Inhibitors Of Eukaryotic Translation Research Articles

The AAA-ATPase MIDASIN 1 Functions in Ribosome Biogenesis and Is Essential for Embryo and Root Development.

Ribosome biogenesis is an orchestrated process that relies on many assembly factors. The AAA-ATPase Midasin 1 (Mdn1) functions as a ribosome assembly factor in yeast (Saccharomyces cerevisiae), but the roles of MDN1 in Arabidopsis (Arabidopsis thaliana) are poorly understood. Here, we showed that the Arabidopsis null mutant of MDN1 is embryo-lethal. Using the weak mutant mdn1-1, which maintains viability, we found that MDN1 is critical for the regular pattern of auxin maxima in the globular embryo and functions in root meristem maintenance. By detecting the subcellular distribution of ribosome proteins, we noted that mdn1-1 impairs nuclear export of the pre-60S ribosomal particle. The processing of ribosomal precusor RNAs, including 35S, 27SB, and 20S, is also affected in this mutant. MDN1 physically interacts with PESCADILLO2 (PES2), an essential assembly factor of the 60S ribosome, and the observed mislocalization of PES2 in mdn1-1 further implied that MDN1 plays an indispensable role in 60S ribosome biogenesis. Therefore, the observed hypersensitivity of mdn1-1 to a eukaryotic translation inhibitor and high-sugar conditions might be associated with the defect in ribosome biogenesis. Overall, this work establishes a role of Arabidopsis MDN1 in ribosome biogenesis, which agrees with its roles in embryogenesis and root development.

Inhibition of Eukaryotic Translation by the Antitumor Natural Product Agelastatin A

Protein synthesis plays an essential role in cell proliferation, differentiation, and survival. Inhibitors of eukaryotic translation have entered the clinic, establishing the translation machinery as a promising target for chemotherapy. A recently discovered, structurally unique marine sponge-derived brominated alkaloid, (-)-agelastatin A (AglA), possesses potent antitumor activity. Its underlying mechanism of action, however, has remained unknown. Using a systematic top-down approach, we show that AglA selectively inhibits protein synthesis. Using a high-throughput chemical footprinting method, we mapped the AglA-binding site to the ribosomal A site. A 3.5Å crystal structure of the 80S eukaryotic ribosome from S.cerevisiae in complex with AglA was obtained, revealing multiple conformational changes of the nucleotide bases in the ribosome accompanying the binding of AglA. Together, these results have unraveled the mechanism of inhibition of eukaryotic translation by AglA at atomic level, paving the way for future structural modifications to develop AglA analogs into novel anticancer agents.

Antimicrobial Activity, AME Resistance, and A-Site Binding Studies of Anthraquinone-Neomycin Conjugates.

The antibacterial effects of aminoglycosides are based on their association with the A-site of bacterial rRNA and interference with the translational process in the bacterial cell, causing cell death. The clinical use of aminoglycosides is complicated by resistance and side effects, some of which arise from their interactions with the human mitochondrial 12S rRNA and its deafness-associated mutations, C1494U and A1555G. We report a rapid assay that allows screening of aminoglycoside compounds to these classes of rRNAs. These screening tools are important to find antibiotics that selectively bind to the bacterial A-site rather than human, mitochondrial A-sites and its mutant homologues. Herein, we report our preliminary work on the optimization of this screen using 12 anthraquinone-neomycin (AMA-NEO) conjugates against molecular constructs representing five A-site homologues, Escherichia coli, human cytosolic, mitochondrial, C1494U, and A1555G, using a fluorescent displacement screening assay. These conjugates were also tested for inhibition of protein synthesis, antibacterial activity against 14 clinically relevant bacterial strains, and the effect on enzymes that inactivate aminoglycosides. The AMA-NEO conjugates demonstrated significantly improved resistance against aminoglycoside-modifying enzymes (AMEs), as compared with NEO. Several compounds exhibited significantly greater inhibition of prokaryotic protein synthesis as compared to NEO and were extremely poor inhibitors of eukaryotic translation. There was significant variation in antibacterial activity and MIC of selected compounds between bacterial strains, with Escherichiacoli, Enteroccocus faecalis, Citrobacter freundii, Shigella flexneri, Serratia marcescens, Proteus mirabilis, Enterobacter cloacae, Staphylococcus epidermidis, and Listeria monocytogenes exhibiting moderate to high sensitivity (50-100% growth inhibition) whereas Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiellla pneumoniae, and MRSA strains expressed low sensitivity, as compared to the parent aminoglycoside NEO.

Hygromycin B hypersensitive (hhy) mutants implicate an intact trans-Golgi and late endosome interface in efficient Tor1 vacuolar localization and TORC1 function.

