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Regulation of NOXO1 activity through reversible interactions with p22 and NOXA1.

Dutta S, Rittinger K - PLoS ONE (2010)

Bottom Line: The source of these chemicals are members of the NOX family of NADPH oxidases that are found in a variety of tissues.Furthermore, we provide data indicating that the molecular details of the interaction between NOXO1 and NOXA1 is significantly different from that between the homologous proteins of the phagocytic oxidase, suggesting that there are important functional differences between the two systems.Taken together, this study provides clear evidence that the assembly of the NOX1 oxidase complex can be regulated through reversible protein-protein interactions.

View Article: PubMed Central - PubMed

Affiliation: National Institute for Medical Research, Medical Research Council, London, United Kingdom.

ABSTRACT
Reactive oxygen species (ROS) have been known for a long time to play important roles in host defense against microbial infections. In addition, it has become apparent that they also perform regulatory roles in signal transduction and cell proliferation. The source of these chemicals are members of the NOX family of NADPH oxidases that are found in a variety of tissues. NOX1, an NADPH oxidase homologue that is most abundantly expressed in colon epithelial cells, requires the regulatory subunits NOXO1 (NOX organizing protein 1) and NOXA1 (NOX activating protein 1), as well as the flavocytochrome component p22(phox) for maximal activity. Unlike NOX2, the phagocytic NADPH oxidase whose activity is tightly repressed in the resting state, NOX1 produces superoxide constitutively at low levels. These levels can be further increased in a stimulus-dependent manner, yet the molecular details regulating this activity are not fully understood. Here we present the first quantitative characterization of the interactions made between the cytosolic regulators NOXO1 and NOXA1 and membrane-bound p22(phox). Using isothermal titration calorimetry we show that the isolated tandem SH3 domains of NOXO1 bind to p22(phox) with high affinity, most likely adopting a superSH3 domain conformation. In contrast, complex formation is severely inhibited in the presence of the C-terminal tail of NOXO1, suggesting that this region competes for binding to p22(phox) and thereby contributes to the regulation of superoxide production. Furthermore, we provide data indicating that the molecular details of the interaction between NOXO1 and NOXA1 is significantly different from that between the homologous proteins of the phagocytic oxidase, suggesting that there are important functional differences between the two systems. Taken together, this study provides clear evidence that the assembly of the NOX1 oxidase complex can be regulated through reversible protein-protein interactions.

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Schematic representation of the domain structures of p47phox, NOXO1, p67phox and NOXA1.(A) Predicted domain structures of NOXO1 and NOXA1 in comparison to p47phox and p67phox, respectively. Human and mouse constructs used in this study are illustrated by black lines. Mouse constructs are identical in length unless otherwise stated in brackets. The autoinhibitory region (AIR) and proline rich region (PPR) are indicated. (B) Alignment of p47phox, human NOXO1 and mouse NOXO1. The PX domains (grey shaded), SH3 domains (cyan shaded), polybasic region (orange shaded ox) and proline rich motif (green shaded) are indicated. (C) Structure of the autoinhibited core of p47phox showing the superSH3 domain conformation. The structure shows the biologically relevant monomeric form of the protein that is also observed in solution, not the domain-swapped crystallized dimer [22]. The SH3 domains and polybasic region are highlighted in cyan and orange, respectively.
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pone-0010478-g001: Schematic representation of the domain structures of p47phox, NOXO1, p67phox and NOXA1.(A) Predicted domain structures of NOXO1 and NOXA1 in comparison to p47phox and p67phox, respectively. Human and mouse constructs used in this study are illustrated by black lines. Mouse constructs are identical in length unless otherwise stated in brackets. The autoinhibitory region (AIR) and proline rich region (PPR) are indicated. (B) Alignment of p47phox, human NOXO1 and mouse NOXO1. The PX domains (grey shaded), SH3 domains (cyan shaded), polybasic region (orange shaded ox) and proline rich motif (green shaded) are indicated. (C) Structure of the autoinhibited core of p47phox showing the superSH3 domain conformation. The structure shows the biologically relevant monomeric form of the protein that is also observed in solution, not the domain-swapped crystallized dimer [22]. The SH3 domains and polybasic region are highlighted in cyan and orange, respectively.

