Posttranslational Modifications in Human Disease
The Pratt Lab is primarily focused on understanding the molecular and physiological consequences of protein posttranslational modifications (PTMs). We are currently focused on the addition of carbohydrates to protein, termed glycosylation, in particular an intracellular version 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 1,000s of potentially O-GlcNAcy-modified proteins. However, the biochemical consequences of the overwhelming majority of these O-GlcNAc 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-GlcNAc proteins.
Chemical Reporters and inhibitors of Glycosylation
In order to characterize PTMs, the Pratt lab continues to develop chemical strategies to visualize, identify, and manipulate glycosylation in living systems. One set of tools we have termed metabolic chemical reporters (MCRs, Fig. 1a). 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. We are also interested in mechanistic inhibitors of glycosylation, or metabolic inhibitors (MIs, Fig. 1b). Currently, we are focused on improving the glycosylation and cell selectivity of these tools to enable increasingly targeted and cutting-edge applications in cell biology.
Figure 1. O-GlcNAc and our chemical strategies to study glycosylation. (a) O-GlcNAc is a dynamic posttranslational modification of serine and threonine residues of many intracellular proteins. (b) Living cells can metabolize chemical reporters and inhibitors to enable the visualization, identification, and manipulation of protein glycosylation.
Synthesis and Biochemical Characterization of glycosylated proteins
With current technologies, the only way to directly test the consequences of site-specific O-GlcNAc 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. We have applied this approach to multiple modifications sites and proteins (Fig. 2) making key discoveries about the functional consequences of O-GlcNAc in protein aggregation, signaling, and DNA repair. We are continuing to apply this approach to a range of O-GlcNAc modified and other glycosylated proteins.
Figure 2. Synthesis of and characterization of glycosylated proteins. We have used expressed protein ligation (EPL) to generate several O-GlcNAc modified proteins and have discovered important effects of this modification on protein aggregation, kinase signaling, and the DNA damage response.