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A Notch Ahead in Treating Alzheimer's Disease

By: Carol A. Rouzer, VICB Communications
Published:  May 1, 2017


Structural studies of the Notch-1 transmembrane domain provide clues for treatment of Alzheimer's disease with reduced toxicity.


γ-Secretase is an integral membrane protein complex comprising the aspartyl protease presenilin-1 and three other subunits (Figure 1). It is most widely known for its role in processing the amyloid precursor protein to yield the Aβ peptides that aggregate to form amyloid deposits, a major step in the pathogenesis of Alzheimer's disease. However, γ-secretase also processes a number of other proteins, including Notch-1, which is an important signaling molecule that regulates development and cell-to-cell interactions. For this reason, attempts to treat or prevent Alzheimer's disease by using γ-secretase inhibitors to block Aβ peptide formation have failed due to serious toxicity. This led Vanderbilt Institute of Chemical Biology member Chuck Sanders and his laboratory to explore the structure of the transmembrane domain of Notch-1 (Notch-TMD), the region of the protein that is cleaved by γ-secretase. Their work was driven by the hypothesis that identification of structural differences between the Notch-TMD and the transmembrane domain of the amyloid precursor protein would provide insight into ways to selectively block the action of γ-secretase-mediated amyloidogenesis while sparing Notch-1 processing [C. L. Deatherage, et al., (2017), Sci. Adv., 3, 1602794].



FIGURE 1. Diagrammatic (left) and surface (right) representation of the structure of the γ-secretase complex comprising presenilin-1 (PS-1, blue, nicastrin (NCT, green), anterior pharynx defective-1 (APH-1, purple), and presenilin enhancer-2 (PEN-2, yellow). Figure reproduced by permission from Macmillan Publishers Ltd. from X. Bai, et al., (2015), Nature, 525, 212. Copyright 2015.



Notch-1 is a 2,500 amino acid protein comprising multiple domains including a single transmembrane domain (Figure 2). Its extracellular domain serves as a receptor, primarily binding ligands that are attached to the surface of an adjacent cell. Ligand binding renders Notch-1 susceptible to proteolytic cleavage, releasing the extracellular domain, which remains bound to the adjacent cell, and leaving behind the transmembrane domain with a short extracellular extension and the complete intracellular domain intact. Intramembrane cleavage by γ-secretase follows, releasing a short peptide (Nβ) into the extracellular space and the Notch-1 intracellular domain (NICD) into the cytoplasm. The NICD translocates to the nucleus where it forms gene transcription regulatory complexes with other proteins. Processing of amyloid precursor protein similarly starts with cleavage of the protein's extracellular domain by β-secretase. The resulting C-terminal peptide, C99, retains the transmembrane domain connected to a short extracellular extension and a larger intracellular domain. Intramembrane cleavage of C99 at variable sites by γ-secretase leads to the release of peptides of 40 or 42 amino acids in length (Aβ40 and Aβ42, respectively). These peptides, particularly Aβ42, are the primary components of amyloid deposits in Alzheimer's disease.




FIGURE 2. (A) Domain structure of Notch 1, showing the repeat epidermal growth factor domain (EGF), three Lin-12/Notch repeats (LNR) and the heterodimerization domain (HD) that together form the negative regulatory region (NRR), the transmembrane domain (TM), the RBP-Jκ-associated molecule (RAM) domain, the ankyrin (ANK) domain, the transcriptional activation domain (TAD), and finally the proline-glutamic acid-, serine- and threonine-rich (PEST) domain. (B) Diagram illustrating the amino acid composition of the TMD plus immediate juxtamembrane regions used in the study. Figure reproduced under the Creative Commons Attribution-NonCommercial 4.0 International License from C. L. Deatherage, et al., (2017), Sci. Adv., 3, 31602794.



Prior work had already yielded a large body of structural information on C99. To obtain comparable data for Notch-TMD, the Sanders lab incorporated the protein into lipid bicelles and used solution NMR to examine its structure. Assignment of amino acid side chains in membrane-associated proteins is particularly challenging in this type of experiment due to the large number of conflicting signals coming from the membrane lipids. Nevertheless, the investigators were able to assign 95% of the protein side chains to their corresponding signals in the NMR data. They then applied XPLOR-NIH calculations to convert the data into structural models of the protein.


Examination of the 10 lowest energy models indicated that the Notch-TMD comprises a fairly straight intra-membrane α-helix spanning amino acids 1732 to 1757 (Figure 3). This distinguishes it from C99, which exhibits a notable bend in its transmembrane domain. Just next to the membrane on the extracellular (N-terminal) side, Notch-TMD is highly disordered, but then order is restored by a stretch of four proline residues that form a type II prolyproline helix. The investigators hypothesized that these structures provide conformational flexibility that is likely required as the extracellular domain establishes contacts with a ligand on an adjacent cell. At the C-terminal end of the transmembrane region a short loop of amino acids extends into the aqueous environment, but this is followed by a hydrophobic tripeptide of leucine, tryptophan, and phenylalanine (LWF motif) that is membrane associated. This LWF motif is required for an interaction between the NICD and a transcriptional activation partner. The researchers proposed that membrane association of the LWF motif prevents this interaction until the NCID is released by γ-secretase cleavage. The C-terminal amino acids distal to the LWF motif extend into the aqueous environment. 


