Editing Notch signaling pathway (section)

== Mechanism ==
{{further|Notch protein}}

The [[Notch protein]] spans the [[cell membrane]], with part of it inside and part outside. [[ligand (biochemistry)|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]].<ref name="pmid11604511">{{cite journal | vauthors = Oswald F, Täuber B, Dobner T, Bourteele S, Kostezka U, Adler G, Liptay S, Schmid RM | display-authors = 6 | title = p300 acts as a transcriptional coactivator for mammalian Notch-1 | journal = Molecular and Cellular Biology | volume = 21 | issue = 22 | pages = 7761–7774 | date = November 2001 | pmid = 11604511 | pmc = 99946 | doi = 10.1128/MCB.21.22.7761-7774.2001 }}</ref>

The cleavage model was first proposed in 1993 based on work done with ''Drosophila'' ''Notch'' and ''C. elegans'' ''lin-12'',<ref name=PMID8406001>{{cite journal | vauthors = Lieber T, Kidd S, Alcamo E, Corbin V, Young MW | title = Antineurogenic phenotypes induced by truncated Notch proteins indicate a role in signal transduction and may point to a novel function for Notch in nuclei | journal = Genes & Development | volume = 7 | issue = 10 | pages = 1949–1965 | date = October 1993 | pmid = 8406001 | doi = 10.1101/gad.7.10.1949 | doi-access = free }}</ref><ref name=PMID8343960>{{cite journal | vauthors = Struhl G, Fitzgerald K, Greenwald I | title = Intrinsic activity of the Lin-12 and Notch intracellular domains in vivo | journal = Cell | volume = 74 | issue = 2 | pages = 331–345 | date = July 1993 | pmid = 8343960 | doi = 10.1016/0092-8674(93)90424-O | s2cid = 27966283 }}</ref> informed by the first oncogenic mutation affecting a human ''Notch'' gene.<ref name=PMID1831692>{{cite journal | vauthors = Ellisen LW, Bird J, West DC, Soreng AL, Reynolds TC, Smith SD, Sklar J | title = TAN-1, the human homolog of the Drosophila notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms | journal = Cell | volume = 66 | issue = 4 | pages = 649–661 | date = August 1991 | pmid = 1831692 | doi = 10.1016/0092-8674(91)90111-B | s2cid = 45604279 }}</ref> Compelling evidence for this model was provided in 1998 by in vivo analysis in ''Drosophila'' by Gary Struhl<ref name=PMID9604939>{{cite journal | vauthors = Struhl G, Adachi A | title = Nuclear access and action of notch in vivo | journal = Cell | volume = 93 | issue = 4 | pages = 649–660 | date = May 1998 | pmid = 9604939 | doi = 10.1016/S0092-8674(00)81193-9 | s2cid = 10828910 | doi-access = free }}</ref> and in cell culture by Raphael Kopan.<ref name=PMID9620803>{{cite journal | vauthors = Schroeter EH, Kisslinger JA, Kopan R | title = Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain | journal = Nature | volume = 393 | issue = 6683 | pages = 382–386 | date = May 1998 | pmid = 9620803 | doi = 10.1038/30756 | s2cid = 4431882 | bibcode = 1998Natur.393..382S }}</ref> Although this model was initially disputed,<ref name="pmid10221902" /> the evidence in favor of the model was irrefutable by 2001.<ref name=PMID22785620>{{cite journal | vauthors = Greenwald I | title = Notch and the awesome power of genetics | journal = Genetics | volume = 191 | issue = 3 | pages = 655–669 | date = July 2012 | pmid = 22785620 | pmc = 3389966 | doi = 10.1534/genetics.112.141812 }}</ref><ref name=PMID11134525>{{cite journal | vauthors = Struhl G, Greenwald I | title = Presenilin-mediated transmembrane cleavage is required for Notch signal transduction in Drosophila | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 98 | issue = 1 | pages = 229–234 | date = January 2001 | pmid = 11134525 | pmc = 14573 | doi = 10.1073/pnas.98.1.229 | doi-access = free | bibcode = 2001PNAS...98..229S }}</ref>

