Tetrahydrobiopterin modulates ubiquitin conjugation to UBC13/UBE2N and proteasome activity by S-nitrosation.

Nitric Oxide (NO) is an intracellular signalling mediator, which affects many biological processes via the posttranslational modification of proteins through S-nitrosation. The availability of NO and NOS-derived reactive oxygen species (ROS) from enzymatic uncoupling are determined by the NO synthase cofactor Tetrahydrobiopterin (BH4). Here, using a global proteomics "biotin-switch" approach, we identified components of the ubiquitin-proteasome system to be altered via BH4-dependent NO signalling by protein S-nitrosation. We show S-nitrosation of ubiquitin conjugating E2 enzymes, in particular the catalytic residue C87 of UBC13/UBE2N, leading to impaired polyubiquitylation by interfering with the formation of UBC13~Ub thioester intermediates. In addition, proteasome cleavage activity in cells also seems to be altered by S-nitrosation, correlating with the modification of cysteine residues within the 19S regulatory particle and catalytic subunits of the 20S complex. Our results highlight the widespread impact of BH4 on downstream cellular signalling as evidenced by the effect of a perturbed BH4-dependent NO-Redox balance on critical processes within the ubiquitin-proteasome system (UPS). These studies thereby uncover a novel aspect of NO associated modulation of cellular homeostasis.

A pivotal role for tryptophan 447 in enzymatic coupling of human endothelial nitric oxide synthase (eNOS): effects on tetrahydrobiopterin-dependent catalysis and eNOS dimerization.

Tetrahydrobiopterin (BH4) is a required cofactor for the synthesis of NO by NOS. Bioavailability of BH4 is a critical factor in regulating the balance between NO and superoxide production by endothelial NOS (eNOS coupling). Crystal structures of the mouse inducible NOS oxygenase domain reveal a homologous BH4-binding site located in the dimer interface and a conserved tryptophan residue that engages in hydrogen bonding or aromatic stacking interactions with the BH4 ring. The role of this residue in eNOS coupling remains unexplored. We overexpressed human eNOS W447A and W447F mutants in novel cell lines with tetracycline-regulated expression of human GTP cyclohydrolase I, the rate-limiting enzyme in BH4 synthesis, to determine the importance of BH4 and Trp-447 in eNOS uncoupling. NO production was abolished in eNOS-W447A cells and diminished in cells expressing W447F, despite high BH4 levels. eNOS-derived superoxide production was significantly elevated in W447A and W447F versus wild-type eNOS, and this was sufficient to oxidize BH4 to 7,8-dihydrobiopterin. In uncoupled, BH4-deficient cells, the deleterious effects of W447A mutation were greatly exacerbated, resulting in further attenuation of NO and greatly increased superoxide production. eNOS dimerization was attenuated in W447A eNOS cells and further reduced in BH4-deficient cells, as demonstrated using a novel split Renilla luciferase biosensor. Reduction of cellular BH4 levels resulted in a switch from an eNOS dimer to an eNOS monomer. These data reveal a key role for Trp-447 in determining NO versus superoxide production by eNOS, by effects on BH4-dependent catalysis, and by modulating eNOS dimer formation.

Analysis of Wnt gene expression in prostate cancer: mutual inhibition by WNT11 and the androgen receptor.

The Wnt signaling pathway is aberrantly activated in many tumor types, including those of the prostate, in which beta-catenin accumulates in cell nuclei and acts as a transcriptional coregulator for the androgen receptor. Because activating mutations in the beta-catenin gene are rare in prostate cancer, we have looked for altered expression of other components of the Wnt signaling pathway in prostate cancer cells. Here we determined the expression levels of Wnt family genes in cultured human prostate cells and prostate cancer cell lines. We found that WNT11 expression is elevated in hormone-independent prostate cancer cell lines. Additional analysis indicated that WNT11 expression is also elevated in high-grade prostatic tumors and in hormone-independent xenografts. Growth of hormone-dependent LNCaP cells in hormone-depleted media led to increased WNT11 expression, which was repressed by the synthetic androgen R1881. This repression was inhibited by the antiandrogen bicalutamide, suggesting that androgens negatively regulate WNT11 expression through the androgen receptor. Expression of WNT11 inhibited androgen receptor transcriptional activity and cell growth in androgen-dependent cells but not in androgen-independent cells. WNT11 inhibited activation of the canonical Wnt pathway by WNT3A in HEK 293 cells and inhibited basal beta-catenin/Tcf transcriptional activity in LNCaP cells. However, expression of stabilized beta-catenin did not prevent the inhibition of androgen receptor transcriptional activity by WNT11. Our observations are consistent with a model in which androgen depletion activates WNT11-dependent signals that inhibit androgen-dependent but not androgen-independent cell growth.

Inhibition of glycogen synthase kinase-3 represses androgen receptor activity and prostate cancer cell growth.

The transcriptional activity of the androgen receptor (AR) is regulated by interaction with various coregulators, one of which is beta-catenin. Interest in the role of beta-catenin in prostate cancer has been stimulated by reports showing that it is aberrantly expressed in the cytoplasm and/or nucleus in up to 38% of hormone-refractory tumours and that overexpression of beta-catenin results in activation of AR transcriptional activity. We have examined the effect of depleting endogenous beta-catenin on AR activity using Axin and RNA interference. Axin, which promotes beta-catenin degradation, inhibited AR transcriptional activity. However, this did not require the beta-catenin-binding domain of Axin. Depletion of beta-catenin using RNA interference increased, rather than decreased, AR activity, suggesting that endogenous beta-catenin is not a transcriptional coactivator for the AR. The glycogen synthase kinase-3 (GSK-3)-binding domain of Axin prevented formation of a GSK-3-AR complex and was both necessary and sufficient for inhibition of AR-dependent transcription. A second GSK-3-binding protein, FRAT, also inhibited AR transcriptional activity, as did the GSK-3 inhibitors SB216763 and SB415286. Finally, inhibition of GSK-3 reduced the growth of AR-expressing prostate cancer cell lines. Our observations suggest a potential new therapeutic application for GSK-3 inhibitors in prostate cancer.