Date Written

Spring 4-11-2016

Document Type

Honors Paper

Degree Name

Biochemistry and Molecular Biology-BS

Department

Biochemistry and Molecular Biology

Advisor

Dr. John Tansey

First Committee Member

Dr. John Tansey

Second Committee Member

Dr. David Sheridan

Third Committee Member

Dr. Karen Steigman

Keywords

Perilipin, Structure, Lipid Droplet, Perilipin-5, Lipid, Protein

Subject Categories

Biochemistry, Biophysics, and Structural Biology

Abstract

The ability of eukaryotic cells to maintain a reservoir of neutral lipids is critical in order maintain the plasma membrane, assist in the production of hormones, and to act as a source of energy if needed. Neutral lipids are stored within a structure known as a lipid droplet coated by members of the PAT family of lipid droplet proteins embedded within its lipid monolayer. The PAT family is comprised of perilipins 1-5 which have been shown to play a roles in regulating lipid droplet metabolism and the droplet’s interaction with lipases. This work seeks to analyze a member of the PAT family, Perilipin-5, using proteolytic mapping in order to determine its domain topology which may provide clues to its function. We hypothesized that delipidation of cellular extracts using either detergents or lipases would alter the conformation of perilipin 5. This would alter the proteolytic cleavage patterns, indicating independently interacting domains of the protein and cleavage sites that were protected from proteases by being buried in lipid. Cellular lysates of Chinese Hamster Ovary cell lines expressing perilipin 5 were used as a source of protein. Following proteolysis, samples were analyzed by western blotting. Triton-X-100 treated samples had more cleavage sites than controls or Tween 20 treated samples. Collectively, these data show at least two major and four minor cleavage products resulting from proteolysis and suggest that perilipin 5 has at least two distinct domains.

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