Preparation and binding study of the START Domain of the Ceramide Transfer Protein (CERT) via Nuclear Magnetic Resonance (NMR)
Öznur Aglar1, Christoph Arenz2 and Heiko M. Möller1
1Institut für Chemie, Universität Potsdam, Karl-Liebknecht-Straße 24-25, 14476, Potsdam, GER
2Institut für Chemie, Humboldt-Universität zu Berlin, Brook-Taylor-Str. 2, 12489, Berlin, GER
Ceramide, an important component in the metabolism of sphingolipids, plays a significant role in proliferation and apoptosis of cells. De novo synthesis of ceramide takes place at the cytosolic surface of the endoplasmic reticulum (ER), and then ceramide is transferred to the Golgi apparatus for conversion into sphingomyelin and glucosphingolipids, mainly by non-vesicular trafficking. Non-vesicular transport of ceramide is carried out by the ceramide transfer protein (CERT) that consists of peptidic motifs and multiple domains. The C-terminal (StAR)-related lipid transfer (START) domain is the most important domain, given the fact that it is capable of extracting and accommodating ceramide in its deep hydrophobic cavity. CERT could be an attractive pharmacological target because of its involvement in common pathological processes such as Alzheimer’s disease, infectious diseases and cancer. A well-known antagonist of CERT is N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl) dodecanamide (HPA-12); however, there is limited structure-activity relationship (SAR) data available.
In this study, we aim to explore the interaction between CERT and HPA-12 and HPA-12 analogs to establish SAR of this compound class by nuclear magnetic resonance spectroscopy (NMR) in order to improve the inhibitory activity of these ligands for a potential drug design.
Herein, we used an optimized expression and purification protocol to prepare an isotopically labeled START domain for getting an idea about the suitability of the protein of interest in receptor-based NMR experiments. The labeled START domain’s monomer in the presence and absence of the ligand yielded promising results in initial 1H-15N HSQC and TROSY NMR spectra. As a next step, sequence specific resonance assignments will be established in order to characterize the binding site and binding mode of HPA-12 and its derivatives.
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