Posttranslational Modifications in Human Disease
The Pratt Lab is primarily focused on understanding the molecular and physiological consequences of protein posttranslational modifications (PTMs). In particular, we are currently focused on the intracellular glycosylation of proteins, a PTM called O-GlcNAc modification (Fig. 1A). This posttranslational modification is the addition of the monosaccharide N-acetyl-glucosamine to serine and threonine side-chains. O-GlcNAc transferase (OGT) adds O-GlcNAc to protein substrates throughout the cytosol, nucleus, and mitochondria, while another enzyme O-GlcNAcase (OGA) can remove it. The action of these two enzymes enables O-GlcNAc to dynamically regulate cellular signaling pathways, similar to phosphorylation. O-GlcNAcylation is absolutely required for development in mammals and insects, and genetic loss of OGT is lethal even at the level of mammalian cell culture in vitro. We and others have identified over 1,000 potentially O-GlcNAcylated proteins in mammals. However, the biochemical consequences of the overwhelming majority of these O-GlcNAcylation events are completely unknown. To address this critical information gap, we develop and apply a variety of chemical tools that enable us to identify and characterize O-GlcNAcylated proteins. Most significantly, we use chemistry to create and apply chemical reporters of glycosylation to investigate the cell biology of glycosylation, and we exploit protein chemistry to build site-specifically glycosylated proteins and understand the biochemistry of substrates is affected.
CHEMICAL Reporters of Glycosylation and other protein modifications
In order to visualize and identify important PTMs, the Pratt lab has developed robust chemical strategies to visualize and identify modified proteins from living systems. We have termed these tools metabolic chemical reporters (MCRs, Fig. 1B). These molecules are structural analogs of naturally occurring metabolites or other small molecules that contain azides or alkynes at different positions. When these MCRs are added to living cells they are incorporated into PTMs and the azides or alkynes can undergo bioorthogonal reactions, such as Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), for the installation of visualization and identification tags. Using this strategy, the lab has created and characterized probes for general protein glycosylation, the first selective MCR for O-GlcNAc modification, and a reporter of aspirin-mediated protein acetylation. In addition to MCRs, we also use chemoenzymatic labeling of native O-GlcNAc modifications (Fig. 1C) This technique, begins with the enzymatic modification of endogenous O-GlcNAcylation events with an azide-containing monosaccharide, GalNAz, followed by the use of CuAAC for tag installation. Chemoenzymatic labeling was originally developed by Linda Hsieh-Wilson (Caltech). We have contributed to this area recently by systematically optimizing this technique in combination with PEG-based “mass tags” to determine the absolute stoichiometry of O-GlcNAcylation on a protein of interest. We are currently applying these chemical tools to identify and characterize key O-GlcNAcylated proteins.
Biochemical characterization of O-GlcNAcylation using synthetic proteins.
Currently, the only way to directly test the consequences of site-specific O-GlcNAcylation is through protein synthesis, as this modification cannot be introduced using other techniques. To accomplish protein synthesis, we use expressed protein ligation (EPL). EPL involves the recombinant production of unmodified portions of the protein in E. coli and the solid-phase synthesis of peptides containing O-GlcNAc. Joining these protein fragments together is accomplished by taking advantage of proteins termed inteins, which are exploited to generate recombinant protein thioesters. Protein thioesters can undergo highly-specific reactions or “native chemical ligations” with other proteins or peptides with N-terminal cysteine residues. Using one or more of these reactions enables the synthesis of large proteins from smaller fragments like molecular puzzle pieces, with the only requirement being cysteine-residues at the sites of ligations (Fig. 2A).
Our published work in this area has focused on the aggregation prone protein α-synuclein. This protein forms toxic amyloids that cause Parkinson’s disease and other synucleinopathies. Therefore, inhibition of its aggregation is one of the most attractive therapeutic approaches. α-Synuclein is subjected to O-GlcNAcylation at up to nine different positions in vivo that could affect its aggregation and the progression of PD (Fig. 2B). We found that different O-GlcNAcylation sites have unique effects on the aggregation, structure, and toxicity of α-synuclein. We are currently continuing to study α-synuclein modifications, as well as the effects of O-GlcNAc on other proteins involved in protein aggregation and the DNA-damage response.