Saccharomyces cerevisiae vacuoles are functionally analogous to mammalian lysosomes. Both also serve as physical platforms for Tor Complex 1 (TORC1) signal transduction, the master regulator of cellular growth and proliferation. Hygromycin B is a eukaryotic translation inhibitor. We recently reported on hygromycin B hypersensitive (hhy) mutants that fail to grow at subtranslation inhibitory concentrations of the drug and exhibit vacuolar defects (Banuelos et al. in Curr Genet 56:121-137, 2010). Here, we show that hhy phenotype is not due to increased sensitivity to translation inhibition and establish a super HHY (s-HHY) subgroup of genes comprised of ARF1, CHC1, DRS2, SAC1, VPS1, VPS34, VPS45, VPS52, and VPS54 that function exclusively or inclusively at trans-Golgi and late endosome interface. Live cell imaging of s-hhy mutants revealed that hygromycin B treatment disrupts vacuolar morphology and the localization of late endosome marker Pep12, but not that of late endosome-independent vacuolar SNARE Vam3. This, along with normal post-late endosome trafficking of the vital dye FM4-64, establishes that severe hypersensitivity to hygromycin B correlates specifically with compromised trans-Golgi and late endosome interface. We also show that Tor1p vacuolar localization and TORC1 anabolic functions, including growth promotion and phosphorylation of its direct substrate Sch9, are compromised in s-hhy mutants. Thus, an intact trans-Golgi and late endosome interface is a requisite for efficient Tor1 vacuolar localization and TORC1 function.

Abstract 2805: LPA-dependent regulation of VCAM-1 in the ovarian cancer metastatic microenvironment and its impact on mesothelial invasion.

Abstract Ovarian cancer is the leading cause of gynecological cancer deaths in the United States due in large part to the advanced stage of disease at diagnosis. The poor prognosis is attributed to ovarian cancer metastasis to the peritoneal cavity and the accumulation of ascites, which contains elevated levels of several growth factors and cytokines including lysophosphatidic acid (LPA), a bioactive lipid that promotes ovarian cancer proliferation and survival as well as activities relevant to metastasis including migration and invasion. Ovarian cancer metastasis is a complex process that involves attachment to and invasion through the mesothelium and has been associated with vascular cell adhesion molecule 1 (VCAM-1). Previously we have shown that VCAM-1 is preferentially expressed on the mesothelium of patients with ovarian cancer where it functions to promote mesothelial invasion. Inhibition of VCAM-1 function reduces tumor burden and increases survival in a mouse model, implicating VCAM-1 as an important target for the treatment of ovarian cancer. Since LPA promotes ovarian cancer migration and invasion, we questioned whether it also influenced the mesothelium to regulate invasion. Using a co-culture system to measure ovarian cancer cell invasion of the mesothelium, treatment with a VPC51299, a LPA1/3 receptor antagonist, which targets both the cancer cells and the mesothelium, resulted a dose dependent decrease in mesothelial invasion. Importantly, specific targeting of the mesothelium using siRNA directed at the LPA1 receptor also diminished ovarian cancer cell invasion of the mesothelium. Additionally, we observed that siRNA knockdown of the LPA receptor diminished VCAM-1 expression; re-expression of VCAM-1 to endogenous levels in cells lacking LPA receptor expression restored mesothelial invasion. To determine how LPA regulated VCAM-1 expression, human mesothelial cells derived from ovarian cancer patients (LP9 or LP3) were subjected to siRNA inhibition of LPA1 receptor expression and protein and mRNA expression were evaluated. The results revealed a significant decrease in VCAM-1 protein expression but no impact on VCAM-1 mRNA levels, indicating that LPA does not have a major role in the regulation of VCAM-1 transcription as has been reported in other systems. Rather, it implicates LPA in the regulation of VCAM-1 protein expression. To investigate whether LPA regulates VCAM-1 protein stability, LP9 cells were treated with a eukaryotic translation inhibitor, cycloheximide, for increasing periods of time. VCAM-1 protein half-life decreased after treatment with cycloheximide and LPA1 knockdown compared to control. Taken together, these observations indicate that in addition to targeting the cancer cells, LPA influences the tumor microenvironment to promote mesothelial invasion in part by regulating VCAM-1 protein stability. Citation Format: Timothy A. Raines, Kevin R. Lynch, Jill K. Slack-Davis. LPA-dependent regulation of VCAM-1 in the ovarian cancer metastatic microenvironment and its impact on mesothelial invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2805. doi:10.1158/1538-7445.AM2013-2805

Biomimetic Photocycloaddition of 3‐Hydroxyflavones: Synthesis and Evaluation of Rocaglate Derivatives as Inhibitors of Eukaryotic Translation

Shedding light on translation: A light-driven, biomimetic [3+2] cycloaddition has been achieved with the synthesis of a series of rocaglate derivatives. Mechanistic data suggest the possibility for donor–acceptor interactions and the involvement of triplet biradicaloids in the photoexcited state. Several new rocaglate derivatives approach the potency of the anticancer agent silvestrol.

