Identification of novel lipid droplet factors that regulate lipophagy and cholesterol efflux in macrophage foam cells.

ABSTRACT

Macrophage autophagy is a highly anti-atherogenic process that promotes the catabolism of cytosolic lipid droplets (LDs) to maintain cellular lipid homeostasis. Selective autophagy relies on tags such as ubiquitin and a set of selectivity factors including selective autophagy receptors (SARs) to label specific cargo for degradation. Originally described in yeast cells, "lipophagy" refers to the degradation of LDs by autophagy. Yet, how LDs are targeted for autophagy is poorly defined. Here, we employed mass spectrometry to identify lipophagy factors within the macrophage foam cell LD proteome. In addition to structural proteins (e.g., PLIN2), metabolic enzymes (e.g., ACSL) and neutral lipases (e.g., PNPLA2), we found the association of proteins related to the ubiquitination machinery (e.g., AUP1) and autophagy (e.g., HMGB, YWHA/14-3-3 proteins). The functional role of candidate lipophagy factors (a total of 91) was tested using a custom siRNA array combined with high-content cholesterol efflux assays. We observed that knocking down several of these genes, including Hmgb1, Hmgb2, Hspa5, and Scarb2, significantly reduced cholesterol efflux, and SARs SQSTM1/p62, NBR1 and OPTN localized to LDs, suggesting a role for these in lipophagy. Using yeast lipophagy assays, we established a genetic requirement for several candidate lipophagy factors in lipophagy, including HSPA5, UBE2G2 and AUP1. Our study is the first to systematically identify several LD-associated proteins of the lipophagy machinery, a finding with important biological and therapeutic implications. Targeting these to selectively enhance lipophagy to promote cholesterol efflux in foam cells may represent a novel strategy to treat atherosclerosis.Abbreviations ADGRL3 adhesion G protein-coupled receptor L3; agLDL aggregated low density lipoprotein; AMPK AMP-activated protein kinase; APOA1 apolipoprotein A1; ATG autophagy related; AUP1 AUP1 lipid droplet regulating VLDL assembly factor; BMDM bone-marrow derived macrophages; BNIP3L BCL2/adenovirus E1B interacting protein 3-like; BSA bovine serum albumin; CALCOCO2 calcium binding and coiled-coil domain 2; CIRBP cold inducible RNA binding protein; COLGALT1 collagen beta(1-O)galactosyltransferase 1; CORO1A coronin 1A; DMA deletion mutant array; Faa4 long chain fatty acyl-CoA synthetase; FBS fetal bovine serum; FUS fused in sarcoma; HMGB1 high mobility group box 1; HMGB2 high mobility group box 2 HSP90AA1 heat shock protein 90 alpha (cytosolic) class A member 1; HSPA5 heat shock protein family A (Hsp70) member 5; HSPA8 heat shock protein 8; HSPB1 heat shock protein 1; HSPH1 heat shock 105kDa/110kDa protein 1; LDAH lipid droplet associated hydrolase; LIPA lysosomal acid lipase A; LIR LC3-interacting region; MACROH2A1 macroH2A.1 histone; MAP1LC3 microtubule-associated protein 1 light chain 3; MCOLN1 mucolipin 1; NBR1 NBR1, autophagy cargo receptor; NPC2 NPC intracellular cholesterol transporter 2; OPTN optineurin; P/S penicillin-streptomycin; PLIN2 perilipin 2; PLIN3 perilipin 3; PNPLA2 patatin like phospholipase domain containing 2; RAB RAB, member RAS oncogene family; RBBP7, retinoblastoma binding protein 7, chromatin remodeling factor; SAR selective autophagy receptor; SCARB2 scavenger receptor class B, member 2; SGA synthetic genetic array; SQSTM1 sequestosome 1; TAX1BP1 Tax1 (human T cell leukemia virus type I) binding protein 1; TFEB transcription factor EB; TOLLIP toll interacting protein; UBE2G2 ubiquitin conjugating enzyme E2 G2; UVRAG UV radiation resistance associated gene; VDAC2 voltage dependent anion channel 2; VIM vimentin.