Nox5 forms a functional oligomer mediated by self-association of its dehydrogenase domain

Biochemistry. 2011 Mar 29;50(12):2013-25. doi: 10.1021/bi1020088. Epub 2011 Mar 4.

Abstract

Nox5 belongs to the calcium-regulated subfamily of NADPH oxidases (Nox). Like other calcium-regulated Noxes, Nox5 has an EF-hand-containing calcium-binding domain at its N-terminus, a transmembrane heme-containing region, and a C-terminal dehydrogenase (DH) domain that binds FAD and NADPH. While Nox1-4 require regulatory subunits, including p22phox, Nox5 activity does not depend on any subunits. We found that inactive point mutants and truncated forms of Nox5 (including the naturally expressed splice form, Nox5S) inhibit full-length Nox5, consistent with formation of a dominant negative complex. Oligomerization of full-length Nox5 was demonstrated using co-immunoprecipitation of coexpressed, differentially tagged forms of Nox5 and occurred in a manner independent of calcium ion. Several approaches were used to show that the DH domain mediates oligomerization: Nox5 could be isolated as a multimer when the calcium-binding domain and/or the N-terminal polybasic region (PBR-N) was deleted, but deletion of the DH domain eliminated oligomerization. Further, a chimera containing the transmembrane domain of Ciona intestinalis voltage sensor-containing phosphatase (CiVSP) fused to the Nox5 DH domain formed a co-immunoprecipitating complex with, and functioned as a dominant inhibitor of, full-length Nox5. Radiation inactivation of Nox5 overexpressed in HEK293 cells and endogenously expressed in human aortic smooth muscle cells indicated molecular masses of ∼350 and ∼300 kDa, respectively, consistent with a tetramer being the functionally active unit. Thus, Nox5 forms a catalytically active oligomer in the membrane that is mediated by its dehydrogenase domain. As a result of oligomerization, the short, calcium-independent splice form, Nox5S, may function as an endogenous inhibitor of calcium-stimulated ROS generation by full-length Nox5.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cell Membrane / enzymology
  • Cell Membrane / metabolism
  • HEK293 Cells
  • Humans
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Molecular Sequence Data
  • Mutagenesis
  • Mutation
  • NADPH Oxidase 5
  • NADPH Oxidases / chemistry*
  • NADPH Oxidases / genetics
  • NADPH Oxidases / metabolism*
  • Protein Multimerization*
  • Protein Structure, Quaternary
  • Protein Structure, Tertiary
  • Protein Subunits / chemistry
  • Protein Subunits / metabolism

Substances

  • Membrane Proteins
  • Protein Subunits
  • NADPH Oxidase 5
  • NADPH Oxidases
  • NOX5 protein, human

Associated data

  • GENBANK/AF353088