Integrating MST, NMR, and negative stain EM to characterize the structure and interactions of Ceramide Transfer Protein (CERT)
Year: 2024
Type: dissertation
Abstract: Ceramide, a pivotal molecule in sphingolipid metabolism, exerts critical regulatory functions in cell proliferation and apoptosis [1]. Following its synthesis at the cytosolic surface of the endoplasmic reticulum (ER), ceramide undergoes both vesicular and non-vesicular transport processes [2]. Within the non-vesicular pathway, ceramide is specifically converted into sphingomyelins and glycosphingolipids within the Golgi apparatus [3]. Facilitating this transport is the ceramide transfer protein (CERT), which encompasses peptidic motifs and multiple domains [3]. Of particular significance is the C-terminal (StAR)-related lipid transfer (START) domain, which exhibits an exceptional ability to selectively bind and transport ceramide within its hydrophobic cavity [3]. The involvement of CERT in various pathological processes, including Alzheimer's disease, infectious diseases, and cancer, positions it as an intriguing target for pharmacological interventions [4,5]. N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl) dodecanamide (HPA-12) has emerged as a well-known antagonist of CERT [6]. Nevertheless, our understanding of the structure-activity relationship (SAR) for this antagonist remains limited. Therefore, a comprehensive investigation into the SAR of HPA-12 and its interactions with the START domain is imperative to uncover profound insights into its pharmacological potential and foster the development of novel therapeutic strategies. The main goal of this project was to gain comprehensive insight into the interaction between the CERT-START domain and small compounds acting as protein inhibitors. To achieve this, a combination of microscale thermophoresis (MST) and nuclear magnetic resonance spectroscopy (NMR) was employed as complementary techniques, allowing these molecular interactions to be characterized carefully. By delving into the intricacies of these interactions, the long-term goal was to enhance the inhibitory activity of the ligands, thereby paving the way for their potential application in future drug design endeavors. To achieve the objectives, an active START domain was obtained through the initial establishment of an efficient expression and purification protocol for subsequent MST and NMR experiments. Furthermore, a biophysical assay using MST was developed to quantify the interaction and evaluate the inhibitory potency of each ligand. However, the stability of the protein posed challenges for NMR experiments. Thus, various optimization strategies were employed to improve the stability and activity of the START domain, allowing for its long-term functionality and potential utilization in NMR resonance assignments of the target protein. In this regard, the utilization of HPA-12 and sequence modifications, including truncation, proved beneficial in improving NMR results and extending the stability of the START domain. In addition to the expression and purification protocol previously established, a specific labeling strategy was implemented using ILV- and LV-labeling approaches to prepare the [15N-uniform and 13C-methyl] truncated- START (t-START) domain for receptor-based NMR investigations of the target protein. Thus, the labeled t-START domain monomers, both in the presence and absence of ligands, exhibited promising results in the initial [1H,15N]-TROSY and [1H,13C]-HMQC NMR spectra, enabling the characterization of their interactions with ligands. Furthermore, the binding constants for each compound were determined utilizing the microscale thermophoresis (MST) technique, providing valuable insights into the strength of the ligand-protein interactions. Simultaneously, an optimized protocol was developed to generate a highly active full-length CERT protein. Due to the absence of a resolved structure for the full-length CERT protein, attempts were made to utilize cryo-electron microscopy (cryo-EM) as a powerful tool to reveal its intricate structure. Consequently, the successful preparation of the CERT protein in its biologically relevant tetrameric form was achieved, and the negative stain electron microscopy (EM) technique was employed to gain insights into its structural characteristics. However, to acquire a high-quality data set necessary for constructing the initial 3D model of the target protein, extensive optimization work was imperative to ensure the suitability of the protein sample for subsequent data collection using the negative stain EM technique. In this regard, our study provides comprehensive and systematic sample optimization steps and initial data processing steps, crucial for constructing the initial 3D model of the protein. Therefore, these are rather preliminary steps on the way toward an experimentally determined structure. Moreover, initial models were generated by combining electron densities from negative stain EM with extensive modeling efforts employing AlphaFold.
Author: Aglar, Öznur
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W6945971102
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