RESUMO
The epithelial-specific splicing regulators Esrp1 and Esrp2 are required for mammalian development, including establishment of epidermal barrier functions. However, the mechanisms by which Esrp ablation causes defects in epithelial barriers remain undefined. We determined that the ablation of Esrp1 and Esrp2 impairs epithelial tight junction (TJ) integrity through loss of the epithelial isoform of Rho GTP exchange factor Arhgef11. Arhgef11 is required for the maintenance of TJs via RhoA activation and myosin light chain (MLC) phosphorylation. Ablation or depletion of Esrp1/2 or Arhgef11 inhibits MLC phosphorylation and only the epithelial Arhgef11 isoform rescues MLC phosphorylation in Arhgef11 KO epithelial cells. Mesenchymal Arhgef11 transcripts contain a C-terminal exon that binds to PAK4 and inhibits RhoA activation byArhgef11. Deletion of the mesenchymal-specific Arhgef11 exon in Esrp1/2 KO epithelial cells using CRISPR/Cas9 restored TJ function, illustrating how splicing alterations can be mechanistically linked to disease phenotypes that result from impaired functions of splicing regulators.
Assuntos
Processamento Alternativo/genética , Células Epiteliais/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Junções Íntimas/metabolismo , Alopecia/patologia , Animais , Animais Recém-Nascidos , Sistemas CRISPR-Cas/genética , Permeabilidade da Membrana Celular , Éxons/genética , Inflamação/patologia , Queratinócitos/metabolismo , Mesoderma/metabolismo , Camundongos Knockout , Cadeias Leves de Miosina/metabolismo , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Quinases Ativadas por p21/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
NADH:ubiquinone oxidoreductase (complex I) pumps protons across the membrane using downhill redox energy. The Escherichia coli complex I consists of 13 different subunits named NuoA-N coded by the nuo operon. Due to the low abundance of the protein and some difficulty with the genetic manipulation of its large ~15-kb operon, purification of E. coli complex I has been technically challenging. Here, we generated a new strain in which a polyhistidine sequence was inserted upstream of nuoE in the operon. This allowed us to prepare large amounts of highly pure and active complex I by efficient affinity purification. The purified complex I contained 0.94 ± 0.1 mol of FMN, 29.0 ± 0.37 mol of iron, and 1.99 ± 0.07 mol of ubiquinone/1 mol of complex I. The extinction coefficient of isolated complex I was 495 mM(-1) cm(-1) at 274 nm and 50.3 mM(-1) cm(-1) at 410 nm. NADH:ferricyanide activity was 219 ± 9.7 µmol/min/mg by using HEPES-Bis-Tris propane, pH 7.5. Detailed EPR analyses revealed two additional iron-sulfur cluster signals, N6a and N6b, in addition to previously assigned signals. Furthermore, we found small but significant semiquinone signal(s), which have been reported only for bovine complex I. The line width was ~12 G, indicating its neutral semiquinone form. More than 90% of the semiquinone signal originated from the single entity with P½ (half-saturation power level) = 1.85 milliwatts. The semiquinone signal(s) decreased by 60% when with asimicin, a potent complex I inhibitor. The functional role of semiquinone and the EPR assignment of clusters N6a/N6b are discussed.
