Oral Presentation Australian and New Zealand Obesity Society Annual Scientific Conference 2023

Phosphorylation-mediated degradation of lipid droplet protein HSD17β11 regulates lipolysis (97575)

Natasha D Suriani 1 , Stacey N Keenan 1 , Gio Fidelito 1 , Ashleigh N Solano 2 3 , Shuai Nie 4 , Ellie H-J Cho 5 , Elizabeth Hinde 2 3 6 , Matthew J Watt 1
  1. Department of Anatomy and Physiology, University of Melbourne, Melbourne, Victoria, Australia
  2. School of Physics, University of Melbourne, Melbourne, Victoria, Australia
  3. Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, Melbourne, Victoria, Australia
  4. Mass Spectrometry and Proteomics Facility, Bio21 Molecular Science and Biotechnology Institute, Melbourne, Victoria, Australia
  5. Biological Optical Microscopy Platform, University of Melbourne, Melbourne, Victoria, Australia
  6. Department of Biochemistry and Pharmacology, University of Melbourne, Melbourne, Victoria, Australia

 

Obesity is characterised by an oversupply of lipids and consequent metabolic dysfunctions, increasing the risk of developing comorbidities such as type 2 diabetes and nonalcoholic fatty liver disease. Lipid droplets (LDs) are dynamic storage organelles for lipids and are enveloped with a wide array of proteins that control lipid metabolism. A subset of LD proteins are involved in lipolysis and are regulated at the posttranslational level by protein kinase A (PKA)-mediated phosphorylation, key of which are adipose triglyceride lipase (ATGL), and perilipin 5 (PLIN5). 17β-hydroxysteroid dehydrogenase 11 (HSD17β11) is a lipid droplet-associated protein whose deletion results in defects in lipolysis and resulting cellular lipid accumulation. Notably, HSD17β11 contains a conserved PKA consensus sites on its serine-33 residue.

Here, we sought to identify PKA phosphorylation sites on HSD17β11 and test whether S33 is a functionally important site for its role in lipid metabolism. To address this aim, we generated a loss of function serine-to-alanine mutant of HSD17β11 at serine-33 (HSD17β11S33A) by re-expression of mutant protein in HSD17β11KO HEK293T cells. Metabolic studies were then performed in HSD17β11S33A and cells expressing wild-type HSD17β11 (HSD17β11FLAG).

Radiometric tracing studies revealed an increase in lipolysis in HSD17β1lS33A cells, with a slight reduction in LD size compared to HSD17β11FLAG cells. Mechanistically, FLIM-FRET analysis revealed a robust interaction between HSD17β11 and PLIN5, which was reduced in response to forskolin stimulation. We then show phosphorylation of HSD17β11 at serine-33 results in ubiquitination and degradation of the protein, and that this effect was ameliorated in HSD17β11S33A cells. Taking this into consideration, our findings suggest that HSD17β11 is a novel regulator of lipolysis, and hence lipid metabolism.