock-in BrafQ241R/+ allele (corresponding to the most frequent human CFC-causing mutation, BRAF p.Q257R), leads to abnormal cell lineage determination and terminal differentiation of hormone-producing cells, causing hypopituitarism. Expression of the BrafV600E/+ allele in embryonic pituitary progenitors leads to an increased expression of cell cycle inhibitors, cell growth arrest and apoptosis, but not tumour formation. Our findings show a critical role of BRAF in hypothalamo-pituitary-axis development both in mouse and human and implicate mutations found in RASopathies as a cause of endocrine deficiencies in humans.


Asunto(s)
Mutación con Ganancia de Función , Hipopituitarismo/genética , Hipotálamo/metabolismo , Hipófisis/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Niño , Preescolar , Corticotrofos/citología , Corticotrofos/metabolismo , Displasia Ectodérmica/genética , Facies , Insuficiencia de Crecimiento/genética , Células HEK293 , Cardiopatías Congénitas/genética , Humanos , Lactante , Sistema de Señalización de MAP Quinasas/genética , Melanotrofos/citología , Melanotrofos/metabolismo , Ratones Noqueados , Ratones Transgénicos , Proteínas Proto-Oncogénicas B-raf/metabolismo , Secuenciación del Exoma Completo/métodos
5.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-33802633

RESUMEN

The current study was designed to investigate the protective role of diosmin against cyclophosphamide-induced premature ovarian insufficiency (POI). Female Swiss albino rats received a single intraperitoneal dose of cyclophosphamide (200 mg/kg) followed by 8 mg/kg/day for the next 15 consecutive days either alone or in combination with oral diosmin at 50 or 100 mg/kg. Histopathological examination of ovarian tissues, hormonal assays for follicle stimulating hormone (FSH), estradiol (E2), and anti-Mullerian hormone (AMH), assessment of the oxidative stress status, as well as measurement of the relative expression of miRNA-145 and its target genes [vascular endothelial growth factor B (VEGF-B) and regulator of cell cycle (RGC32)] were performed. Diosmin treatment ameliorated the levels of E2, AMH, and oxidative stress markers. Additionally, both low and high diosmin doses significantly reduced the histopathological alterations and nearly preserved the normal ovarian reserve. MiRNA-145 expression was upregulated after treatment with diosmin high dose. miRNA-145 target genes were over-expressed after both low and high diosmin administration. Based on our findings, diosmin has a dose-dependent protective effect against cyclophosphamide-induced ovarian toxicity in rats.


Asunto(s)
Diosmina/uso terapéutico , Insuficiencia Ovárica Primaria/inducido químicamente , Insuficiencia Ovárica Primaria/tratamiento farmacológico , Animales , Biomarcadores/metabolismo , Peso Corporal/efectos de los fármacos , Caspasa 3/metabolismo , Catalasa/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Colágeno/metabolismo , Ciclofosfamida , Diosmina/farmacología , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hormonas/sangre , Malondialdehído/sangre , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Tamaño de los Órganos/efectos de los fármacos , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/crecimiento & desarrollo , Folículo Ovárico/patología , Estrés Oxidativo/efectos de los fármacos , Insuficiencia Ovárica Primaria/sangre , Insuficiencia Ovárica Primaria/patología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo
6.
Proc Natl Acad Sci U S A ; 118(17)2021 04 27.
Artículo en Inglés | MEDLINE | ID: mdl-33827988

RESUMEN

In order to understand the transmission and virulence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is necessary to understand the functions of each of the gene products encoded in the viral genome. One feature of the SARS-CoV-2 genome that is not present in related, common coronaviruses is ORF10, a putative 38-amino acid protein-coding gene. Proteomic studies found that ORF10 binds to an E3 ubiquitin ligase containing Cullin-2, Rbx1, Elongin B, Elongin C, and ZYG11B (CRL2ZYG11B). Since CRL2ZYG11B mediates protein degradation, one possible role for ORF10 is to "hijack" CRL2ZYG11B in order to target cellular, antiviral proteins for ubiquitylation and subsequent proteasomal degradation. Here, we investigated whether ORF10 hijacks CRL2ZYG11B or functions in other ways, for example, as an inhibitor or substrate of CRL2ZYG11B While we confirm the ORF10-ZYG11B interaction and show that the N terminus of ORF10 is critical for it, we find no evidence that ORF10 is functioning to inhibit or hijack CRL2ZYG11B Furthermore, ZYG11B and its paralog ZER1 are dispensable for SARS-CoV-2 infection in cultured cells. We conclude that the interaction between ORF10 and CRL2ZYG11B is not relevant for SARS-CoV-2 infection in vitro.


