Studio di una glutation perossidasi del reticolo endoplasmatico
Year: 2012
Type: dissertation
Abstract: Cells are continuously facing an oxidative challenge induced by free radicals. These may be generated in different way, i. e. by electron escaping the mitochondrial transport chain, activity of the NADPH oxidase and nitric oxide synthase family of enzymes and autoxidation of favoenzymes. However, the radicals superoxide (*O2- ) and nitric oxide (NO*) do not appear so destructive when produced in tissues. The abundance of superoxide dismutases (SODs) assures that O2*- is immediately dismutated to O2 and the apparently less dangerous H2O2. In pathologic conditions, however, H2O2 is used to produce hypoclorous acid (HOCl) and similarly reactive compounds by the heme peroxidases of leukocytes, or the hydroxyl radical (OH*) by Fenton-like chemistry. Similarly, under these conditions, peroxynitrite (ONOO-) -the reaction product between NO* and *O2- - is transformed into more toxic species. Interestingly, no enzyme has so far been discovered that could detoxify OH*, RO*, singlet oxygen, or HOCl. Evidently, nature was sage enough not to embark in this effort, due to the difficulty to compete with the diffusion-limited reaction of these species on biological targets. However living cells are continuously facing an oxidative challenge and self-protection appears mandatory. This is essentially achieved by preventing radical formation from hydroperoxides (R-OOH) and ONOO-. Interestingly, this appears particularly relevant on the light of the observation that these species may act as mediators of diverse physiological responses such as cell proliferation, differentiation and migration. The enzymes involved in mammals to handle R-OOH and ONOO-, belong to two distinct protein families, the glutathione peroxidases (GPxs) and the peroxiredoxins (Prxs). Both act on H2O2 and on ONOO- -although ONOO- is generally believed to be a better substrate for Prx than GPxs- and, also, on a wide spectrum of alkyl hydroperoxides comprising the products of lipoxygenases. GPxs are widely distributed in nature, from bacteria to humans. They use either selenium or sulfur in the redox center that is oxidized by the hydroperoxide, thus forming an oxidized enzyme intermediate, which is regenerated in its reduced form by a reductant. This is not always glutathione (GSH), as the name of the family should suggest. The redox center is remarkably conserved in the whole family and it is made of a catalytic tetrad containing Sec or Cys, Gln, Trp and Asn. When it contains selenium, as it typically occurs in mammalian GPx -the ‘SecGPxs’- the oxidized intermediate formed upon reaction of the catalytic moiety with the R-OOH -believed to be a selenol (SeO-)- is reduced by GSH. In this reaction a selenodisulfide intermediate is formed, that is displaced by a second GSH, yielding GSSG and reduced enzyme. When it contains sulphur, instead, as in the GPxs of non-vertebrates and plants (CysGPxs), the reducing substrate is a Trx or a ‘redoxin’ of the Trx family, and an intrachain disulphide is the oxidized intermediate of the enzyme. This mechanism requires, besides the ‘peroxidatic Cys ‘(CP), a second ‘resolving Cys’ (CR). The sole monomeric SecGPx of vertebrates -GPx4- does not bear the CR residue, is active on GSH and, obviously, does not form the intermediate intrachain selenodisulfide. The ‘canonic mechanism’ of GPxs, involving a CR when the redox center is a CP instead of a ‘peroxidatic Sec’ (UP) and the consequent use of Trx or ‘redoxins’ as the reducing substrate for the intermediate disulphide (CP - CR) has been challenged by the discovery of GPx7 and GPx8 in mammals. These are two monomeric GPxs of the endoplasmic reticulum (ER), containing a CP but missing a Cys residue –located within the aminoacid stretch of the alpha 4 helix of the GPx thioredoxin fold- where the CR residue of non-vertebrate and plants GPxs is found. Here, GPx7 has been characterized as the prototype of these unusual 1CysGPx. Although at low levels, GPx7 is widely distributed in tissues. Both, its NH2-terminal peptide and the C terminal KREDL motif proved to be functional to ER entrance and retention respectively. Kinetic studies on GPx7 have been carried out using phospholipid hydroperoxides, the most appropriate for monomeric GPxs, using the enzyme expressed in E. coli, fused with synuclein to overcome an otherwise very poor expression. From steady state kinetics the rate constant for the oxidation of the active site resulted rather high (k+1=9.5 x E03 M-1sec-1), whilst the rate of the regeneration of the catalyst by GSH remarkably low (k’+2=12.6 M-1sec-1). Unexpectedly, the oxidized active site is instead rapidly reduced by a ‘redoxin’, protein disulfide isomerase (PDI) (k’+2=3.5 x E03 M-1sec-1), but not by Trx. This evidence was corroborated by the affinity constant for PDI measured by SPR (KD= 5.2 μM). Furthermore the presence in GPx7 of a Cys residue replacing the resolving function of the CR of non-vertebrates and plants GPxs, was ruled out by mutagenesis. Thus, the ‘canonical mechanism’ by which in GPx a CR is required for being reduced by a ‘redoxin’ does not apply to GPx7. This enzyme indeed accepts electrons from a ‘redoxin’ such as PDI by a mechanism involving the CP only. GPx7, and the structurally similar GPx8, therefore, represent rare exceptions within the family of glutathione peroxidases, for being 1CysGPx preferentially reduced by a ‘redoxin’. In fact the GPxs family is mostly contributed by the non-vertebrates ‘2CysGPx’ that are functionally Trx peroxidases. Although these data frame the physiological function of GPx7 in the area of oxidative folding of proteins in ER, no evidence was obtained in this respect. Searching for adaptive response to ER stress alternative to the unfolded protein response (UPR), we observed increased phosphorylation of the extracellular signal regulated kinase 1-2 (ERK 1-2) by GPx7 silencing following a proliferative stimulus by the fibroblast growth factor (FGF). These data finalize the enzymatic characterization of GPx7 as a ‘non-canonical’ member of the GPxs family, do not support the role in ER stress but envisage a role in the cross talk between the redox status of ER and growth factor signaling.
Author: Silvia Quartesan
Language: Italian
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2274937281