RESUMO
To date, there is a lack of research into the vaginal and sperm microbiome and its bearing on artificial insemination (AI) success in the ovine species. Using hypervariable regions V3-V4 of the 16S rRNA, we describe, for the first time, the combined effect of the ovine microbiome of both females (50 ewes belonging to five herds) and males (five AI rams from an AI center) on AI outcome. Differences in microbiota abundance between pregnant and non-pregnant ewes and between ewes carrying progesterone-releasing intravaginal devices (PRID) with or without antibiotic were tested at different taxonomic levels. The antibiotic treatment applied with the PRID only altered Streptobacillus genus abundance, which was significantly lower in ewes carrying PRID with antibiotic. Mageebacillus, Histophilus, Actinobacilllus and Sneathia genera were significantly less abundant in pregnant ewes. In addition, these genera were more abundant in two farms with higher AI failure. Species of these genera such as Actinobacillus seminis and Histophilus somni have been associated with reproductive disorders in the ovine species. These genera were not present in the sperm samples of AI rams, but were found in the foreskin samples of rams belonging to herd 2 (with high AI failure rate) indicating that their presence in ewes' vagina could be due to prior transmission by natural mating with rams reared in the herd.
RESUMO
AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that acts as a sensor of cellular energy charge. Once activated it switches on catabolic pathways and switches off many ATP-consuming processes (anabolic pathways) to preserve the energy status of the cell. In order to identify new targets of AMPK action we have performed a two-hybrid screening of a human pancreas cDNA library. As a result, we have identified TRIP6 as a novel target of AMPK action. This protein belongs to the zyxin family of proteins located at the focal adhesion plaques in the plasma membrane, although they may also travel to the nucleus, where they have regulatory properties. We confirmed the physical interaction between the catalytic subunit (AMPK-alpha2) of the AMPK complex and TRIP6 in mammalian cells by two-hybrid and co-immunoprecipitation assays. We also showed that AMPK was able to phosphorylate in vitro TRIP6 at the N-terminus. Finally, we present evidence that transcriptional co-activator properties of TRIP6 were enhanced by AMPK action.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Quinases Ativadas por AMP , ATPases Associadas a Diversas Atividades Celulares , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Adesões Focais/metabolismo , Humanos , Imunoprecipitação , Proteínas com Domínio LIM , Dados de Sequência Molecular , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ribonucleotídeos/farmacologia , Especificidade por Substrato , Fatores de Tempo , Fatores de Transcrição/química , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
R5/PTG is one of the glycogen targeting subunits of type 1 protein phosphatase, a master regulator of glycogen synthesis. R5/PTG recruits the phosphatase to the places where glycogen synthesis occurs, allowing the activation of glycogen synthase and the inactivation of glycogen phosphorylase, thus increasing glycogen synthesis and decreasing its degradation. In this report, we show that the activity of R5/PTG is regulated by AMP-activated protein kinase (AMPK). We demonstrate that AMPK interacts physically with R5/PTG and modifies its basal phosphorylation status. We have also mapped the major phosphorylation sites of R5/PTG by mass spectrometry analysis, observing that phosphorylation of Ser-8 and Ser-268 increased upon activation of AMPK. We have recently described that the activity of R5/PTG is down-regulated by the laforin-malin complex, composed of a dual specificity phosphatase (laforin) and an E3-ubiquitin ligase (malin). We now demonstrate that phosphorylation of R5/PTG at Ser-8 by AMPK accelerates its laforin/malin-dependent ubiquitination and subsequent proteasomal degradation, which results in a decrease of its glycogenic activity. Thus, our results define a novel role of AMPK in glycogen homeostasis.