RESUMEN
Despite acting as a barrier for the organs they encase, epithelial cells turn over at some of the fastest rates in the body. However, epithelial cell division must be tightly linked to cell death to preserve barrier function and prevent tumour formation. How does the number of dying cells match those dividing to maintain constant numbers? When epithelial cells become too crowded, they activate the stretch-activated channel Piezo1 to trigger extrusion of cells that later die. However, it is unclear how epithelial cell division is controlled to balance cell death at the steady state. Here we show that mammalian epithelial cell division occurs in regions of low cell density where cells are stretched. By experimentally stretching epithelia, we find that mechanical stretch itself rapidly stimulates cell division through activation of the Piezo1 channel. To stimulate cell division, stretch triggers cells that are paused in early G2 phase to activate calcium-dependent phosphorylation of ERK1/2, thereby activating the cyclin B transcription that is necessary to drive cells into mitosis. Although both epithelial cell division and cell extrusion require Piezo1 at the steady state, the type of mechanical force controls the outcome: stretch induces cell division, whereas crowding induces extrusion. How Piezo1-dependent calcium transients activate two opposing processes may depend on where and how Piezo1 is activated, as it accumulates in different subcellular sites with increasing cell density. In sparse epithelial regions in which cells divide, Piezo1 localizes to the plasma membrane and cytoplasm, whereas in dense regions in which cells extrude, it forms large cytoplasmic aggregates. Because Piezo1 senses both mechanical crowding and stretch, it may act as a homeostatic sensor to control epithelial cell numbers, triggering extrusion and apoptosis in crowded regions and cell division in sparse regions.
Asunto(s)
Recuento de Células , Células Epiteliales/citología , Canales Iónicos/metabolismo , Mecanotransducción Celular/fisiología , Mitosis , Proteínas de Pez Cebra/metabolismo , Animales , Apoptosis , Calcio/metabolismo , Membrana Celular/metabolismo , Ciclina B/genética , Citoplasma/metabolismo , Perros , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Homeostasis , Humanos , Canales Iónicos/deficiencia , Canales Iónicos/genética , Células de Riñón Canino Madin Darby , Fosforilación , Transporte de Proteínas , Transcripción Genética , Pez Cebra , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genéticaRESUMEN
BACKGROUND: Acute ingestion of bitter melon (BM) has been shown to suppress the postprandial glycemic response in diabetics, but its impact on glucose regulation among individuals with impaired glucose tolerance is unclear. Moreover, one's glucose tolerance level may influence the effectiveness of BM. This study aimed to examine the acute effects of a beverage containing BM extract on blood glucose regulation during an oral glucose tolerance test (OGTT) among prediabetics. METHODS: Ten prediabetic adults completed two OGTTs-glucose only (D2) and glucose+BM (D3). Responders were identified as subjects whose area under the glucose curve (AUCglu) during D3 was lower than D2. To compare the acute effects of the beverage among individuals with varying glucose tolerance levels, subjects were grouped by their glucose response pattern-Fastpeak (peak glucose (Glupeak) at 30 min postglucose (30P)) and Slowpeak (Glupeak after 30P). RESULTS: During D3, responders (n=5) experienced a 13.2% reduction in AUCglu (95% confidence interval (CI): -18.1% to -8.3%), 12.2% reduction in mean glucose (95% CI: -17.3% to -7.0%) and 10.6% reduction in Glupeak (95% CI: -17.5% to -3.7%); plasma glucose was reduced by 9.1% at 30P (95% CI: -15.6% to -2.6%), -24.0% at 60P (95% CI: -36.8% to -11.2%) and -20.0% at 90P (95% CI: -35.8% to -4.2%) during D3. No between-trial differences were noted for Fastpeak or Slowpeak. CONCLUSIONS: Acute ingestion of BM prior to the second OGTT (D3) led to a reduced postprandial glucose response in 50% of the subjects but did not affect the insulin response. Furthermore, the effectiveness of the beverage was seemingly uninfluenced by the subjects' glucose tolerance level. Although BM has shown to aid blood glucose management in diabetics, it remains uncertain why only a portion of subjects responded positively to the BM extract in the current study.
Asunto(s)
Glucemia/análisis , Momordica charantia , Extractos Vegetales/administración & dosificación , Periodo Posprandial/efectos de los fármacos , Estado Prediabético/sangre , Anciano , Bebidas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Periodo Posprandial/fisiología , Resultado del TratamientoRESUMEN
The Tup1-Ssn6 corepressor complex regulates the expression of several sets of genes, including genes that specify mating type in the yeast Saccharomyces cerevisiae. Repression of mating-type genes occurs when Tup1-Ssn6 is brought to the DNA by the Matalpha2 DNA-binding protein and assembled upstream of a- and haploid-specific genes. We have determined the 2.3 A X-ray crystal structure of the C-terminal domain of Tup1 (accesion No. 1ERJ), a 43 kDa fragment that contains seven copies of the WD40 sequence motif and binds to the Matalpha2 protein. Moreover, this portion of the protein can partially substitute for full-length Tup1 in bringing about transcriptional repression. The structure reveals a seven-bladed beta propeller with an N-terminal subdomain that is anchored to the side of the propeller and extends the beta sheet of one of the blades. Point mutations in Tup1 that specifically affect the Tup1-Matalpha2 interaction cluster on one surface of the propeller. We identified regions of Tup1 that are conserved among the fungal Tup1 homologs and may be important in protein-protein interactions with additional components of the Tup1-mediated repression pathways.