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.<ref name=PMID22785620 />

The '''Notch cascade''' consists of Notch and Notch [[ligand (biochemistry)|ligands]], as well as intracellular proteins transmitting the notch signal to the cell's nucleus. The Notch/Lin-12/Glp-1 receptor family<ref name="pmid7716513">{{cite journal | vauthors = Artavanis-Tsakonas S, Matsuno K, Fortini ME | title = Notch signaling | journal = Science | volume = 268 | issue = 5208 | pages = 225–232 | date = April 1995 | pmid = 7716513 | doi = 10.1126/science.7716513 | bibcode = 1995Sci...268..225A }}</ref> was found to be involved in the specification of cell fates during development in ''Drosophila'' and ''C. elegans''.<ref name="pmid9546393">{{cite journal | vauthors = Singson A, Mercer KB, L'Hernault SW | title = The C. elegans spe-9 gene encodes a sperm transmembrane protein that contains EGF-like repeats and is required for fertilization | journal = Cell | volume = 93 | issue = 1 | pages = 71–79 | date = April 1998 | pmid = 9546393 | doi = 10.1016/S0092-8674(00)81147-2 | s2cid = 17455442 | doi-access = free }}</ref>

The intracellular domain of Notch forms a complex with [[RBPJ|CBF1]] and [[MAML1|Mastermind]] to activate transcription of target genes. The structure of the complex has been determined.<ref name=PMID16530044>{{cite journal | vauthors = Nam Y, Sliz P, Song L, Aster JC, Blacklow SC | title = Structural basis for cooperativity in recruitment of MAML coactivators to Notch transcription complexes | journal = Cell | volume = 124 | issue = 5 | pages = 973–983 | date = March 2006 | pmid = 16530044 | doi = 10.1016/j.cell.2005.12.037 | s2cid = 17809522 | doi-access = free | author-link5 = Stephen Blacklow }}</ref><ref name=PMID16530045>{{cite journal | vauthors = Wilson JJ, Kovall RA | title = Crystal structure of the CSL-Notch-Mastermind ternary complex bound to DNA | journal = Cell | volume = 124 | issue = 5 | pages = 985–996 | date = March 2006 | pmid = 16530045 | doi = 10.1016/j.cell.2006.01.035 | s2cid = 9224353 | doi-access = free }}</ref>

=== Pathway ===

Maturation of the notch receptor involves cleavage at the prospective extracellular side during intracellular trafficking in the Golgi complex.<ref name="pmid10899003">{{cite journal | vauthors = Munro S, Freeman M | title = The notch signalling regulator fringe acts in the Golgi apparatus and requires the glycosyltransferase signature motif DXD | journal = Current Biology | volume = 10 | issue = 14 | pages = 813–820 | date = July 2000 | pmid = 10899003 | doi = 10.1016/S0960-9822(00)00578-9 | s2cid = 13909969 | doi-access = free | bibcode = 2000CBio...10..813M }}</ref> 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.{{citation needed|date=May 2007}} The notch ligands are also single-pass transmembrane proteins and are members of the DSL (Delta/Serrate/LAG-2) family of proteins. In ''[[Drosophila melanogaster]]'' (the fruit fly), there are two ligands named [[Delta (ligand)|Delta]] and [[Serrate (ligand)|Serrate]]. In mammals, the corresponding names are [[Delta-like]] and [[Jagged (ligand)|Jagged]]. In mammals there are multiple Delta-like and Jagged ligands, as well as possibly a variety of other ligands, such as F3/contactin.<ref name="Lai" />

In the nematode ''[[Caenorhabditis elegans|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.{{citation needed|date=June 2007}}

The notch extracellular domain is composed primarily of small cystine-rich motifs called [[Epidermal growth factor|EGF]]-like repeats.<ref name="pmid16973733">{{cite journal | vauthors = Ma B, Simala-Grant JL, Taylor DE | title = Fucosylation in prokaryotes and eukaryotes | journal = Glycobiology | volume = 16 | issue = 12 | pages = 158R–184R | date = December 2006 | pmid = 16973733 | doi = 10.1093/glycob/cwl040 | doi-access =  }}</ref>