FIGURE 3. (A) One of the 10 lowest energy structures for the Notch-1 TMD obtained from NMR data using XPLOR-NIH calculations. The amino acid shown in space-filling format is the tryptophan residue of the LWF motif. (B) One of the 10 lowest energy structures obtained from MD simulations of the Notch-TMD placed in a hydrated DMPC bilayer.  Figure reproduced under the Creative Commons Attribution-NonCommercial 4.0 International License from C. L. Deatherage, et al., (2017), Sci. Adv., 3, 31602794.



To further explore the Notch-TMD structure, the investigators used molecular dynamics simulations to evaluate the conformation of their ten lowest energy models following solvation in a disordered dimyristoylphosphatidylcholine (DMPC) bilayer. The results mostly confirmed the conclusions drawn from the NMR data; however, they also revealed that the transmembrane domain has a tendency to tilt within the bilayer. Additional NMR studies focused on differences in the Notch-TMD structure as a result of embedding the protein in bicelles formed from lipids of varying chain lengths. The purpose of these studies was to determine the impact of membrane thickness on the protein. The data suggested that the Notch-TMD exhibits very little conformational variability as a result of changes in membrane thickness, a property it shares with C99. In these same studies, the researchers used paramagnetic probes to assess water accessibility of the various regions of the protein. As previously reported for C99, they found almost no effect of membrane thickness on water accessibility at the C-terminus of the intra-membrane region of the protein, which is demarcated by a series of five basic residues. Unlike C99, however, which exhibits considerable variability in water accessibility with membrane thickness at the N-terminus of its membrane binding domain, only slight variability was observed in this region for Notch-TMD.


A particularly intriguing property of C99 is its ability to bind cholesterol due to the presence of a tandem glycine zipper (GXXXG motif) that provides a flat surface near the C-terminus of the transmembrane domain. Evidence suggests that cholesterol binding plays a role in amyloidogenesis, though the precise mechanisms are not fully understood. To determine if cholesterol binding is also a property of the Notch-TMD, the investigators used NMR to search for signal changes upon addition of cholesterol to the protein in lipid bicelles. They found no evidence for a protein-cholesterol interaction in the case of the Notch-TMD.


The data acquired by the Sanders group allow a direct comparison of the Notch-TMD to C99 (Figure 4). An immediate conclusion is that both structures share a short N-terminal extracellular domain and a transmembrane domain, which are prerequisites for γ-secretase cleavage. However, they exhibit few other obvious similarities, suggesting that γ-secretase may lack additional structural requirements. The differences between the two proteins are interesting and may be of both biochemical and therapeutic significance. One of these differences lies in the response of the proteins to changes in membrane thickness. As described above, it appears that Notch-TMD primarily accommodates such changes by increasing or decreasing its tilt angle. In contrast, C99 possesses a number of residues at its N-terminal end that can slide into or out of the membrane, thereby altering the length of the transmembrane domain in response to changes in membrane thickness. This phenomenon has implications for amyloidogenesis, as the resultant shift of the transmembrane domain within the bilayer affects the cleavage site of γ-secretase. Thus, cleavage of C99 in a thicker bilayer leads to a higher Aβ40/Aβ42 ratio than when cleavage occurs in a thinner bilayer. Because a change in tilt would not dramatically alter the relative position of amino acids within the membrane for the Notch-TMD, the investigators hypothesize that membrane thickness is unlikely to affect the cleavage site of this protein. Further experimentation will be required to test that hypothesis.




FIGURE 4. Diagrammatic comparison of the structures of the Notch-TMD and C99. The cholesterol-binding domain of C99 is shown in purple. Asterisks indicate the position of basic residues that determine the end of the trans-membrane helix in each case. Note that the Notch-TMD is straight, while the trans-membrane region of C99 is bent. Note also that C99 has two membrane-associated regions one in the intracellular domain (ICD) and one in the extracellular domain (ECD) connected to the trans-membrane domain by a fairly lengthy loop, whereas the membrane-associated LWF motif of the Notch-TMD is much closer to the C-terminus of the transmembrane helix. Figure reproduced under the Creative Commons Attribution-NonCommercial 4.0 International License from C. L. Deatherage, et al., (2017), Sci. Adv., 3, 31602794.



Perhaps the most important distinction, from a therapeutic point of view, between Notch-TMD and C99 is seen in their differential ability to bind cholesterol. Small molecules are currently available that block γ-secretase-mediated processing of C99 through binding to the glycine zipper motifs of the protein. These γ-secretase modulators (GSMs) have no direct effect on processing of Notch-1. Although it remains to be seen if GSMs will have clinical value in the treatment or prevention of Alzheimer's disease, they clearly demonstrate that selective inhibition of C99 processing, based on distinct structural features of the protein, is possible.



ViewScience Advances article: Structural and biochemical differences between the Notch and the amyloid precursor protein transmembrane domains






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