At high resolution, we determined the crystal structures of copper-bound and metal-free tyrosinase in a complex with ORF378 designated as a "caddie" protein because it assists with transportation of two Cu(II) ions into the tyrosinase catalytic center. These structures suggest that the caddie protein covers the hydrophobic molecular surface of tyrosinase and interferes with the binding of a substrate tyrosine to the catalytic site of tyrosinase. The caddie protein, which consists of one six-stranded ␤-sheet and one ␣-helix, has no similarity with all proteins deposited into the Protein Data Bank. Although tyrosinase and catechol oxidase are classified into the type 3 copper protein family, the latter enzyme lacks monooxygenase activity. The difference in catalytic activity is based on the structural observations that a large vacant space is present just above the active center of tyrosinase and that one of the six His ligands for the two copper ions is highly flexible. These structural characteristics of tyrosinase suggest that, in the reaction that catalyzes the ortho-hydroxylation of monophenol, one of the two Cu(II) ions is coordinated by the peroxide-originated oxygen bound to the substrate. Our crystallographic study shows evidence that the tyrosinase active center formed by dinuclear coppers is flexible during catalysis.

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Capsaicin is the major pungent ingredient in red peppers. Here, we report that it has a profound antiproliferative effect on prostate cancer cells, inducing the apoptosis of both androgen receptor (AR)-positive (LNCaP) and -negative (PC-3, DU-145) prostate cancer cell lines associated with an increase of p53, p21, and Bax. Capsaicin down-regulated the expression of not only prostate-specific antigen (PSA) but also AR. Promoter assays showed that capsaicin inhibited the ability of dihydrotestosterone to activate the PSA promoter/enhancer even in the presence of exogenous AR in LNCaP cells, suggesting that capsaicin inhibited the transcription of PSA not only via downregulation of expression of AR, but also by a direct inhibitory effect on PSA transcription. Capsaicin inhibited NF-K activation by preventing its nuclear migration. In further studies, capsaicin inhibited tumor necrosis factor-A-stimulated degradation of IKBA in PC-3 cells, which was associated with the inhibition of proteasome activity. Taken together, capsaicin inhibits proteasome activity which suppressed the degradation of IKBA, preventing the activation of NF-KB. Capsaicin, when given orally, significantly slowed the growth of PC-3 prostate cancer xenografts as measured by size [75 F 35 versus 336 F 123 mm 3 (FSD); P = 0.017] and weight [203 F 41 versus 373 F 52 mg (FSD); P = 0.0006; capsaicin-treated versus vehicletreated mice, respectively]. In summary, our data suggests that capsaicin, or a related analogue, may have a role in the management of prostate cancer. (Cancer Res 2006; 66(6): 3222-9)