Ubiquitin metabolism in Chlamydomonas reinhardtii

This work characterizes parameters of ubiquitin metabolism in Chlamydomonas reinhardtii Dangeard growing under constant conditions and after an exposure to cold shock. Ratio of free and conjugated ubiquitin to total protein, and rate constant of ubiquitin synthesis and conjugation increased about two-fold during first 4 hours after cold treatment, whereas rate constant of ubiquitin degradation reached its maximum 9 hours after treatment. Half-life of ubiquitin calculated from the constant of degradation decreased from 6 hours to 3.5 hours during first four hours after the cold treatment. Rate constant of ubiquitin deconjugation did not change after cold treatment. Ratio of free to conjugated ubiquitin decreased temporarily to approx. 8 immediately after cold treatment and raised back to its original value at 2 h after cold treatment. These observations raise questions regarding the regulatory mechanisms of ubiquitin synthesis and hydrolysis

Expression and Function of Androgen Receptor Coactivator p44/Mep50/WDR77 in Ovarian Cancer

Hormones, including estrogen and progesterone, and their receptors play an important role in the development and progression of ovarian carcinoma. Androgen, its receptor and coactivators have also been implicated in these processes. p44/Mep50/WDR77 was identified as a subunit of the methylosome complex and lately characterized as a steroid receptor coactivator that enhances androgen receptor as well as estrogen receptor-mediated transcriptional activity in a ligand-dependent manner. We previously described distinct expression and function of p44 in prostate, testis, and breast cancers. In this report, we examined the expression and function of p44 in ovarian cancer. In contrast to findings in prostate and testicular cancer and similar to breast cancer, p44 shows strong cytoplasmic localization in morphologically normal ovarian surface and fallopian tube epithelia, while nuclear p44 is observed in invasive ovarian carcinoma. We observed that p44 can serve as a coactivator of both androgen receptor (AR) and estrogen receptor (ER) in ovarian cells. Further, overexpression of nuclear-localized p44 stimulates proliferation and invasion in ovarian cancer cells in the presence of estrogen or androgen. These findings strongly suggest that p44 plays a role in mediating the effects of hormones during ovarian tumorigenesis

A Newly Identified Essential Complex, Dre2-Tah18, Controls Mitochondria Integrity and Cell Death after Oxidative Stress in Yeast

A mutated allele of the essential gene TAH18 was previously identified in our laboratory in a genetic screen for new proteins interacting with the DNA polymerase delta in yeast [1]. The present work shows that Tah18 plays a role in response to oxidative stress. After exposure to lethal doses of H2O2, GFP-Tah18 relocalizes to the mitochondria and controls mitochondria integrity and cell death. Dre2, an essential Fe/S cluster protein and homologue of human anti-apoptotic Ciapin1, was identified as a molecular partner of Tah18 in the absence of stress. Moreover, Ciapin1 is able to replace yeast Dre2 in vivo and physically interacts with Tah18. Our results are in favour of an oxidative stress-induced cell death in yeast that involves mitochondria and is controlled by the newly identified Dre2-Tah18 complex

Apoptose in der Hefe Saccharomyces cerevisiae : ein neuer Zelltod Prozess reguliert durch das Ubiquitin-Proteasome System