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 [[glycans|''O''-linked glycans]] at specific sites.<ref name="pmid12460944">{{cite journal | vauthors = Shao L, Luo Y, Moloney DJ, Haltiwanger R | title = O-glycosylation of EGF repeats: identification and initial characterization of a UDP-glucose: protein O-glucosyltransferase | journal = Glycobiology | volume = 12 | issue = 11 | pages = 763–770 | date = November 2002 | pmid = 12460944 | doi = 10.1093/glycob/cwf085 | doi-access =  }}</ref> An [[glycosylation|''O''-glucose]] sugar may be added between the first and second conserved cysteines, and an [[glycosylation|''O''-fucose]] may be added between the second and third conserved cysteines. These sugars are added by an as-yet-unidentified [[glycosylation|''O''-glucosyltransferase]] (except for [https://www.ncbi.nlm.nih.gov/pubmed/18243100 Rumi]), and [[GDP-fucose Protein O-fucosyltransferase 1|GDP-fucose Protein ''O''-fucosyltransferase 1]] ([[GDP-fucose Protein O-fucosyltransferase 1|POFUT1]]), respectively. The addition of [[glycosylation|''O''-fucose]] by [[GDP-fucose Protein O-fucosyltransferase 1|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 [[xylose|xylosyltransferases]], and the [[glycosylation|''O''-fucose]] can be elongated to a tetrasaccharide by the ordered addition of an [[N-acetylglucosamine]] (GlcNAc) sugar by an [[N-acetylglucosamine|N-Acetylglucosaminyltransferase]] called [[Fringe Genes|Fringe]], the addition of a [[galactose]] by a [[galactose|galactosyltransferase]], and the addition of a [[sialic acid]] by a [[sialic acid|sialyltransferase]].<ref name="pmid17132502">{{cite book | vauthors = Lu L, Stanley P | chapter = Roles of O-Fucose Glycans in Notch Signaling Revealed by Mutant Mice | title = Functional Glycomics | series = Methods in Enzymology | volume = 417 | pages = 127–136 | year = 2006 | pmid = 17132502 | doi = 10.1016/S0076-6879(06)17010-X | isbn = 9780121828226 }}</ref>

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.<ref name="pmid17215308">{{cite journal | vauthors = Thomas GB, van Meyel DJ | title = The glycosyltransferase Fringe promotes Delta-Notch signaling between neurons and glia, and is required for subtype-specific glial gene expression | journal = Development | volume = 134 | issue = 3 | pages = 591–600 | date = February 2007 | pmid = 17215308 | doi = 10.1242/dev.02754 | doi-access = free }}</ref> If Fringe adds a GlcNAc to the [[glycosylation|''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.<ref name="pmid12826675">{{cite journal | vauthors = LaVoie MJ, Selkoe DJ | title = The Notch ligands, Jagged and Delta, are sequentially processed by alpha-secretase and presenilin/gamma-secretase and release signaling fragments | journal = The Journal of Biological Chemistry | volume = 278 | issue = 36 | pages = 34427–34437 | date = September 2003 | pmid = 12826675 | doi = 10.1074/jbc.M302659200 | doi-access = free }}</ref> 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.<ref name="pmid19726682">{{cite journal | vauthors = van Tetering G, van Diest P, Verlaan I, van der Wall E, Kopan R, Vooijs M | title = Metalloprotease ADAM10 is required for Notch1 site 2 cleavage | journal = The Journal of Biological Chemistry | volume = 284 | issue = 45 | pages = 31018–31027 | date = November 2009 | pmid = 19726682 | pmc = 2781502 | doi = 10.1074/jbc.M109.006775 | doi-access = free }}</ref> This releases the extracellular portion of notch (NECD), which continues to interact with the ligand. The ligand plus the notch extracellular domain is then [[endocytosis|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.{{citation needed|reason=This claim needs a reliable source; Bogart was a famous actor, and his major biographies don't mention snooker.|date=August 2016}} After this first cleavage, an enzyme called [[Gamma-secretase|γ-secretase]] (which is implicated in [[Alzheimer's disease]]) 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 (NICD), which then moves to the [[Cell nucleus|nucleus]], where it can regulate gene expression by activating the [[transcription factor]] [[RBPJ|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.<ref>{{cite book |doi=10.1038/npg.els.0004194 |chapter=Drosophila ''Patterning'': Delta-Notch Interactions |title=Encyclopedia of Life Sciences |year=2006 | vauthors = Desbordes S, López-Schier H |isbn=0470016175 }}</ref> Other proteins also participate in the intracellular portion of the notch signaling cascade.<ref name="pmid22223095">{{cite journal | vauthors = Borggrefe T, Liefke R | title = Fine-tuning of the intracellular canonical Notch signaling pathway | journal = Cell Cycle | volume = 11 | issue = 2 | pages = 264–276 | date = January 2012 | pmid = 22223095 | doi = 10.4161/cc.11.2.18995 | doi-access = free }}</ref>

