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Title: C-terminal tail of NADPH oxidase organizer 1 (Noxo1) mediates interaction with NADPH oxidase activator (Noxa1) in the NOX1 complex

Abstract

NOX1 (NADPH oxidase) similar to phagocyte NADPH oxidase, is expressed mainly in the colon epithelium and it is responsible for host defense against microbial infections by generating ROS (reactive oxygen species). NOX1 is activated by two regulatory cytosolic proteins that form a hetero-dimer, Noxo1 (NOX organizer 1) and Noxa1 (NOX activator 1). The interaction between Noxa1 and Noxo1 is critical for activating NOX1. However no structural studies for interaction between Noxa1 and Noxo1 has not been reported till date. Here, we studied the inter-molecular interaction between the SH3 domain of Noxa1 and Noxo1 using pull-down assay and NMR spectroscopy. {sup 15}N/{sup 13}C-labeled SH3 domain of Noxa1 has been purified for hetero-nuclear NMR experiments (HNCACB, CBCACONH, HNCA, HNCO, and HSQC). TALOS analysis using backbone assignment data of the Noxa1 SH3 domain showed that the structure primarily consists of β-sheets. Data from pull-down assay between the Noxo1 and Noxa1 showed that the SH3 domains (Noxa1) is responsible for interaction with Noxo1 C-terminal tail harboring proline rich region (PRR). The concentration-dependent titration of the Noxo1 C-terminal tail to Noxa1 shows that Noxo1 particularly in the RT loop: Q407*, H408, S409, A412*, G414*, E416, D417, L418, and F420; n-Src loop: C430, E431*, V432*, A435,more » W436, and L437; and terminal region: I447; F448*, F452* and V454 interact with Noxa1. Our results will provide a detailed understanding for interaction between Noxa1 and Noxo1 at the molecular level, providing insights into their cytoplasmic activity-mediated functioning as well as regulatory role of C-terminal tail of Noxo1 in the NOX1 complex. - Highlights: • C-terminal of Noxo1 harboring PRR motif is important for Noxa1 interaction. • Noxa1 SH3 binds C-terminal region of Noxo1with K{sub d} of 8.2 ± 1.41 μM. • Data from NMR experiments determined secondary structures of Noxa1 SH3. • Noxa1 interacts with PRR region of Noxo1, however, binding affinity is weaker than C-terminal tail of Noxo1. • Our results provide insights into regulatory role of C-terminal tail of Noxo1 in the NOX1 complex.« less

Authors:
 [1];  [1];  [2];  [1];  [3];  [1]
  1. Structural Biochemistry & Molecular Biophysics Laboratory, Department of Biochemistry, College of Life Sciences & Biotechnology, Yonsei University, Seoul 03722 (Korea, Republic of)
  2. Nuclear Magnetic Resonance Laboratory, National Center for Inter-University Research Facilities, Seoul National University, Seoul 08826 (Korea, Republic of)
  3. Laboratory of Cellular Signal Transduction and Drug Discovery, Department of Life Science, Ewha Womans University, Seoul 03760 (Korea, Republic of)
Publication Date:
OSTI Identifier:
22719043
Resource Type:
Journal Article
Journal Name:
Biochemical and Biophysical Research Communications
Additional Journal Information:
Journal Volume: 490; Journal Issue: 3; Other Information: Copyright (c) 2017 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0006-291X
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; CARBON 13; CONCENTRATION RATIO; LARGE INTESTINE; NITROGEN 15; NUCLEAR MAGNETIC RESONANCE; OXIDASES; OXYGEN; PHAGOCYTES; PROLINE; TITRATION; TRACER TECHNIQUES