Mentions: The phagocytic oxidase is still the best characterized member of this protein family and consists of a heterodimeric flavocytochrome (containing NOX2 and p22phox) that makes up the catalytic core of the enzyme plus the cytosolic regulatory subunits p40phox, p47phox and p67phox and the small GTPase Rac [8], [16]–[18]. Because excessive ROS production is cytotoxic and can induce a number of pathological processes, complicated regulatory mechanisms have evolved that tightly control the activity of the phagocytic oxidase. In the resting state, inappropriate activation is prevented through the partitioning of its subunits between the cytosol (p40phox, p47phox, p67phox and Rac) and the membrane (NOX2, p22phox). The resting, cytoplasmic location of a trimeric p40-p67-p47phox complex is maintained due to an autoinhibitory conformation of p47phox that prevents the interaction with the membrane-bound flavocytochrome [18]. Cell activation induces phosphorylation of p47phox at a number of serine residues leading to conformational changes and ultimately relieving autoinhibition. This in turn allows p47phox to interact with a consensus PxxP motif within the cytoplasmic portion of p22phox, thereby promoting membrane translocation of the p40-p67-p47phox complex and assembly of the active NADPH oxidase enzyme [19]–[22]. Due to these multiple activities of p47phox this subunit plays a key role in NADPH oxidase function, acting simultaneously as a regulator of oxidase activity and an adaptor protein promoting enzyme assembly. In the autoinhibited state its tandem SH3 (Src homology 3) domains are engaged in an intramolecular interaction with a region rich in basic residues (polybasic region, also referred to as autoinhibitory region AIR) and hence are prevented from interacting with p22phox [21], [23](Figure 1). The crystal structure of the autoinhibited core of p47phox revealed that the tandem SH3 domains adopt an unusual, novel conformation, termed the superSH3 domain, in which the two SH3 domains co-operate to form a single ligand binding site that is occupied by a non-canonical motif located in the N-terminal portion of the polybasic region [22], [24]. Extensive, additional contacts are made between the polybasic region and the reminder of p47phox, outside of the core superSH3 domain ligand binding site that are important to enforce the resting state. Multiple serine residues within this polybasic region become phosphorylated upon activation, a process that weakens the intramolecular interactions and instead favours complex formation with the membrane-bound cytochrome. Additionally, a proline-rich region in the C-terminal portion of p47phox mediates the interaction with p67phox via specific recognition of its C-terminal SH3 domain. This interaction, which occurs with an unusually high affinity of 20 nM [25]–[27], is absolutely required for translocation of p67phox to the membrane.


Regulation of NOXO1 activity through reversible interactions with p22 and NOXA1.

Dutta S, Rittinger K - PLoS ONE (2010)

Schematic representation of the domain structures of p47phox, NOXO1, p67phox and NOXA1.(A) Predicted domain structures of NOXO1 and NOXA1 in comparison to p47phox and p67phox, respectively. Human and mouse constructs used in this study are illustrated by black lines. Mouse constructs are identical in length unless otherwise stated in brackets. The autoinhibitory region (AIR) and proline rich region (PPR) are indicated. (B) Alignment of p47phox, human NOXO1 and mouse NOXO1. The PX domains (grey shaded), SH3 domains (cyan shaded), polybasic region (orange shaded ox) and proline rich motif (green shaded) are indicated. (C) Structure of the autoinhibited core of p47phox showing the superSH3 domain conformation. The structure shows the biologically relevant monomeric form of the protein that is also observed in solution, not the domain-swapped crystallized dimer [22]. The SH3 domains and polybasic region are highlighted in cyan and orange, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2864300&req=5

