New Paper Describes Dual Rac2 Regulatory Role

By Jason Socrates Bardi

In a new Nature Immunology paper that is both the culmination of several years of research and a roadmap for future inquiries, The Scripps Research Institute (TSRI) Professor Gary Bokoch and Research Associate Becky Diebold describe the molecular details of the regulation of NADPH oxidase by the protein Rac2.

“For the past 10 years we’ve been trying to find the role of Rac2 in regulating NADPH oxidase function,” says Bokoch, whose immunology laboratory primarily studies GTP binding proteins involved in signal transduction. "Understanding this mechanism will have a lot of different implications for many areas of research."

NADPH oxidase is a multisubunit enzyme that assembles on the surface of various cell types, including phagocytic leukocytes, the blood cells that destroy foreign pathogens in an innate immune response. The pathogens are engulfed by the leukocytes and then are destroyed by the NADPH oxidases, which blast them with highly reactive superoxide anions.

These NADPH oxidases must be tightly regulated or else they will "shoot" indiscriminately, oxidizing normal cells and damaging healthy tissue. Phagocytic leukocytes regulate the NADPH oxidase complexes by segregating the individual components, bringing the subunits together only when needed during an immune response. Rac2 apparently provides an additional level of regulation, controlling subsequent oxidant formation by a two-step molecular mechanism.

The active subunit of the NADPH complex is the heterodimer protein cytochrome b, which binds a heme and facilitates an electron cascade that produces the superoxide molecules. When the cell is not destroying foreign bodies, the cytochrome b subunit stands alone in the membrane, unloaded as it were.

But when it engulfs a pathogen, the small GTP binding protein Rac2 associates with p67, another piece of the NADPH complex in the cytosol, and they translocate to the cellular membrane where they associate with the cytochrome b, activating it.

In their paper, Diebold and Bokoch demonstrate that Rac2 regulates the activation of the NADPH oxidase complex by binding to p67 and bringing it to the membrane where it assembles with cytochrome b, turning on the electron flux.

But when it engulfs a pathogen, the small GTP binding protein Rac2 is activated and, separately but simultaneously, translocates to the cellular membrane along with p47 and p67, other cytosolic oxidase components, to "load" the oxidase.

Diebold and Bokoch also demonstrate that Rac2 regulates cytochrome b directly through a physical interaction involving a unique Rac "insert domain." This results in the first step in the overall electron transfer reaction. The second step requires Rac2 to additionally interact with p67 to finally generate superoxide. This dual regulatory role for Rac2 had not been previously known.

The researchers believe the findings are most relevant to inflammation, atherosclerosis, and even cancer because non-inflammatory cells also carry NADPH oxidases. The capability to modulate oxidant production without totally disarming the protective leukocyte immune response could provide an improved therapeutic approach to diseases such as arthritis, cardiac tissue damage associated with heart attack, and atherosclerosis. The present work may also provide clues to elucidating the mechanisms operative in other signaling systems in which the Rac2 protein plays a direct regulatory role.

The article, "Molecular basis for Rac2 regulation of phagocyte NADPH oxidase," by Becky A. Diebold and Gary M. Bokoch, appears in the March 2001 issue of the journal Nature Immunology.

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“Understanding this mechanism will have different implications for many areas of research.”

—Gary Bokoch