title: Basic leucine zipper protein Cnc-C is a substrate and transcriptional regulator of the Drosophila 26S proteasome.
abstract: While the 26S proteasome is a key proteolytic complex, little is known about how proteasome levels are maintained in higher eukaryotic cells. Here we describe an RNA interference (RNAi) screen of Drosophila melanogaster that was used to identify transcription factors that may play a role in maintaining levels of the 26S proteasome. We used an RNAi library against 993 Drosophila transcription factor genes to identify genes whose suppression in Schneider 2 cells stabilized a ubiquitin-green fluorescent protein reporter protein. This screen identified Cnc (cap 'n' collar [CNC]; basic region leucine zipper) as a candidate transcriptional regulator of proteasome component expression. In fact, 20S proteasome activity was reduced in cells depleted of cnc. Immunoblot assays against proteasome components revealed a general decline in both 19S regulatory complex and 20S proteasome subunits after RNAi depletion of this transcription factor. Transcript-specific silencing revealed that the longest of the seven transcripts for the cnc gene, cnc-C, was needed for proteasome and p97 ATPase production. Quantitative reverse transcription-PCR confirmed the role of Cnc-C in activation of transcription of genes encoding proteasome components. Expression of a V5-His-tagged form of Cnc-C revealed that the transcription factor is itself a proteasome substrate that is stabilized when the proteasome is inhibited. We propose that this single cnc gene in Drosophila resembles the ancestral gene family of mammalian nuclear factor erythroid-derived 2-related transcription factors, which are essential in regulating oxidative stress and proteolysis.
authors.affiliation: Department of Molecular Biology and Functional Genomics, Stockholm University, Stockholm, Sweden.
authors.forename: Kristian Björk
authors.initials: KB
authors.lastname: Grimberg
authors.forename: Anne
authors.initials: A
authors.lastname: Beskow
authors.forename: Daniel
authors.initials: D
authors.lastname: Lundin
authors.forename: Monica M
authors.initials: MM
authors.lastname: Davis
authors.forename: Patrick
authors.initials: P
authors.lastname: Young
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D029721:Drosophila Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism*; D019143:Evolution, Molecular; D055785:Gene Knockdown Techniques; D017344:Genes, Insect; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000072019:Kelch-Like ECH-Associated Protein 1; D008322:Mammals / Q000235:genetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D010802:Phylogeny; D046988:Proteasome Endopeptidase Complex / Q000235:genetics* / Q000378:metabolism*; D034622:RNA Interference; D012097:Repressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D002352:Carrier Proteins; D029721:Drosophila Proteins; D047908:Intracellular Signaling Peptides and Proteins; C527886:Keap1 protein, Drosophila; D000072019:Kelch-Like ECH-Associated Protein 1; D012097:Repressor Proteins; C498505:Rpn10 protein, Drosophila; C116463:cnc protein, Drosophila; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease