Vertebrate eggs are arrested at the metaphase stage of meiosis II. Only upon fertilization will the metaphase-II-arrested eggs exit meiosis II and enter interphase. In 1971, Masui and Markert ...injected egg extracts into a two-cell-stage embryo and found that the injected blastomere arrested at the next mitosis. On the basis of these observations, they proposed the existence of an activity present in the eggs that is responsible for meiosis-II arrest and can induce mitotic arrest, and named this activity cytostatic factor (CSF). Although the existence of CSF was hypothesized more than 35 years ago, its precise identity remained unclear until recently. The discovery of the Mos-MAPK pathway and characterization of the anaphase-promoting complex/cyclosome (APC/C) as a central regulator of M-phase exit provided the framework for a molecular understanding of CSF. These pathways have now been linked by the discovery and characterization of the protein Emi2, a meiotic APC/C inhibitor, the activity and stability of which are controlled by the Mos-MAPK pathway. Continued investigation into the mechanism of action and mode of regulation of Emi2 promises to shed light not only on CSF function, but also on the general principles of APC/C regulation and the control of protein function by MAPK pathways.
Loss of cell division cycle 2 (Cdc2, also known as Cdk1) activity after cyclin B degradation is necessary, but not sufficient, for mitotic exit. Proteins phosphorylated by Cdc2 and downstream mitotic ...kinases must be dephosphorylated. We report here that protein phosphatase-1 (PP1) is the main catalyst of mitotic phosphoprotein dephosphorylation. Suppression of PP1 during early mitosis is maintained through dual inhibition by Cdc2 phosphorylation and the binding of inhibitor-1. Protein kinase A (PKA) phosphorylates inhibitor-1, mediating binding to PP1. As Cdc2 levels drop after cyclin B degradation, auto-dephosphorylation of PP1 at its Cdc2 phosphorylation site (Thr 320) allows partial PP1 activation. This promotes PP1-regulated dephosphorylation at the activating site of inhibitor-1 (Thr 35) followed by dissociation of the inhibitor-1-PP1 complex and then full PP1 activation to promote mitotic exit. Thus, Cdc2 both phosphorylates multiple mitotic substrates and inhibits their PP1-mediated dephosphorylation.
The human cytochrome P450 (P450) superfamily consists of membrane‐bound proteins that metabolize a myriad of xenobiotics and endogenous compounds. Quantification of P450 expression in various tissues ...under normal and induced conditions has an important role in drug safety and efficacy. Conventional immunoquantification methods have poor dynamic range, low throughput, and a limited number of specific antibodies. Recent advances in MS‐based quantitative proteomics enable absolute protein quantification in a complex biological mixture. We have developed a gel‐free MS‐based protein quantification strategy to quantify CYP3A enzymes in human liver microsomes (HLM). Recombinant protein‐derived proteotypic peptides and synthetic stable isotope‐labeled proteotypic peptides were used as calibration standards and internal standards, respectively. The lower limit of quantification was ˜20 fmol P450. In two separate panels of HLM examined (n = 11 and n = 22), CYP3A, CYP3A4 and CYP3A5 concentrations were determined reproducibly (CV ≤27%). The MS‐based method strongly correlated with the immunoquantification method (r2≥0.87) and marker activities (r2≥0.88), including testosterone 6β‐hydroxylation (CYP3A), midazolam 1′‐hydroxylation (CYP3A), itraconazole 6‐hydroxylation (CYP3A4) and CYP3A5‐mediated vincristine M1 formation (CYP3A5). Taken together, our MS‐based method provides a specific, sensitive and reliable means of P450 protein quantification and should facilitate P450 characterization during drug development, especially when specific substrates and/or antibodies are unavailable.
CYP4F enzymes, including CYP4F2 and CYP4F3B, were recently shown to be the major enzymes catalyzing the initial oxidative O-demethylation of the antiparasitic prodrug pafuramidine (DB289) by human ...liver microsomes. As suggested by a low oral bioavailability, DB289 could undergo first-pass biotransformation in the intestine, as well as in the liver. Using human intestinal microsomes (HIM), we characterized the enteric enzymes that catalyze the initial O-demethylation of DB289 to the intermediate metabolite, M1. M1 formation in HIM was catalyzed by cytochrome P450 (P450) enzymes, as evidenced by potent inhibition by 1-aminobenzotriazole and the requirement for NADPH. Apparent K(m) and V(max) values ranged from 0.6 to 2.4 microM and from 0.02 to 0.89 nmol/min/mg protein, respectively (n = 9). Of the P450 chemical inhibitors evaluated, ketoconazole was the most potent, inhibiting M1 formation by 66%. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, inhibited M1 formation in a concentration-dependent manner (up to 95%). Immunoinhibition with an antibody raised against CYP4F2 showed concentration-dependent inhibition of M1 formation (up to 92%), whereas antibodies against CYP3A4/5 and CYP2J2 had negligible to modest effects. M1 formation rates correlated strongly with arachidonic acid omega-hydroxylation rates (r(2) = 0.94, P < 0.0001, n = 12) in a panel of HIM that lacked detectable CYP4A11 protein expression. Quantitative Western blot analysis revealed appreciable CYP4F expression in these HIM, with a mean (range) of 7 (3-18) pmol/mg protein. We conclude that enteric CYP4F enzymes could play a role in the first-pass biotransformation of DB289 and other xenobiotics.
