Abstract
The exposure of human endothelial cells to 3-morpholinosydnonimine (SIN-1) induced the expression of cyclooxygenase-2 (COX-2) in a dose- and time-dependent manner. Interestingly, after a prolonged incubation (>8 h) several proteoforms were visualized by Western blot, corresponding to different states of glycosylation of the protein. This effect was specific for SIN-1 that generates peroxynitrite and it was not detected with other nitric oxide-donors. Metabolic labeling experiments using 35S or cycloheximide suggested that the formation of hypoglycosylated COX-2 was dependent on de novo synthesis of the protein rather than the deglycosylation of the native protein. Moreover, SIN-1 reduced the activity of the hexokinase, the enzyme responsible for the first step of glycolysis. The hypoglycosylated COX-2 induced by SIN-1 showed a reduced capacity to generate prostaglandins and the activity was only partially recovered after immunoprecipitation. Finally, hypoglycosylated COX-2 showed a more rapid rate of degradation compared to COX-2 induced by IL-1α and an alteration in the localization with an accumulation mainly detected in the nuclear membrane. Our results have important implication to understand the effect of peroxynitrite on COX-2 expression and activity, and they may help to identify new pharmacological tools direct to increase COX-2 degradation or to inhibit its activity.
Highlights
Cyclooxygenase (COX), known as prostaglandin (PG)H synthase, is the first enzyme in the conversion of fatty acid substrates, most notably arachidonic acid, to PGH2 .PGH2 is further metabolized by downstream synthase enzymes to a range of prostanoids.PGH2 is an unstable endoperoxide, the substrate for the terminal synthases as prostacyclin (PGI2 )-synthase, PGE2 -synthase or thromboxane A2 (TXA2 )-synthase, which are responsible for the formation of the different prostanoids
We have previously shown that SIN-1 dose-dependently induces in human umbilical vein endothelial cells (HUVEC), up to 6 h, an increase in the level of COX-2 protein, recognized as a double band (70–72 kDa), by a monoclonal antibody direct against the carboxyl-terminal region of the protein [23]
We showed that prolonged (>8 h) treatment of HUVEC with SIN-1, induced a de novo synthesis of COX-2 mainly localized in the nuclear membrane, and characterized by hypoglycosylation, and loss of activity
Summary
Cyclooxygenase (COX), known as prostaglandin (PG)H synthase, is the first enzyme in the conversion of fatty acid substrates, most notably arachidonic acid, to PGH2 .PGH2 is further metabolized by downstream synthase enzymes to a range of prostanoids.PGH2 is an unstable endoperoxide, the substrate for the terminal synthases as prostacyclin (PGI2 )-synthase, PGE2 -synthase or thromboxane A2 (TXA2 )-synthase, which are responsible for the formation of the different prostanoids. Cyclooxygenase (COX), known as prostaglandin (PG)H synthase, is the first enzyme in the conversion of fatty acid substrates, most notably arachidonic acid, to PGH2. PGH2 is further metabolized by downstream synthase enzymes to a range of prostanoids. COX is the major enzyme involved in their biosynthesis and two different isoforms, COX-1 and COX-2, with similar structure and catalytic activity, have been described [1]. Both isoforms are heme-containing glycoproteins mainly located on the inner and outer membranes of the nuclear envelope and on the lumenal surface of the endoplasmic reticulum [2]. The N-linked glycosylation, which occurs in the endoplasmic reticulum, is required for proper folding, activity, degradation, and turnover of the two proteins [3,4,5]
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