Crystal structure of the alpha(6)beta(6) holoenzyme of propionyl-coenzyme A carboxylase.

Propionyl-coenzyme A carboxylase (PCC), a mitochondrial biotin-dependent enzyme, is essential for the catabolism of the amino acids Thr, Val, Ile and Met, cholesterol and fatty acids with an odd number of carbon atoms. Deficiencies in PCC activity in humans are linked to the disease propionic acidaemia, an autosomal recessive disorder that can be fatal in infants. The holoenzyme of PCC is an alpha(6)beta(6) dodecamer, with a molecular mass of 750 kDa. The alpha-subunit contains the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains, whereas the beta-subunit supplies the carboxyltransferase (CT) activity. Here we report the crystal structure at 3.2-A resolution of a bacterial PCC alpha(6)beta(6) holoenzyme as well as cryo-electron microscopy (cryo-EM) reconstruction at 15-A resolution demonstrating a similar structure for human PCC. The structure defines the overall architecture of PCC and reveals unexpectedly that the alpha-subunits are arranged as monomers in the holoenzyme, decorating a central beta(6) hexamer. A hitherto unrecognized domain in the alpha-subunit, formed by residues between the BC and BCCP domains, is crucial for interactions with the beta-subunit. We have named it the BT domain. The structure reveals for the first time the relative positions of the BC and CT active sites in the holoenzyme. They are separated by approximately 55 A, indicating that the entire BCCP domain must translocate during catalysis. The BCCP domain is located in the active site of the beta-subunit in the current structure, providing insight for its involvement in the CT reaction. The structural information establishes a molecular basis for understanding the large collection of disease-causing mutations in PCC and is relevant for the holoenzymes of other biotin-dependent carboxylases, including 3-methylcrotonyl-CoA carboxylase (MCC) and eukaryotic acetyl-CoA carboxylase (ACC).

CYTOLOGICAL, ULTRASTRUCTURAL, AND BIOCHEMICAL STUDIES OF THE STRUCTURE OF PREMATURELY CONDENSED CHROMOSOMES

The phenomenon of premature chromosome condensation, resulting from fusion between mitotic and interphase cells, includes dissolution of the interphase nuclear framework, thus allowing a direct visualization of interphase chromosomes. Light microscope morphology of prematurely condensed chromosomes (PCC) from synchronized HeLa cells supports the model of an interphase "chromosome condensation cycle". PCC are increasingly attenuated as cells progress through G(,1). A maximum degree of decondensation is observed at active sites of DNA replication during S phase, and a condensed morphology is rapidly resumed following completion of replication of a chromosome segment.^ To permit ultrastructural and biochemical studies of PCC, a procedure was developed to induce premature chromosome condensation at high frequency. This was achieved by polyethylene glycol (PEG)-mediated fusion of a dense monolayer of mitotic and interphase cells induced by centrifugation onto lectin-coated culture dishes. Using this method, PCC induction frequencies of 60-90% are routinely obtained.^ Scanning electron microscope analysis of PCC spreads revealed that the extension of PCC during progression through G(,1) is accompanied by a transition of the basic 30 nm chromatin fiber from tightly packed looping fibers to extended longitudinal fibers. Sites of active DNA replication is S-PCC were indicated to be organized a single longitudinal fibers. Following replication of a chromosome segment, a rapid reorganization from the extended longitudinal fiber to packed looping fibers occurs. The postreplication maturation process appears to include the assembly of a chromosome core consisting of multiple longitudinal fibers.^ The role of histone H1 phosphorylation in PCC formation was investigated by acidurea polyacrylamide gel electrophoresis of total histone extracted from metaphase chromosomes and PCC following high frequency fusion. This investigation failed to demonstrate an extensive phosphorylation of H1 associated with PCC formation. However, significant dephosphorylation of superphosphorylated metaphase chromosome H1 was observed, indicating that interphase H1-phosphatase activity is dominant over metaphase H1 kinase activity. These observations provide evidence against models suggesting a role for H1 superphosphorylation in triggering mitotic condensation of chromosomes. ^

Pharmacogenetics of the human glutathione S-transferase P1 gene in the metabolism and therapeutic efficacy of cis-diamminedichloroplatinum

The human GSTP1 gene has been shown, conclusively, to be polymorphic. The three main GSTP1 alleles, GSTP1*A, GSTP1*B, and GSTP1*C, encode proteins which differ in the 3-dimensional structure of their active sites and in their function in phase II metabolism of carcinogens, mutagens, and anticancer agents. Although, it is well established that GSTP1 is over expressed in many human tumors and that the levels of GSTP1 expression correlate directly with tumor resistance to chemotherapy and inversely with patient survival, the significance of the polymorphic GSTP1 gene locus on tumor response to chemotherapy remains unclear. The goal of this project was to define the role and significance of the polymorphic GSTP1 gene locus in GSTP1-based tumor drug resistance and as a determinant of patient response to chemotherapy. The hypothesis to be tested was that the polymorphic GSTP1 gene locus will confer to tumors a differential ability to metabolize cisplatin resulting in a GSTP1 genotype-based sensitivity to cisplatin. The study examined: (a) whether the different GSTP 1 alleles confer different levels of cellular protection against cisplatin-induced cytotoxicity, (b) whether the allelic GSTP1 proteins metabolize cisplatin with different efficiencies, and (c) whether the GSTP1 genotype is a determinant of tumor response to cisplatin therapy. The results demonstrate that the GSTP1 alleles differentially protect tumors against cisplatin-induced apoptosis and clonogenic cell kill in the rank order: GSTP1*C > GSTP1*B > GSTP1*A. The same rank order was observed for the kinetics of GSTP1-catalyzed cisplatin metabolism, both in cell-free and cellular systems, to the rate-limiting monoglutathionyl-platinum metabolite, which was characterized, for the first time, by mass spectral analysis. Finally, this study demonstrates that both GSTP1 genotype and the level of GSTP1 expression significantly contribute to tumor sensitivity to cisplatin treatment. Overall, the results of this project show that the polymorphic GSTP1 gene locus plays a significant role in tumor sensitivity to cisplatin treatment. Furthermore, these studies have contributed to the overall understanding of the significance of the polymorphic GSTP1 gene locus in tumor resistance to cancer chemotherapy and have provided the basis for further investigations into how this can be utilized to optimize and individualize cancer chemotherapy for cancer patients. ^