Abstract
We have previously identified prenylated Rab acceptor 1 (PRA1) as a novel cellular interacting partner for Epstein-Barr virus-encoded oncoprotein, latent membrane protein 1 (LMP1). The intracellular trafficking and full signaling of LMP1 requires its interaction with PRA1. To further explore the role of PRA1 in Epstein-Barr virus-associated nasopharyngeal carcinoma (NPC) cells, we generated several PRA1-knockdown cell clones, which exhibited altered cell morphology and increased cell motility. We identified proteins differentially expressed in the knockdown clones by means of isobaric mass tags labeling coupled with multidimensional liquid chromatography-mass spectrometry. We validated a panel of proteins, which showed consistent up-regulation in PRA1-knockdown clones and participated in regulating lipid homeostasis and cell migration. Immunofluorescence staining further revealed altered localization of these proteins and accumulation of intracellular cholesterol in PRA1-knockdown clones. These effects were phenocopied by treatment with a cholesterol transport inhibitor, U18666A. Moreover, overexpressed PRA1 was able to alleviate the dysregulation of these affected proteins either from PRA1 knockdown or U18666A treatment, implying a role for PRA1 in regulating the levels of these affected proteins in response to altered cholesterol homeostasis. We further demonstrated that LMP1 expression caused PRA1 sequestration in NPC cells, leading to a consequence reminiscent of PRA1 knockdown. Finally, the immunohistochemistry showed a physiological relevance of the PRA1-associated proteome-wide changes in NPC biopsy tissues. In sum, our findings delineated novel roles of PRA1 in lipid transport and cell migration, and provided additional insights into the molecular basis of NPC morphogenesis, namely a consequence of LMP1-PRA1 interaction.
Highlights
From the ‡Molecular Medicine Research Center, §Department of Medical Biotechnology and Laboratory Science, ¶Graduate Institute of Biomedical Sciences, and ʈDepartment of Cell and Molecular Biology, Chang Gung University, Tao-Yuan 333, Taiwan
We discovered a panel of dysregulated proteins in Prenylated Rab acceptor 1 (PRA1)-knockdown clones, which participate in lipid metabolism and transport and cell adhesion and migration, by using isobaric mass tags labeling approaches combined with multidimensional liquid chromatography-mass spectrometry (LC-MS/MS)
Identification of Proteins Differentially Expressed in PRA1knockdown Cells by iTRAQ-Liquid Chromatography (LC)-MS/MS Analysis—We sought to analyze the morphological changes of PRA1-knockdown cells in the aspect of protein expression
Summary
Each of the selected proteins, including LAMC2, ITGA6, ITGB4, FABP5, CAV1, TIP47, PFN2, and annexin A3, showed elevated protein levels, albeit at varied magnitudes, in four PRA1-knockdown clones (K3–2, K3–14, K3– 8, and K3–7) compared with three control clones (C13, C15, and C15–3) and the parental NPC-TW04 cells. These results confirmed the trend reported by the iTRAQ experiments (Table I), which implicated the elevated levels of these proteins in PRA1-knockdown cells.
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