Asunto(s)
Proteínas Fúngicas/química , Proteínas Nucleares , Proteínas Represoras/química , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Secuencia Conservada , Cristalografía por Rayos X , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Homeodominio/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Fragmentos de Péptidos/química , Unión Proteica , Estructura Secundaria de Proteína , Secuencias Repetitivas de Aminoácido , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiaeRESUMEN
The Saccharomyces cerevisiae Tup1 protein is a member of a family of WD repeat containing proteins that are involved in repression of transcription. Tup1, along with the Ssn6 protein, represses a wide variety of genes in yeast including cell type-specific and glucose-repressed genes. Tup1 and Ssn6 are recruited to these specific gene sets by interaction with sequence-specific DNA binding proteins. In this work, a protein complex containing Ssn6 and Tup1 was purified to determine its composition. The size of the complex is estimated to be 440 kDa. Tup1 and Ssn6, which are both phosphoproteins, are the only proteins present in stoichiometric amounts in the complex. We also demonstrate that this purified complex represses transcription in an in vitro assay.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas Nucleares , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae , Transcripción Genética , Sistema Libre de Células , Proteínas Fúngicas/aislamiento & purificación , Peso Molecular , Fosfoproteínas/aislamiento & purificación , Fosfoproteínas/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/aislamiento & purificación , Saccharomyces cerevisiaeRESUMEN
Yeast Rox3 protein, implicated by genetic evidence in both negative and positive transcriptional regulation, is identified as a mediator subunit by peptide sequence determination and is shown to copurify and co-immunoprecipitate with RNA polymerase II holoenzyme.
Asunto(s)
Proteínas Fúngicas/química , ARN Polimerasa II/química , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/química , Secuencia de Aminoácidos , Regulación Fúngica de la Expresión Génica , Sustancias Macromoleculares , Complejo Mediador , Datos de Secuencia Molecular , Pruebas de Precipitina , Saccharomyces cerevisiae/químicaRESUMEN
It has been proposed that eukaryotic repressors of transcription can act by organizing chromatin, thereby preventing the accessibility of nearby DNA to activator proteins required for transcription initiation. In this study, we test this idea for the yeast alpha 2 repressor using a simple, artificial promoter that contains a single binding site for the activator protein Gal4 and a single binding site for the repressor alpha 2. When both the repressor and the activator are expressed in the same cell, the artificial promoter is efficiently repressed. In vivo footprinting experiments demonstrate that Gal4 can occupy its binding site even when the promoter is repressed. This result indicates that alpha 2-directed repression must result from interference with some stage in transcription initiation other than activator binding to DNA.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas de Homeodominio/genética , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción , Secuencia de Bases , Sitios de Unión/genética , Cartilla de ADN/genética , ADN de Hongos/genética , ADN de Hongos/metabolismo , Proteínas de Unión al ADN , Genes Reporteros , Datos de Secuencia Molecular , Nucleosomas/genética , Transcripción GenéticaRESUMEN
The tetratricopeptide repeat (TPR) is a 34-amino-acid degenerate sequence motif that is found in a large variety of proteins, both prokaryotic and eukaryotic. TPRs are usually found in tandem arrays of up to 16 copies. In this paper we identify a direct interaction between the TPRs of Ssn6, a general transcriptional repressor, and alpha 2, a cell-type regulator in Saccharomyces cerevisiae. Five of the Ssn6 TPRs were tested individually, and all were found to interact specifically with alpha 2. These results suggest a model for TPR-protein interactions and for the role that a tandem array of TPRs may have in mediating transcriptional repression.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Homeodominio , Proteínas Nucleares , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cromatografía de Afinidad , Secuencia Conservada , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Glutatión Transferasa , Modelos Químicos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Proteínas tau/metabolismoRESUMEN
Tup1 and Ssn6 transcriptionally repress a wide variety of genes in yeast but do not appear to bind DNA. We provide genetic and biochemical evidence that the DNA-binding protein alpha 2, a regulator of cell-type-specific genes, recruits the Tup1/Ssn6 repressor by directly interacting with Tup1. This interaction is mediated by a region of Tup1 containing seven copies of the WD repeat, a 40 amino acid motif of unknown function found in many other proteins. We have found that a single WD repeat will interact with alpha 2, indicating that the WD repeat is a protein-protein interaction domain. Furthermore, a fragment of Tup1 containing primarily WD repeats provides at least partial repression in the absence of Ssn6, suggesting that the repeats also mediate interaction between Tup1 and other components of the repression machinery.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Nucleares , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Secuencia de Consenso , Cruzamientos Genéticos , Proteínas de Unión al ADN/química , Escherichia coli/metabolismo , Proteínas Fúngicas/química , Datos de Secuencia Molecular , Mutagénesis , Plásmidos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Mapeo Restrictivo , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Transcripción Genética , Transformación Genética , beta-Galactosidasa/metabolismoRESUMEN
The homeodomain protein alpha 2 and the SRF-like protein Mcm1 are required to establish cell type in the yeast Saccharomyces cerevisiae. Together, these regulatory proteins recognize a specific DNA operator, marking a set of genes for transcriptional repression. In this paper, we show that occupancy of the operator by alpha 2-Mcm1 is not sufficient to bring about repression. Rather, repression is effected only when Ssn6 (a TPR protein) and Tup1 (a beta-transducin repeat protein) are also present in the cell. We show that Ssn6 represses transcription when brought to a promoter by a bacterial DNA-binding domain and that Tup1 is required for this repression. Based on these and other results, we propose that Ssn6-Tup1 is a general repressor of transcription in yeast, recruited to target promoters by a variety of sequence-specific DNA-binding proteins.