Family Report for: LIDHydrolase

Variations in the gene LDAH (C2ORF43), which encodes lipid droplet-associated hydrolase (LDAH), are among few loci associated with human prostate cancer. Homologs of LDAH have been identified as proteins of lipid droplets (LDs). LDs are cellular organelles that store neutral lipids, such as triacylglycerols and sterol esters, as precursors for membrane components and as reservoirs of metabolic energy. LDAH is reported to hydrolyze cholesterol esters and to be important in macrophage cholesterol ester metabolism. Here, we confirm that LDAH is localized to LDs in several model systems. We generated a murine model in which Ldah is disrupted but found no evidence for a major function of LDAH in cholesterol ester or triacylglycerol metabolism in vivo, nor a role in energy or glucose metabolism. Our data suggest that LDAH is not a major cholesterol ester hydrolase, and an alternative metabolic function may be responsible for its possible effect on development of prostate cancer.

OBJECTIVE: Lipid-laden macrophages or foam cells are characterized by massive cytosolic lipid droplet (LD) deposition containing mostly cholesterol ester (CE) derived from the lipoproteins cleared from the arterial wall. Cholesterol efflux from foam cells is considered to be atheroprotective. Because cholesterol is effluxed as free cholesterol, CE accumulation in LDs may limit free cholesterol efflux. Our objective was to identify proteins that regulate cholesterol trafficking through LDs. APPROACH AND
RESULTS: In a proteomic analysis of the LD fraction of RAW 264.7 macrophages, we identified an evolutionarily conserved protein with a canonical GXSXG lipase catalytic motif and a predicted alpha/beta-hydrolase fold, the RIKEN cDNA 1110057K04 gene, which we named LD-associated hydrolase (LDAH). LDAH association with LDs was confirmed by immunoblotting and immunocytochemistry. LDAH was labeled with a probe specific for active serine hydrolases. LDAH showed relatively weak in vitro CE hydrolase activity. However, cholesterol measurements in intact cells supported a significant role of LDAH in CE homeostasis because LDAH upregulation and downregulation decreased and increased, respectively, intracellular cholesterol and CE in human embryonic kidney-293 cells and RAW 264.7 macrophages. Mutation of the putative nucleophilic serine impaired active hydrolase probe binding, in vitro CE hydrolase activity, and cholesterol-lowering effect in cells, whereas this mutant still localized to the LD. LDAH upregulation increased CE hydrolysis and cholesterol efflux from macrophages, and, interestingly, LDAH is highly expressed in macrophage-rich areas within mouse and human atherosclerotic lesions.
CONCLUSIONS: The data identify a candidate target to promote reverse cholesterol transport from atherosclerotic lesions.

Lipid bodies (LBs), multifunctional organelles present in most eukaryotic cells, are sites of eicosanoid formation in leukocytes; but little is known about the composition of leukocyte LBs or the biogenesis and internal structures of LBs from mammalian cells. Proteomic analyses of LBs purified from human monocytic U937 cells detected, common to LBs in other cells, proteins involved in cholesterol and triglyceride metabolism, Rab GTPases, and many membrane and endoplasmic reticulum (ER)-associated proteins. Newly lipid body (LB)-associated proteins included MRP-14, potentially involved in arachidonate transport, and ribosomal subunit proteins and translation regulatory proteins. Ultrastructurally, in U937 cells as well as human neutrophils and eosinophils, ribosomes are attached to and distributed within LBs, and LBs contain extensive ER-like membranes. The presence of ribosomes, ER-like membranes and many membrane-associated and ER luminal proteins within LBs, supports a new model by which enveloped ER-membranes and domains form LBs and indicates that LBs may be sites of protein synthesis.

