Propiedades Electr´ onicas del Material de Banda Intermedia

i) The electron transport system

The NADPH oxidase is a multi-protein enzyme complex composed of a number of cytosolic and membrane bound proteins. The NADPH oxidase is dormant in resting cells and its protein components are separated into cytoplasmic and plasma membrane components (Figure 1.3) but after activation all the components assemble as an enzyme complex at the membrane. During the respiratory burst electrons are transferred from NADPH (electron donor) to molecular oxygen (electron acceptor) producing superoxide anions. The redox centre o f this enzyme com plex is a heterodimeric membrane-associated flavocytochrome b, also known as cytochrome b.

245 (to indicate its low oxidation-reduction mid-point potential) or cytochrome b^ss (to indicate the absorbance maximum of its a band in the reduced state). Flavocytochrome

b consists of three subunits (Segal, et a l, 1987), two small a subunits (also known as p2 1Phox) and a larger p subunit of approximately 76-92 kDa (designated gp91P^°^). The gene for these subunits of cytochrome b have been cloned and sequenced (Royer- Pokora é'f c//., 1986).

The large subunit of cytochrome b (gp91P'^°^) is heavily glycosylated and has two major domains (Segal et a l , 1992). The N-terminal domain is hydrophobic whereas, the C-terminal domain is much more hydrophilic and lies on the cytosolic side of the membrane, overlying the transmembrane structure. This hydrophilic region of the molecule has a cleft that accomodates the substrate, NADPH, as well as flavin adenine dinucleotide (FAD), to which electrons are passed. From FAD electrons pass onto haems located within the transmembrane domains, attached to one or both of the subunits (Segal et a l , 1992; Quinn et a l, 1992). It has been shown that CHO cell lines transfected with and expressing the N -term inal fragment containing the transmembrane domains of gp91P^°^, exhibit an arachidonate-activatable H+ flux, indicating a role for gp91Pho^ as the NADPH oxidase H+ channel (Henderson et a l,

1997). The structure of gp91P^^°^ has strong homology with a number o f electron- transporting proteins from different origins such as ferredoxin-NADP"*" reductase (FNR) which is an important photosynthetic protein (Segal et a l, 1992).

Vacuole 2e-j I7 p l^ Cytoplasm J ( F A D ITpho^ ITphox i7phox iphox _Active Iphox gp91 Inactive NADP+ + H+ p21 raci “(gd p V

Figure 1.3; Model of phagocyte NADPH oxidase activation during phagocytosis of microorganisms. In the inactive state p40Phox, and p67Ph«' are present in the cytosol as a complex and p2H^‘^ and GDI are in another complex. Upon activation p2irac ig separated from GDI, converting it to active form. Both p47P^^°' and p67P"^°^ are phosphorylated and upon activation all these components attach to flavocytochrome b in the vacuole membrane.

phox = phagocyte oxidase, GDI = GDP dissociation inhibitors, O = Haem FAD = Flav in adenine dinucleotide, (Adapted from Segal, 1996)

ii) C yto so lic com ponents

Activation of the oxidase is regulated by several cytosolic proteins, some of which attach to the flavocytochrome b. p47P^°^ (Segal et a i, 1985), p67P^°^ (Leto et al., 1990) and p4QP^'o^ (Wientjes et a i , 1993) are specialised components of the NADPH oxidase of "professional" phagocytic cells. They all contain SH3 (Src homology 3) domains, which provide attachment sites to proline rich regions on the other proteins. In resting cells p47P^°^ and p67P^°^ reside in the cytosol in a 240-kDa complex with a third component, p40P^o^ (Someya et al., 1993; Fuchs et al., 1995). Upon activation, the p47P^°^ and p6 7P^ox are all phosphorylated and, along with p4 0Phox^ translocate to the region of plasma membrane forming the phagocytic vacuole, where they associate with hydrophilic regions o f flavocytochrom e b

