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2.
BMC Genomics ; 22(1): 771, 2021 Oct 28.
Artículo en Inglés | MEDLINE | ID: mdl-34711176

RESUMEN

BACKGROUND: Temperature change affects the myriad of concurrent cellular processes in a non-uniform, disruptive manner. While endothermic organisms minimize the challenge of ambient temperature variation by keeping the core body temperature constant, cells of many ectothermic species maintain homeostatic function within a considerable temperature range. The cellular mechanisms enabling temperature acclimation in ectotherms are still poorly understood. At the transcriptional level, the heat shock response has been analyzed extensively. The opposite, the response to sub-optimal temperature, has received lesser attention in particular in animal species. The tissue specificity of transcriptional responses to cool temperature has not been addressed and it is not clear whether a prominent general response occurs. Cis-regulatory elements (CREs), which mediate increased transcription at cool temperature, and responsible transcription factors are largely unknown. RESULTS: The ectotherm Drosophila melanogaster with a presumed temperature optimum around 25 °C was used for transcriptomic analyses of effects of temperatures at the lower end of the readily tolerated range (14-29 °C). Comparative analyses with adult flies and cell culture lines indicated a striking degree of cell-type specificity in the transcriptional response to cool. To identify potential cis-regulatory elements (CREs) for transcriptional upregulation at cool temperature, we analyzed temperature effects on DNA accessibility in chromatin of S2R+ cells. Candidate cis-regulatory elements (CREs) were evaluated with a novel reporter assay for accurate assessment of their temperature-dependency. Robust transcriptional upregulation at low temperature could be demonstrated for a fragment from the pastrel gene, which expresses more transcript and protein at reduced temperatures. This CRE is controlled by the JAK/STAT signaling pathway and antagonizing activities of the transcription factors Pointed and Ets97D. CONCLUSION: Beyond a rich data resource for future analyses of transcriptional control within the readily tolerated range of an ectothermic animal, a novel reporter assay permitting quantitative characterization of CRE temperature dependence was developed. Our identification and functional dissection of the pst_E1 enhancer demonstrate the utility of resources and assay. The functional characterization of this CoolUp enhancer provides initial mechanistic insights into transcriptional upregulation induced by a shift to temperatures at the lower end of the readily tolerated range.


Asunto(s)
Drosophila melanogaster , Drosophila , Animales , Frío , Drosophila melanogaster/genética , Secuencias Reguladoras de Ácidos Nucleicos , Temperatura
3.
Development ; 144(24): 4573-4587, 2017 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-29084803

RESUMEN

Cells in ectotherms function normally within an often wide temperature range. As temperature dependence is not uniform across all the distinct biological processes, acclimation presumably requires complex regulation. The molecular mechanisms that cope with the disruptive effects of temperature variation are still poorly understood. Interestingly, one of five different ß-tubulin paralogs, ßTub97EF, was among the genes upregulated at low temperature in cultured Drosophila cells. As microtubules are known to be cold sensitive, we analyzed whether ßTub97EF protects microtubules at low temperatures. During development at the optimal temperature (25°C), ßTub97EF was expressed in a tissue-specific pattern primarily in the gut. There, as well as in hemocytes, expression was increased at low temperature (14°C). Although ßTub97EF mutants were viable and fertile at 25°C, their sensitivity within the well-tolerated range was slightly enhanced during embryogenesis specifically at low temperatures. Changing ß-tubulin isoform ratios in hemocytes demonstrated that ß-Tubulin 97EF has a pronounced microtubule stabilizing effect. Moreover, ßTub97EF is required for normal microtubule stability in the gut. These results suggest that ßTub97EF upregulation at low temperature contributes to acclimation by stabilizing microtubules.


