Characterization of PNPLA5/GS2‐like neutral lipase and identification its lipid droplet targeting motif

Abstract

Recently, a new family of cytoplasmic lipases named Patatin-like Phospholipase Domain Containing Proteins (PNPLA), whose members include ATGL/PNPLA2, Adiponutrin/PNPLA3, and PNPLA5/GS2-like, has been discovered to play a key role in the hydrolysis of stored triglycerides and regulation of lipid droplet (LD) metabolism. Our studies have focused on the role of PNPLA5 (a.k.a. GS2-like) and its targeting to LDs. Using affinity-purified antibodies, we found that PNPLA5 is present in many murine tissues, with highest expression in kidney and heart, and much less in adipose. However, PNPLA5 expression was induced >11-fold during differentiation of NIH 3T3-L1 cells to adipocytes. Expression of GFP-labeled PNPLA5 in HeLa cells revealed that it was localized to LDs, along with ATGL/PNPLA2, ADRP, and TIP47. Importantly, over-expression of PNPLA5 reduced the volume of LDs, with an associated decrease in stored triglycerides, comparable to that achieved by over-expression of ATGL/PNPLA2. Interestingly, over-expression of a catalytically inactive mutant version of PNPLA5 resulted in the generation of significantly enlarged, but fewer, LDs. Analysis of truncated and mutant versions of PNPLA5 revealed a four amino acid LD targeting motif (LTM) in the C-terminal third of the protein, whose sequence is conserved among members of the PNPLA family. This work supported by Hatch Project NYC-165415 and NIH grant DK51596 to WJB