Albany 2015:Book of Abstracts
June 9-13 2015
©Adenine Press (2012)
Structural Basis of the Effect of Systemic Lupus Erythematosus (SLE)-Associated Mutations on the Activity of NADPH Oxidase
NADPH oxidase is a multiple subunits complex, important for the generation of reactive oxygen species (ROS). ROS formed in phagocytes promote the killing of microbes but in immune cells ROS production is limited and reduced production may influence antigen processing, immunoregulation and other functions that contribute to the development of autoimmune diseases. Two NADPH subunits, CYBA and CYBB, are localized in cellular membranes. The three cytosolic subunits NCF1, NCF2 and NCF4 translocate upon activation to the membrane where together with RAC-GTP they form the active NOX2 complex1. Dissociation of RAC-GDP and formation of RAC-GTP is regulated by guanine nucleotide exchange factors (GEFs) such as the VAV proteins. NCF2 interacts directly with VAV1, enhances its GEF activity and amplifies RAC nucleotide exchange and NADPH oxidase activation (2)
SLE is a multisystem autoimmune disorder with extensive immune dysregulation and chronic inflammation affecting multiple organs. Its development is associated with mutations in multiple genes. In this study we tested the candidacy of NCF2 as a risk factor for SLE. We found that association between NCF2 and SLE in North Americans of European descent could be attributed to a single non-synonymous coding mutation in the PB1 domain of NCF2, H389->Q (3) The same mutation is also a risk factor in Northern American Hispanic population. Another NCF2-PB1 mutation, R395->W, is independently associated with SLE in Hispanics but not in Americans of European descent (4).
The interaction of NCF2 PB1 domain with VAV1 was studied by computational docking and confirmed experimentally. NCF2 H389 interacts with the zinc finger of VAVI (3). NCF2 R395 stabilizes the conformation of NCF2 loop 395-402, interacts with the C-terminal tail of NCF4 and together they bind to the DH domain of VAV1. The interactions in both sites exploit evolutionarily conserved residues. The two-point interaction of NCF2/NCF4 with VAV1 acts to stabilize VAV1 in a conformation adequate for nucleotide-free RAC1 binding. Our model of the quaternary complex NCF2/NCF4/VAV1/RAC1 further predicts that NCF2 loop 395-402 interacts with the insertion domain of RAC1 (residues 120-137) and with switch-II loop 60-66(4)Switch-II changes conformation between the GDP-bound and nucleotide-free RAC1 promoting release of GDP. Its proximity to NCF2 loop 395-402 suggests that NCF2 contributes to the stabilization of nucleotide-free RAC1, thus enhancing the nucleotide exchange process.
Support for this study was provided by the National Institutes of Health grant [RO1AR0571720 to COJ] and by a grant from the Alliance for Lupus Research to COJ.
2. Ming, W.; Li, S.; Billadeau, D. D.; Quilliam, L. A.; Dinauer, M. C. Molecular and cellular biology 2007, 27, 312.
3. Jacob, C. O.; Eisenstein, M.; Dinauer, M. C.; Ming, W.; Liu, Q.; John, S.; Quismorio, F. P., Jr.; Reiff, A.; Myones, B. L.; Kaufman, K. M.; McCurdy, D.; Harley, J. B.; Silverman, E.; Kimberly, R. P.; Vyse, T. J.; Gaffney, P. M.; Moser, K. L.; Klein-Gitelman, M.; Wagner-Weiner, L.; Langefeld, C. D.; Armstrong, D. L.; Zidovetzki, R. Proceedings of the National Academy of Sciences of the United States of America 2012, 109, E59.
4. Armstrong, D. L.; Eisenstein, M.; Zidovetzki, R.; Jacob, C. O. The Journal of biological chemistry 2015.
Don L. Armstrong2
1 Department of Chemical Research Support