Integral repeats (in the Golgi complex) before it

Integral membrane proteins are understood to be membrane-bound proteins that have some or all parts of them interacting with the lipid core of the membrane bilayer (Lodish et al, 2000). Known to be involved in highly conserved intercellular pathways, the Notch receptor is a latent transcription regulatory protein that is involved in the regulation and determination of the cell’s fate (e.g. apoptosis or proliferation) (Monoley et al, 2000). This essay will be placing a focus on the Notch-1 receptor. Notch-1 is a single-pass transmembrane receptor domain. It has cysteine-rich EGF (epidermal growth factor)-like repeats that make up the receptor’s extracellular domain, and they are mostly constructed from ?-strands. Each EGF-like repeat is composed of forty amino acids and each receptor has thirty-six of these repeats. Three disulfide bridges uphold the structure due to the presence of six cysteine residues on them. In addition, the EGF repeats also have calcium ion (Ca2+) binding regions (Shao et al, 2003) (Campbell et al, 1993).O-linked glycosylation modification usually takes place on these EGF repeats (in the Golgi complex) before it is fused with the plasma membrane, in which O-glucose and O-fucose get added to precise sites of the first and second cysteine residue of the structure. They are attached by the hydroxyl (-OH) group of serine or threonine residues (Campbell et al, 1993) through the catalysis by unknown O-glucosyltransferase and O-fucosyltransferase 1 (POFUT-1) (Shao et al, 2012).It is unknown with regards to how O-glycosylation affects the overall Notch signaling pathway, but it was noted that O-fucose improves the efficiency an enzyme called Fringe, which catalyzes the addition of N-acetylglucosamine (GlcNAc) to the receptor, followed by the orderly addition of galactose by galactosyltransferase, and sialic acid/NANA by sialyltransferase (Lu et al, 2006). There is a link between the absence of O-fucose and the percentage of inefficient Notch-1 receptors, in which an investigation conducted on mouse Notch-1 postulated that O-fucose is involved in several EGF-repeats (Lu, 2006). A detailed study suggested the EGF-12, in specific, undergoes significant modification by O-fucose, where this specificity is revealed in the primary structure of the EGF repeats (Shao et al, 2003). On the other hand, the stabilization of Notch-1’s extracellular domain is maintained by O-glucose, this relates to the ability of the receptor to couple ligand-binding and in turn induces conformational changes required for proteolysis (Rana et al, 2012).A Notch-1 receptor is kept in its dormant state by the negative regulatory region (NRR), which is made of three LNR (LIN12/Notch repeats) and an HD (heterodimerization domain). There are three disulfide bridges that maintain the LNR structures, and calcium-binding sites on it. Whereas there are four ?-sheet on the base of HD (Greenwald et al, 2005).On the NRR, a non-polar plug covers the tumour necrosis alpha factor converting enzyme (TACE) cleavage site as illustrated by an atomic structure model. Upon the receptor activation, the structure changes conformation to expose the metalloprotease site and allows the endocytosed Notch-ligand complex to bind to it (Chillakuri et al, 2012) (Hambleton, 2014). As a result, the extracellular domain of the Notch-1 receptor is cleaved off (Crodle et al, 2008).Meanwhile, the intracellular domain of Notch goes through two proteolytic steps: firstly, a ligand from the DSL (Delta/Serrate/LAG-2) binds to the ectodomain, exposing the cleavage site “site 2” to a protease from the ADAM family. A second cleavage follows, in which ?-secretase cleaves the protein at the “site 3” transmembrane domain and this results in the release of the intracellular domain from the Notch-1 structure. This Notch-1 intracellular domain, NICD, contains a nuclear localization signal (NLS) that directs its translocation to the nucleus (Greenwald et al, 2005).The NICD binds with CSL (CBF1, Suppressor of Hairless, Lag 1) and co-activator MAM (Mastermind family) in the nucleus. This process is assisted by the RAM and ANK domain, whereby the RAM domain boosts NICD’s interaction with CSL, while ANK repeat forms temporary interactions with CSL and other proteins (Deregowski et al, 2006). As a result, CSL is converted from a transcriptional repressor to a transcriptional activator, which in turn activates gene transcription depending on the cell’s need (Greenwald et al, 2005).Knowledge of the stability and interaction of the DSL-Notch complex helps in our understanding of the regulation of the Notch-1 signalling pathway. At the beginning of the process, Delta binds to the ligand-binding site (consist of three EGF-repeats) of the receptor, and as revealed