Saccharomyces cerevisiae Ypr147cp was found localized to lipid droplets but the physiological role of Ypr147cp remains unknown. Sequence analysis of Ypr147cp revealed an alpha/beta hydrolase domain along with the conserved GXSXG lipase motif. Recombinant Ypr147cp showed both triacylglycerol lipase and ester hydrolase activities. Knock out of YPR147C led to accumulation of TAG in ypr147cDelta when compared to wild type (WT). In addition, transmission electron microscopic analysis of ypr147cDelta cells revealed a greater number of lipid bodies, justifying the increase in TAG content, and the phenotype was rescued upon overexpression of YPR147C in ypr147cDelta. Moreover, the lipid profiling confirmed the accumulation of fatty acids derived from neutral and phospholipids in ypr147cDelta cells. Based on these results, Ypr147cp is identified as a lipid droplet associated triacylglycerol lipase along with an ester hydrolyzing capacity.

Variations in the gene LDAH (C2ORF43), which encodes lipid droplet-associated hydrolase (LDAH), are among few loci associated with human prostate cancer. Homologs of LDAH have been identified as proteins of lipid droplets (LDs). LDs are cellular organelles that store neutral lipids, such as triacylglycerols and sterol esters, as precursors for membrane components and as reservoirs of metabolic energy. LDAH is reported to hydrolyze cholesterol esters and to be important in macrophage cholesterol ester metabolism. Here, we confirm that LDAH is localized to LDs in several model systems. We generated a murine model in which Ldah is disrupted but found no evidence for a major function of LDAH in cholesterol ester or triacylglycerol metabolism in vivo, nor a role in energy or glucose metabolism. Our data suggest that LDAH is not a major cholesterol ester hydrolase, and an alternative metabolic function may be responsible for its possible effect on development of prostate cancer.

OBJECTIVE: Lipid-laden macrophages or foam cells are characterized by massive cytosolic lipid droplet (LD) deposition containing mostly cholesterol ester (CE) derived from the lipoproteins cleared from the arterial wall. Cholesterol efflux from foam cells is considered to be atheroprotective. Because cholesterol is effluxed as free cholesterol, CE accumulation in LDs may limit free cholesterol efflux. Our objective was to identify proteins that regulate cholesterol trafficking through LDs. APPROACH AND
RESULTS: In a proteomic analysis of the LD fraction of RAW 264.7 macrophages, we identified an evolutionarily conserved protein with a canonical GXSXG lipase catalytic motif and a predicted alpha/beta-hydrolase fold, the RIKEN cDNA 1110057K04 gene, which we named LD-associated hydrolase (LDAH). LDAH association with LDs was confirmed by immunoblotting and immunocytochemistry. LDAH was labeled with a probe specific for active serine hydrolases. LDAH showed relatively weak in vitro CE hydrolase activity. However, cholesterol measurements in intact cells supported a significant role of LDAH in CE homeostasis because LDAH upregulation and downregulation decreased and increased, respectively, intracellular cholesterol and CE in human embryonic kidney-293 cells and RAW 264.7 macrophages. Mutation of the putative nucleophilic serine impaired active hydrolase probe binding, in vitro CE hydrolase activity, and cholesterol-lowering effect in cells, whereas this mutant still localized to the LD. LDAH upregulation increased CE hydrolysis and cholesterol efflux from macrophages, and, interestingly, LDAH is highly expressed in macrophage-rich areas within mouse and human atherosclerotic lesions.
CONCLUSIONS: The data identify a candidate target to promote reverse cholesterol transport from atherosclerotic lesions.

Lipid bodies (LBs), multifunctional organelles present in most eukaryotic cells, are sites of eicosanoid formation in leukocytes; but little is known about the composition of leukocyte LBs or the biogenesis and internal structures of LBs from mammalian cells. Proteomic analyses of LBs purified from human monocytic U937 cells detected, common to LBs in other cells, proteins involved in cholesterol and triglyceride metabolism, Rab GTPases, and many membrane and endoplasmic reticulum (ER)-associated proteins. Newly lipid body (LB)-associated proteins included MRP-14, potentially involved in arachidonate transport, and ribosomal subunit proteins and translation regulatory proteins. Ultrastructurally, in U937 cells as well as human neutrophils and eosinophils, ribosomes are attached to and distributed within LBs, and LBs contain extensive ER-like membranes. The presence of ribosomes, ER-like membranes and many membrane-associated and ER luminal proteins within LBs, supports a new model by which enveloped ER-membranes and domains form LBs and indicates that LBs may be sites of protein synthesis.