Poster Presentation 4th Metabolic Diseases; Breakthrough Discoveries in Diabetes & Obesity Meeting 2024

Phosphorylation-Mediated Degradation of HSD17β11 Regulates Lipolysis. (#189)

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

Obesity is characterised by an oversupply of lipids and consequent metabolic dysfunction, increasing the risk of developing comorbidities such as type 2 diabetes and metabolic dysfunction-associated steatotic 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, such as adipose triglyceride lipase (ATGL) and perilipin 5 (PLIN5). Here, we identify a lipid droplet protein 17β-hydroxysteroid dehydrogenase 11 (HSD17β11) as a novel interacting partner of PLIN5 and demonstrate that HSD17β11 facilitates the regulation of lipolysis.

 

Deletion of HSD17β11 (HSDKO) led to an accumulation of triglycerides and cholesterol esters, which coincided with increased lipid droplet size and number. Through radiometric 14C-tracing, we showed that HSDKO led to a reduction in lipolysis and increased fatty acid uptake and incorporation into cholesterol esters, triglycerides, and phospholipids, without impacting fatty acid oxidation. Since HSD17β11 expression is highest in the liver, we then repeated these experiments in a human liver cell line, THLE-2s, which recapitulated key metabolic defects of a HSDKO. Overall, these studies add HSD17β11 to a growing list of lipolytic regulators.

 

Building upon this work, we sought to identify PKA phosphorylation sites in HSD17β11 and identified serine-33 as a PKA consensus site. To test whether serine-33 is a functionally important site in lipid metabolism, we generated a phosphorylation-defective mutant of HSD17β11 at serine-33 (HSDS33A) by re-expressing the phosphomutant protein in HSDKO HEK293T cells. Metabolic studies were performed in HSDS33A and cells expressing wild-type HSD17β11 (HSDFLAG). 14C-tracing studies revealed an increase in lipolysis in HSDS33A cells without significantly impacting lipid storage or fatty acid metabolism. Mechanistically, FLIM-FRET analysis revealed a robust interaction between HSD17β11 and PLIN5 but not HSD17β11 and ATGL, which was independent to forskolin stimulation or serine-33 mutation. Intriguingly, cycloheximide assay revealed that phosphorylation of HSD17β11 results in degradation of the protein, and this degradation was ameliorated in HSDS33A cells, suggesting that serine-33 is responsible for regulating stability of HSD17β11. We postulate that HSD17β11 degradation is important to release PLIN5 to inhibit ATGL activity, which could be a novel mechanism to prevent unrestricted lipolysis and facilitate the recovery of triglyceride stores in cells.