=== Ligand interactions ===
[[File:N1 dll4 cells.png|thumb|455x455px|Crystal structure of the Notch1-DLL4 complex depicted as the interaction is predicted to occur between two cells (PDB ID: 4XLW)]]
Notch signaling is initiated when Notch receptors on the cell surface engage ligands presented ''in trans'' on opposing cells''.'' Despite the expansive size of the Notch extracellular domain, it has been demonstrated that EGF domains 11 and 12 are the critical determinants for interactions with Delta.<ref>{{cite journal | vauthors = Rebay I, Fleming RJ, Fehon RG, Cherbas L, Cherbas P, Artavanis-Tsakonas S | title = Specific EGF repeats of Notch mediate interactions with Delta and Serrate: implications for Notch as a multifunctional receptor | journal = Cell | volume = 67 | issue = 4 | pages = 687–699 | date = November 1991 | pmid = 1657403 | doi = 10.1016/0092-8674(91)90064-6 | s2cid = 12643727 }}</ref> Additional studies have implicated regions outside of Notch EGF11-12 in ligand binding. For example, Notch EGF domain 8 plays a role in selective recognition of Serrate/Jagged<ref>{{cite journal | vauthors = Rebay I, Fleming RJ, Fehon RG, Cherbas L, Cherbas P, Artavanis-Tsakonas S | title = Specific EGF repeats of Notch mediate interactions with Delta and Serrate: implications for Notch as a multifunctional receptor | journal = Cell | volume = 67 | issue = 4 | pages = 687–699 | date = November 1991 | pmid = 1657403 | doi = 10.1016/0092-8674(91)90064-6 | s2cid = 12643727 | bibcode = 2012Sci...338.1229Y }}</ref> and EGF domains 6-15 are required for maximal signaling upon ligand stimulation.<ref>{{Cite journal|date=2013|title=Intrinsic selectivity of Notch 1 for Delta-like 4 over Delta-like 1|journal=Journal of Biological Chemistry}}</ref> A crystal structure of the interacting regions of Notch1 and Delta-like 4 (Dll4) provided a molecular-level visualization of Notch-ligand interactions, and revealed that the N-terminal MNNL (or C2) and DSL domains of ligands bind to Notch EGF domains 12 and 11, respectively.<ref name="Luca_2015">{{cite journal | vauthors = Luca VC, Jude KM, Pierce NW, Nachury MV, Fischer S, Garcia KC | title = Structural biology. Structural basis for Notch1 engagement of Delta-like 4 | journal = Science | volume = 347 | issue = 6224 | pages = 847–853 | date = February 2015 | pmid = 25700513 | pmc = 4445638 | doi = 10.1126/science.1261093 | bibcode = 2015Sci...347..847L }}</ref> The Notch1-Dll4 structure also illuminated a direct role for Notch O-linked fucose and glucose moieties in ligand recognition, and rationalized a structural mechanism for the glycan-mediated tuning of Notch signaling.<ref name="Luca_2015" />

=== Synthetic Notch signaling ===

It is possible to engineer synthetic Notch receptors by replacing the extracellular receptor and intracellular transcriptional domains with other domains of choice. This allows researchers to select which ligands are detected, and which genes are upregulated in response. Using this technology, cells can report or change their behavior in response to contact with user-specified signals, facilitating new avenues of both basic and applied research into cell-cell signaling.<ref name="Harmansa">{{cite journal | vauthors = Harmansa S, Affolter M | title = Protein binders and their applications in developmental biology | journal = Development | volume = 145 | issue = 2 | pages = dev148874 | date = January 2018 | pmid = 29374062 | doi = 10.1242/dev.148874 | doi-access = free }}</ref> Notably, this system allows multiple synthetic pathways to be engineered into a cell in parallel.<ref>{{cite journal | vauthors = Themeli M, Sadelain M | title = Combinatorial Antigen Targeting: Ideal T-Cell Sensing and Anti-Tumor Response | journal = Trends in Molecular Medicine | volume = 22 | issue = 4 | pages = 271–273 | date = April 2016 | pmid = 26971630 | pmc = 4994806 | doi = 10.1016/j.molmed.2016.02.009 }}</ref><ref>{{cite journal | vauthors = Sadelain M | title = Chimeric antigen receptors: driving immunology towards synthetic biology | journal = Current Opinion in Immunology | volume = 41 | pages = 68–76 | date = August 2016 | pmid = 27372731 | pmc = 5520666 | doi = 10.1016/j.coi.2016.06.004 }}</ref>