Caroline Smet-Nocca, Ph.D., Université Lille-Nord de France, Lille, France
Caroline Smet-Nocca, Ph.D.
Université Lille-Nord de France

Caroline Smet-Nocca has studied Organic and Medicinal Chemistry at the University of Sciences and Technology in Lille where she obtained an engineering degree and a Master thesis in Organic Chemistry in 2001. Her research interests are focused on the regulation of protein structure and function by posttranslational modifications. During her Masters and PhD training, she worked on deciphering molecular interactions between the peptidyl-prolyl cis/trans isomerase Pin1 and the neuronal Tau protein in order to understand the role of this complex in Alzheimer’s disease. She has tackled the molecular mechanisms of Pin1 functional interactions with phosphorylated Tau using NMR spectroscopy and modified peptides as substrates. She was also involved in the search of small-molecule inhibitors of protein-protein interactions targeting Pin1 binding to its phosphorylated substrates. As a postdoctoral fellow at the Interdisciplinary Research Institute, Dr. Smet-Nocca investigated by NMR functional interactions between the general transcriptional coactivator CBP (Creb-binding protein) and the human Thymine-DNA Glycosylase in the decoupling of epigenetic signaling, DNA base excision repair and transcriptional regulation mechanisms potentially involved in genetic diseases and myeloid leukemia. Dr. Smet-Nocca then moved to the “Structural and Functional Glycobiology Unit” in Lille where she has been an Assistant Professor in Biochemistry since 2009. She continued her work on the molecular investigations of Tau protein in Alzheimer’s disease. She is now focusing on the role of acetylation and O-β-linked N-acetylglucosaminylation (O-GlcNAcylation) on Tau structure and pathophysiological functions. She is applying both enzymatic and semisynthetic approaches to incorporate posttranslational modifications on Tau protein. The patterns of modification obtained by enzymatic activities are characterized by NMR spectroscopy which provides a complete overview of the modification sites (identification and quantification) in a direct manner (i.e., without the use of a third part, such as antibodies). The use of semisynthetic proteins obtained by native chemical ligation allows investigating the role of specific posttranslational modifications at structural and functional levels. These well-characterized samples are then involved in functional assays – tubulin polymerization and aggregation into PHF-like fibers- to understand the mechanisms of regulation of Tau functions by posttranslational modifications.

Investigations of interactions of lysine tweezer CLR01 with Tau protein by NMR spectroscopy.

Our group at UMR CNRS 8576 Université Lille-Nord de France, Lille, France, is involved in the molecular investigations of functional interactions of lysine-specific molecular tweezers with the microtubule-associated protein Tau. Our main methodology to investigate protein-protein or protein-ligand interactions is high-resolution NMR spectroscopy that allows to map protein interactions at a per-residue resolution. The central experiment of protein NMR spectroscopy is the two-dimensional 1H-15N HSQC (heteronuclear single quantum coherence, Figure 1). In this experiment, it is possible to visualize in principle one peak (or resonance) for each amino acid of the protein sequence (except proline residues). Each resonance detected in 1H-15N HSQC corresponds to an amide group in the backbone of the protein (other H-N groups found in some amino acid side chains, such as Asn, Gln, Arg, and Trp can also be detected). Thus, the 1H-15N HSQC is a kind of a fingerprint for a single protein. These amide resonances are very sensitive to modifications of the chemical environment. Therefore, H-N groups are useful probes to detect, map and quantify binding of another protein or small ligand. In such cases, the resonances of several H-N groups are perturbed (their chemical shift and/or intensity change) upon ligand binding. This method allows detecting binding events in a large range of dissociation constants.

We use uniformly 15N-labeled or selective 15N-Lys-labeled Tau protein recombinantly produced in E. coli to get proteins in sufficient amount (10-20 mg per liter of culture) and purity (> 95%) for NMR analyses (Figure 2) and functional assays. With these labeled Tau proteins, we  monitor interactions at per-residue resolution with unlabeled partners such as heparin (Figure 3) or the molecular tweezer, CLR01. We can thus map the interface occupied by molecules on Tau.

We also aim to understand the mechanism by which the molecular tweezers interferes with formation of paired helical filament (PHF)-like fibers. We use an in vitro assay in which aggregation of Tau proteins is induced by a polyanion such as heparin. Kinetics of fiber formation can be monitored by measuring the fluorescence of Thioflavin T (ThT), a small-molecule ligand that specifically binds to β-sheet structures generated during the aggregation process. As detected by electron microscopy, the morphology of Tau fibrils obtained with heparin is similar to those of PHFs isolated from AD brains (Figure 3).