Citation Formats

Shrestha, Pravesh, Yun, Ji-hye, Ko, Yoon-Joo, Kim, Myeongkyu, Bae, Yun Soo, and Lee, Weontae. C-terminal tail of NADPH oxidase organizer 1 (Noxo1) mediates interaction with NADPH oxidase activator (Noxa1) in the NOX1 complex. United States: N. p., 2017. Web. doi:10.1016/J.BBRC.2017.06.083.
Shrestha, Pravesh, Yun, Ji-hye, Ko, Yoon-Joo, Kim, Myeongkyu, Bae, Yun Soo, & Lee, Weontae. C-terminal tail of NADPH oxidase organizer 1 (Noxo1) mediates interaction with NADPH oxidase activator (Noxa1) in the NOX1 complex. United States. doi:10.1016/J.BBRC.2017.06.083.
Shrestha, Pravesh, Yun, Ji-hye, Ko, Yoon-Joo, Kim, Myeongkyu, Bae, Yun Soo, and Lee, Weontae. Sat . "C-terminal tail of NADPH oxidase organizer 1 (Noxo1) mediates interaction with NADPH oxidase activator (Noxa1) in the NOX1 complex". United States. doi:10.1016/J.BBRC.2017.06.083.
@article{osti_22719043,
title = {C-terminal tail of NADPH oxidase organizer 1 (Noxo1) mediates interaction with NADPH oxidase activator (Noxa1) in the NOX1 complex},
author = {Shrestha, Pravesh and Yun, Ji-hye and Ko, Yoon-Joo and Kim, Myeongkyu and Bae, Yun Soo and Lee, Weontae},
abstractNote = {NOX1 (NADPH oxidase) similar to phagocyte NADPH oxidase, is expressed mainly in the colon epithelium and it is responsible for host defense against microbial infections by generating ROS (reactive oxygen species). NOX1 is activated by two regulatory cytosolic proteins that form a hetero-dimer, Noxo1 (NOX organizer 1) and Noxa1 (NOX activator 1). The interaction between Noxa1 and Noxo1 is critical for activating NOX1. However no structural studies for interaction between Noxa1 and Noxo1 has not been reported till date. Here, we studied the inter-molecular interaction between the SH3 domain of Noxa1 and Noxo1 using pull-down assay and NMR spectroscopy. {sup 15}N/{sup 13}C-labeled SH3 domain of Noxa1 has been purified for hetero-nuclear NMR experiments (HNCACB, CBCACONH, HNCA, HNCO, and HSQC). TALOS analysis using backbone assignment data of the Noxa1 SH3 domain showed that the structure primarily consists of β-sheets. Data from pull-down assay between the Noxo1 and Noxa1 showed that the SH3 domains (Noxa1) is responsible for interaction with Noxo1 C-terminal tail harboring proline rich region (PRR). The concentration-dependent titration of the Noxo1 C-terminal tail to Noxa1 shows that Noxo1 particularly in the RT loop: Q407*, H408, S409, A412*, G414*, E416, D417, L418, and F420; n-Src loop: C430, E431*, V432*, A435, W436, and L437; and terminal region: I447; F448*, F452* and V454 interact with Noxa1. Our results will provide a detailed understanding for interaction between Noxa1 and Noxo1 at the molecular level, providing insights into their cytoplasmic activity-mediated functioning as well as regulatory role of C-terminal tail of Noxo1 in the NOX1 complex. - Highlights: • C-terminal of Noxo1 harboring PRR motif is important for Noxa1 interaction. • Noxa1 SH3 binds C-terminal region of Noxo1with K{sub d} of 8.2 ± 1.41 μM. • Data from NMR experiments determined secondary structures of Noxa1 SH3. • Noxa1 interacts with PRR region of Noxo1, however, binding affinity is weaker than C-terminal tail of Noxo1. • Our results provide insights into regulatory role of C-terminal tail of Noxo1 in the NOX1 complex.},
doi = {10.1016/J.BBRC.2017.06.083},
journal = {Biochemical and Biophysical Research Communications},
issn = {0006-291X},
number = 3,
volume = 490,
place = {United States},
year = {2017},
month = {8}
}