Asunto(s)
Frío , Drosophila melanogaster/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/biosíntesis , Aclimatación , Animales , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Tracto Gastrointestinal/metabolismo , Dominios Proteicos/fisiología , Isoformas de Proteínas/metabolismo , Activación Transcripcional/genética , Tubulina (Proteína)/genética
4.
Proc Natl Acad Sci U S A ; 111(15): 5592-7, 2014 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-24706800

RESUMEN

Effects of temperature on biological processes are complex. Diffusion is less affected than the diverse enzymatic reactions that have distinct individual temperature profiles. Hence thermal fluctuations pose a formidable challenge to ectothermic organisms in which body temperature is largely dictated by the ambient temperature. How cells in ectotherms cope with the myriad disruptive effects of temperature variation is poorly understood at the molecular level. Here we show that nucleocytoplasmic posttranslational modification of proteins with O-linked GlcNAc (O-GlcNAc) is closely correlated with ambient temperature during development of distantly related ectotherms ranging from the insect Drosophila melanogaster to the nematode Caenorhabditis elegans to the fish Danio rerio. Regulation seems to occur at the level of activity of the only two enzymes, O-GlcNAc transferase and O-GlcNAcase, that add and remove, respectively, this posttranslational modification in nucleus and cytoplasm. With genetic approaches in D. melanogaster and C. elegans, we demonstrate the importance of high levels of this posttranslational modification for successful development at elevated temperatures. Because many cytoplasmic and nuclear proteins in diverse pathways are O-GlcNAc targets, temperature-dependent regulation of this modification might contribute to an efficient coordinate adjustment of cellular processes in response to thermal change.


Asunto(s)
Aclimatación/fisiología , Acetilglucosamina/metabolismo , Caenorhabditis elegans/embriología , Drosophila melanogaster/embriología , Procesamiento Proteico-Postraduccional/fisiología , Temperatura , Pez Cebra/embriología , Animales , Tamaño de la Nidada , Cruzamientos Genéticos , Técnica del Anticuerpo Fluorescente , Immunoblotting , Especificidad de la Especie
5.
PLoS One ; 8(3): e60014, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23555865

RESUMEN

Lymphangioleiomyomatosis (LAM), a multisystem disease of women, is manifest by the proliferation of smooth muscle-like cells in the lung resulting in cystic lung destruction. Women with LAM can also develop renal angiomyolipomas. LAM is caused by mutations in the tuberous sclerosis complex genes (TSC1 or TSC2), resulting in hyperactive mammalian Target of Rapamycin (mTOR) signaling. The mTOR inhibitor, Rapamycin, stabilizes lung function in LAM and decreases the volume of renal angiomyolipomas, but lung function declines and angiomyolipomas regrow when treatment is discontinued, suggesting that factors induced by mTORC1 inhibition may promote the survival of TSC2-deficient cells. Whether microRNA (miRNA, miR) signaling is involved in the response of LAM to mTORC1 inhibition is unknown. We identified Rapamycin-dependent miRNA in LAM patient angiomyolipoma-derived cells using two separate screens. First, we assayed 132 miRNA of known significance to tumor biology. Using a cut-off of >1.5-fold change, 48 microRNA were Rapamycin-induced, while 4 miRs were downregulated. In a second screen encompassing 946 miRNA, 18 miRs were upregulated by Rapamycin, while eight were downregulated. Dysregulation of miRs 29b, 21, 24, 221, 106a and 199a were common to both platforms and were classified as candidate "RapamiRs." Validation by qRT-PCR confirmed that these microRNA were increased. miR-21, a pro-survival miR, was the most significantly increased by mTOR-inhibition (p<0.01). The regulation of miR-21 by Rapamycin is cell type independent. mTOR inhibition promotes the processing of the miR-21 transcript (pri-miR-21) to a premature form (pre-miR-21). In conclusion, our findings demonstrate that Rapamycin upregulates multiple miRs, including pro-survival miRs, in TSC2-deficient patient-derived cells. The induction of miRs may contribute to the response of LAM and TSC patients to Rapamycin therapy.