Asunto(s)
/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cullin/metabolismo , Complejos Multiproteicos/metabolismo , Sistemas de Lectura Abierta , Proteínas Virales/metabolismo , /genética , Proteínas de Ciclo Celular/genética , Proteínas Cullin/genética , Células HEK293 , Humanos , Complejos Multiproteicos/genética , Proteínas Virales/genética
7.
Mol Cell ; 81(7): 1367-1369, 2021 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-33798413

RESUMEN

Mutations in ataxia telangiectasia mutated (ATM) kinase lead to cerebellar neurodegeneration. In this issue of Molecular Cell, Lee et al. (2021) revealed how transcription-induced reactive oxygen species and DNA-RNA hybrids activate PARP enzymes, generating the nucleic acid poly-ADP-ribose, which promotes the accumulation of protein aggregates in A-T-like disorders.


Asunto(s)
Ataxia Telangiectasia , Ácidos Nucleicos , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Poli(ADP-Ribosa) Polimerasa-1 , Poli ADP Ribosilación , Poli(ADP-Ribosa) Polimerasas/metabolismo , Agregado de Proteínas , Proteostasis , Proteínas Supresoras de Tumor/genética
8.
Nat Commun ; 12(1): 2111, 2021 04 08.
Artículo en Inglés | MEDLINE | ID: mdl-33833229

RESUMEN

Smc5/6 is essential for genome structural integrity by yet unknown mechanisms. Here we find that Smc5/6 co-localizes with the DNA crossed-strand processing complex Sgs1-Top3-Rmi1 (STR) at genomic regions known as natural pausing sites (NPSs) where it facilitates Top3 retention. Individual depletions of STR subunits and Smc5/6 cause similar accumulation of joint molecules (JMs) composed of reversed forks, double Holliday Junctions and hemicatenanes, indicative of Smc5/6 regulating Sgs1 and Top3 DNA processing activities. We isolate an intra-allelic suppressor of smc6-56 proficient in Top3 retention but affected in pathways that act complementarily with Sgs1 and Top3 to resolve JMs arising at replication termination. Upon replication stress, the smc6-56 suppressor requires STR and Mus81-Mms4 functions for recovery, but not Srs2 and Mph1 helicases that prevent maturation of recombination intermediates. Thus, Smc5/6 functions jointly with Top3 and STR to mediate replication completion and influences the function of other DNA crossed-strand processing enzymes at NPSs.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Replicación del ADN/genética , Genoma Fúngico/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Endonucleasas de ADN Solapado/metabolismo , RecQ Helicasas/metabolismo , Saccharomyces cerevisiae/metabolismo
9.
Cell ; 184(8): 2167-2182.e22, 2021 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-33811809

RESUMEN

Cardiac injury and dysfunction occur in COVID-19 patients and increase the risk of mortality. Causes are ill defined but could be through direct cardiac infection and/or inflammation-induced dysfunction. To identify mechanisms and cardio-protective drugs, we use a state-of-the-art pipeline combining human cardiac organoids with phosphoproteomics and single nuclei RNA sequencing. We identify an inflammatory "cytokine-storm", a cocktail of interferon gamma, interleukin 1ß, and poly(I:C), induced diastolic dysfunction. Bromodomain-containing protein 4 is activated along with a viral response that is consistent in both human cardiac organoids (hCOs) and hearts of SARS-CoV-2-infected K18-hACE2 mice. Bromodomain and extraterminal family inhibitors (BETi) recover dysfunction in hCOs and completely prevent cardiac dysfunction and death in a mouse cytokine-storm model. Additionally, BETi decreases transcription of genes in the viral response, decreases ACE2 expression, and reduces SARS-CoV-2 infection of cardiomyocytes. Together, BETi, including the Food and Drug Administration (FDA) breakthrough designated drug, apabetalone, are promising candidates to prevent COVID-19 mediated cardiac damage.


