News and Publications

Division of Biochemistry

Bernard M. Babior, M.D., Ph.D., Division Head

Oxygen-Dependent Bacterial Killing by Neutrophils and Role of Mitochondria in Apoptosis

B.M. Babior

OXYGEN-DEPENDENT KILLING BY NEUTROPHILS

Neutrophils are small, highly motile cells that seek out and destroy invading pathogens. Among their microbicidal weapons is a mixture of reactive oxidants that kill the invaders by chemical combustion. These oxidants are all formed from superoxide (O₂^-), a compound generated by the 1-electron reduction of oxygen. Superoxide is produced by the leukocyte NADPH oxidase, an enzyme that is dormant in resting neutrophils but is activated when the cells encounter invading microorganisms. The activated oxidase catalyzes the production of O₂^- from oxygen and NADPH:

The leukocyte NADPH oxidase is a complicated enzyme with a group of 5 core subunits that transfer electrons from NADPH to oxygen and an unknown number of auxiliary subunits that regulate the activity of the enzyme. Two of the core subunits are in the plasma membrane, and 3 are in the cytosol. When the cell is activated, the cytosolic subunits together with one of the auxiliary subunits migrate to the membrane to assemble the working oxidase (Fig. 1).

The chief interest of this laboratory is the mechanism of activation of the oxidase. The phosphorylation of serines on the cytosolic subunit p47^PHOX is a key event in this activation. Although p47^PHOX contains 30 serines, phosphorylation is restricted to 9--10 serines in the C-terminal quarter of the molecule. The only indispensable phosphorylated serine is Ser379, whose state of phosphorylation is uncertain at this time. We have detected pairs of serines for which phosphorylation of at least 1 member of the pair is necessary for oxidase activity. In earlier work, we showed that Ser303 and Ser304 are such a pair. We recently found that Ser359 and Ser370 are another such pair. A p47^PHOX mutant in which these serines are replaced by alanine not only does not support production of O₂^- but also does not translocate and is not even phosphorylated either in vitro or in whole cells. These serines must therefore be the first to be phosphorylated when the oxidase is activated; their phosphorylation must somehow alter the conformation of the p47^PHOX molecule so that the remaining serines become available to the phosphorylating kinase.

The 6 serines between Ser310 and Ser348 inclusive are not required for oxidase activity, although some evidence suggests that the phosphorylation of one or more of these serines may be involved in the regulation of the oxidase. Most puzzling of all is Ser379. If this serine is converted to an alanine, transfer of p47^PHOX to the membrane is prevented. The state of phosphorylation of p47^PHOX in the active enzyme, however, is curiously ambiguous. We are working to resolve this ambiguity.

We are also investigating the mechanism of activation of the oxidase by using a kinase-dependent cell-free oxidase activating system that we developed during the preceding 2 years. Oxidase activation in this system requires only 3 cytosolic oxidase components: p47^PHOX and p67^PHOX, which are 2 of the 5 core oxidase components, and Rac2, a low molecular weight G protein. Neutrophil membranes are also required, not only because they contain the 2 membrane-associated core components but also because they also contain a third protein whose phosphorylation is required for oxidase activation.

We think that this third protein is Rap1A, a low molecular weight G protein whose participation in oxidase activity has been shown by other investigators. We found that incubating neutrophil membranes with ATP results in the phosphorylation of Rap1A and that inhibitors that interfere with the phosphorylation of Rap1A interfere with oxidase activity to a similar extent. Studies to better define the role of Rap1A in oxidase activation in this system are in progress.

In earlier studies, we showed that the core cytosolic component p67^PHOX has an NADPH-binding site whose properties suggest that it could be the substrate-binding site for the oxidase. We have now found that p67^PHOX has NADPH dehydrogenase activity, transporting electrons from NADPH to Fe(CN)₆^-3. The activity is low, however, and we are trying to determine whether NADPH dehydrogenation can be accelerated by adding other cytosolic oxidase components.

MITOCHONDRIA AND APOPTOSIS

Studies on the role of mitochondria in apoptosis are continuing in collaboration with R. Gottlieb, Department of Molecular and Experimental Medicine. We have shown that the release of cytochrome c, an essential element in mitochondria-dependent apoptosis, requires permeabilization of the outer membrane of the mitochondria of apoptotic cells. We are attempting to purify the protein responsible for the loss of interaction of cytochrome c with the rest of the mitochondrial electron transport chain and are looking at other aspects of mitochondrial function in apoptosis.

PUBLICATIONS

Adachi, S., Cross, A.R., Babior, B.M., Gottlieb, R.A. Bcl-2 and the outer mitochondrial membrane in the activation of cytochrome c during Fas-mediated apoptosis. J. Biol. Chem. 272:21878, 1997.

Babior, B.M., Matzner, Y. The FMF gene: Cloned at last. N. Engl. J. Med. 337:1548, 1997.

Inanami, O., Johnson, J.L., Babior, B.M. The leukocyte NADPH oxidase subunit p47^phox: The role of the cysteine residues. Arch. Biochem. Biophys. 350:36, 1998.

Inanami, O., Johnson, J.L., McAdara, J.K., El Benna, J., Faust, L.P., Newburger, P.E., Babior, B.M. Activation of the leukocyte NADPH oxidase by phorbol ester requires the phosphorylation of p47^phox on serine S303 or S304. J. Biol. Chem., in press.

Niessen, H., Meisenholder, G.W., Li, H.-L., Gluck, S.L., Lee, B.S., Forgac, M., Bowman, B., Engler, R.L., Babior, B.M., Gottlieb, R.A. G-CSF upregulates the vacuolar proton ATPase in human neutrophils. Blood 90:4598, 1997.

Park, J.-W., Scott, K.E., Babior, B.M. Activation of the leukocyte NADPH oxidase in a cell-free system: Phosphorylation vs amphiphiles. Exp. Hematol. 26:37, 1998.