Investigating the Role of O-GlcNAc Glycosylation in Neurodegeneration

O-GlcNAc glycosylation of nuclear and cytosolic proteins is an essential post-translational modification implicated in many diseases, from cancer to diabetes. Importantly, many important neuronal proteins are also O-GlcNAc modified, and aberrant O-GlcNAcylation of these proteins may contribute to the pathology of neurodegenerative diseases although these mechanisms have not been well defined. Here we investigated the role of O-GlcNAc glycosylation in the brain, utilizing both chemistry and molecular biology to study O-GlcNAc transferase (OGT), the enzyme that adds the sugar modification. To evaluate the role of OGT in adult neurons, we generated a forebrain-specific conditional knockout of OGT (OGT cKO) in mice. Although indistinguishable from wild-type littermates at birth, after three weeks we observe progressive neurodegeneration in OGT cKO mice. Hallmarks of Alzheimer’s disease, including neuronal loss, neuroinflammation, behavioral deficits, hyperphosphorylated tau, and amyloid beta peptide accumulation, are observed. Furthermore, decreases in OGT protein levels were found in human AD brain tissue, suggesting that altered O-GlcNAcylation likely contributes to neurodegenerative diseases in humans. This model is one of a few mouse models that recapitulate AD phenotypes without mutating and overexpressing human tau, amyloid precursor protein, or presenilin, highlighting the essential role of OGT in neurodegenerative pathways.

Given the importance of OGT in the brain, we further investigated the regulation of the OGT enzyme by phosphorylation. We found that phosphorylation of OGT near its C-terminus reduces its activity in cancer cells, and have developed phosphorylation-specific antibodies to aid mechanistic studies. Furthermore, mutation of this phosphorylation site on OGT, followed by overexpression in neurons was shown to enhance neurite outgrowth, demonstrating a functional consequence for this site. Thus phosphorylation of OGT inhibits its activity and enhances neurite outgrowth, and current studies aim to characterize the signaling pathway that regulates OGT phosphorylation in neurons.

Designing conformational control of human tissue transglutaminase

Understanding the mechanisms of enzymes is crucial for our understanding of their role in biology and for designing methods to perturb or harness their activities for medical treatments, industrial processes, or biological engineering. One aspect of enzymes that makes them difficult to fully understand is that they are in constant motion, and these motions and the conformations adopted throughout these transitions often play a role in their function.

Traditionally, it has been difficult to isolate a protein in a particular conformation to determine what role each form plays in the reaction or biology of that enzyme. A new technology, computational protein design, makes the isolation of various conformations possible, and therefore is an extremely powerful tool in enabling a fuller understanding of the role a protein conformation plays in various biological processes.

One such protein that undergoes large structural shifts during different activities is human type II transglutaminase (TG2). TG2 is an enzyme that exists in two dramatically different conformational states: (1) an open, extended form, which is adopted upon the binding of calcium, and (2) a closed, compact form, which is adopted upon the binding of GTP or GDP. TG2 possess two separate active sites, each with a radically different activity. This open, calcium-bound form of TG2 is believed to act as a transglutaminse, where it catalyzes the formation of an isopeptide bond between the sidechain of a peptide-bound glutamine and a primary amine. The closed, GTP-bound conformation is believed to act as a GTPase. TG2 is also implicated in a variety of biological and pathological processes.

To better understand the effects of TG2’s conformations on its activities and pathological processes, we set out to design variants of TG2 isolated in either the closed or open conformations. We were able to design open-locked and closed-biased TG2 variants, and use these designs to unseat the current understanding of the activities and their concurrent conformations of TG2 and explore each conformation’s role in celiac disease models. This work also enabled us to help explain older confusing results in regards to this enzyme and its activities. The new model for TG2 activity has immense implications for our understanding of its functional capabilities in various environments, and for our ability to understand which conformations need to be inhibited in the design of new drugs for diseases in which TG2’s activities are believed to elicit pathological effects.