July
28,
2023
Jinshan Xie, Pharmaceutical Sciences graduate student (Jiang Research Group), will be defending her PhD research thesis:
O-linked β-N-acetyl glucosaminylation (O‐GlcNAcylation) is an essential and abundant post-translational modification occurring on serine or threonine residues of over 9,000 intracellular proteins. This modification is governed by a single pair of enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). A long-standing question in the field is how the sole enzymes recognize such a vast number of protein substrates that lack a local consensus sequence motif. This thesis addresses this knowledge gap by investigating novel protein-protein interactions on non-catalytic domains that contribute to substrate recognition in OGT and OGA.
Firstly, we have identified the intervening domain (Int-D), a poorly understood protein fold found only in metazoan OGTs, as a specific regulator of OGT protein-protein interactions and substrate modification. Utilizing proteomic peptide phage display (ProP-PD) coupled with structural, biochemical, and cellular characterizations, we discovered a strongly enriched peptide motif, employed by the Int-D to facilitate specific O-GlcNAcylation. We further show that disruption of Int-D binding dysregulates important cellular programs including response to nutrient deprivation and glucose metabolism.
Secondly, we elucidated the structural basis for OGA’s substrate recognition through the discovery of a plastic exosite located on its non-catalytic stalk domain. The high-resolution crystal structure of OGA in complex with an unmodified peptide ligand derived from PDLIM7 protein substrate, revealed a U-shaped binding mode, in which the peptide wraps around a flexible loop in OGA stalk domain, inducing a significant conformational change. Combined with biochemical analyses, we further demonstrated that the exosite exhibits differential recognition and activity regulation towards distinct protein substrates.
In summary, these findings provide valuable insights into the intricate mechanisms of OGT/OGA substrate recognition and functional regulation, paving the way for the development of innovative therapeutic interventions for targeting O-GlcNAc disorder in diseases.
