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