Identification of major proteins in the lipid droplet-enriched fraction isolated from the human hepatocyte cell line HuH7.
Recent studies have revealed the presence of intracellular lipid droplets in wide variety of species. In mammalian cells, there exist proteins specifically localize in lipid droplets. However, the protein profile in the droplet remains yet to be clarified. In this study, a fraction enriched with lipid droplets was isolated from a human hepatocyte cell line HuH7 using sucrose density gradient centrifugation, and 17 major proteins in the fraction were identified using nano LC-MS/MS techniques. Adipose differentiation-related protein (ADRP) was the most abundant protein in the fraction. The secondary abundant proteins were identified to be acyl-CoA synthetase 3 (ACS3) and 17beta-hydroxysteroid dehydrogenase 11 (17betaHSD11). Included in the identified proteins were five lipid-metabolizing enzymes as well as two lipid droplet-specific proteins. When HuH7 cell lysate was fractionated by a density gradient, most of 17betaHSD11 was found in the droplet-enriched fraction. In immunocytochemical analysis, 17betaHSD11 showed ring-shaped images which overlapped with those for ADRP. These results suggest that a specific set of proteins is enriched in the lipid droplet-enriched fraction and that 17betaHSD11 localizes specifically in the fraction.
Fujimoto
Yasuyuki
Y
Department of Molecular Pathology, Faculty of Pharmaceutical Sciences, Teikyo University, 1091-1 Suarashi, Sagamiko, Tsukui, Kanagawa 199-0195, Japan.
Itabe
Hiroyuki
H
Sakai
Jun
J
Makita
Minoru
M
Noda
Junich
J
Mori
Masahiro
M
Higashi
Yusuke
Y
Kojima
Shinichi
S
Takano
Tatsuya
T
D015067:17-Hydroxysteroid Dehydrogenases / Q000032:analysis; D000445:Aldehyde Oxidoreductases; D000595:Amino Acid Sequence; D001391:Azo Compounds; D045744:Cell Line, Tumor; D002499:Centrifugation, Density Gradient; D002855:Chromatography, Thin Layer; D003066:Coenzyme A Ligases / Q000032:analysis; D004396:Coloring Agents; D004591:Electrophoresis, Polyacrylamide Gel; D006416:Hematoxylin; D022781:Hepatocytes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008055:Lipids / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D000070780:Perilipin-2; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism*; D014357:Trypsin; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
D001391:Azo Compounds; D004396:Coloring Agents; D008055:Lipids; D008565:Membrane Proteins; C000602982:PLIN2 protein, human; D010446:Peptide Fragments; D000070780:Perilipin-2; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D015067:17-Hydroxysteroid Dehydrogenases; C118226:HSD17B11 protein, human; D000445:Aldehyde Oxidoreductases; D014357:Trypsin; D003066:Coenzyme A Ligases; C090799:FAA2 protein, S cerevisiae; D000098859:Long-Chain-Fatty-Acid-CoA Ligase; C011049:oil red O; D006416:Hematoxylin