Apoptosis is co-regulated by the conserved family of Bcl-2-related proteins, which includes both its agonists (Bax) and antagonists (Bcl-XL). A mutant strain of the yeast Saccharomyces cerevisiae has been shown to express all morphological signs of apoptosis. Overexpression of Bax is lethal in S. cerevisiae, whereas simultaneous overexpression of Bcl-XL rescues the cells. We report that overexpression of mammalian Bax in a S. cerevisiae wild type strain triggers morphological changes similar to those of apoptotic metazoan cells: the loss of asymmetric distribution of plasma membrane phosphatidylserine, plasma membrane blebbing, chromatin condensation and margination, and DNA fragmentation. Simultaneous overexpression of Bcl-XL prevents these changes. We demonstrate that Bax triggers phenotypic alterations in yeast strongly resembling those it causes in metazoan apoptotic cells. Oxygen radicals are important components of metazoan apoptosis. Oxygen radicals accumulate in yeast cells overexpressing Bax, whereas radical depletion or hypoxia prevents apoptosis. This suggests that the generation of oxygen radicals is a key event in the ancestral apoptotic pathway and offer an explanation for the mechanism of Bax-induced apoptosis in the absence of any established apoptotic gene in yeast. I have identified the yeast gene STM1 in an overexpression screen for new proteasomal substrates. Stm1 is a bona fide substrate of the proteasome. It is localized in the perinuclear region and is required for growth in the presence of mutagens. Overexpression in cells with impaired proteasomal degradation leads to cell death accompanied with cytological markers of apoptosis. Cells lacking Stm1 display deficiency in the apoptosis-like cell death process induced by treatment with low concentrations of H2O2. I suggest that Stm1 is involved in the control of the apoptosis-like cell death in yeast.Das Apoptoseprogramm wird unter anderem reguliert durch eine Reihe von Bcl-2 verwandten Proteinen, wie Bax oder Bcl-XL. In einer vorausgehenden Arbeit war gezeigt worden, dass eine Mutation in Hefezellen einen Zelltod auslösen kann, der mit den in Säugetierzellen Apoptose-typischen morphologischen Veränderungen einhergeht. Bax Protein bei Expression in Hefezellen einen Zelltod induziert, der die typischen Anzeichen der Apoptose in Säugerzellen aufweist: So wurde ein Verlust der asymmetrischen Verteilung von Phosphatidylserin in der Plasmamembran sowie das Auftreten von Vesikeln an der Plasmamembran beobachtet. Darüber hinaus war ein Kondensieren des Chromatins sowie eine anschließende Anlagerung an die Kernhülle feststellbar. Außerdem konnte mit Hilfe des TUNEL Tests eindeutig eine Fragmentierung der DNA nachgewiesen werden. Diese Untersuchungen zeigten klar, dass der durch Bax in S. cerevisiae Zellen induzierte Zelltod klar apoptotischer Natur ist. Sauerstoffradikale sind wichtige Mediatoren der Apoptose in Metazoen. Die Sauerstoffradikale akkumulieren in Zellen denen Bax überexprimiert wurde. Bei Verminderung der Radikale durch Einsatz von Radikalfängern oder bei Anziehen der Zellen in sauerstofffreiem Medium konnte der durch Bax induzierte Zelltod verhindert werden. Diese Befunde sprechen für das Vorliegen eines konservierten Mechanismus, der durch die Apoptoseregulatoren aus Säugetieren angesprochen werden kann. Um herauszufinden ob Ubiquitin-abhängige Proteolyse in S. cerevisae eine Rolle in der Regulation der Apoptose spielen kann, wurde in einem zweistufigen Ansatz nach potenziell beteiligten Proteinen gesucht. STM1 war durch dieses Verfahren identifiziert. Stm1 konnte als in vivo Substrat des proteasomalen Systems charakterisiert werden. Lokalisationsstudien zeigten, dass Stm1 in der Peripherie des Zellkerns lokalisiert ist. Zellen denen Stm1 fehlt zeigen einen Defekt in der Induktion der Apoptose durch niedrige Konzentrationen von H2O2

Gene Expression From Random Libraries of Yeast Promoters

Genomewide techniques to assay gene expression and transcription factor binding are in widespread use, but are far from providing predictive rules for the function of regulatory DNA. To investigate more intensively the grammar rules for active regulatory sequence, we made libraries from random ligations of a very restricted set of sequences. Working with the yeast Saccharomyces cerevisiae, we developed a novel screen based on the sensitivity of ascospores lacking dityrosine to treatment with lytic enzymes. We tested two separate libraries built by random ligation of a single type of activator site either for a well-characterized sporulation factor, Ndt80, or for a new sporulation-specific regulatory site that we identified and several neutral spacer elements. This selective system achieved up to 1:10(4) enrichment of the artificial sequences that were active during sporulation, allowing a high-throughput analysis of large libraries of synthetic promoters. This is not practical with methods involving direct screening for expression, such as those based on fluorescent reporters. There were very few false positives, since active promoters always passed the screen when retested. The survival rate of our libraries containing roughly equal numbers of spacers and activators was a few percent that of libraries made from activators alone. The sequences of ∼100 examples of active and inactive promoters could not be distinguished by simple binary rules; instead, the best model for the data was a linear regression fit of a quantitative measure of gene activity to multiple features of the regulatory sequence