Asunto(s)
/complicaciones , Cardiotónicos/uso terapéutico , Proteínas de Ciclo Celular/antagonistas & inhibidores , Cardiopatías/tratamiento farmacológico , Quinazolinonas/uso terapéutico , Factores de Transcripción/antagonistas & inhibidores , /metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Citocinas/metabolismo , Femenino , Cardiopatías/etiología , Células Madre Embrionarias Humanas , Humanos , Inflamación/complicaciones , Inflamación/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Factores de Transcripción/metabolismo
10.
Mol Cell ; 81(6): 1126-1127, 2021 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-33740471
11.
Mol Cell ; 81(6): 1128-1129, 2021 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-33740472

RESUMEN

Huang et al. (2021) identified a mechanism acting through the arginine methyltransferase PRMT6 that stabilizes the interaction of RCC1 with chromatin, promoting cell proliferation and tumorigenicity. Targeting this mechanism might enhance the treatment of tumors such as glioblastoma.


Asunto(s)
Glioblastoma , Proteínas Nucleares , Carcinogénesis/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Cromosomas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Metilación , Mitosis , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Células Madre/metabolismo
12.
Int J Mol Sci ; 22(5)2021 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-33669056

RESUMEN

Warsaw breakage syndrome (WABS) is a genetic disorder characterized by sister chromatid cohesion defects, growth retardation, microcephaly, hearing loss and other variable clinical manifestations. WABS is due to biallelic mutations of the gene coding for the super-family 2 DNA helicase DDX11/ChlR1, orthologous to the yeast chromosome loss protein 1 (Chl1). WABS is classified in the group of "cohesinopathies", rare hereditary diseases that are caused by mutations in genes coding for subunits of the cohesin complex or protein factors having regulatory roles in the sister chromatid cohesion process. In fact, among the cohesion regulators, an important player is DDX11, which is believed to be important for the functional coupling of DNA synthesis and cohesion establishment at the replication forks. Here, we will review what is known about the molecular and cellular functions of human DDX11 and its role in WABS etiopathogenesis, even in light of recent findings on the role of cohesin and its regulator network in promoting chromatin loop formation and regulating chromatin spatial organization.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ARN Helicasas DEAD-box/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Enfermedades Raras/metabolismo , Anomalías Múltiples/genética , Animales , Ciclo Celular/genética , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Cromátides/patología , Cromatina/patología , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica , ARN Helicasas DEAD-box/genética , Replicación del ADN/genética , Regulación de la Expresión Génica/genética , Humanos , Mutación , Filogenia , Enfermedades Raras/congénito , Enfermedades Raras/enzimología , Enfermedades Raras/fisiopatología
13.
Gene ; 784: 145584, 2021 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-33753149

RESUMEN

Sister chromatid cohesion (SCC) is essential for the maintenance of genome integrity. The establishment of SCC is coupled to DNA replication, and this is achieved in budding yeast Saccharomyces cerevisiae by a mechanism that is dependent on the interaction between Eco1 acetyltransferase and PCNA in the DNA replication complex. In vertebrates, the Eco1 homolog ESCO2 has been reported to interact with MCM complex in the DNA replication complex to establish DNA replication-dependent cohesion. Here we show that budding yeast Eco1 is also physically interacted with the MCM complex. We found that Eco1 was specifically bound to Mcm2 subunit in the MCM complex and they interacted via their N-terminal regions, using yeast two-hybrid system. The underlying mechanism of the interaction was different between yeast and vertebrates. Intensive molecular dissection of Eco1 identified residues important for interaction with Mcm2 and/or PCNA. Mutant forms of Eco1 (Eco1mWW and Eco1mGRK), where sets of the identified residues were substituted with alanine, resulted in impaired SCC, decreased level of acetylation of Smc3, and a reduction of Eco1 protein amount in yeast cells. We, hence, suggest that Eco1 is stabilized by its interactions with MCM complex and PCNA, which allows it to promote DNA replication-coupled SCC establishment.


