title: High hydrostatic pressure inhibits the biosynthesis of eukaryotic elongation factor-2.
abstract: High continuous hydrostatic pressure is known to inhibit the total cellular protein synthesis. In this study, our goal was to identify pressure-regulated proteins by using two dimensional gel electrophoresis and mass spectrometry. This analysis showed that under 30 MPa continuous hydrostatic pressure the biosynthesis of eukaryotic elongation factor-2 (eEF-2) was inhibited both in HeLa carcinoma and T/C28a4 chondrocytic cell lines. Western blot analysis of HeLa cells revealed that the cellular protein level of eEF-2 decreased by 40%-50% within 12 h of the pressure treatment. However, the steady-state mRNA level of eEF-2 was not affected by the pressure. Cycloheximide addition after 4 h-pressure treatment suggested that the half-life of eEF-2 protein was shorter in pressurized cells. eEF-2 is responsible for the translocation of ribosome along the specific mRNA during translation, and its phosphorylation prevents the ribosomal translocation. Therefore, increased phosphorylation of eEF-2 was considered as one mechanism that could explain the reduced level of protein synthesis in pressurized HeLa cell cultures. However, Western blot analysis with an antibody recognizing the Thr56-phosphorylated form of eEF-2 showed that phosphorylation of eEF-2 was not elevated in pressurized samples. In conclusion, the inhibition of protein synthesis under high pressure occurs independent of the phosphorylation of eEF-2. However, this inhibition may result from the decrease of cellular eEF-2 protein.
authors.affiliation: Department of Anatomy, University of Kuopio, 70211 Kuopio, Finland.
authors.forename: Mika A
authors.initials: MA
authors.lastname: Elo
authors.forename: Hannu M
authors.initials: HM
authors.lastname: Karjalainen
authors.forename: Reijo K
authors.initials: RK
authors.lastname: Sironen
authors.forename: Leena
authors.initials: L
authors.lastname: Valmu
authors.forename: Nicholas T
authors.initials: NT
authors.lastname: Redpath
authors.forename: Gareth J
authors.initials: GJ
authors.lastname: Browne
authors.forename: Nisse
authors.initials: N
authors.lastname: Kalkkinen
authors.forename: Heikki J
authors.initials: HJ
authors.lastname: Helminen
authors.forename: Mikko J
authors.initials: MJ
authors.lastname: Lammi
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D017931:DNA Primers; D015180:Electrophoresis, Gel, Two-Dimensional; D005057:Eukaryotic Cells / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D020652:Peptide Elongation Factor 2 / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011312:Pressure; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D017931:DNA Primers; D020652:Peptide Elongation Factor 2