Oral Presentation AUS-oMicS 2025

Identifying the Regulatory Network of the O-GlcNAc-transferase in the Ischemic Heart (#79)

Fiddia Zahra 1 , Kamau Fahie 1 , Bhargavi Narayanan 1 , Robert N O’Meally 2 , Mark Kohr 3 , Brian Foster 4 , Natasha E Zachara 1
  1. Johns Hopkins University, Baltimore, MARYLAND, United States
  2. The Mass Spectrometry and Proteomics Facility, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
  3. Department of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA
  4. Division of Cardiology, The Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

In response to injury, cells and tissues remodel their cellular environment to repair damaged structures and if necessary to initiate apoptosis. This process, known as the cellular stress response, includes robust and dynamic changes in the modification of nuclear, cytoplasmic, and mitochondrial proteins by monosaccharides of O-linked β-N-acetylglucosamine (O-GlcNAc). Acute enhancement of O-GlcNAc reduces apoptosis and necrosis in models of injury that include oxidative stress and myocardial ischemia reperfusion (I/R) injury. O-GlcNAc-cycling is regulated by the O-GlcNAc transferase (OGT) and O-GlcNAcase, which add and remove O-GlcNAc, respectively. While ischemia alters OGT activity and O-GlcNAcylation, the regulatory mechanisms underpinning these changes in the heart remain unclear. As OGT’s protein interactions influence both substrate targeting and activity, we hypothesized that ischemia alters these interactions and thereby modulates OGT function and downstream signaling. Using hearts exposed to sham or ischemia (20min), we defined the basal and ischemia-induced interactome of OGT using immunoprecipitation, tandem mass tags (TMT), and mass spectrometry. These studies identified ~400 proteins per treatment that were enriched by an OGT specific antibody when compared to a non-specific antibody. Furthermore, quantitative comparisons revealed proteins with altered interactions during ischemia (99 upregulated and 84 downregulated). OGT interacting proteins included established partners such as HCF-1 and Carm1. Among the OGT interactors were key proteins involved in regulation of the cardioprotective process of autophagy: AMPK, ATG7, p62/Sequestosome, and Rab7. Of these, AMPK, ATG7, and p62/Sequestosome are direct targets of OGT. Biochemical analysis demonstrates that enhancing O-GlcNAc levels results in an upregulation of autophagy and that blocking autophagy though inhibition of AMPK abrogates the impact of O-GlcNAc on autophagy and survival. Collectively, these findings begin to define OGTs regulators and substrates in the ischemic heart, as well as identify key proteins and pathways that promote cardioprotection.