Proteomic changes in response to lipin1 overexpression in 293T human renal epithelial cells

Abstract

Lipin1, a member of the lipin family, serves as a phospholipid phosphatase or a co-transcriptional regulator in lipid metabolism. Recent studies also show that lipin1 is involved in many other cellular metabolism processes. However, the clear regulatory mechanism for lipin1 is unknown. The 293T human renal epithelial cell line represents a commonly used and well established expression system for recombinant proteins. Herein, we used two-dimensional polyacrylamide gel electrophoresis (2D-GE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to explore the changes in protein expression induced by lipin1 overexpression in 293T cells. Western blotting was used to confirm one of the expression changes of related proteins. Subsequently, the function and relationship of these proteins were analyzed by bioinformatics approach. By using 2D-PAGE, approximately 152 proteins were separated and eleven proteins were found to be significantly affected by lipin1 overexpression compared to the control. Among them, three proteins (eEF-1B γ, CCT1 and CCT3) were up-regulated and other eight proteins (NDKA, Stathmin, HNRNP A1, TK, KRT1, PKM, RanBP1 and LDHB) were down-regulated. These proteins were successfully identified with peptide mass fingerprinting using MALDI-TOF-MS after in-gel trypsin digestion. The bioinformatic analysis showed that these proteins are classified into seven protein species, including transferase, cleavage enzyme, cytoskeleton protein, chaperone protein, regulatory protein, structural protein and oxidoreductase. The results highlight the potential roles of lipin1 involved in many cellular metabolism processes, including myelin synthesis, extracellular domain formation, membrane bound vesicle synthesis and companion protein T complex synthesis.