Why human cytochrome P450c21 is a progesterone 21-hydroxylase.

Human cytochrome P450c21 (steroid 21-hydroxylase, CYP21A2) catalyzes the 21-hydroxylation of progesterone (P4) and its preferred substrate 17α-hydroxyprogestrone (17OHP4). CYP21A2 activities, which are required for cortisol and aldosterone biosynthesis, involve the formation of energetically disfavored primary carbon radicals. Therefore, we hypothesized that the binding of P4 and 17OHP4 to CYP21A2 restricts access of the reactive heme-oxygen complex to the C-21 hydrogen atoms, suppressing oxygenation at kinetically more favorable sites such as C-17 and C-16, which are both hydroxylated by cytochrome P450c17 (CYP17A1). We reasoned that expansion of the CYP21A2 substrate-binding pocket would increase substrate mobility and might yield additional hydroxylation activities. We built a computer model of CYP21A2 based principally on the crystal structure of CYP2C5, which also 21-hydroxylates P4. Molecular dynamics simulations indicate that binding of the steroid nucleus perpendicular to the plane of the CYP21A2 heme ring limits access of the heme oxygen to the C-21 hydrogen atoms. Residues L107, L109, V470, I471, and V359 were found to contribute to the CYP21A2 substate-binding pocket. Mutation of V470 and I471 to alanine or glycine preserved P4 21-hydroxylase activity, and mutations of L107 or L109 were inactive. Mutations V359A and V359G, in contrast, acquired 16α-hydroxylase activity, accounting for 40% and 90% of the P4 metabolites, respectively. We conclude that P4 binds to CYP21A2 in a fundamentally different orientation than to CYP17A1 and that expansion of the CYP21A2 substrate-binding pocket allows additional substrate trajectories and metabolic switching.

Characterization of nuclear betaII-tubulin in tumor cells: a possible novel target for taxol.

As the subunits of microtubules, alpha- and beta-tubulins have been thought to only exist in the cytoplasm where they are incorporated into microtubules. However, the beta(II) isotype of tubulin has recently been observed in the nuclei of rat kidney mesangial cells [Walss et al., 1999: Cell Motil. Cytoskeleton 42:274-284]. In this study, we detected nuclear beta(II)-tubulin in rat C6 glioma cells, human T98G glioma cells, human MCF-7 breast carcinoma cells, human MDA-MB-435 breast carcinoma cells, and human Hela cervix carcinoma cells. In addition, nuclear beta(II)-tubulin in these cells was found to exist as alphabeta(II) dimers instead of assembled microtubules and appeared to be particularly concentrated in the nucleoli. Several anti-tubulin drugs were used to treat C6 cells to determine their influence on nuclear beta(II)-tubulin. Taxol, a tubulin drug with higher specificity for beta(II)-tubulin than for other beta-tubulin isotypes, irreversibly decreased nuclear beta(II) content in a concentration-dependent manner in C6 cells. Meanwhile, cells were found to be apoptotic as was suggested by the presence of multiple micronuclei and DNA fragmentation. On the other hand, no depletion of nuclear beta(II)-tubulin was observed when C6 cells were incubated with colchicine or nocodazole, two anti-tubulin drugs with higher specificity for the alphabeta(IV) isotype, supporting the hypothesis that drugs with higher specificity for beta(II)-tubulin deplete nuclear beta(II)-tubulin.

Occurrence of nuclear beta(II)-tubulin in cultured cells.

Microtubules are cylindrical organelles that play critical roles in cell division. Their subunit protein, tubulin, is a target for various antitumor drugs. Tubulin exists as various forms, known as isotypes. In most normal cells, tubulin occurs only in the cytosol and not in the nucleus. However, we have recently reported the finding of the beta(II) isotype of tubulin in the nuclei of cultured rat kidney mesangial cells. Mesangial cells, unlike most normal cell lines, have the ability to proliferate rapidly in culture. In efforts to determine whether nuclear beta(II)-tubulin occurred in other cell lines, we examined the distribution of the beta(I), beta(II), and beta(IV) mammalian tubulin isotypes in a variety of normal and cancer human cell lines by immunofluorescence microscopy. We have found that, in the normal cell lines, all three isotypes are present only in the cytoplasm. However, the beta(II) isotype of tubulin is located not only in the cytoplasm, but also in the nuclei of the following cell lines: LNCaP prostate carcinoma, MCF-7, MDA-MB-231, MDA-MB-435, and Calc18 breast carcinoma, C6 and T98G glioma, and HeLa cells. In contrast, the beta(I) and beta(IV) isotypes, which are also synthesized in cancer cells, are not localized to the nucleus but are restricted to the cytoplasm. We have also seen beta(II) in breast cancer excisions. In most of these cells, beta(II) appears to be concentrated in the nucleoli. These results suggest that transformation may lead to localization of beta(II)-tubulin in cell nuclei, serving an as yet unknown function, and that nuclear beta(II) may be a useful marker for detection of tumor cells.