Helmut Bergler

Diazaborine resistance in the yeast Saccharomyces cerevisiae reveals a link between YAP1 and the pleiotropic drug resistance genes PDR1 and PDR3

We have investigated the mechanisms underlying resistance to the drug diazaborine in Saccharomyces cerevisiae. We used UV mutagenesis to generate resistant mutants, which were divided into three different complementation groups. The resistant phenotype in these groups was found to be caused by allelic forms of the genes AFG2, PDR1, andPDR3. The AFG2 gene encodes an AAA (ATPases associated to a variety of cellularactivities) protein of unknown function, whilePDR1 and PDR3 encode two transcriptional regulatory proteins involved in pleiotropic drug resistance development. The isolated PDR1–12 and PDR3–33alleles carry mutations that lead to a L1044Q and a Y276H exchange, respectively. In addition, we report that overexpression of Yap1p, the yeast homologue of the transcription factor AP1, results in a diazaborine-resistant phenotype. The YAP1-mediated diazaborine resistance is dependent on the presence of functionalPDR1 and PDR3 genes, although PDR3had a more pronounced effect. These results provide the first evidence for a functional link between the Yap1p-dependent stress response pathway and Pdr1p/Pdr3p-dependent development of pleiotropic drug resistance.

Cytoplasmic recycling of 60S preribosomal factors depends on the AAA protein Drg1.

ABSTRACT Allelic forms of DRG1/AFG2 confer resistance to the drug diazaborine, an inhibitor of ribosome biogenesis in Saccharomyces cerevisiae. Our results show that the AAA-ATPase Drg1 is essential for 60S maturation and associates with 60S precursor particles in the cytoplasm. Functional inactivation of Drg1 leads to an increased cytoplasmic localization of shuttling pre-60S maturation factors like Rlp24, Arx1, and Tif6. Surprisingly, Nog1, a nuclear pre-60S factor, was also relocalized to the cytoplasm under these conditions, suggesting that it is a previously unsuspected shuttling preribosomal factor that is exported with the precursor particles and very rapidly reimported. Proteins that became cytoplasmic under drg1 mutant conditions were blocked on pre-60S particles at a step that precedes the association of Rei1, a later-acting preribosomal factor. A similar cytoplasmic accumulation of Nog1 and Rlp24 in pre-60S-bound form could be seen after overexpression of a dominant-negative Drg1 variant mutated in the D2 ATPase domain. We conclude that the ATPase activity of Drg1 is required for the release of shuttling proteins from the pre-60S particles shortly after their nuclear export. This early cytoplasmic release reaction defines a novel step in eukaryotic ribosome maturation.

Transcriptional Activation of ZEB1 by Slug Leads to Cooperative Regulation of the Epithelial–Mesenchymal Transition-Like Phenotype in Melanoma

The E-box-binding zinc finger transcription factors Slug and ZEB1 are important repressors of E-cadherin, contributing to epithelial-mesenchymal transition (EMT) in primary epithelial cancers. Activator or repressor status of EMT transcription factors defines consequences for tumorigenesis. We show that changes in expression levels of Slug in melanoma cell lines lead to concomitant alterations of ZEB1 expression. Electrophoretic mobility shift, luciferase reporter, and chromatin immunoprecipitation assays identified Slug as a direct transcriptional activator at E-boxes of the ZEB1 promoter. Transcriptional activation of ZEB1 was demonstrated to be specific for Slug, as EMT regulators Snail and Twist failed to influence ZEB1 expression. Slug and ZEB1 cooperatively repressed E-cadherin expression resulting in decreased adhesion to human keratinocytes, but promoted migration of melanoma cells. Our results show that the transcriptional activity of ZEB1 is increased by Slug, suggesting a hierarchical organized expression of EMT transcription factors through directed activation, triggering an EMT-like process in melanoma.

The power of AAA-ATPases on the road of pre-60S ribosome maturation--molecular machines that strip pre-ribosomal particles.

