RESUMEN

The histone deacetylase HDAC3 is associated with the NCoR/SMRT co-repressor complex, and its canonical function is in transcriptional repression, but it can also activate transcription. Here, we show that the repressor and activator functions of HDAC3 can be genetically separated in Drosophila. A lysine substitution in the N terminus (K26A) disrupts its catalytic activity and activator function, whereas a combination of substitutions (HEBI) abrogating the interaction with SMRTER enhances repressor activity beyond wild type in the early embryo. We conclude that the crucial functions of HDAC3 in embryo development involve catalytic-dependent gene activation and non-enzymatic repression by several mechanisms, including tethering of loci to the nuclear periphery.

Asunto(s)

Proteínas de Drosophila , Drosophila , Histona Desacetilasas , Proteínas Represoras , Animales , Drosophila/metabolismo , Regulación de la Expresión Génica , Proteínas Represoras/metabolismo , Proteínas de Drosophila/metabolismo , Histona Desacetilasas/metabolismo

RESUMEN

Many protein condensates can convert to fibrillar aggregates, but the underlying mechanisms are unclear. Liquid-liquid phase separation (LLPS) of spider silk proteins, spidroins, suggests a regulatory switch between both states. Here, we combine microscopy and native mass spectrometry to investigate the influence of protein sequence, ions, and regulatory domains on spidroin LLPS. We find that salting out-effects drive LLPS via low-affinity stickers in the repeat domains. Interestingly, conditions that enable LLPS simultaneously cause dissociation of the dimeric C-terminal domain (CTD), priming it for aggregation. Since the CTD enhances LLPS of spidroins but is also required for their conversion into amyloid-like fibers, we expand the stickers and spacers-model of phase separation with the concept of folded domains as conditional stickers that represent regulatory units.

Asunto(s)

Fibroínas , Seda , Seda/química , Fibroínas/química , Proteínas de Artrópodos , Secuencia de Aminoácidos

RESUMEN

Spider silk is the toughest fiber found in nature, and bulk production of artificial spider silk that matches its mechanical properties remains elusive. Development of miniature spider silk proteins (mini-spidroins) has made large-scale fiber production economically feasible, but the fibers' mechanical properties are inferior to native silk. The spider silk fiber's tensile strength is conferred by poly-alanine stretches that are zipped together by tight side chain packing in ß-sheet crystals. Spidroins are secreted so they must be void of long stretches of hydrophobic residues, since such segments get inserted into the endoplasmic reticulum membrane. At the same time, hydrophobic residues have high ß-strand propensity and can mediate tight inter-ß-sheet interactions, features that are attractive for generation of strong artificial silks. Protein production in prokaryotes can circumvent biological laws that spiders, being eukaryotic organisms, must obey, and the authors thus design mini-spidroins that are predicted to more avidly form stronger ß-sheets than the wildtype protein. Biomimetic spinning of the engineered mini-spidroins indeed results in fibers with increased tensile strength and two fiber types display toughness equal to native dragline silks. Bioreactor expression and purification result in a protein yield of ≈9 g L-1 which is in line with requirements for economically feasible bulk scale production.

RESUMEN

Recombinant spider silk proteins (spidroins) have multiple potential applications in development of novel biomaterials, but their multimodal and aggregation-prone nature have complicated production and straightforward applications. Here, we report that recombinant miniature spidroins, and importantly also the N-terminal domain (NT) on its own, rapidly form self-supporting and transparent hydrogels at 37 °C. The gelation is caused by NT α-helix to ß-sheet conversion and formation of amyloid-like fibrils, and fusion proteins composed of NT and green fluorescent protein or purine nucleoside phosphorylase form hydrogels with intact functions of the fusion moieties. Our findings demonstrate that recombinant NT and fusion proteins give high expression yields and bestow attractive properties to hydrogels, e.g., transparency, cross-linker free gelation and straightforward immobilization of active proteins at high density.

Asunto(s)

Fibroínas , Arañas , Animales , Fibroínas/química , Hidrogeles , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Seda/química , Arañas/metabolismo

RESUMEN

The proper functional interaction between different tissues represents a key component in systemic metabolic control. Indeed, disruption of endocrine inter-tissue communication is a hallmark of severe metabolic dysfunction in obesity and diabetes. Here, we show that the FNDC4-GPR116, liver-white adipose tissue endocrine axis controls glucose homeostasis. We found that the liver primarily controlled the circulating levels of soluble FNDC4 (sFNDC4) and lowering of the hepatokine FNDC4 led to prediabetes in mice. Further, we identified the orphan adhesion GPCR GPR116 as a receptor of sFNDC4 in the white adipose tissue. Upon direct and high affinity binding of sFNDC4 to GPR116, sFNDC4 promoted insulin signaling and insulin-mediated glucose uptake in white adipocytes. Indeed, supplementation with FcsFNDC4 in prediabetic mice improved glucose tolerance and inflammatory markers in a white-adipocyte selective and GPR116-dependent manner. Of note, the sFNDC4-GPR116, liver-adipose tissue axis was dampened in (pre) diabetic human patients. Thus our findings will now allow for harnessing this endocrine circuit for alternative therapeutic strategies in obesity-related pre-diabetes.

