Notch3 in Development, Health and Disease

Notch3 is one of four mammalian Notch proteins, which act as signalling receptors to control cell fate in many developmental and adult tissue contexts. Notch signalling continues to be important in the adult organism for tissue maintenance and renewal and mis-regulation of Notch is involved in many diseases. Genetic studies have shown that Notch3 gene knockouts are viable and have limited developmental defects, focussed mostly on defects in the arterial smooth muscle cell lineage. Additional studies have revealed overlapping roles for Notch3 with other Notch proteins, which widen the range of developmental functions. In the adult, Notch3, in collaboration with other Notch proteins, is involved in stem cell regulation in different tissues in stem cell regulation in different tissues, and it also controls the plasticity of the vascular smooth muscle phenotype involved in arterial vessel remodelling. Overexpression, gene amplification and mis-activation of Notch3 are associated with different cancers, in particular triple negative breast cancer and ovarian cancer. Mutations of Notch3 are associated with a dominantly inherited disease CADASIL (cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy), and there is further evidence linking Notch3 misregulation to hypertensive disease. Here we discuss the distinctive roles of Notch3 in development, health and disease, different views as to the underlying mechanisms of its activation and misregulation in different contexts and potential for therapeutic intervention

Alternative mechanisms of Notch activation by partitioning into distinct endosomal domains

Different membrane microdomain compositions provide unique environments that can regulate signaling receptor function. We identify microdomains on the endosome membrane of Drosophila endosomes, enriched in lipid-raft or clathrin/ESCRT-0, which are associated with Notch activation by distinct, ligand-independent mechanisms. Transfer of Notch between microdomains is regulated by Deltex and Suppressor of deltex ubiquitin ligases and is limited by a gate-keeper role for ESCRT complexes. Ubiquitination of Notch by Deltex recruits it to the clathrin/ESCRT-0 microdomain and enhances Notch activation by an ADAM10-independent/TRPML-dependent mechanism. This requirement for Deltex is bypassed by the downregulation of ESCRT-III. In contrast, while ESCRT-I depletion also activates Notch, it does so by an ADAM10-dependent/TRPML-independent mechanism and Notch is retained in the lipid raft-like microdomain. In the absence of such endosomal perturbation, different activating Notch mutations also localize to different microdomains and are activated by different mechanisms. Our findings demonstrate the interplay between Notch regulators, endosomal trafficking components, and Notch genetics, which defines membrane locations and activation mechanisms

E3 Ubiquitin Ligase Regulators of Notch Receptor Endocytosis: From Flies to Humans

Notch is a developmental receptor, conserved in the evolution of the metazoa, which regulates cell fate proliferation and survival in numerous developmental contexts, and also regulates tissue renewal and repair in adult organisms. Notch is activated by proteolytic removal of its extracellular domain and the subsequent release of its intracellular domain, which then acts in the nucleus as part of a transcription factor complex. Numerous regulatory mechanisms exist to tune the amplitude, duration and spatial patterning of this core signalling mechanism. In Drosophila, Deltex (Dx) and Suppressor of dx (Su(dx)) are E3 ubiquitin ligases which interact with the Notch intracellular domain to regulate its endocytic trafficking, with impacts on both ligand-dependent and ligand-independent signal activation. Homologues of Dx and Su(dx) have been shown to also interact with one or more of the four mammalian Notch proteins and other target substrates. Studies have shown similarities, specialisations and diversifications of the roles of these Notch regulators. This review collates together current research on vertebrate Dx and Su(dx)-related proteins, provides an overview of their various roles, and discusses their contributions to cell fate regulation and disease

The First Defined Null Allele of the Notch Regulator, a Suppressor of Deltex: Uncovering Its Novel Roles in <i>Drosophila melanogaster</i> Oogenesis

Suppressor of deltex (Su(dx)) is a Drosophila melanogaster member of the NEDD4 family of the HECT domain E3 ubiquitin ligases. Su(dx) acts as a regulator of Notch endocytic trafficking, promoting Notch lysosomal degradation and the down-regulation of both ligand-dependent and ligand-independent signalling, the latter involving trafficking through the endocytic pathway and activation of the endo/lysosomal membrane. Mutations of Su(dx) result in developmental phenotypes in the Drosophila wing that reflect increased Notch signalling, leading to gaps in the specification of the wing veins, and Su(dx) functions to provide the developmental robustness of Notch activity to environmental temperature shifts. The full developmental functions of Su(dx) are unclear; however, this is due to a lack of a clearly defined null allele. Here we report the first defined null mutation of Su(dx), generated by P-element excision, which removes the complete open reading frame. We show that the mutation is recessive-viable, with the Notch gain of function phenotypes affecting wing vein and leg development. We further uncover new roles for Su(dx) in Drosophila oogenesis, where it regulates interfollicular stalk formation, egg chamber separation and germline cyst enwrapment by the follicle stem cells. Interestingly, while the null allele exhibited a gain in Notch activity during oogenesis, the previously described Su(dx)SP allele, which carries a seven amino acid in-frame deletion, displayed a Notch loss of function phenotypes and an increase in follicle stem cell turnover. This is despite both alleles displaying similar Notch gain of function in wing development. We attribute this unexpected context-dependent outcome of Su(dx)sp being due to the partial retention of function by the intact C2 and WW domain regions of the protein. Our results extend our understanding of the developmental role of Su(dx) in the tissue renewal and homeostasis of the Drosophila ovary and illustrate the importance of examining an allelic series of mutations to fully understand developmental functions