Proteome-wide dysregulation by PRA1 depletion delineates a role of PRA1 in lipid transport and cell migration.
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.
Liu
Hao-Ping
HP
Molecular Medicine Research Center, Department of Medical Biotechnology and Laboratory Science, Graduate Institute of Biomedical Sciences, Chang Gung University, Tao-Yuan, Taiwan.
Wu
Chih-Ching
CC
Kao
Hung-Yi
HY
Huang
Yi-Chuan
YC
Liang
Ying
Y
Chen
Chia-Chun
CC
Yu
Jau-Song
JS
Chang
Yu-Sun
YS
D000736:Androstenes / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D002784:Cholesterol / Q000378:metabolism; D002853:Chromatography, Liquid; D019204:GTP-Binding Proteins / Q000172:deficiency* / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D007553:Isotope Labeling; D050356:Lipid Metabolism / Q000187:drug effects; D013058:Mass Spectrometry; D008954:Models, Biological; D009303:Nasopharyngeal Neoplasms / Q000378:metabolism / Q000473:pathology; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results; D033921:Vesicular Transport Proteins / Q000172:deficiency* / Q000378:metabolism*; D014763:Viral Matrix Proteins / Q000378:metabolism
D000736:Androstenes; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D009363:Neoplasm Proteins; D020543:Proteome; D033921:Vesicular Transport Proteins; D014763:Viral Matrix Proteins; C006261:3-beta-(2-(diethylamino)ethoxy)androst-5-en-17-one; D002784:Cholesterol; D019204:GTP-Binding Proteins; C108941:RABAC1 protein, human