9 research outputs found
Reactive oxygen species in phagocytic leukocytes
Phagocytic leukocytes consume oxygen and generate reactive oxygen species in response to appropriate stimuli. The phagocyte NADPH oxidase, a multiprotein complex, existing in the dissociated state in resting cells becomes assembled into the functional oxidase complex upon stimulation and then generates superoxide anions. Biochemical aspects of the NADPH oxidase are briefly discussed in this review; however, the major focus relates to the contributions of various modes of microscopy to our understanding of the NADPH oxidase and the cell biology of phagocytic leukocytes
A Regulated Adaptor Function of p40(phox): Distinct p67(phox) Membrane Targeting by p40(phox) and by p47(phox)
In the phagocytic cell, NADPH oxidase (Nox2) system, cytoplasmic regulators (p47(phox), p67(phox), p40(phox), and Rac) translocate and associate with the membrane-spanning flavocytochrome b(558), leading to activation of superoxide production. We examined membrane targeting of phox proteins and explored conformational changes in p40(phox) that regulate its translocation to membranes upon stimulation. GFP-p40(phox) translocates to early endosomes, whereas GFP-p47(phox) translocates to the plasma membrane in response to arachidonic acid. In contrast, GFP-p67(phox) does not translocate to membranes when expressed alone, but it is dependent on p40(phox) and p47(phox) for its translocation to early endosomes or the plasma membrane, respectively. Translocation of GFP-p40(phox) or GFP-p47(phox) to their respective membrane-targeting sites is abolished by mutations in their phox (PX) domains that disrupt their interactions with their cognate phospholipid ligands. Furthermore, GFP-p67(phox) translocation to either membrane is abolished by mutations that disrupt its interaction with p40(phox) or p47(phox). Finally, we detected a head-to-tail (PX–Phox and Bem1 [PB1] domain) intramolecular interaction within p40(phox) in its resting state by deletion mutagenesis, cell localization, and binding experiments, suggesting that its PX domain is inaccessible to interact with phosphatidylinositol 3-phosphate without cell stimulation. Thus, both p40(phox) and p47(phox) function as diverse p67(phox) “carrier proteins” regulated by the unmasking of membrane-targeting domains in distinct mechanisms
Phospholipase A 2
Phospholipases represent one of the earliest enzyme activities to be identified and studied, and the phospholipase A2 superfamily traces its roots to the identification of lytic actions of snake venom at the end of the 19th century. Both electrostatic and hydrophobic interactions contribute to the interfacial binding of sPLA2 to anionic phospholipid membranes. The interaction between basic residues on the binding surface with anionic vesicles plays an important role in interfacial binding. The major functions will be summarized below and include the ability to kill Gram-positive and Gram-negative bacteria, thereby affecting host defense against bacterial infections. sPLA2 may be involved in the pathogensis of inflammatory bowel disease including Crohn's disease and ulcerative colitis. GIIA sPLA2 protein and mRNA were detected in Paneth cells of the small intestinal mucosa in the intestine in Crohn's disease patients