title: Identification of albumin precursor protein, Phi AP3, and alpha-smooth muscle actin as novel components of redox sensing machinery in vascular smooth muscle cells.
abstract: Aerobic organisms are continually subjected to environmental stressors that compromise redox homeostasis and induce cellular injury. In vascular smooth muscle cells (vSMCs), the activation/repression of redox-regulated genes after environmental stress often involves protein binding to cis-acting antioxidant response elements (AREs). The present study was conducted to identify proteins that participate in redox-regulated protein binding to human c-Ha-ras and mouse glutathione S-transferase A1 AREs in vSMCs after oxidant injury. Challenge of vSMCs with 0.3 or 3 microM hydrogen peroxide, 3-methylcholanthrene, benzo[a]pyrene-7,8-diol, 3-hydroxy benzo[a]pyrene, and benzo[a]pyrene-3,6-quinone induced concentration-related increases in ARE protein binding. The profiles of ARE complex assembly were comparable, but exhibited chemical specificity. Pretreatment with 0.5 mM N-acetylcysteine inhibited activation of ARE protein binding in hydrogen peroxide-treated cells. Preparative electrophoretic mobility shift assays coupled to Western analysis identified NF-E2-related proteins 1 and 2 and JunD in complexes assembled on AREs. Polyethylenimine affinity and sequence-specific serial immobilized DNA affinity chromatography followed by N-terminal sequencing identified albumin precursor protein, phi AP3, and alpha-smooth muscle actin as members of the ARE signaling pathway. Sequence analysis of albumin protein revealed homology to the redox-regulated transcription factors Bach1 and 2, as well as cytoskeletal and molecular motor proteins. These results implicate albumin precursor protein, phi AP3, and alpha-smooth muscle actin as participants in redox sensing in vSMCs, and suggest that protein complex assembly involves interactions between leucine zipper and zinc finger transcription factors with cytoskeletal proteins.
authors.affiliation: Department of Physiology and Pharmacology, Center for Environmental and Rural Health, Texas A&M University, College Station, Texas 77843-4455, USA.
authors.forename: M T
authors.initials: MT
authors.lastname: Holderman
authors.forename: K P
authors.initials: KP
authors.lastname: Miller
authors.forename: L J
authors.initials: LJ
authors.lastname: Dangott
authors.forename: K S
authors.initials: KS
authors.lastname: Ramos
mesh_terms: D000199:Actins / Q000378:metabolism*; D000418:Albumins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D051267:NF-E2-Related Factor 2; D051264:Nuclear Respiratory Factors; D009943:Organophosphorus Compounds / Q000737:chemistry; D010084:Oxidation-Reduction; D011094:Polyethyleneimine / Q000737:chemistry; D011498:Protein Precursors / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism
chemical_list: C043517:2-(4-isothiocyanatophenoxy)-1,3,2-dioxaphosphinene 2-oxide; D000199:Actins; D000418:Albumins; D004268:DNA-Binding Proteins; D051267:NF-E2-Related Factor 2; C495636:Nfe2l2 protein, mouse; D051264:Nuclear Respiratory Factors; D009943:Organophosphorus Compounds; D011498:Protein Precursors; D015534:Trans-Activators; D011094:Polyethyleneimine; D004247:DNA