Splice variants of protein disulfide isomerase - identification, distribution and functional characterization in the rat.

BACKGROUND: Protein Disulfide Isomerase (PDI) enzyme is an emerging therapeutic target in oncology and hematology. Although PDI reductase activity has been studied with isolated fragments of the protein, natural structural variations affecting reductase activity have not been addressed. METHODS: In this study, we discovered four coding splice variants of the Pdi pre-mRNA in rats. In vitro Michaelis constants and apparent maximum steady-state rate constants after purification and distribution in different rat tissues were determined. RESULTS: The consensus sequence was found to be the most expressed splice variant while the second most expressed variant represents 15 to 35% of total Pdi mRNA. The third variant shows a quasi-null expression profile and the fourth was not quantifiable. The consensus sequence splice variant and the second splice variant are widely expressed (transcription level) in the liver and even more present in males. Measurements of the reductase activity of recombinant PDI indicate that the consensus sequence and third splice variant are fully active variants. The second most expressed variant, differing by a lack of signal peptide, was found active but less than the consensus sequence. GENERAL SIGNIFICANCE: Our work emphasizes the importance of taking splice variants into account when studying PDI-like proteins to understand the full biological functionalities of PDI.

Impacts of vesicular environment on Nox2 activity measurements in vitro.

BACKGROUND: The production of superoxide anions (O2•-) by the phagocyte NADPH oxidase complex has a crucial role in the destruction of pathogens in innate immunity. Majority of in vitro studies on the functioning of NADPH oxidase indirectly follows the enzymatic reaction by the superoxide reduction of cytochrome c (cyt c). Only few reports mention the alternative approach consisting in measuring the NADPH consumption rate. When using membrane vesicles of human neutrophils, the enzyme specific activity is generally found twice higher by monitoring the NADPH oxidation than by measuring the cyt c reduction. Up to now, the literature provides only little explanations about such discrepancy despite the critical importance to quantify the exact enzyme activity. METHODS: We deciphered the reasons of this disparity in studying the role of key parameters, including. cyt c and arachidonic acid concentrations, in conjunction with an ionophore, a detergent and using Clark electrode to measure the O2 consumption rates. RESULTS: Our results show that the O2•- low permeability of the vesicle membrane as well as secondary reactions (O2•- and H2O2 disproportionations) are strong clues to shed light on this inconsistency. CONCLUSION AND GENERAL SIGNIFICANCE: These results altogether indicate that the cyt c reduction method underestimates the accurate Nox2 activity.

Phagocyte NADPH oxidase, oxidative stress and lipids: Anti- or pro ageing?

The role of NADPH oxidase in ageing is debated because of the dual roles of free radicals, toxic though necessary. In this paper we summarize some results about two aspects linked to the regulation of the activity of phagocyte NADPH oxidase (Nox2), encountered frequently in elderly people: inflammation and hypercholesterolemia. In the presence of a high amount of reactive oxygen species (ROS) created by itself or by any other source, the enzyme activity is mostly lowered. Oxidation of the membrane and/or of one of the cytosolic partners could be responsible for this loss of activity. However using a cell free system, we had also shown that a low amount of ROS could activate this enzyme. Similarly, cholesterol has a similar dual role, either activating or inhibiting. In in vitro cell free system with neutrophil membranes from healthy donors, the addition, as well as the removal of cholesterol, diminishes the Nox2 activity. The activity of Nox2 is lowered in neutrophils of untreated hypercholesterolemic patients. Finally oxysterols (25-hydroxy-cholesterol or 5α, 6α - epoxy-cholesterol) do not induce effects different from that of non-oxidized cholesterol. These findings are in agreement with the Janus role of NADPH oxidase, the main source of non-mitochondrial ROS.