Asunto(s)
Acetiltransferasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Acetilación , Acetiltransferasas/química , Acetiltransferasas/genética , Sitios de Unión , Cromosomas Fúngicos/metabolismo , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Unión Proteica , Estabilidad Proteica , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
14.
Nat Commun ; 12(1): 1899, 2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33771996

RESUMEN

Polo-like kinase 1 (Plk1) is instrumental for mitotic entry and progression. Plk1 is activated by phosphorylation on a conserved residue Thr210 in its activation segment by the Aurora A kinase (AURKA), a reaction that critically requires the co-factor Bora phosphorylated by a CyclinA/B-Cdk1 kinase. Here we show that phospho-Bora is a direct activator of AURKA kinase activity. We localize the key determinants of phospho-Bora function to a 100 amino acid region encompassing two short Tpx2-like motifs and a phosphoSerine-Proline motif at Serine 112, through which Bora binds AURKA. The latter substitutes in trans for the Thr288 phospho-regulatory site of AURKA, which is essential for an active conformation of the kinase domain. We demonstrate the importance of these determinants for Bora function in mitotic entry both in Xenopus egg extracts and in human cells. Our findings unveil the activation mechanism of AURKA that is critical for mitotic entry.


Asunto(s)
Aurora Quinasa A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Mitosis , Treonina/metabolismo , Secuencias de Aminoácidos/genética , Animales , Aurora Quinasa A/genética , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Ciclina A2/genética , Ciclina A2/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Activación Enzimática , Femenino , Humanos , Oocitos/metabolismo , Fosforilación , Prolina/genética , Prolina/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Serina/genética , Serina/metabolismo , Treonina/genética , Xenopus laevis
15.
Nat Commun ; 12(1): 1920, 2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33772001

RESUMEN

Adipogenesis associated Mth938 domain containing (AAMDC) represents an uncharacterized oncogene amplified in aggressive estrogen receptor-positive breast cancers. We uncover that AAMDC regulates the expression of several metabolic enzymes involved in the one-carbon folate and methionine cycles, and lipid metabolism. We show that AAMDC controls PI3K-AKT-mTOR signaling, regulating the translation of ATF4 and MYC and modulating the transcriptional activity of AAMDC-dependent promoters. High AAMDC expression is associated with sensitization to dactolisib and everolimus, and these PI3K-mTOR inhibitors exhibit synergistic interactions with anti-estrogens in IntClust2 models. Ectopic AAMDC expression is sufficient to activate AKT signaling, resulting in estrogen-independent tumor growth. Thus, AAMDC-overexpressing tumors may be sensitive to PI3K-mTORC1 blockers in combination with anti-estrogens. Lastly, we provide evidence that AAMDC can interact with the RabGTPase-activating protein RabGAP1L, and that AAMDC, RabGAP1L, and Rab7a colocalize in endolysosomes. The discovery of the RabGAP1L-AAMDC assembly platform provides insights for the design of selective blockers to target malignancies having the AAMDC amplification.


Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Antineoplásicos/farmacología , Neoplasias de la Mama/genética , Proteínas de Ciclo Celular/genética , Everolimus/farmacología , Femenino , Proteínas Activadoras de GTPasa/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Imidazoles/farmacología , Proteínas del Tejido Nervioso/metabolismo , Oncogenes/genética , Unión Proteica , Quinolinas/farmacología , Receptores Estrogénicos/metabolismo , Transducción de Señal/efectos de los fármacos
16.
Nat Commun ; 12(1): 1908, 2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33772005

