Functional Proteomic Analysis of Lipases and Esterases in Cultured Human Adipocytes
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- 作者：Maximilian Schicher ; Maria Morak ; Ruth Birner-Gruenberger ; Heidemarie Kayer ; Bojana Stojcic ; Gerald Rechberger ; Manfred Kollroser ; Albin Hermetter
- 刊名：Journal of Proteome Research
- 出版年：2010
- 出版时间：December 3, 2010
- 年：2010
- 卷：9
- 期：12
- 页码：6334-6344
- 全文大小：454K
- 年卷期：v.9,no.12(December 3, 2010)
- ISSN：1535-3907
文摘
This study reports on the analysis of the lipolytic proteome of cultured human fat cells. We used specific affinity tags to detect and identify the lipolytic and esterolytic enzymes in human subcutaneous (Sc) and visceral (Visc) adipocytes. For this purpose, differentiated fat cells were incubated with a fluorescent suicide inhibitor followed by protein separation using one- or two-dimensional gel electrophoresis. After detection by fluorescence laser scanning, the labeled proteins were tryptically digested and peptides were identified by mass spectrometry. In addition, a biotinylated probe was used for specific enzyme labeling with subsequent avidin affinity isolation of the tagged proteins. Finally, we determined the quantitative differences in protein expression levels between subcutaneous and visceral adipocytes using differential activity-based gel electrophoresis (DABGE). We found that the lipase/esterase patterns of both cell types are very similar, except for some proteins that were only found in Sc cells. Two novel enzyme candidates identified in this study were overexpressed and characterized using biologically relevant glycerolipid substrates in vitro. Both of them showed pronounced hydrolytic activities on hydrophobic acylglycerols and therefore may be considered lipases. The physiological functions of the novel lipolytic proteins in vivo are currently subject to investigation.