A pathogenesis-related 10 protein catalyzes the final step in thebaine biosynthesis.

The ultimate step in the formation of thebaine, a pentacyclic opiate alkaloid readily converted to the narcotic analgesics codeine and morphine in the opium poppy, has long been presumed to be a spontaneous reaction. We have detected and purified a novel enzyme from opium poppy latex that is capable of the efficient formation of thebaine from (7S)-salutaridinol 7-O-acetate at the expense of labile hydroxylated byproducts, which are preferentially produced by spontaneous allylic elimination. Remarkably, thebaine synthase (THS), a member of the pathogenesis-related 10 protein (PR10) superfamily, is encoded within a novel gene cluster in the opium poppy genome that also includes genes encoding the four biosynthetic enzymes immediately upstream. THS is a missing component that is crucial to the development of fermentation-based opiate production and dramatically improves thebaine yield in engineered yeast.

CDC5 inhibits the hyperphosphorylation of the checkpoint kinase Rad53, leading to checkpoint adaptation.

The Saccharomyces cerevisiae polo-like kinase Cdc5 promotes adaptation to the DNA damage checkpoint, in addition to its numerous roles in mitotic progression. The process of adaptation occurs when cells are presented with persistent or irreparable DNA damage and escape the cell-cycle arrest imposed by the DNA damage checkpoint. However, the precise mechanism of adaptation remains unknown. We report here that CDC5 is dose-dependent for adaptation and that its overexpression promotes faster adaptation, indicating that high levels of Cdc5 modulate the ability of the checkpoint to inhibit the downstream cell-cycle machinery. To pinpoint the step in the checkpoint pathway at which Cdc5 acts, we overexpressed CDC5 from the GAL1 promoter in damaged cells and examined key steps in checkpoint activation individually. Cdc5 overproduction appeared to have little effect on the early steps leading to Rad53 activation. The checkpoint sensors, Ddc1 (a member of the 9-1-1 complex) and Ddc2 (a member of the Ddc2/Mec1 complex), properly localized to damage sites. Mec1 appeared to be active, since the Rad9 adaptor retained its Mec1 phosphorylation. Moreover, the damage-induced interaction between phosphorylated Rad9 and Rad53 remained intact. In contrast, Rad53 hyperphosphorylation was significantly reduced, consistent with the observation that cell-cycle arrest is lost during adaptation. Thus, we conclude Cdc5 acts to attenuate the DNA damage checkpoint through loss of Rad53 hyperphosphorylation to allow cells to adapt to DNA damage. Polo-like kinase homologs have been shown to inhibit the ability of Claspin to facilitate the activation of downstream checkpoint kinases, suggesting that this function is conserved in vertebrates.

Damage in transition.

Double-stranded DNA breaks (DSBs) are a particularly dangerous form of DNA damage because they can lead to chromosome loss, translocations or truncations. When DSBs occur, many proteins are recruited to the break site; these proteins serve to both initiate DNA repair and to activate a checkpoint response. Repair occurs via one of two pathways: non-homologous end-joining (NHEJ), in which broken DNA ends are directly ligated; or homologous recombination (HR), in which a homologous chromosome is used as a template in a replicative repair process. The checkpoint response is mediated by the phosphatidyl inositol 3-kinase-like kinases, Mec1 and Tel1 (ATR and ATM in humans, respectively). Two recent studies in yeast have significantly increased our understanding of when each of the proteins involved in these processes is localized to a break and, in addition, how their sequential localization is achieved. Specifically, these studies support and expand upon a model in which Tel1 and the NHEJ proteins are the first proteins to localize to the break to initiate signaling and attempt repair, but are subsequently replaced by Mec1 and the HR proteins. This transition is mediated by a cyclin-dependent kinase-dependent initiation of 5'-->3' processing (resection) of the DSB. Thus, the cell-cycle stage at which DSBs occur affects the way in which the DSBs are processed and recognized.

Conserved and divergent roles of Bcr1 and CFEM proteins in Candida parapsilosis and Candida albicans.

