RESUMO
Selenoprotein T (SelT) is a recently characterized thioredoxin-like protein whose expression is very high during development, but is confined to endocrine tissues in adulthood where its function is unknown. We report here that SelT is required for adaptation to the stressful conditions of high hormone level production in endocrine cells. Using immunofluorescence and TEM immunogold approaches, we find that SelT is expressed at the endoplasmic reticulum membrane in all hormone-producing pituitary cell types. SelT knockdown in corticotrope cells promotes unfolded protein response (UPR) and ER stress and lowers endoplasmic reticulum-associated protein degradation (ERAD) and hormone production. Using a screen in yeast for SelT-membrane protein interactions, we sort keratinocyte-associated protein 2 (KCP2), a subunit of the protein complex oligosaccharyltransferase (OST). In fact, SelT interacts not only with KCP2 but also with other subunits of the A-type OST complex which are depleted after SelT knockdown leading to POMC N-glycosylation defects. This study identifies SelT as a novel subunit of the A-type OST complex, indispensable for its integrity and for ER homeostasis, and exerting a pivotal adaptive function that allows endocrine cells to properly achieve the maturation and secretion of hormones.
Assuntos
Hormônio Adrenocorticotrópico/metabolismo , Corticotrofos/metabolismo , Degradação Associada com o Retículo Endoplasmático , Hexosiltransferases/genética , Proteínas de Membrana/genética , Selenoproteínas/genética , Transdução de Sinais , Hormônio Adrenocorticotrópico/genética , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Corticotrofos/citologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Edição de Genes , Regulação da Expressão Gênica , Glicosilação , Hexosiltransferases/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Microssomos/metabolismo , Hipófise/citologia , Hipófise/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno , Selenoproteínas/antagonistas & inibidores , Selenoproteínas/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
The bacterial strain producing thermostable, alklophilic alpha-amylase was identified as Bacillus amyloliquefaciens KCP2 using 16S rDNA gene sequencing data (NCBI Accession No: KF112071). Medium components were optimized through the statistical approach for the synthesis of alpha-amylase by the organism under solid-state fermentation using wheat bran as the substrate. The medium components influencing the enzyme production were identified using a two-level fractional factorial Plackett-Burman design. Among the various variables screened, starch, ammonium sulphate and calcium chloride were found to be most significant medium components. The optimum levels of these significant parameters were determined employing the response surface Central Composite design which significantly increased the enzyme production with the supplementation of starch 0.01 g, ammonium sulphate 0.2 g and 5 mM calcium chloride in the production medium. Temperature and pH stability of the alpha-amylase suggested its wide application in the food and pharmaceutical industries.