Before fertilization, vertebrate eggs are arrested in meiosis II by cytostatic factor (CSF), which holds the anaphase-promoting complex (APC) in an inactive state. It was recently reported that Mos, ...an integral component of CSF, acts in part by promoting the Rsk-mediated phosphorylation of the APC inhibitor Emi2/Erp1. We report here that Rsk phosphorylation of Emi2 promotes its interaction with the protein phosphatase PP2A. Emi2 residues adjacent to the Rsk phosphorylation site were important for PP2A binding. An Emi2 mutant that retained Rsk phosphorylation but lacked PP2A binding could not be modulated by Mos. PP2A bound to Emi2 acted on two distinct clusters of sites phosphorylated by Cdc2, one responsible for modulating its stability during CSF arrest and one that controls binding to the APC. These findings provide a molecular mechanism for Mos action in promoting CSF arrest and also define an unusual mechanism, whereby protein phosphorylation recruits a phosphatase for dephosphorylation of distinct sites phosphorylated by another kinase.
Vertebrate eggs are arrested at Metaphase II by Emi2, the meiotic anaphase-promoting complex/cyclosome (APC/C) inhibitor. Although the importance of Emi2 during oocyte maturation has been widely ...recognized and its regulation extensively studied, its mechanism of action remained elusive. Many APC/C inhibitors have been reported to act as pseudosubstrates, inhibiting the APC/C by preventing substrate binding. Here we show that a previously identified zinc-binding region is critical for the function of Emi2, whereas the D-box is largely dispensable. We further demonstrate that instead of acting through a "pseudosubstrate" mechanism as previously hypothesized, Emi2 can inhibit Cdc20-dependent activation of the APC/C substoichiometrically, blocking ubiquitin transfer from the ubiquitin-charged E2 to the substrate. These findings provide a novel mechanism of APC/C inhibition wherein the final step of ubiquitin transfer is targeted and raise the interesting possibility that APC/C is inhibited by Emi2 in a catalytic manner.
The transition of oocytes from meiosis I (MI) to meiosis II (MII) requires partial cyclin B degradation to allow MI exit without S phase entry. Rapid reaccumulation of cyclin B allows direct ...progression into MII, producing a cytostatic factor (CSF)-arrested egg. It has been reported that dampened translation of the anaphase-promoting complex (APC) inhibitor Emi2 at MI allows partial APC activation and MI exit. We have detected active Emi2 translation at MI and show that Emi2 levels in MI are mainly controlled by regulated degradation. Emi2 degradation in MI depends not on Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), but on Cdc2-mediated phosphorylation of multiple sites within Emi2. As in MII, this phosphorylation is antagonized by Mos-mediated recruitment of PP2A to Emi2. Higher Cdc2 kinase activity in MI than MII allows sufficient Emi2 phosphorylation to destabilize Emi2 in MI. At MI anaphase, APC-mediated degradation of cyclin B decreases Cdc2 activity, enabling Cdc2-mediated Emi2 phosphorylation to be successfully antagonized by Mos-mediated PP2A recruitment. These data suggest a model of APC autoinhibition mediated by stabilization of Emi2; Emi2 proteins accumulate at MI exit and inhibit APC activity sufficiently to prevent complete degradation of cyclin B, allowing MI exit while preventing interphase before MII entry.
Clinical pharmacogenomics is an expanding area in healthcare that relies heavily on pharmacists for advocacy and implementation. To support pharmacists' significant roles in clinical ...pharmacogenomics, pharmacy schools and colleges in the United States (US) have strived to incorporate pharmacogenomics education into their curricula, and various teaching strategies have been employed in recent years to meet pharmacogenomics educational outcomes. The six major strategies reported in the literature are described and compared in this review, which culminates in a proposed longitudinal curriculum design for pharmacogenomics education.
Publications focused on pharmacogenomics education to pharmacy students within the US in the past decade were evaluated and summarized.
The major education strategies that have been studied are didactic lecture, personal genotyping or personal genomic testing, simulation laboratory activity, interprofessional education, practice-based activity such as clinical rotation, and combinational courses. Strengths and limitations of each teaching strategy are summarized and discussed.
Based upon each education strategy's strengths and weaknesses, the authors propose a longitudinal curriculum design to ensure that pharmacogenomics is taught multiple times to pharmacy students with diverse formats and teaching objectives conducive to long-term knowledge retention and practice readiness. Through this longitudinal curriculum design, pharmacy graduates will be well equipped to lead clinical pharmacogenomics in practice.
Cytochrome P450 1B1 (CYP1B1) is an anticancer therapeutic target due to its overexpression in a number of steroid hormone-related cancers. One anticancer drug discovery strategy is to develop ...prodrugs specifically activated by CYP1B1 in malignant tissues to cytotoxic metabolites. Here, we aimed to develop an in vitro screening model for CYP1B1-targeted anticancer prodrugs using the KLE human endometrial carcinoma cell line. KLE cells demonstrated superior stability of CYP1B1 expression relative to transiently transfected cells and did not express any appreciable amount of cognate CYP1A1 or CYP1A2, which would have compromised the specificity of the screening assay. The effect of two CYP1B1-targeted probe prodrugs on KLE cells was evaluated in the absence and presence of a CYP1B1 inhibitor to chemically "knock out" CYP1B1 activity (CYP1B1 inhibited). Both probe prodrugs were more toxic to KLE cells than to CYP1B1-inhibited KLE cells and significantly induced G0/G1 arrest and decreased the S phase in KLE cells. They also exhibited pro-apoptotic effects in KLE cells, which were attenuated in CYP1B1-inhibited KLE cells. In summary, a KLE cell-based model has been characterized to be suitable for identifying CYP1B1-targeted anticancer prodrugs and should be further developed and employed for screening chemical libraries.
In a recent issue of
Molecular Cell,
Enquist-Newman et al. (2008) demonstrate that Acm1 is ubiquitinated by APC
Cdc20. By contrast, the high-affinity interaction between Acm1 and APC
Cdh1 renders it ...a poor substrate, but a specific inhibitor, of the APC
Cdh1 complex.
PDF
