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Characterizing the Domains of Perilipin 5 Using Proteolytic Mapping
Author(s) -
Bailey Hannah M,
Huggins Pearson N,
Tansey John T
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1134.8
Subject(s) - perilipin , lipid droplet , biochemistry , proteolysis , adipose triglyceride lipase , oleic acid , chemistry , trypsin , microbiology and biotechnology , biology , lipase , adipocyte , enzyme , adipose tissue
Cellular lipid storage occurs in lipid droplets, subcellular organelles coated with perilipin proteins. Members of the perilipin family have been found to play key roles in the regulation and metabolism of cellular lipid stores. Perilipin 5 is known to interact with adipocyte triacylglycerol lipase (ATGL) and hormone sensitive lipase as well as the ATGL activator CGI‐58, yet little is known about the structure of perilipin 5. Based on comparisons of perilipin 5 sequence to other family members we hypothesized that there would be discrete domains of perilipin 5 which would account for the different observed interactions. Chinese hamster ovary (CHO) cell lines stably expressing perilipin 5 with a carboxy terminal 3X FLAG epitope tag were used as a source of protein. Cells were treated with oleic acid to promote triacylglycerol storage. To identify perilipin domains, partial proteolysis was performed using trypsin and samples were analyzed by western blotting using either an amino terminal perilipin 5 specific antibody or an anti 3XFLAG antibody. Proteolytic digestion patterns differed based on oleic acid treatment. In oleic acid treated cells, a breakdown product of approximately 40 kDa was observed indicating two potential domains, an amino terminal one of ~20 kDa and a carboxy terminal of ~40 kDa. These data indicate that the perilipin proteins may fold into at least two domains with an exposed linker between them.