title: Conditional knockdown of proteasomes results in cell-cycle arrest and enhanced expression of molecular chaperones Hsp70 and Hsp40 in chicken DT40 cells.
abstract: The 26 S proteasome is an evolutionarily conserved ATP-dependent protease complex that degrades poly-ubiquitinated proteins and plays essential roles in a critical part of cellular regulation. In vertebrates, the roles of the proteasome have been widely studied by use of specific inhibitors, but not genetically. Here, we generated a cell line Z(-/-/-)/Z-HA, in which the expression of the catalytic subunit of the proteasome, Z (beta2) could be manipulated. This cell line expresses exogenous Z protein under the control of a tetracycline-repressible promoter in a Z-nullizygous genetic background. Treatment of these cells with doxycycline inhibited Z expression and, hence, the function of the proteasome. The latter resulted in accumulation of poly-ubiquitinated proteins and concomitant induction of molecular chaperones Hsp70 and Hsp40. These results suggest a synergistic role for the proteasome with these molecular chaperones to eliminate misfolded or damaged proteins in vivo. Furthermore, knockdown of the proteasome induced apoptotic cell death following cell-cycle arrest at G(2)/M phase. Our Z(-/-/-)/Z-HA cell line would be useful for evaluating proteolytic processes catalyzed by the proteasome in many biological events in vertebrate cells.
authors.affiliation: Department of Molecular Oncology, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Tanahashi-Hori
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Tanahashi
authors.forename: Keiji
authors.initials: K
authors.lastname: Tanaka
authors.forename: Tomoki
authors.initials: T
authors.lastname: Chiba
mesh_terms: D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle; D002470:Cell Survival; D002478:Cells, Cultured; D002645:Chickens; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis*; D006360:Heat-Shock Proteins / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D025821:Polyubiquitin / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D021122:Protein Subunits
chemical_list: D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D009097:Multienzyme Complexes; D021122:Protein Subunits; D025821:Polyubiquitin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex