Structural and Functional Analysis of NADPH Oxidase 4 Public
Jackson, Heather Marie (2010)
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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