Candida parapsilosis is a pathogenic fungus that is major cause of hospital-acquired infection, predominantly due to growth as biofilms on indwelling medical devices. It is related to Candida albicans, which remains the most common cause of candidiasis disease in humans. The transcription factor Bcr1 is an important regulator of biofilm formation in vitro in both C. parapsilosis and C. albicans. We show here that C. parapsilosis Bcr1 is required for in vivo biofilm development in a rat catheter model, like C. albicans. By comparing the transcription profiles of a bcr1 deletion in both species we found that regulation of expression of the CFEM family is conserved. In C. albicans, three of the five CFEM cell wall proteins (Rbt5, Pga7 and Csa1) are associated with both biofilm formation and acquisition of iron from heme, which is an important virulence characteristic. In C. parapsilosis, the CFEM family has undergone an expansion to 7 members. Expression of three genes (CFEM2, CFEM3, and CFEM6) is dependent on Bcr1, and is induced in low iron conditions. All three are involved in the acquisition of iron from heme. However, deletion of the three CFEM genes has no effect on biofilm formation in C. parapsilosis. Our data suggest that the role of the CFEM family in iron acquisition is conserved between C. albicans and C. parapsilosis, but their role in biofilm formation is not.

Cdc5 blocks in vivo Rad53 activity, but not in situ activity (ISA).

DNA damage promotes the activation of a signal transduction cascade referred to as the DNA damage checkpoint. This pathway initiates with the Mec1/ATR kinase, which then phosphorylates the Rad53/Chk2 kinase. Mec1 phosphorylation of Rad53 is then thought to promote Rad53 autophosphorylation, ultimately leading to a fully active Rad53 molecule that can go on to phosphorylate substrates important for DNA damage resistance. In the absence of DNA repair, this checkpoint is eventually downregulated in a Cdc5-dependent process referred to as checkpoint adaptation. Recently, we showed that overexpression of Cdc5 leads to checkpoint inactivation and loss of the strong electrophoretic shift associated with Rad53 inactivation. Interestingly, this same overexpression did not strongly inhibit Rad53 autophosphorylation activity as measured by the in situ assay (ISA). The ISA involves incubating the re-natured Rad53 protein with γ ³²P labeled ATP after electrophoresis and western blotting. Using a newly identified Rad53 target, we show that despite strong ISA activity, Rad53 does not maintain phosphorylation of this substrate. We hypothesize that, during adaptation, Rad53 may be in a unique state in which it maintains some Mec1 phosphorylation, but does not have the auto-phosphorylations required for full activity towards exogenous substrates.

Complicated tails: histone modifications and the DNA damage response.

In recent years, several ATP-dependent chromatin-remodeling complexes and covalent histone modifications have been implicated in the response to double-stranded DNA breaks (DSBs). When a DSB occurs, cells must identify the DSB, activate the DNA damage checkpoint, and repair the break. Chromatin modification appears to be important but not essential for each of these processes, yet its precise mechanistic roles are only beginning to come into focus. Here, we discuss the role of chromatin in signaling by the DNA damage checkpoint pathway.

Silencing of pi-class glutathione S-transferase in MDA PCa 2a and MDA PCa 2b cells.

BACKGROUND: Loss of expression of the glutathione S-transferase-pi (GSTP1) is the most common genetic alteration described in human prostate cancer, occurring in virtually all tumors regardless of grade or stage. Of the available human prostate cancer cell lines, only LNCaP mirrors this phenotype. We investigated whether the prostate cancer cell lines MDA PCa 2a and MDA PCa 2b share this phenotype. METHODS: GSTP1 protein and mRNA levels were assessed in the MDA PCa 2a and MDA PCa 2b cell lines by Western and Northern blot. DNA methylation was evaluated by Southern blot analysis of genomic DNA digested with the methylation-sensitive restriction enzymes BssHII, NotI, and SacII. Re-expression of GSTP1 was determined by RT-PCR following treatment with 5-azacytidine, a DNA methyltransferase inhibitor, and/or the histone deacetylase inhibitor trichostatin A (TSA). RESULTS: Like all human prostatic carcinomas in vivo, both the MDA PCa 2a and 2b cell lines lack protein and mRNA expression of GSTP1. This lack of expression is associated with methylation in the GSTP1 gene promoter. Treatment with the methyltransferase inhibitor 5-azacytidine resulted in re-expression of GSTP1. By itself, TSA did not result in re-expression of GSTP1, nor did it augment expression induced by 5-azacytidine. CONCLUSIONS: MDA PCa 2a and 2b appear to be useful models of human prostatic carcinoma in that they lack expression of GSTP1 due to gene silencing via promoter methylation. Inhibition of histone acetylation does not appear to affect GSTP1 expression.