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Structural and Functional Analysis of NADPH Oxidase 4

Jackson, Heather Marie (2010)
Dissertation (210 pages)
Committee Chair / Thesis Adviser: Lambeth, David J
Committee Members: Cheng, Xiaodong ; Matsumura, Ichiro ; Hartzell Jr., Criss ; Hepler, John R
Research Fields: Chemistry, Biochemistry; Biology, Bioinformatics
Keywords: NADPH Oxidase
Program: Laney Graduate School, Biological and Biomedical Sciences (Biochemistry, Cell & Developmental Biology)
Permanent url: http://pid.emory.edu/ark:/25593/8jmr8

Abstract

NADPH oxidases (Nox) are integral membrane oxidoreductases that
catalyze the transfer of electrons from NADPH to molecular oxygen producing
superoxide anion and secondary reactive oxygen species (ROS). These ROS react with
proteins or microbes linking Nox enzymes to important roles in cell signaling and innate
immunity. All seven mammalian isoforms (Nox1-5, Duox1-2) are composed of a heme-
binding transmembrane domain and an NADPH- and FAD-binding dehydrogenase
domain. Unlike the regulatory subunit-dependent Nox1-3 or EF-hand containing Nox5
and Duox1/2, Nox4 displays spontaneous activity. We hypothesize that Nox4 provides a
likely model for the active conformation of other Nox isoforms.
In an effort to understand the structural features of these enzymes that control
activity, we used a combination of computational modeling and molecular evolution
analysis to identify regions of the enzymes of possible functional significance and tested
these predictions. A Nox4 dehydrogenase domain homology model was constructed
using crystal structures of the ferredoxin reductase super-family as templates. This
model along with a model of the heme-binding domain predicted the B-loop of the
transmembrane domain is in contact with the dehydrogenase domain in the holo-enzyme.
We used fluorescence polarization to detect an interaction between
recombinant Nox4 dehydrogenase domain and Nox4 B-loop peptides, consistent with the
model prediction. This interaction is also detected in Nox2 and mutations in the B-loop
that abolish these interactions also inhibit Nox2 or Nox4 activity, implying a functional
role for the B-loop: dehydrogenase domain interaction.
Comparisons between the Nox4 dehydrogenase domain model and similar
structures identified important ligand-binding sites and insertions in the Nox/Duox family
absent in the other structures. We hypothesize that these differences in the Nox/Duox
family sequences mediate the regulatory mechanisms seen with the various Nox/Duox
isoforms. Chimera proteins of Nox2 and Nox4 identify the dehydrogenase domain as
being the part of the protein responsible for determining subunit-dependent or
spontaneous activity. Furthermore, recombinant Nox4 dehydrogenase domain exhibits constitutive electron transferase activity. This structural
and functional analysis of the Nox enzymes improves our understanding of how these
enzymes work and provides a framework to aid in specific drug design.

Table of Contents

Chapter 1.
Introduction to the Nox family of NADPH Oxidases...............................................………………….1
1.1. NADPH Oxidases and Reactive Oxygen Species….……..…………………...............................…1
1.2. Nox/Duox Enzymes and their roles in Physiology and Pathophysiology……...........................8
Nox1……………………………………………………………………….....................................................8
Nox2……………………………………………………………….............................................................11
Nox3…………………………………………………………....…............................................................14
Nox4……………………………………………………….………............................................................15
Nox5………………………………………………….……………............................................................18
Duox1/ Duox2…………………………...…………………………........................................................20
1.3. Activity Regulation of Nox/Duox Enzymes…………………..………………................................22
p22- phox dependent Nox subfamily..…………………………….....................................................23
Regulatory-subunit dependent subfamily Nox1-3………………....................................................23
Organizer subunits……………...………………………....................................................................23
Activator subunits……...……………………………….....................................................................28
Small GTPases…………………………………………........................................................................29
Oxidase Assembly……………………………………….....................................................................32
Constitutively Active Nox4………………………...….....................................................................34
EF-hand dependent Nox subfamily……………………………..............................................….........36
Nox5……………………………………………………..........................................................................36
Duox1/ Duox2………………………………………...........................................................….............36
1.4. Scope of Dissertation…………………………………………………...................................………...38
Chapter 2.
Homology Model of the Nox4 Dehydrogenase domain………………..............................................39
2.1 Introduction…………………………………………………………………….......................................40
2.2 Model Construction…………………………….…………………………….....................................…43
2.2.1 Basis Set Proteins……………………………………………………..................................….........43
2.2.2 FAD-binding domain……………………………………………………...........................................48
2.2.3 NADPH-binding domain…………………………………………...................................................53
2.3 Results and Discussion…………………………………………………….……....................................64
2.3.1 FAD and NADPH binding sites……………………………………….....................................…......64
2.3.2 Conserved Regions in all Nox/Duox Sequences……………………….......................................78
2.3.3 Insertions…………………………………………………….........................................…………......83
2.3.4 Homology models of the transmembrane and dehydrogenase domains of Nox4 ………..........89
2.4 Conclusions and Future Directions…....................................…………………………………………91

Chapter 3.
The Nox4 B-loop interacts with the dehydrogenase domain……................................................…93
3.1 Introduction…………………………………………………………………….........................................94
3.2 Experimental Procedures………………………………………………......................................………96
3.3 Results…………………………………………………………………………..........................................104
3.3.1 Conservation of the B-loop region in Vertebrate Nox Enzymes……..................................….104
3.3.2 Amino acids in the Nox4 B-loop are important for Nox4 Activity…….....................................106
3.3.3 Nox4 B-loop binds to recombinant Nox4 dehydrogenase (DH) domain.....…...........…………...109
3.3.4 Truncations of the Nox4 DH domain identify a B-loop-binding sub-domain……...........…........117

3.3.5 Isoform specificity of Nox DH domain: B-loop interaction....................................................126
3.3.6 Effect of B-loop peptides on Nox4 Activity…………………………….........................................133
3.4 Discussion and Future Directions…………………………………………….......................................137

Chapter 4.
The dehydrogenase domain of Nox 1-4 confers subunit-dependent or independent activity…....…..147
4.1 Introduction…………………………………………………………………............................................148
4.2 Experimental Procedures……………………………………….......................................……...….….150
4.3 Results………………………………………………………………..........................................……….…154
4.3 1 ROS Generation by Nox2, Nox4, and Nox2/4 and Nox4/2 Chimeric Proteins…………………......154
4.3.2 The Nox4 DH domain displays constitutive electron transferring activity…………...……..........157
4.3.3 Residues conserved exclusively in Nox1-3 affect activity……………......................................161
4.4 Discussion and Future directions…………………………………………….......................................165
Overall Conclusions………………………………………………………………….......................................174
References…………………………………………………………………....................................…………...176

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