Year: 1996
Type: dissertation
Abstract: Human neutrophils respond to a variety of soluble and particulate factors that recruit these cells to areas of inflammation and infection.At these sites, neutrophils phagocytose foreign particles and destroy them using a combination of microbicidal mechanisms including the production of microbicidal oxidants such as superoxide anion (02^-).The generation of 02^-by neutrophils requires the assembly of a membrane-bound complex known as the NADPH oxidase, which is composed of a plasma membrane flavocytochrome b, a membrane-associated low molecular weight GTP-binding protein (Rap1A), and four cytosolic proteins (p47-phox, p67-phox, p40-phox, and Rac2).During activation, cytosolic proteins p47-phox, p67-phox, Rac2, and possibly p40-phox translocate to the plasma membrane and associate with flavocytochrome b to form the active complex.The activation and assembly of the NADPH oxidase is regulated by signaling events and by molecular interaction between its components.These processes are not well characterized, and an understanding of how these protein components interact is essential to understanding its regulation.Several biochemical and molecular biological approaches were used in the current studies to define and subsequently confirm novel interactions occurring between flavocytochrome b and p47-phox and between p47 -phox and p67-phox.Two novel regions of p47-phox:flavocytochrome b interaction were mapped to gp91-phox residues 86-93 and 450-457.The mapping also confirmed previously reported binding domains on gp91-phox (554-564) and putative SH3 domain binding sites on p22-phox (158-162 and 177-183).Using a complimentary approach, p47-phox residues 323-342 were identified as a flavocytochrome b binding site.Interestingly, a novel region in p47-phox encompassing residues 323-332 (which is within the flavocytochrome b binding site) and a previously identified SH3-binding domain encompassing p47-phox residues 361-370 were identified as p61-phox binding sites.P67-phox and flavocytochrome b were found to compete for the binding of p47-phox after activation, suggesting that both p67-phox and flavocytochrome b utilize a common binding site in p47-phox, presumably at distinct stages during the activation process.This p47-phox region appears to play a key role in regulating NADPH oxidase assembly.The identification of these novel interactions has provided a better understanding of the mechanism of NADPH oxidase assembly and activation.
Author: Frank R. DeLeo
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W171640760