Proteomic analysis of the Notch interactome.

Recent large-scale studies have provided a global description of the interactome-the whole network of protein interactions in a cell or an organism-for several model organisms. Defining protein interactions on a proteome-wide scale has led to a better understanding of the cellular functions of many proteins, especially those that have not been studied by classical molecular genetic approaches. Here we describe the resources, methods, and techniques necessary for generation of such a proteome-scale interactome in a high throughput manner. These procedures will also be applicable to low or medium throughput focused studies aimed at understanding interactions between members of specific pathways such as Notch signaling.

Bacterial expression and in vitro refolding of limited fragments of the Notch receptor and its ligands.

Prokaryotic expression of limited fragments of the Notch receptor and its ligands followed by in vitro refolding has been used for the production of the significant amounts of protein required for structure determination by X-ray crystallography or nuclear magnetic resonance spectroscopy. As an illustration of the protocol for the production of these EGF-containing constructs we have focused on a limited fragment of human Notch 1 that contains three calcium-binding EGF domains, hNotch-111-13. Following characterization by the methods described here, this construct has been shown to be functionally competent in a range of assays and the structure has been solved by X-ray crystallography and NMR.

CADASIL: comunicación de una familia con una nueva mutación p. G296C en el exón 6 del gen Notch- 3 / CADASIL: brief report on a family with a new p. G296C mutation in exon 6 of the Notch- 3 gene

Introducción. CADASIL es una arteriopatía sistémica de herencia autosómica dominante, cuya anomalía genética se sitúa en el gen Notch-3 del cromosoma 19, y se caracteriza clínicamente por migraña con aura, ictus de repetición y deterioro cognitivo, y es causante de la ocurrencia de ictus en la población joven. Caso clínico. El caso propositus es un varón de 57 años que presentó un cuadro de disartria, pérdida de fuerza en las extremidades izquierdas y alteración del equilibrio con dismetría en las extremidades izquierdas, en relación con un ictus isquémico agudo en el pedúnculo cerebeloso. El estudio ultraestructural de la biopsia de piel identificó los depósitos de material electrón densos típicos del CADASIL. El análisis genético identificó una nueva mutación para esta enfermedad en el codón 296 del exón 6 del gen Notch-3 que produce un cambio aminoacídico de glicina a cisteína en la proteína (p.G296C). Conclusión. Se comunica el caso de una familia afectada de CADASIL, portadora de una nueva mutación p.G296C situada en el exón 6 del gen Notch-3 (AU)

Introduction. CADASIL is a dominant autosomal inborn systemic arteriopathy, whose genetic anomaly is located in the Notch-3 gene of chromosome 19. It is clinically characterised by migraine with aura, recurrent stroke and cognitive deterioration, and is one of the causes of strokes among the young. Case report. The propositus was a 57-year-old male who presented a clinical picture of dysarthria, loss of strength in the left extremities and alterations affecting balance with dysmetry in the left extremities, related with an acute ischaemic stroke in the cerebellar peduncle. An ultrastructural study of a biopsy specimen of the skin revealed the electron-dense deposits that characterise CADASIL. The genetic analysis identified a new mutation for this disease in codon 296 of exon 6 in the Notch-3 gene that produces a change of amino acid, from glycine to cysteine in protein (p.G296C). Conclusions. This communication reports the case of a family with CADASIL that was a carrier of a new p.G296C mutation located in exon 6 of the Notch-3 gene (AU)

O-GlcNAc modification of the extracellular domain of Notch receptors.

Epidermal growth factor (EGF) domains are posttranslationally modified with unique O-linked glycans. The classical types of O-glycans on EGF domains are O-fucose and O-glucose glycans, found on many plasma glycoproteins and signaling molecules, whose biological functions have been demonstrated especially in the context of the Notch signaling pathway. We recently discovered O-GlcNAc modification as a new modification of the EGF domain that occurs on the conserved Ser/Thr residue located between the fifth and sixth cysteine residues within the EGF domain of Notch receptors in Drosophila. Here, we describe the methods employed to detect the O-GlcNAc modification of EGF repeats of Notch receptors. These methods include mass spectrometric analysis, galactosyltransferase labeling, immunoblotting with a specific antibody, and beta-N-acetyl-hexosaminidase digestion experiments. We also describe a method to detect O-GlcNAc transferase activity from crude membrane fraction proteins prepared from cultured S2 cells.

Regulation of notch signaling via O-glucosylation insights from Drosophila studies.

Recent work using Drosophila melanogaster has shown that a protein O-glucosyltransferase called Rumi regulates Notch signaling. Studies on several alleles of rumi identified in a forward genetic screen indicated that Rumi is a temperature-sensitive regulator of Notch signaling in flies. Further genetic and rescue experiments demonstrated that Rumi is a general regulator of Drosophila Notch signaling. Biochemical analyses showed that Rumi adds glucose to specific EGF repeats in the extracellular domain of Notch receptor in the Drosophila S2 cell line. Furthermore, RNAi-mediated knockdown of Rumi in this cell line resulted in a severe decrease in the level of O-linked glucose on Notch. In this chapter, we discuss the genetic and biochemical methods used to determine the role of Rumi in the regulation of Notch signaling in flies.