The biogenesis of ribosomes is a fundamental cellular process, which provides the molecular machines that synthesize all cellular proteins. The assembly of eukaryotic ribosomes is a highly complex multi-step process that requires more than 200 ribosome biogenesis factors, which mediate a broad spectrum of maturation reactions. The participation of many energy-consuming enzymes (e.g. AAA-type ATPases, RNA helicases, and GTPases) in this process indicates that the expenditure of energy is required to drive ribosome assembly. While the precise function of many of these enzymes remains elusive, recent progress has revealed that the three AAA-type ATPases involved in 60S subunit biogenesis are specifically dedicated to the release and recycling of distinct biogenesis factors. In this review, we will highlight how the molecular power of yeast Drg1, Rix7, and Rea1 is harnessed to promote the release of their substrate proteins from evolving pre-60S particles and, where appropriate, discuss possible catalytic mechanisms. This article is part of a Special Issue entitled: AAA ATPases: structure and function.

Inhibition of lipid biosynthesis induces the expression of the pspA gene.

Treatment of Escherichia coli with diazaborine strongly induces the synthesis of a 28 kDa protein which is associated with the cytoplasmic membrane. The partial amino acid sequence proved that this protein is identical to the phage shock protein PspA. The kinetics of the expression of the pspA gene were determined in an E. coli strain which carried a pspA-lacZ fusion in the chromosome. PspA synthesis is independent of the growth phase. It is, however, strongly induced when fatty acid biosynthesis is inhibited by diazaborine or cerulenin. Treatment with either compound also causes dose-dependent inhibition of phospholipid biosynthesis whose degree correlates with the induction of PspA. Another cause of induction of PspA synthesis is treatment of E. coli with globomycin, which is an inhibitor of the processing of lipoproteins.

Yar1 Protects the Ribosomal Protein Rps3 from Aggregation

Background: Because of their biochemical properties, newly synthesized ribosomal proteins are prone to aggregation. Results: Yar1 directly interacts with free Rps3, accompanies it from the cytoplasm to the nucleus and maintains its solubility. Conclusion: Yar1 acts as an anti-aggregation factor for Rps3. Significance: Ribosomal proteins require protection from aggregation. 2000 ribosomes have to be synthesized in yeast every minute. Therefore the fast production of ribosomal proteins, their efficient delivery to the nucleus and correct incorporation into ribosomal subunits are prerequisites for optimal growth rates. Here, we report that the ankyrin repeat protein Yar1 directly interacts with the small ribosomal subunit protein Rps3 and accompanies newly synthesized Rps3 from the cytoplasm into the nucleus where Rps3 is assembled into pre-ribosomal subunits. A yar1 deletion strain displays a similar phenotype as an rps3 mutant strain, showing an accumulation of 20S pre-rRNA and a 40S export defect. The combination of an rps3 mutation with a yar1 deletion leads to an enhancement of these phenotypes, while increased expression of RPS3 suppresses the defects of a yar1 deletion strain. We further show that Yar1 protects Rps3 from aggregation in vitro and increases its solubility in vivo. Our data suggest that Yar1 is a specific chaperone for Rps3, which serves to keep Rps3 soluble until its incorporation into the pre-ribosome.

Rlp24 activates the AAA-ATPase Drg1 to initiate cytoplasmic pre-60S maturation

Rlp24 recruits Drg1 to pre-60S particles and stimulates its ATP hydrolysis to promote downstream maturation through specific extraction of Rlp24.

The conserved Bud20 zinc finger protein is a new component of the ribosomal 60S subunit export machinery.