by an NMR structure model, this forms a very flexible complex. An analysis on the complex using GRAMM-X (a protein docking software) predicted ten possible structures, however, with a more detailed study, researchers were able to present Model-I and Model-X as being the most probable structure. This is based on the structures’ involved surface area and its interaction energy (Majumder et al, 2012).It was then eventually concluded that Model-X serves as the superior structure over Model-I due to a set of evidence. This includes a theory proposing that heterocomplexes have a more plant interface than homodimers. Knowing the Notch-1 receptors are heterodimers and Model-X has a more planar structure than Model-I, we can thus deduce that Model-X has a better structure (Jones et al, 1996). Taking into account that both models are set in a thermodynamically closed system (a system that allows the exchange of heat and work with its surroundings, but not matter) (Drickamer, 2017), analysis can be done on the effectiveness of both models in solvation energy gain. This involves the use of the Gibbs free energy formula: ?G = ?H – T?S, is used:•    G is the Gibbs free energy•    H is the enthalpy (heat content)•    T is the temperature (in Kelvins)•    S is the entropy (can be thought as ‘randomness’)Gibbs free energy is defined as the calculation of the maximum amount of work done in a thermodynamically closed system. All chemical systems favour states of minimum Gibbs free energy. Therefore, a reduction in G increases the spontaneity of reactions at a constant temperature and pressure (Drickamer, 2017).Calculations were done on the G used for the complex formation. Recorded data revealed that in Model-I, the energy gain for Delta is -6.28 kJ/mole and -7.53 kJ/mole for Notch. Whereas for Model-X, the energy gain is -13.81 kJ/mole and -13.39 kJ/mole for Delta and Notch respectively. Thus, the study suggests that Model-X also has a more favourable (more negative) solvation energy gain compared to Model-I, making it a better structure (Majumder, Roy & Thakur, 2012).Furthermore, researchers analyzed the distance between V453 of Notch and Delta residues of Drosophila Notch-1. Results indicated that the Model-X has a shorter distance between the residues than Model-I. This could be explained by Model-X having nine interface residues (with four hydrogen bonds and salt bridges) in comparison to Model-I (only one salt bridge). So, we can conclude that Model-X offers better interactions than Model-I (Majumder, Roy & Thakur, 2012).The amino acid sequence of the Notch-1 receptor for its transmembrane region, FMYVAAAAFVLLFFVGCGVLL (UniProt, 2018), clearly shows that it contains hydrophobic amino acid residues, namely phenylalanine, methionine, tyrosine, valine, alanine, glycine, and cysteine. The transmembrane amino acid residues interact with the phospholipid tails via hydrophobic interactions in the membrane bilayer. A nuclear magnetic resonance (NMR) study conducted by Deatherage et al. (2015) on the transmembrane domain of the Notch-1 receptor proved that the transmembrane domain has a ?-helix structure which spreads from residue 1732 to residue 1761, with the first turn at the water-bilayer interface (includes residues 1732 to residue 1736) (Deatherage et al, 2015). Its hydrophobic side chains of the structure interact via van der Waals’ forces with fatty acyl chains, and the peptide bond is shielded away. The positive-charged lysine and arginine interact with the negatively-charged phospholipid head groups, and this anchors the structure to the plasma membrane (Lodish et al, 2000). In addition, water is in a more energetically-favourable condition when the non-polar/hydrophobic residues are packed away (Drickamer, 2017). Taking into account with regards to the concept of entropy, S, the Notch-1 receptor is embedded into the plasma membrane in a way that this allows a reduction in entropically-unfavourable processes such as hydrogen-bonding between free water molecules. This, in turn, leads to a more negative Gibbs free energy value, G, and allows Notch-1 receptor to exist in the lipid bilayer (Lodish et al, 2000).The Notch-1 receptor plays an important role in several phases of embryonic development and the dysfunction of Notch-1 activity is recognized to be a key factor in many diseases such as T-cell leukaemia and Alzheimer’s disease. An insight into the structure of Notch-1 and how it works reveals an intricate series of steps. Although much of its structure and function is well understood, there is still much uncertainty on the Notch-1 receptor, for example, the manner by which O-linked glycosylation of Notch-1 affects its function. Thus, further research is highly encouraged while more is being discovered about this integral membrane protein.