Notch signaling pathway
The Notch signaling pathway is a highly conserved cell signaling system present in most animals. Mammals possess four different notch receptors, referred to as NOTCH1, NOTCH2, NOTCH3, and NOTCH4. The notch receptor is a single-pass transmembrane receptor protein. It is a hetero-oligomer composed of a large extracellular portion, which associates in a calcium-dependent, non-covalent interaction with a smaller piece of the notch protein composed of a short extracellular region, a single transmembrane-pass, and a small intracellular region.
Notch signaling promotes proliferative signaling during neurogenesis, and its activity is inhibited by Numb to promote neural differentiation. It plays a major role in the regulation of embryonic development.
Notch signaling is dysregulated in many cancers, and faulty notch signaling is implicated in many diseases, including T-cell acute lymphoblastic leukemia, cerebral autosomal-dominant arteriopathy with sub-cortical infarcts and leukoencephalopathy, multiple sclerosis, Tetralogy of Fallot, and Alagille syndrome. Inhibition of notch signaling inhibits the proliferation of T-cell acute lymphoblastic leukemia in both cultured cells and a mouse model.
Discovery
In 1914, John S. Dexter noticed the appearance of a notch in the wings of the fruit fly Drosophila ampelophila. The alleles of the gene were identified in 1917 by American evolutionary biologist Thomas Hunt Morgan. Its molecular analysis and sequencing was independently undertaken in the 1980s by Spyros Artavanis-Tsakonas and Michael W. Young. Alleles of the two C. elegans ''Notch genes were identified based on developmental phenotypes: lin-12 and glp-1. The cloning and partial sequence of lin-12 was reported at the same time as Drosophila Notch'' by Iva Greenwald.Mechanism
The Notch protein spans the cell membrane, with part of it inside and part outside. Ligand proteins binding to the extracellular domain induce proteolytic cleavage and release of the intracellular domain, which enters the cell nucleus to modify gene expression.The cleavage model was first proposed in 1993 based on work done with Drosophila ''Notch and C. elegans lin-12, informed by the first oncogenic mutation affecting a human Notch gene. Compelling evidence for this model was provided in 1998 by in vivo analysis in Drosophila by Gary Struhl and in cell culture by Raphael Kopan. Although this model was initially disputed, the evidence in favor of the model was irrefutable by 2001.
The receptor is normally triggered via direct cell-to-cell contact, in which the transmembrane proteins of the cells in direct contact form the ligands that bind the notch receptor. The Notch binding allows groups of cells to organize themselves such that, if one cell expresses a given trait, this may be switched off in neighbouring cells by the intercellular notch signal. In this way, groups of cells influence one another to make large structures. Thus, lateral inhibition mechanisms are key to Notch signaling. lin-12 and Notch mediate binary cell fate decisions, and lateral inhibition involves feedback mechanisms to amplify initial differences.
The Notch cascade consists of Notch and Notch ligands, as well as intracellular proteins transmitting the notch signal to the cell's nucleus. The Notch/Lin-12/Glp-1 receptor family was found to be involved in the specification of cell fates during development in Drosophila and C. elegans''.
The intracellular domain of Notch forms a complex with CBF1 and Mastermind to activate transcription of target genes. The structure of the complex has been determined.
Pathway
Maturation of the notch receptor involves cleavage at the prospective extracellular side during intracellular trafficking in the Golgi complex. This results in a bipartite protein, composed of a large extracellular domain linked to the smaller transmembrane and intracellular domain. Binding of ligand promotes two proteolytic processing events; as a result of proteolysis, the intracellular domain is liberated and can enter the nucleus to engage other DNA-binding proteins and regulate gene expression.Notch and most of its ligands are transmembrane proteins, so the cells expressing the ligands typically must be adjacent to the notch expressing cell for signaling to occur. The notch ligands are also single-pass transmembrane proteins and are members of the DSL family of proteins. In Drosophila melanogaster, there are two ligands named Delta and Serrate. In mammals, the corresponding names are Delta-like and Jagged. In mammals there are multiple Delta-like and Jagged ligands, as well as possibly a variety of other ligands, such as F3/contactin.
In the nematode C. elegans, two genes encode homologous proteins, glp-1 and lin-12. There has been at least one report that suggests that some cells can send out processes that allow signaling to occur between cells that are as much as four or five cell diameters apart.
The notch extracellular domain is composed primarily of small cystine-rich motifs called EGF-like repeats.
Notch 1, for example, has 36 of these repeats. Each EGF-like repeat is composed of approximately 40 amino acids, and its structure is defined largely by six conserved cysteine residues that form three conserved disulfide bonds. Each EGF-like repeat can be modified by O-linked glycans at specific sites. An O-glucose sugar may be added between the first and second conserved cysteines, and an O-fucose may be added between the second and third conserved cysteines. These sugars are added by an as-yet-unidentified O-glucosyltransferase, and GDP-fucose Protein O-fucosyltransferase 1, respectively. The addition of O-fucose by POFUT1 is absolutely necessary for notch function, and, without the enzyme to add O-fucose, all notch proteins fail to function properly. As yet, the manner by which the glycosylation of notch affects function is not completely understood.
The O-glucose on notch can be further elongated to a trisaccharide with the addition of two xylose sugars by xylosyltransferases, and the O-fucose can be elongated to a tetrasaccharide by the ordered addition of an N-acetylglucosamine sugar by an N-Acetylglucosaminyltransferase called Fringe, the addition of a galactose by a galactosyltransferase, and the addition of a sialic acid by a sialyltransferase.
To add another level of complexity, in mammals there are three Fringe GlcNAc-transferases, named lunatic fringe, manic fringe, and radical fringe. These enzymes are responsible for something called a "fringe effect" on notch signaling. If Fringe adds a GlcNAc to the O-fucose sugar then the subsequent addition of a galactose and sialic acid will occur. In the presence of this tetrasaccharide, notch signals strongly when it interacts with the Delta ligand, but has markedly inhibited signaling when interacting with the Jagged ligand. The means by which this addition of sugar inhibits signaling through one ligand, and potentiates signaling through another is not clearly understood.
Once the notch extracellular domain interacts with a ligand, an ADAM-family metalloprotease called ADAM10, cleaves the notch protein just outside the membrane. This releases the extracellular portion of notch, which continues to interact with the ligand. The ligand plus the notch extracellular domain is then endocytosed by the ligand-expressing cell. There may be signaling effects in the ligand-expressing cell after endocytosis; this part of notch signaling is a topic of active research. After this first cleavage, an enzyme called γ-secretase cleaves the remaining part of the notch protein just inside the inner leaflet of the cell membrane of the notch-expressing cell. This releases the intracellular domain of the notch protein, which then moves to the nucleus, where it can regulate gene expression by activating the transcription factor CSL. It was originally thought that these CSL proteins suppressed Notch target transcription. However, further research showed that, when the intracellular domain binds to the complex, it switches from a repressor to an activator of transcription. Other proteins also participate in the intracellular portion of the notch signaling cascade.