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Conserved Cysteine Residues Provide a Protein-Protein Interaction Surface in Dual Oxidase (DUOX) Proteins
Journal article   Open access   Peer reviewed

Conserved Cysteine Residues Provide a Protein-Protein Interaction Surface in Dual Oxidase (DUOX) Proteins

Jennifer L. Meitzler, Sara Hinde, Botond Bánfi, William M. Nauseef and Paul R. Ortiz de Montellano
The Journal of biological chemistry, Vol.288(10), pp.7147-7157
03/08/2013
DOI: 10.1074/jbc.M112.414797
PMCID: PMC3591624
PMID: 23362256
url
https://doi.org/10.1074/jbc.M112.414797View
Published (Version of record) Open Access

Abstract

Intramolecular disulfide bond formation is promoted in oxidizing extracellular and endoplasmic reticulum compartments and often contributes to protein stability and function. DUOX1 and DUOX2 are distinguished from other members of the NOX protein family by the presence of a unique extracellular N-terminal region. These peroxidase-like domains lack the conserved cysteines that confer structural stability to mammalian peroxidases. Sequence-based structure predictions suggest that the thiol groups present are solvent-exposed on a single protein surface and are too distant to support intramolecular disulfide bond formation. To investigate the role of these thiol residues, we introduced four individual cysteine to glycine mutations in the peroxidase-like domains of both human DUOXs and purified the recombinant proteins. The mutations caused little change in the stabilities of the monomeric proteins, supporting the hypothesis that the thiol residues are solvent-exposed and not involved in disulfide bonds that are critical for structural integrity. However, the ability of the isolated hDUOX1 peroxidase-like domain to dimerize was altered, suggesting a role for these cysteines in protein-protein interactions that could facilitate homodimerization of the peroxidase-like domain or, in the full-length protein, heterodimeric interactions with a maturation protein. When full-length hDUOX1 was expressed in HEK293 cells, the mutations resulted in decreased H2O2 production that correlated with a decreased amount of the enzyme localized to the membrane surface rather than with a loss of activity or with a failure to synthesize the mutant proteins. These results support a role for the cysteine residues in intermolecular disulfide bond formation with the DUOX maturation factor DUOXA1. Background: Dual oxidases (DUOXs) are membrane-bound ROS-generating enzymes. Results: Conserved DUOX cysteines localized in an N-terminal domain contribute to enzymatic maturation, independent of structural stabilization. Conclusion: Intermolecular disulfides support the interaction between DUOX enzymes and their maturation factors. Significance: This study reflects a complex profile of protein interactions required for activity and localization of the DUOX enzymes.
 Protein Folding Cell Surface Protein Chaperone Chaperonin Cysteine-mediated Cross-linking Disulfide DUOX Maturation NADPH Oxidase NOX

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