RESUMEN

DNA replication in eukaryotes initiates at many origins distributed across each chromosome. Origins are bound by the origin recognition complex (ORC), which, with Cdc6 and Cdt1, recruits and loads the Mcm2-7 (MCM) helicase as an inactive double hexamer during G1 phase. The replisome assembles at the activated helicase in S phase. Although the outline of replisome assembly is understood, little is known about the dynamics of individual proteins on DNA and how these contribute to proper complex formation. Here we show, using single-molecule optical trapping and confocal microscopy, that yeast ORC is a mobile protein that diffuses rapidly along DNA. Origin recognition halts this search process. Recruitment of MCM molecules in an ORC- and Cdc6-dependent fashion results in slow-moving ORC-MCM intermediates and MCMs that rapidly scan the DNA. Following ATP hydrolysis, salt-stable loading of MCM single and double hexamers was seen, both of which exhibit salt-dependent mobility. Our results demonstrate that effective helicase loading relies on an interplay between protein diffusion and origin recognition, and suggest that MCM is stably loaded onto DNA in multiple forms.


Asunto(s)
Proteínas de Ciclo Celular/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Mantenimiento de Minicromosoma/genética , Complejo de Reconocimiento del Origen/genética , Origen de Réplica/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Algoritmos , Sitios de Unión/genética , Proteínas de Ciclo Celular/metabolismo , ADN de Hongos/genética , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Modelos Genéticos , Complejo de Reconocimiento del Origen/metabolismo , Unión Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
17.
Int J Mol Sci ; 22(4)2021 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-33671248

RESUMEN

Cyclin-dependent kinases (CDKs) are crucial regulators of the eukaryotic cell cycle. The critical role of CDK2 in the progression of meiosis was demonstrated in a single mammalian species, the mouse. We used immunocytochemistry to study the localization of CDK2 during meiosis in seven rodent species that possess hetero- and homomorphic male sex chromosomes. To compare the distribution of CDK2 in XY and XX male sex chromosomes, we performed multi-round immunostaining of a number of marker proteins in meiotic chromosomes of the rat and subterranean mole voles. Antibodies to the following proteins were used: RAD51, a member of the double-stranded DNA break repair machinery; MLH1, a component of the DNA mismatch repair system; and SUN1, which is involved in the connection between the meiotic telomeres and nuclear envelope, alongside the synaptic protein SYCP3 and kinetochore marker CREST. Using an enhanced protocol, we were able to assess the distribution of as many as four separate proteins in the same meiotic cell. We showed that during prophase I, CDK2 localizes to telomeric and interstitial regions of autosomes in all species investigated (rat, vole, hamster, subterranean mole voles, and mole rats). In sex bivalents following synaptic specificity, the CDK2 signals were distributed in three different modes. In the XY bivalent in the rat and mole rat, we detected numerous CDK2 signals in asynaptic regions and a single CDK2 focus on synaptic segments, similar to the mouse sex chromosomes. In the mole voles, which have unique XX sex chromosomes in males, CDK2 signals were nevertheless distributed similarly to the rat XY sex chromosomes. In the vole, sex chromosomes did not synapse, but demonstrated CDK2 signals of varying intensity, similar to the rat X and Y chromosomes. In female mole voles, the XX bivalent had CDK2 pattern similar to autosomes of all species. In the hamster, CDK2 signals were revealed in telomeric regions in the short synaptic segment of the sex bivalent. We found that CDK2 signals colocalize with SUN1 and MLH1 signals in meiotic chromosomes in rats and mole voles, similar to the mouse. The difference in CDK2 manifestation at the prophase I sex chromosomes can be considered an example of the rapid chromosome evolution in mammals.


Asunto(s)
Quinasa 2 Dependiente de la Ciclina/metabolismo , Mamíferos/metabolismo , Profase Meiótica I , Cromosomas Sexuales/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Femenino , Masculino , Modelos Biológicos , Fase Paquiteno , Ratas , Espermatocitos/metabolismo
18.
Nat Commun ; 12(1): 1746, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741931