Asunto(s)
Linfangioleiomiomatosis/genética , MicroARNs/metabolismo , Sirolimus/farmacología , Esclerosis Tuberosa/genética , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Immunoblotting , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
6.
Proc Natl Acad Sci U S A ; 108(30): 12455-60, 2011 Jul 26.
Artículo en Inglés | MEDLINE | ID: mdl-21746920

RESUMEN

Tuberous sclerosis complex (TSC) is a tumor suppressor syndrome characterized by benign tumors in multiple organs, including the brain and kidney. TSC-associated tumors exhibit hyperactivation of mammalian target of rapamycin complex 1 (mTORC1), a direct inhibitor of autophagy. Autophagy can either promote or inhibit tumorigenesis, depending on the cellular context. The role of autophagy in the pathogenesis and treatment of the multisystem manifestations of TSC is unknown. We found that the combination of mTORC1 and autophagy inhibition was more effective than either treatment alone in inhibiting the survival of tuberin (TSC2)-null cells, growth of TSC2-null xenograft tumors, and development of spontaneous renal tumors in Tsc2(+/-) mice. Down-regulation of Atg5 induced extensive central necrosis in TSC2-null xenograft tumors, and loss of one allele of Beclin1 almost completely blocked macroscopic renal tumor formation in Tsc2(+/-) mice. Surprisingly, given the finding that lowering autophagy blocks TSC tumorigenesis, genetic down-regulation of p62/sequestosome 1 (SQSTM1), the autophagy substrate that accumulates in TSC tumors as a consequence of low autophagy levels, strongly inhibited the growth of TSC2-null xenograft tumors. These data demonstrate that autophagy is a critical component of TSC tumorigenesis, suggest that mTORC1 inhibitors may have autophagy-dependent prosurvival effects in TSC, and reveal two distinct therapeutic targets for TSC: autophagy and the autophagy target p62/SQSTM1.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Choque Térmico/metabolismo , Esclerosis Tuberosa/metabolismo , Esclerosis Tuberosa/patología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/genética , Autofagia/genética , Autofagia/fisiología , Proteína 5 Relacionada con la Autofagia , Beclina-1 , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Genes p53 , Proteínas de Choque Térmico/genética , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Ratones SCID , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Asociadas a Microtúbulos/genética , Complejos Multiproteicos , Proteínas/genética , Proteínas/metabolismo , Proteína Sequestosoma-1 , Serina-Treonina Quinasas TOR , Esclerosis Tuberosa/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética
7.
Adv Exp Med Biol ; 680: 481-8, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20865533

RESUMEN

The inactive porphobilinogen synthase (PBGS) hexamer has an oligomer-specific and phylogenetically variable surface cavity that is not present in the active octamer. The octamer and hexamer are components of a dynamic quaternary structure equilibrium characteristic of morpheeins. Small molecules that bind to the hexamer-specific surface cavity, which is at the interface of three subunits, are predicted to act as allosteric inhibitors that function by drawing the oligomeric equilibrium toward the hexamer. We used GLIDE as a tool to enrich a 250,000 molecule library for molecules with enhanced probability of acting as hexamer-stabilizing allosteric inhibitors of PBGS from Yersinia enterocolitica. Eighty-six compounds were tested in vitro and five showed hexamer stabilization. We discuss the application of computational docking to surface cavities as an approach to find allosteric modulators of protein function with specific reference to morpheeins that function as an equilibrium of non-additive quaternary structure assemblies.


Asunto(s)
Proteínas/química , Sitio Alostérico , Biología Computacional , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Estabilidad de Enzimas/efectos de los fármacos , Modelos Moleculares , Porfobilinógeno Sintasa/antagonistas & inhibidores , Porfobilinógeno Sintasa/química , Porfobilinógeno Sintasa/metabolismo , Estructura Cuaternaria de Proteína , Subunidades de Proteína , Proteínas/metabolismo , Yersinia enterocolitica/enzimología
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