Document Type

Dissertation

Degree

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor's Name

Jaroslava Miksovska

First Advisor's Committee Title

Committee chair

Second Advisor's Name

Xiaotang Wang

Second Advisor's Committee Title

committee member

Third Advisor's Name

Yuan Liu

Third Advisor's Committee Title

committee member

Fourth Advisor's Name

John P. Berry

Fourth Advisor's Committee Title

committee member

Fifth Advisor's Name

Prem P. Chapagain

Fifth Advisor's Committee Title

committee member

Keywords

DREAM, KChIP3, casenilin, presenilin, Alzheimer’s disease, pain modulation, fluorescence spectroscopy, circular dichroism, isothermal titration calorimetry, photothermal spectroscopy, computational MD simulation, dynamics network analysis, binding interface, drug discovery

Date of Defense

6-23-2016

Abstract

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.

Identifier

FIDC000735

 

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