title: C-terminal truncation of Noxa1 greatly enhances its ability to activate Nox2 in a pure reconstitution system.
abstract: Noxa1 activates Nox2 together with Noxo1 and Rac in a pure reconstitution system, but the resulting activity is considerably lower than that induced by p67(phox) and p47(phox). In this study, we found that C-terminal-truncated forms of Noxa1 exhibited higher activities than full-length Noxa1. Of the truncations examined, Noxa1(1-225) showed the highest ability for activation. Kinetic studies revealed that Noxa1(1-225) had a threefold higher Vmax value than full-length Noxa1 with a similar EC50 value. The affinities of Noxo1 and RacQ61L were not much altered by the truncation. Conversely, the affinity of FAD for the Nox2 complex was enhanced after the truncation. In the absence of Noxo1, Noxa1(1-225) showed much higher activity with a lower EC50 than full-length Noxa1. Noxa1(1-225) showed comparable activity to that of p67(phox) with either Noxo1 or p47(phox), although the stability was lower than that with p67(phox) and p47(phox). These findings indicate that the role of the C-terminal half of Noxa1 is autoinhibition. The data suggest a two-step autoinhibition mechanism, comprising self-masking to interrupt the binding to the oxidase, and holding of the activation domain in a suboptimal position to the oxidase. This study reveals that when both types of inhibition are released, Noxa1 achieves high-level superoxide production.
authors.affiliation: Department of Applied Chemistry, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan.
authors.forename: Masahito
authors.initials: M
authors.lastname: Kawano
authors.forename: Rey
authors.initials: R
authors.lastname: Ishii
authors.forename: Yuki
authors.initials: Y
authors.lastname: Yoshioka
authors.forename: Takehito
authors.initials: T
authors.lastname: Fukuda
authors.forename: Minoru
authors.initials: M
authors.lastname: Tamura
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D019255:NADPH Oxidases / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; D005182:Flavin-Adenine Dinucleotide; D019255:NADPH Oxidases
keywords: Autoinhibition; NADPH oxidase; Nox; Noxa1; Noxo1; Superoxide; Truncation