Integral of serine or threonine residues (Campbell et al,

Integral membrane proteins are membrane-bound proteins. The lipid core of the membrane bilayer interacts with some or all parts of the protein (Lodish et al, 2000). Known to be a latent transcription regulatory protein, the Notch receptor is involved in several highly conserved intercellular pathways. It is also involved in determining the cell’s fate, i.e.

apoptosis or proliferation (Monoley et al, 2000). My essay will be placing a focus on the Notch-1 receptor.Notch-1 is understood to be a single-pass transmembrane receptor domain. This protein contains cysteine-rich EGF (epidermal growth factor)-like repeats which makes up the extracellular structure of the Notch-1 receptor. These EGF-repeats are mainly constructed from ?-strands, and they are made up forty amino acids. Each Notch-1 receptor has thirty-six EGF-repeats and there are three disulfide bridges that uphold the entire structure due to the presence of six cysteine residues. Furthermore, there are calcium ion (Ca2+) binding regions on each EGF-repeats (Shao et al, 2003) (Campbell et al, 1993).

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The EGF-repeats are usually transported to the Golgi complex to undergo O-linked glycosylation modification before it is transported in Clathrin-coated vesicle to the plasma membrane (to eventually fuse and become a component of the membrane). The modification involves the addition of O-glucose and O-fucose to precise regions of the first and second cysteine residues in the EGF-repeat. These are attached through the hydroxyl (-OH) group of serine or threonine residues (Campbell et al, 1993), and the process is catalyzed by an unknown O-glucosyltransferase and O-fucosyltransferase 1 (POFUT-1) (Shao et al, 2012).It has not be determined with regards to how O-glycosylation affects the overall signaling pathway of the Notch-1 receptor. However, it was noted that O-fucose is needed to increases the efficiency of an enzyme known as Fringe. Fringe catalyzes the addition of N-acetylglucosamine (GlcNAc) to the receptor, which is followed by the orderly addition of f galactose by galactosyltransferase, and sialic acid/NANA by sialyltransferase (Lu et al, 2006). There is also an observed correlation between the absence of O-fucose and the percentage of inefficient Notch-1 receptors, where an investigation conducted on mouse Notch-1 postulated that O-fucose is involved in the modification of several EGF-repeats (Lu, 2006).

A detailed study suggested the EGF-12, in particular, is significantly modified by O-fucose, and this specificity is revealed in the primary structure of the EGF repeats (Shao et al, 2003). On the other hand, O-glucose is responsible for the stabilization of Notch-1’s extracellular domain, which relates to the receptor’s ability to couple ligand-binding, and leads to conformational changes required for proteolysis 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), which are stabilized by three disulphide bridges, and an HD (heterodimerization domain). The LNR also contains calcium-binding sites, while the base of HD contains four ?-sheets (Greenwald et al, 2005).A non-polar plug covers the tumour necrosis alpha factor converting enzyme (TACE) cleavage site on the NRR, which was illustrated by a model of its atomic structure.

When the receptor is activated, the structure changes conformation and exposes its metalloprotease site, allowing the endocytosed Notch-ligand complex to bind to it (Chillakuri et al, 2012) (Hambleton, 2014). This results in the cleavage of the extracellular domain of the Notch-1 receptor (Crodle et al, 2008).The remaining intercellular domain, on the other hand, undergoes two steps of proteolysis. Firstly, a DSL (Delta/Serrate/LAG-2) ligand binds to the receptor’s ectodomain and exposes the “site 2” cleavage site to a protease from the ADAM 10/17 family (Deatherage, 2015). A second cleavage follows with the enzyme ?-secretase cleaving the protein at the “site 3” transmembrane domain.

As a result, the intracellular domain of the Notch-1 receptor is released from the overall structure. This Notch-1 intracellular domain, NICD, contains a nuclear localization signal (NLS) that allows recognition by the importins and eventually the NICD’s translocation to the nucleus through the nuclear pore complex (NPC) (Greenwald et al, 2005).When the NICD is in the nucleus, it binds with transcription regulator CSL (CBF1, Suppressor of Hairless, Lag 1) and co-activator MAM (Mastermind family). The RAM and ANK domain of the NICD assist this procedure. RAM improves the interaction between NICD and CSL, while ANK forms temporary bonds with CSL and other proteins (Deregowski et al, 2006). This ultimately leads to the conversion of CSL from a transcriptional repressor to a transcriptional activator, resulting in the activation of gene transcription.

This process is completely dependent on the cellular requirements (Greenwald et al, 2005).Knowledge of the stability and interaction of the DSL ligand-Notch complex can improve our understanding of the regulation of the Notch-1 signalling pathway. The process begins with the DSL-ligand interact and binding to the binding site (made of three EGF-repeats) on the Notch-1 receptor to form a flexible complex, as reveal by an NMR structure model.

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 permits heat and work exchange 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.

Gibbs free energy is defined as the calculation of the maximum amount of work done in a thermodynamically closed system, and minimum Gibbs free energy is favoured by all systems. Therefore, a reduction in G increases the spontaneity of reactions at a constant temperature and pressure (Drickamer, 2017).Calculations were done on the total Gibbs free energy needed 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. Hydrophobic interactions occur between the transmembrane amino acid residues and the phospholipid tails. 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 twist at the water-bilayer interface (includes residues 1732 to residue 1736) (Deatherage et al, 2015).

Its peptide bond is buried and hydrophobic side chains of the structure interact via van der Waals’ forces with fatty acyl chains. The entire structure is anchored to the plasma membrane through the interaction of the positive-charged lysine and arginine with the negatively-charged phospholipid head groups (Lodish et al, 2000). With the hydrophobic residues packed away, water is in a more energetically-favourable condition (Drickamer, 2017). Taking into account the concept of entropy (disorder), S, the Notch-1 receptor is embedded into the plasma membrane in a way that this disrupts entropically-unfavourable processes like 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 embryonic development, and the dysfunction of this particular receptor 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 effect of O-linked glycosylation on function of Notch-1. Thus, further research is highly encouraged while more is being discovered about this integral membrane protein.