Asunto(s)

Tejido Adiposo Blanco/metabolismo , Proteínas de la Membrana/metabolismo , Estado Prediabético/metabolismo , Proteínas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células 3T3-L1 , Adipocitos/metabolismo , Tejido Adiposo Blanco/citología , Adolescente , Adulto , Anciano , Animales , Células CHO , Estudios de Cohortes , Cricetulus , Estudios Transversales , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/prevención & control , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Femenino , Técnicas de Silenciamiento del Gen , Glucosa/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Insulina/metabolismo , Resistencia a la Insulina , Islotes Pancreáticos/metabolismo , Hígado/metabolismo , Masculino , Proteínas de la Membrana/administración & dosificación , Proteínas de la Membrana/sangre , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Persona de Mediana Edad , Células 3T3 NIH , Estado Prediabético/sangre , Estado Prediabético/tratamiento farmacológico , Estado Prediabético/etiología , Cultivo Primario de Células , Proteínas/análisis , Receptores Acoplados a Proteínas G/sangre , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Adulto Joven

RESUMEN

Adipocytes, once considered simple lipid-storing cells, are rapidly emerging as complex cells with many biologically diverse functions. A powerful high-throughput method for analyzing single cells is flow cytometry. Several groups have attempted to analyze and sort freshly isolated adipocytes; however, using an adipocyte-specific reporter mouse, we demonstrate that these studies fail to detect the majority of white adipocytes. We define critical settings required for adipocyte flow cytometry and provide a rigid strategy for analyzing and sorting white and brown adipocyte populations. The applicability of our protocol is shown by sorting mouse adipocytes based on size or UCP1 expression and demonstrating that a subset of human adipocytes lacks the ß2-adrenergic receptor, particularly in the insulin-resistant state. In conclusion, the present study confers key technological insights for analyzing and sorting mature adipocytes, opening up numerous downstream research applications.

Asunto(s)

Adipocitos/citología , Adipocitos/metabolismo , Citometría de Flujo/métodos , Tejido Adiposo/citología , Tejido Adiposo/metabolismo , Tejido Adiposo Pardo/citología , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/citología , Tejido Adiposo Blanco/metabolismo , Animales , Humanos , Ratones , Proteína Desacopladora 1/metabolismo

RESUMEN

FNDC4 is a secreted factor sharing high homology with the exercise-associated myokine irisin (FNDC5). Here we report that Fndc4 is robustly upregulated in several mouse models of inflammation as well as in human inflammatory conditions. Specifically, FNDC4 levels are increased locally at inflamed sites of the intestine of inflammatory bowel disease patients. Interestingly, administration of recombinant FNDC4 in the mouse model of induced colitis markedly reduces disease severity compared with mice injected with a control protein. Conversely, mice lacking Fndc4 develop more severe colitis. Analysis of binding of FNDC4 to different immune cell types reveals strong and specific binding to macrophages and monocytes. FNDC4 treatment of bone marrow-derived macrophages in vitro results in reduced phagocytosis, increased cell survival and reduced proinflammatory chemokine expression. Hence, treatment with FNDC4 results in a state of dampened macrophage activity, while enhancing their survival. Thus, we have characterized FNDC4 as a factor with direct therapeutic potential in inflammatory bowel disease and possibly other inflammatory diseases.

Asunto(s)

Antiinflamatorios/metabolismo , Colitis/metabolismo , Macrófagos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Colitis/genética , Colitis/patología , Sulfato de Dextran , Progresión de la Enfermedad , Regulación de la Expresión Génica , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Fagocitosis/efectos de los fármacos , Proteínas/química , Proteínas/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

Positive Transcription Elongation Factor b (P-TEFb) is a kinase consisting of Cdk9 and Cyclin T that releases RNA Polymerase II (Pol II) into active elongation. It can assemble into a larger Super Elongation Complex (SEC) consisting of additional elongation factors. Here, we use a miRNA-based approach to knock down the maternal contribution of P-TEFb and SEC components in early Drosophila embryos. P-TEFb or SEC depletion results in loss of cells from the embryo posterior and in cellularization defects. Interestingly, the expression of many patterning genes containing promoter-proximal paused Pol II is relatively normal in P-TEFb embryos. Instead, P-TEFb and SEC are required for expression of some non-paused, rapidly transcribed genes in pre-cellular embryos, including the cellularization gene Serendipity-α. We also demonstrate that another P-TEFb regulated gene, terminus, has an essential function in embryo development. Similar morphological and gene expression phenotypes were observed upon knock down of Mediator subunits, providing in vivo evidence that P-TEFb, the SEC and Mediator collaborate in transcription control. Surprisingly, P-TEFb depletion does not affect the ratio of Pol II at the promoter versus the 3' end, despite affecting global Pol II Ser2 phosphorylation levels. Instead, Pol II occupancy is reduced at P-TEFb down-regulated genes. We conclude that a subset of non-paused, pre-cellular genes are among the most susceptible to reduced P-TEFb, SEC and Mediator levels in Drosophila embryos.

Asunto(s)

Desarrollo Embrionario , Factor B de Elongación Transcripcional Positiva/genética , ARN Polimerasa II/genética , Transcripción Genética , Animales , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Complejo Mediador/genética , Fenotipo , Fosforilación , Factor B de Elongación Transcripcional Positiva/metabolismo , Regiones Promotoras Genéticas