Protein profiling and identification of modulators regulated by human papillomavirus 16 E7 oncogene in HaCaT keratinocytes by proteomics.
OBJECTIVE
Viral oncogenes E6 and E7 are selectively retained and expressed in carcinoma cells infected with human papillomavirus type 16 and cooperated with each other in immortalization and transformation of primary keratinocytes. This study was performed to identify proteins to be bound or modulated by high risk HPV E7 oncogene by using a proteomics.


METHODS
HaCaT normal keratinocyte was prepared to establish a stable cell line expressing E7. The E7-affinity column was also prepared to obtain E7-interacting proteins. In order to search the target molecules modulated by E7 expression, we used 2-dimensional electrophoresis and matrix-assisted laser desorption/ionization time of flight (MALDI/TOF) mass spectrometry. Pull down assay was also performed in order to confirm the E7-interacting proteins.


RESULTS
We identified 28 spots that are modulated by E7 in HaCaT/E7 using 2-dimensional electrophoresis (2-DE) and MALDI/TOF mass spectrometry. Proteomics analyses showed that actin and leukocyte elastase inhibitor were down-regulated, whereas stress-induced phosphoprotein 1, CD2 binding protein 1, catalase, T-complex protein 1, Ku70-binding protein, heat shock 60 kDa protein 1, G1/S-specific cyclin E1 and peroxiredoxin 2 were up-regulated. Western blot revealed that heat shock 60 kDa protein, catalase and peroxiredoxin 2 were also up-regulated. Pull down assay also showed that leukocyte elastase inhibitor (LEI) and Ku70-binding protein were bound to the E7 oncoprotein. By using E7-affinity column and 2-DE/MALDI-TOF, 22 spots were found to interact with E7 recombinant protein. MG11-like proteins, livin inhibitor-of-apoptosis, protein serine kinase c17, CD2 binding protein 1, cyclin E1, TATA box binding protein-associated factor and uridine-cytidine kinase 2 were up-regulated by E7 oncogene and also bound to E7 oncoprotein.


CONCLUSIONS
It is presumed that E7 can influence cell status by modulating the factors related to cell signaling, apoptosis and cell cycle regulation.
Lee
Kyung-Ae
KA
Laboratory of Cellular Biology, Korea Research Institute of Bioscience and Biotechnology, Yuseong, P.O. Box 115, Daejeon 305-600, South Korea.
Kang
Jeong-Woo
JW
Shim
Jung-Hyun
JH
Kho
Chang Won
CW
Park
Sung Goo
SG
Lee
Hee Gu
HG
Paik
Sang-Gi
SG
Lim
Jong-Seok
JS
Yoon
Do-Young
DY
D034961:Antigens, Nuclear / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D002472:Cell Transformation, Viral / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism* / Q000473:pathology / Q000821:virology*; D000072200:Ku Autoantigen; D018832:Molecular Chaperones / Q000378:metabolism; D009856:Oncogene Proteins, Viral / Q000235:genetics* / Q000378:metabolism; D027383:Papillomaviridae / Q000235:genetics*; D050725:Papillomavirus E7 Proteins; D030361:Papillomavirus Infections / Q000378:metabolism / Q000821:virology; D011485:Protein Binding; D040901:Proteomics; D015843:Serpins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection; D015854:Up-Regulation; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000378:metabolism / Q000821:virology
D034961:Antigens, Nuclear; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D018832:Molecular Chaperones; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D015843:Serpins; C059731:oncogene protein E7, Human papillomavirus type 16; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen