Downstream regulatory element antagonist modulator (DREAM) is involved in various interactions with targets both inside and outside of the nucleus. In the cytoplasm, DREAM interacts with the C-terminal fragments of presenilins to facilitate the production of β-amyloid plaques in Alzheimer’s disease. In the nucleus, Ca2+ free DREAM directly binds to specific downstream regulatory elements of prodynorphin/c-fos gene to repress the gene transcription in pain modulation. These interactions are regulated by Ca2+ and/or Mg2+ association at the EF-hands in DREAM. Therefore, understanding the conformational dynamics and stability associated with Ca2+ and/or Mg2+ binding to DREAM is crucial for elucidating the mechanisms of interactions of DREAM with DNA or presenilins. The critical barrier for envisioning the mechanisms of these interactions lies in the lack of NMR/crystal structures of Apo and Mg2+DREAM.
Using a combination of fluorescence spectroscopy, circular dichroism, isothermal titration calorimetry, photothermal spectroscopy, and computational approaches, I showed that Mg2+ association at the EF-hand 2 structurally stabilizes the N-terminal alpha-helices 1, 2, and 5, facilitating the interaction with DNA. Binding of Ca2+ at the EF-hand 3 induces significant structural changes in DREAM, mediated by several hydrophobic residues in both the N- and C-domains. These findings illustrate the critical role of EF-hand 3 for Ca2+ signal transduction from the C- to N-terminus in DREAM. The Ca2+ association at the EF-hand 4 stabilizes the C-terminus by forming a cluster consisting of several hydrophobic residues in C-terminal domain. I also demonstrated that association of presenilin-1 carboxyl peptide with DREAM is Ca2+ dependent and the complex is stabilized by aromatic residues F462 and F465 from presenilin-1 and F252 from DREAM. Stabilization is also provided by residues R200 and R207 in the loop connecting a7 and a8 in DREAM and the residues D450 and D458 in presenilin-1.
These findings provide a structural basis for the development of new drugs for chronic pain and Alzheimer’s disease treatments.
| Identifer | oai:union.ndltd.org:fiu.edu/oai:digitalcommons.fiu.edu:etd-3768 |
| Date | 23 June 2016 |
| Creators | Pham, Khoa Ngoc |
| Publisher | FIU Digital Commons |
| Source Sets | Florida International University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | FIU Electronic Theses and Dissertations |
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