RESUMEN

Origin licensing ensures precise once per cell cycle replication in eukaryotic cells. The Origin Recognition Complex, Cdc6 and Cdt1 load Mcm2-7 helicase (MCM) into a double hexamer, bound around duplex DNA. The complex formed by ORC-Cdc6 bound to duplex DNA (OC) recruits the MCM-Cdt1 complex into the replication origins. Through the stacking of both complexes, the duplex DNA is inserted inside the helicase by an unknown mechanism. In this paper we show that the DNA insertion comes with a topological problem in the stacking of OC with MCM-Cdt1. Unless an essential, conserved C terminal winged helix domain (C-WHD) of Cdt1 is present, the MCM splits into two halves. The binding of this domain with the essential C-WHD of Mcm6, allows the latching between the MCM-Cdt1 and OC, through a conserved Orc5 AAA-lid interaction. Our work provides new insights into how DNA is inserted into the eukaryotic replicative helicase, through a series of synchronized events.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Replicación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Componente 6 del Complejo de Mantenimiento de Minicromosoma/química , Componente 6 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Mantenimiento de Minicromosoma/química , Proteínas de Mantenimiento de Minicromosoma/genética , Proteínas Nucleares/metabolismo , Complejo de Reconocimiento del Origen/química , Complejo de Reconocimiento del Origen/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
19.
Molecules ; 26(4)2021 Feb 23.
Artículo en Inglés | MEDLINE | ID: mdl-33672137

RESUMEN

Bacillus subtilis spore display has become a field of increasing interest in the past two decades. To improve the efficiency of B. subtilis spore display, its directed modification was performed based on the cellulosome architecture by introducing onto them divergent cohesin (Coh) modules that can specifically bind to the target enzyme bearing the matching dockerins (Doc). In this study, five different pairs of cohesins and dockerins, selected from four cellulolytic microbes, were examined for their capabilities in displaying a tetrameric enzyme ß-galactosidase from Bacillus stearothermophilus IAM11001 on the surface of B. subtilis WB600 spores. Immunofluorescence microscopy, western blotting, dot blotting, and enzyme assay was applied to confirm its surface expression. All the resultant five Coh-Doc based spore display can hydrolyze o-nitrophenyl-ß-D-galactopyranoside. Further, the optimized Coh-Doc based spore display exhibited the highest display efficiency. Overall, the results of current study may open new perspectives on the use of Coh-Doc interaction, which will find application in improving the efficiency of B. subtilis spore display.


Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Esporas Bacterianas/metabolismo , Bacillus subtilis/química , Proteínas Bacterianas/química , Proteínas de Ciclo Celular/química , Proteínas Cromosómicas no Histona/química , Esporas Bacterianas/química
20.
Life Sci ; 274: 119330, 2021 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-33711383

RESUMEN

AIMS: The functions and molecular mechanisms of miR-340-3p in lung adenocarcinoma (LUAD) progression remain unclear. On the other hand, the role of HUS1 in LUAD progression should be further explored. MAIN METHODS: Data from cancer database were subjected to bioinformatics analysis. Quantitative real-time PCR and western blot were performed to detect gene expression. Colony formation and MTT assay were performed to examine cell growth in vitro. Wound healing assays and transwell assays were performed to examine cell migration. KEY FINDINGS: Here, our results showed that miR-340-3p was lower expressed in LUAD tissues and LUAD-derived cell lines. And miR-340-3p suppressed the proliferation and migration ability of LUAD cells. Further, miR-340-3p inhibits HUS1 expression, which was higher expressed in LUAD tissues and promoted the proliferation and migration ability of LUAD cells. Moreover, higher HUS1 expression was associated with poor survival rate and shorter survival time in patients with LUAD, and HUS1 expression was negative correlated with that of miR-340-3p in clinical samples. In addition, overexpression of HUS1 counteracted the downregulation of cell growth by miR-340-3p. SIGNIFICANCE: The study mainly indicated that miR-340-3p may play a tumor suppressor role in the progression of LUAD, with the function of restraining HUS1 expression, highlighting a potential therapeutic target for LUAD.


Asunto(s)
Adenocarcinoma del Pulmón/patología , Biomarcadores de Tumor/metabolismo , Proteínas de Ciclo Celular/metabolismo , Movimiento Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/metabolismo , Apoptosis , Biomarcadores de Tumor/genética , Proteínas de Ciclo Celular/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Pronóstico , Células Tumorales Cultivadas
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