(H eyw orth et al., 1991; Tyagi et al., 1992). It has been shown that p40P^°^ is responsible for electron transfer from FAD to the haem centre o f flavocytochrome b

(Cross and Curnutte, 1995). p6 7P^^®x appears to be associated with the cytoskeleton as it remains in the insoluble pellet after the cell has been permeabilised with detergents (Woodman et al., 1991).

p 2 1 rac is a member of the family of small GTP-binding proteins, which act as molecular switches. It is required for activity of the NADPH oxidase in a cell free, in vitro system (Abo et al., 1991). The activity o f small GTP-binding proteins is regulated by the guanine nucleotide to which they bind. In the cytosol of resting phagocytes, rac is complexed with an inhibitory protein Rho-GDI (Rho GDP- dissociation inhibitor), and when cells are activated rac translocates to the plasma membrane independently of other cytoplasmic components (Heyworth et al., 1994).

Rapl is a small GTPase which associates with flavocytochrome b in neutrophils (Quinn et al., 1989). It is not necessary for the assembly and the functioning of the oxidase system but it has been shown that a dominant negative mutant o f R apl inhibited the oxidative burst in differentiated HL-60 cells and in an Epstein-Barr virus (EBV)-transformed B-cell line (Gabig et al., 1995; Maly et al., 1994). It has been demonstrated that a variety of stimuli that activate the neutrophil, including FMLP (7V- formylmethionyl-leucyl-phenylalanine, a chemotactic tripeptide of bacterial origin), GM -CSF and platelet activating factor (PAF), induce a rapid and transient R apl activation. In addition, Rapl is normally activated in neutrophils from patients with CGD that lack cytochrome b or p47P^°^ and have a defective NADPH oxidase system, suggesting an independent activation of Rapl from the respiratory burst (M'Rabet et

o/., 1998).

Hi) Signal transduction components

Activation of NADPH oxidase involves translocation of cytosolic proteins from the cytosol to the m em brane (review ed by R obinson and B adw ey, 1995). Translocation is initiated by a series of highly regulated signalling events which involves kinases such as phosphoinositol 3-kinase (PI3-k) (Arcaro and W ymann, 1993; Vlahos et al., 1995), mitogen-activated protein kinases (MAPK), protein kinase C and protein kinase A (Dusi et a i , 1996; El Benna et al., 1994; El Benna et al.,

1996a). The flavocytochrome b, p40P^°^, p47P^°^ and p67P^®^ components of the oxidase all become phosphorylated during activation of the respiratory burst. PAK (p21-activated kinase, p65P^*^) is a potential downstream target for Rac-1 signalling and it was initially found as a brain serine/threonine kinase (Manser et al., 1994). PAK is activated in phagocytes as a result of its interaction with Rac-GTP and it can phosphorylate the p47P^°^ in a Rac-GTP dependent manner (Knaus et al., 1995). It has been shown that amongst 100 amino acid residues in the C-terminal domain of p4 7phox there are number of serines flanked with basic amino acids and this is a favourable structure for phosphorylation by protein kinase C (PKC) (Volpp et al.,

1989). It has also been shown that stimulation of the neutrophil’s respiratory burst is accompanied by the phosphorylation of multiple serine residues in p47P^®^ (El Benna

et al., 1994), and this protein is a target for proline directed mitogen activated protein kinases (MAPK) such as extracellular signal-regulated kinase (ERK) and p38 MAPK when neutrophils are exposed to an appropriate stimulus (El Benna et al., 1996b). p4 7phox binds to flavocytochrom e b, then p67P^°^ attaches to the p47P^°^- flavocytochrom e com plex. The sites o f attachm ent o f these proteins to flavocytochrome b have been partially defined. One of these is the C-terminus of p2ipliox and the other is a short helix overlying the nucleotide-binding groove of gp9 1phox (Leusen et al., 1994). Activation might involve moving of this helix and allowing the substrate, NADPH, access to FAD, the first redox cofactor.