Date of Degree

9-2017

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor(s)

Ruth E. Stark

Committee Members

Ranajeet Ghose

Sébastien Poget

Judith Storch

Cédric Bernard

Subject Categories

Biochemistry | Biophysics | Molecular Biology | Structural Biology

Keywords

AFABP, PPAR𝛾, NMR, protein-protein interaction, ligand-binding

Abstract

The Adipocyte Fatty Acid-Binding Protein (AFABP) is mainly expressed in fat cells. It can bind fatty acids and other lipophilic substances such as eicosanoids and retinoids. The peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor protein that requires ligand binding to regulate the specific gene transcription. PPARγ is expressed at extremely high levels in adipose tissue, macrophages, and the large intestine, where it controls lipid adipogenesis and energy conversion. Moreover, it has been found that AFABP and PPARγ can form a complex in vivo. It is proposed that AFABP carries the ligand and enters into the nucleus where it transfers the ligand to PPARγ by binding and macromolecular interaction.

The goal of this project is to study the interaction between these two proteins in vitro, including their binding affinity, the location of the binding interface on the AFABP protein, and the dependence of these phenomena on specific ligands. New protocols were developed to obtain pure monomeric AFABP protein in a delipidated state. The formation of a disulfide-linked dimer connected through an N-terminal cysteine was demonstrated and minimized by the exclusion of oxygen, though it is not known whether this dimer exists under physiological conditions. A corresponding protocol was developed to isolate delipidated PPARγ and to optimize conditions for the acquisition of its high-resolution NMR spectra.

Making sequence-specific backbone NH assignments and exploiting the local changes in the chemical environment that accompany interactions between the AFABP and PPARγ proteins, TROSY-HSQC NMR was used to determine which AFABP amino acid residues are located at their interface by investigating the perturbation of backbone NH NMR chemical shifts. The perturbed residues are located primarily in the portal region; weak binding between AFABP and PPARγ (Kd~ 500 𝜇M) is estimated from chemical shift changes during titration. Fluorescence competition assays also provide insight on protein-ligand dissociation constants and show that the ligand prefers to bind AFABP rather than PPARγ. Nonetheless, titration of PPARγ with holo-AFABP demonstrates the partial transfer of the ligand from AFABP to PPARγ, which could be sufficient for the regulation of targeted gene transcription.

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