Regulation of pteridine biosynthesis by bacterial products

Nitric oxide (NO) is an important signalling molecule involved in neurotransmission, regulation of blood pressure and the immune response. An essential cofactor for NO production is tetrahydrobiopterin. Formation of this cofactor is regulated by a number of proteins (cytokines) formed in the course of an immune response as well as by compounds from bacterial cell walls playing a role in infections. Another regulator of tetrahydrobiopterin biosynthesis is GFRP (GTP cyclohydrolase I feedback regulatory protein), which mediates the inhibition of tetrahydrobiopterin biosynthesis by its end product and its stimulation by the amino acid phenylalanine. This control mechanism avoids over-production of tetrahydrobiopterin and, vice versa, supply of optimal tetrahydrobiopterin levels for conversion of phenylalanine to tyrosine, an important precursor for neurotransmitter synthesis. In previous work, we found that expression of the regulator protein GFRP is efficiently down-regulated in monocytes and endothelial cells by a bacterial cell wall component (lipopolysaccharide, LPS) causing release of pteridine formation from metabolic control. In the proposed project we would like to identify further compounds from bacteria or cytokines able to regulate GFRP expression and some of the major receptors and signalling molecules being involved in this response. In addition, we would like to test the impact of regulating GFRP expression on NO formation in cells capable to express endothelial or cytokine-inducible NO synthase. This is particularly interesting in connection with sepsis and related diseases, an exaggerated reaction of the immune system with a high incidence of mortality caused by bacterial infection. Among other things, these pathological states are characterized by an over-production of NO. Conceivably, uncontrolled formation of tetrahydrobiopterin by down-regulation of GFRP further supports the deleterious NO over-production observed in this condition.

Nitric oxide (NO) is an important signalling molecule involved in neurotransmission, regulation of blood pressure and the immune response. An essential cofactor for NO production is tetrahydrobiopterin. Formation of this cofactor is regulated by a number of cytokines formed in the course of an immune response as well as by compounds from bacterial cell walls playing a role in infections, which primarily affect the enzyme GTP cyclohydrolase I, the first and rate-limiting enzyme in tetrahydrobiopterin biosynthesis. GTP cyclohydrolase I, which is active as a decamer of identical subunits, is inhibited by tetrahydrobiopterin and stimulated by the amino acid phenylalanine, a process which is mediated by a protein called GFRP (GTP cyclohydrolase I feedback regulatory protein). This control mechanism avoids over-production of tetrahydrobiopterin and assures supply of optimal tetrahydrobiopterin levels for conversion of phenylalanine to tyrosine, an important precursor for neurotransmitter synthesis. In the present project, we first characterized regulation of GTP cyclohydrolase I by GFRP in endothelial cells and found that at low intracellular tetrahydrobiopterin levels, also the activity of endothelial NOS (eNOS) was affected via feed-forward stimulation or feed-back inhibition of tetrahydrobiopterin biosynthesis. This effect was not seen with high intracellular tetrahydrobiopterin levels since eNOS was then saturated with its cofactor. Furthermore, we studied the effect of antioxidants, particularly of alpha-tocopherol on eNOS. In contrast to ascorbic acid, which acts primarily via improving tetrahydrobiopterin availability, alpha- tocopherol also had a direct effect on eNOS, i.e. activation via serine 1177 phosphorylation. Investigating the background for the extremely low tetrahydrobiopterin biosynthetic capacity of human monocytes as compared to endothelial cells, we found that this is caused by skipping of exon 3 of the second enzyme of tetrahydrobiopterin biosynthesis, i.e. 6-pyruvoyl tetrahydropterin synthase, which occurs to a high extent in monocytes but only to a minor extent in endothelial cells. As we previously found, also GTP cyclohydrolase I is alternatively spliced yielding proteins with various C-termini. Since GTP cyclohydrolase I is active as a decamer, we co-expressed wild-type and variant GTP cyclohydrolase I proteins and found that the inactive variants were incorporated into decamers and thereby decreased enzyme activity. In summary, we investigated mechanisms affecting tetrahydrobiopterin availability which is crucial for optimal NOS function. Our findings thus contribute to the general understanding of a pathway that is of major importance for proper immune and endothelium function.