ABSTRACT The nuclear export of the preribosomal 60S (pre-60S) subunit is coordinated with late steps in ribosome assembly. Here, we show that Bud20, a conserved C2H2-type zinc finger protein, is an unrecognized shuttling factor required for the efficient export of pre-60S subunits. Bud20 associates with late pre-60S particles in the nucleoplasm and accompanies them into the cytoplasm, where it is released through the action of the Drg1 AAA-ATPase. Cytoplasmic Bud20 is then reimported via a Kap123-dependent pathway. The deletion of Bud20 induces a strong pre-60S export defect and causes synthetic lethality when combined with mutant alleles of known pre-60S subunit export factors. The function of Bud20 in ribosome export depends on a short conserved N-terminal sequence, as we observed that mutations or the deletion of this motif impaired 60S subunit export and generated the genetic link to other pre-60S export factors. We suggest that the shuttling Bud20 is recruited to the nascent 60S subunit via its central zinc finger rRNA binding domain to facilitate the subsequent nuclear export of the preribosome employing its N-terminal extension.

The Cadherin Switch in Melanoma Instigated by HGF is Mediated through Epithelial-Mesenchymal Transition Regulators

Dear Sir, A crucial step in melanoma initiation is the progressive loss of E-cadherin expression, followed by an upregulation of N-cadherin in melanoma cells (Hsu et al., 1996; Tang et al., 1994; Hsu et al., 2000). This process is essentially accomplished by epithelial-mesenchymal transition regulators (EMTRs). Representatives of EMTRs are Snail and Slug of the Snail family of zinc finger transcription factors and Twist of the basic helix-loop-helix (bHLH) family (Cano et al., 2000; Yang et al., 2004). Growth factors govern the continuous repression of E-cadherin via EMTRs to facilitate tumor progression (Thiery and Chopin, 1999; Fuxe et al., 2010). Grotegut et al. (2006) demonstrated that HGF induced scattering and motility in epithelial cells is related to Snail upregulation. We have shown previously that overexpression of HGF downregulates E-cadherin in melanocytic cells (Li et al., 2001). Here we report that HGF leads to stage dependent changes in EMTR expression. Protein expression levels of Slug, Snail and Twist were determined over a period of 24h after stimulation with 50ng/ml of rhHGF in three representative melanocytic cell lines. In melanocytes (FOM101) an increase in Slug and Snail expression was observed (Fig. 1A), in WM35 (Fig. S1A) and WM164 (Fig. 1B) melanoma cells, Slug was profoundly downregulated, whereas Twist expression increased in the nucleus and the cytoplasm (Fig. 1B). To simulate the cadherin switch in melanoma, WM164 cells, which express E- and N-cadherin, were transduced with an adenoviral vector encoding the cDNA of HGF (Ad.CMV.rhHGF) or a mock vector control (Ad.CMV.LacZ) at 20pfu/cell. A profound decrease of E-cadherin and an increase of N-cadherin expression was observed (Fig. 1C). Further, to investigate on signaling pathways involved in EMTR regulation after HGF exposure, we used specific inhibitors of MAPK (PD 98059), PI3K (LY 294002), GSK3β (GSK3β-inhibitor VIII and lithium chloride [LiCl]) or NF-κB (BAY 11-7082). In FOM101, addition of BAY 11-7082 completely abolished protein levels of Slug and Snail in the nucleus (Fig. 1D, left panel) and inhibition of GSK3β led to decreased Slug levels (Fig.1D, right panel). In melanoma cells exposure to PD 98059 led to a significant increase in Slug expression, which resulted in a downregulation of E-cadherin in WM164 (Fig. 1E, left panel and S1B, left panel). Like in melanocytes, inhibition of GSK3β decreased Slug levels (Fig. 1E, right panel and S1B, right panel). Inhibition of MAPK, PI3K or GSK3β leads to downregulation of Twist in the nucleus (Fig. 1E, left panel). Figure 1 HGF mediated stage specific changes of EMTR expression in melanocytic cells are dependent on NF-κB, MAPK, PI3K, and GSK3β. Melanocytic cells were either stimulated with rhHGF (50ng/ml) up to 24h (A, B) or transduced with Ad.CMV.hHGF (20pfu/cell) ... Since HGF led to differential expression levels of Slug and Twist in melanoma cell lines, we determined changes in cadherin levels of both EMTRs after silencing in WM164 cells with consequences for adhesion and migration. Silencing of Slug led to a doubling of E-cadherin levels, whereas expression of N-cadherin was significantly downregulated after Twist silencing with minor changes in E-cadherin levels (Fig. 2A). Correspondingly, we observed a highly significant increase in adhesion of WM164 cells to keratinocytes after silencing of Slug (Fig. 2B) and a profound reduction of adhesion to fibroblasts after silencing of Twist, but not Slug (Fig. 2C). Silencing of both EMTRs resulted in a significant reduction of migration, although silencing of Twist showed a more pronounced effect in scratch assays (Fig. 2D,E). Slug overexpression in WM9 and WM164 cells led to a downregulation of E-cadherin but no changes in N-cadherin levels (Fig. S2A), adhesion to keratinocytes, but not fibroblasts, was clearly reduced (Fig. S2B,C) and migration was enhanced (Fig. S2D,E). Taken together, these results demonstrate Slug to be important for the loss of epithelial properties and a predominant effect of Twist on N-cadherin regulation and adhesion to fibroblasts. Figure 2 Twist promotes adhesion of WM164 cells to fibroblasts. Slug (siSlug) and Twist (siTwist) were silenced and expression levels compared to control siRNA (scRNA). (A) Silencing of Slug led to increased E-cadherin levels whereas silencing of Twist resulted ... Based on the differential regulation of Slug and Twist we examined staining patterns in melanocytic tissue by immunohistochemistry. Examination revealed a highly positive staining for Twist in the cytoplasm of nevi but nearly absence in the nucleus (Fig. 2F and S3A,B). In primary as well as in metastatic lesions cytoplasmatic and nuclear staining of Twist was observed (Fig. 2F and S3A). In contrast, Slug was already found in the nucleus and cytoplasm of nevi (Fig. 2F and S3A). These data suggest an inverse nuclear expression of Slug and Twist in the progression of melanoma. Indeed, probing tissue microarrays with anti-Twist antibodies indicated an association with worse patient survival (Hoek et al., 2004). Together we show that the cadherin switch mediated by HGF is accomplished through stage specific changes in expression levels of Snail, Slug and Twist, suggesting a hierarchical activation of Slug and Twist in melanoma progression. Our data further suggest that Twist is driving melanoma conversion after the initial de-coupling from keratinocytes initiating binding to fibroblasts.