pone-0010478-g001: Schematic representation of the domain structures of p47phox, NOXO1, p67phox and NOXA1.(A) Predicted domain structures of NOXO1 and NOXA1 in comparison to p47phox and p67phox, respectively. Human and mouse constructs used in this study are illustrated by black lines. Mouse constructs are identical in length unless otherwise stated in brackets. The autoinhibitory region (AIR) and proline rich region (PPR) are indicated. (B) Alignment of p47phox, human NOXO1 and mouse NOXO1. The PX domains (grey shaded), SH3 domains (cyan shaded), polybasic region (orange shaded ox) and proline rich motif (green shaded) are indicated. (C) Structure of the autoinhibited core of p47phox showing the superSH3 domain conformation. The structure shows the biologically relevant monomeric form of the protein that is also observed in solution, not the domain-swapped crystallized dimer [22]. The SH3 domains and polybasic region are highlighted in cyan and orange, respectively.
Mentions: The phagocytic oxidase is still the best characterized member of this protein family and consists of a heterodimeric flavocytochrome (containing NOX2 and p22phox) that makes up the catalytic core of the enzyme plus the cytosolic regulatory subunits p40phox, p47phox and p67phox and the small GTPase Rac [8], [16]–[18]. Because excessive ROS production is cytotoxic and can induce a number of pathological processes, complicated regulatory mechanisms have evolved that tightly control the activity of the phagocytic oxidase. In the resting state, inappropriate activation is prevented through the partitioning of its subunits between the cytosol (p40phox, p47phox, p67phox and Rac) and the membrane (NOX2, p22phox). The resting, cytoplasmic location of a trimeric p40-p67-p47phox complex is maintained due to an autoinhibitory conformation of p47phox that prevents the interaction with the membrane-bound flavocytochrome [18]. Cell activation induces phosphorylation of p47phox at a number of serine residues leading to conformational changes and ultimately relieving autoinhibition. This in turn allows p47phox to interact with a consensus PxxP motif within the cytoplasmic portion of p22phox, thereby promoting membrane translocation of the p40-p67-p47phox complex and assembly of the active NADPH oxidase enzyme [19]–[22]. Due to these multiple activities of p47phox this subunit plays a key role in NADPH oxidase function, acting simultaneously as a regulator of oxidase activity and an adaptor protein promoting enzyme assembly. In the autoinhibited state its tandem SH3 (Src homology 3) domains are engaged in an intramolecular interaction with a region rich in basic residues (polybasic region, also referred to as autoinhibitory region AIR) and hence are prevented from interacting with p22phox [21], [23](Figure 1). The crystal structure of the autoinhibited core of p47phox revealed that the tandem SH3 domains adopt an unusual, novel conformation, termed the superSH3 domain, in which the two SH3 domains co-operate to form a single ligand binding site that is occupied by a non-canonical motif located in the N-terminal portion of the polybasic region [22], [24]. Extensive, additional contacts are made between the polybasic region and the reminder of p47phox, outside of the core superSH3 domain ligand binding site that are important to enforce the resting state. Multiple serine residues within this polybasic region become phosphorylated upon activation, a process that weakens the intramolecular interactions and instead favours complex formation with the membrane-bound cytochrome. Additionally, a proline-rich region in the C-terminal portion of p47phox mediates the interaction with p67phox via specific recognition of its C-terminal SH3 domain. This interaction, which occurs with an unusually high affinity of 20 nM [25]–[27], is absolutely required for translocation of p67phox to the membrane.

Bottom Line: The source of these chemicals are members of the NOX family of NADPH oxidases that are found in a variety of tissues.Furthermore, we provide data indicating that the molecular details of the interaction between NOXO1 and NOXA1 is significantly different from that between the homologous proteins of the phagocytic oxidase, suggesting that there are important functional differences between the two systems.Taken together, this study provides clear evidence that the assembly of the NOX1 oxidase complex can be regulated through reversible protein-protein interactions.

View Article: PubMed Central - PubMed

Affiliation: National Institute for Medical Research, Medical Research Council, London, United Kingdom.

ABSTRACT
Reactive oxygen species (ROS) have been known for a long time to play important roles in host defense against microbial infections. In addition, it has become apparent that they also perform regulatory roles in signal transduction and cell proliferation. The source of these chemicals are members of the NOX family of NADPH oxidases that are found in a variety of tissues. NOX1, an NADPH oxidase homologue that is most abundantly expressed in colon epithelial cells, requires the regulatory subunits NOXO1 (NOX organizing protein 1) and NOXA1 (NOX activating protein 1), as well as the flavocytochrome component p22(phox) for maximal activity. Unlike NOX2, the phagocytic NADPH oxidase whose activity is tightly repressed in the resting state, NOX1 produces superoxide constitutively at low levels. These levels can be further increased in a stimulus-dependent manner, yet the molecular details regulating this activity are not fully understood. Here we present the first quantitative characterization of the interactions made between the cytosolic regulators NOXO1 and NOXA1 and membrane-bound p22(phox). Using isothermal titration calorimetry we show that the isolated tandem SH3 domains of NOXO1 bind to p22(phox) with high affinity, most likely adopting a superSH3 domain conformation. In contrast, complex formation is severely inhibited in the presence of the C-terminal tail of NOXO1, suggesting that this region competes for binding to p22(phox) and thereby contributes to the regulation of superoxide production. Furthermore, we provide data indicating that the molecular details of the interaction between NOXO1 and NOXA1 is significantly different from that between the homologous proteins of the phagocytic oxidase, suggesting that there are important functional differences between the two systems. Taken together, this study provides clear evidence that the assembly of the NOX1 oxidase complex can be regulated through reversible protein-protein interactions.

Show MeSH
Related in: MedlinePlus