RESUMEN
Protein glycosylation is an essential post-translational modification in all domains of life. Its impairment in humans can result in severe diseases named congenital disorders of glycosylation (CDGs). Most of the glycosyltransferases (GTs) responsible for proper glycosylation are polytopic membrane proteins that represent challenging targets in proteomics. We established a multiple reaction monitoring (MRM) assay to comprehensively quantify GTs involved in the processes of N-glycosylation and O- and C-mannosylation in the endoplasmic reticulum. High robustness was achieved by using an enriched membrane protein fraction of isotopically labeled HEK 293T cells as an internal protein standard. The analysis of primary skin fibroblasts from eight CDG type I patients with impaired ALG1, ALG2, and ALG11 genes, respectively, revealed a substantial reduction in the corresponding protein levels. The abundance of the other GTs, however, remained unchanged at the transcript and protein levels, indicating that there is no fail-safe mechanism for the early steps of glycosylation in the endoplasmic reticulum. The established MRM assay was shared with the scientific community via the commonly used open source Skyline software environment, including Skyline Batch for automated data analysis. We demonstrate that another research group could easily reproduce all analysis steps, even while using different LC-MS hardware.
Asunto(s)
Trastornos Congénitos de Glicosilación , Glicosiltransferasas , Humanos , Glicosilación , Glicosiltransferasas/genética , Trastornos Congénitos de Glicosilación/genética , Proteómica , Procesamiento Proteico-Postraduccional , Proteínas de la Membrana/genética , ManosiltransferasasRESUMEN
The heat shock transcription factors heat shock transcription factor 1 and Hsf2 have been studied for many years, mainly in the context of stress response and in malignant cells. Their physiological function in nonmalignant human cells under nonstress conditions is still largely unknown. To approach this important issue, Joutsen et al. present immunohistochemical staining data on Hsf1 and Hsf2 in 80 nonpathological human tissue samples. The wealth of these data elicits many interesting questions that will spur many future research projects.
Asunto(s)
Factores de Transcripción del Choque Térmico , Inmunohistoquímica , Humanos , Factores de Transcripción del Choque Térmico/metabolismo , Factores de Transcripción del Choque Térmico/genética , Inmunohistoquímica/métodos , Factores de Transcripción/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Choque Térmico/metabolismoRESUMEN
J-domain proteins (JDPs) are the largest family of chaperones in most organisms, but much of how they function within the network of other chaperones and protein quality control machineries is still an enigma. Here, we report on the latest findings related to JDP functions presented at a dedicated JDP workshop in Gdansk, Poland. The report does not include all (details) of what was shared and discussed at the meeting, because some of these original data have not yet been accepted for publication elsewhere or represented still preliminary observations at the time.
Asunto(s)
Proteínas HSP70 de Choque Térmico , Chaperonas Moleculares , Proteínas HSP70 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Polonia , Proteínas del Choque Térmico HSP40/metabolismoRESUMEN
Preserving and regulating cellular homeostasis in the light of changing environmental conditions or developmental processes is of pivotal importance for single cellular and multicellular organisms alike. To counteract an imbalance in cellular homeostasis transcriptional programs evolved, called the heat shock response, unfolded protein response, and integrated stress response, that act cell-autonomously in most cells but in multicellular organisms are subjected to cell-nonautonomous regulation. These transcriptional programs downregulate the expression of most genes but increase the expression of heat shock genes, including genes encoding molecular chaperones and proteases, proteins involved in the repair of stress-induced damage to macromolecules and cellular structures. Sixty-one years after the discovery of the heat shock response by Ferruccio Ritossa, many aspects of stress biology are still enigmatic. Recent progress in the understanding of stress responses and molecular chaperones was reported at the 12th International Symposium on Heat Shock Proteins in Biology, Medicine and the Environment in the Old Town Alexandria, VA, USA from 28th to 31st of October 2023.