eIF3a is over-expressed in urinary bladder cancer and influences its phenotype independent of translation initiation

PurposeThe eukaryotic translation initiation factor (eIF) 3a, the largest subunit of the eIF3 complex, is a key functional entity in ribosome establishment and translation initiation. In the past, aberrant eIF3a expression has been linked to the pathology of various cancer types but, so far, its expression has not been investigated in transitional cell carcinomas. Here, we investigated the impact of eIF3 expression on urinary bladder cancer (UBC) cell characteristics and UBC patient survival.Methods and resultseIF3a expression was reduced through inducible knockdown in the UBC-derived cell lines RT112, T24, 5637 and HT1197. As a consequence of eIF3a down-regulation, UBC cell proliferation, clonogenic potential and motility were found to be decreased and, concordantly, UBC tumour cell growth rates were found to be impaired in xenotransplanted mice. Polysomal profiling revealed that reduced eIF3a levels increased the abundance of 80S ribosomes, rather than impairing translation initiation. Microarray-based gene expression and ontology analyses revealed broad effects of eIF3a knockdown on the transcriptome. Analysis of eIF3a expression in primary formalin-fixed paraffin embedded UBC samples of 198 patients revealed that eIF3a up-regulation corresponds to tumour grade and that high eIF3a expression corresponds to longer overall survival rates of patients with low grade tumours.ConclusionsFrom our results we conclude that eIF3a expression may have a profound effect on the UBC phenotype and, in addition, may serve as a prognostic marker for low grade UBCs.