Biomimetic Photocycloaddition of 3‐Hydroxyflavones: Synthesis and Evaluation of Rocaglate Derivatives as Inhibitors of Eukaryotic Translation

Durch Licht geschaffen: Die Titelreaktion wurde zur Synthese einer Reihe von Rocaglat-Derivaten genutzt. Mechanistischen Studien zufolge könnten Donor-Akzeptor-Wechselwirkungen und Triplettdiradikal-artige Zustände am photochemisch angeregten Zustand beteiligt sein. Einige neue Rocaglat-Derivate reichen in ihrer Wirksamkeit an das Tumortherapeutikum Silvestrol heran.

A genome-wide screen in Saccharomyces cerevisiae reveals a critical role for the mitochondria in the toxicity of a trichothecene mycotoxin

Trichothecene mycotoxins synthesized by Fusarium species are potent inhibitors of eukaryotic translation. They are encountered in both the environment and in food, posing a threat to human and animal health. They have diverse roles in the cell that are not limited to the inhibition of protein synthesis. To understand the trichothecene mechanism of action, we screened the yeast knockout library to identify genes whose deletion confers resistance to trichothecin (Tcin). The largest group of resistant strains affected mitochondrial function, suggesting a role for fully active mitochondria in trichothecene toxicity. Tcin inhibited mitochondrial translation in the wild-type strain to a greater extent than in the most resistant strains, implicating mitochondrial translation as a previously unrecognized site of action. The Tcin-resistant strains were cross-resistant to anisomycin and chloramphenicol, suggesting that Tcin targets the peptidyltransferase center of mitochondrial ribosomes. Tcin-induced cell death was partially rescued by mutants that regulate mitochondrial fusion and maintenance of the tubular morphology of mitochondria. Treatment of yeast cells with Tcin led to the fragmentation of the tubular mitochondrial network, supporting a role for Tcin in disruption of mitochondrial membrane morphology. These results provide genome-wide insight into the mode of action of trichothecene mycotoxins and uncover a critical role for mitochondrial translation and membrane maintenance in their toxicity.

Satratoxin G interaction with 40S and 60S ribosomal subunits precedes apoptosis in the macrophage

Satratoxin G (SG) and other macrocyclic trichothecene mycotoxins are potent inhibitors of eukaryotic translation that are potentially immunosuppressive. The purpose of this research was to test the hypothesis that SG-induced apoptosis in the macrophage correlates with binding of this toxin to the ribosome. Exposure of RAW 264.7 murine macrophages to SG at concentrations of 10 to 80 ng/ml induced DNA fragmentation within 4 h that was indicative of apoptosis. To relate these findings to ribosome binding of SG, RAW cells were exposed to different toxin concentrations for various time intervals, ribosomal fractions isolated by sucrose density gradient ultracentrifugation and resultant fractions analyzed for SG by competitive ELISA. SG was found to specifically interact with 40S and 60S ribosomal subunits as early as 5 min and that, at high concentrations or extended incubation times, the toxin induced polysome disaggregation. While co-incubation with the simple Type B trichothecene DON had no effect on SG uptake into cell cytoplasm, it inhibited SG binding to the ribosome, suggesting that the two toxins bound to identical sites and that SG binding was reversible. Although both SG and DON induced mobilization of p38 and JNK 1/2 to the ribosome, phosphorylation of ribosomal bound MAPKs occurred only after DON treatment. SG association with the 40S and 60S subunits was also observed in the PC-12 neuronal cell model which is similarly susceptible to apoptosis. To summarize, SG rapidly binds small and large ribosomal subunits in a concentration- and time-dependent manner that was consistent with induction of apoptosis.

A putative protein translation inhibitory factor encoded by Cotesia plutellae bracovirus suppresses host hemocyte-spreading behavior

An endoparasitoid, Cotesia plutellae (Hymenoptera: Braconidae), possesses a mutualistic bracovirus (CpBV), which plays significant roles in the parasitized host, Plutella xylostella (Lepidoptera: Plutellidae). CpBV15 β, a viral gene encoded by CpBV, is expressed at early and late parasitization periods, suggesting that it functions to manipulate the physiology of the parasitized host. This paper reports a physiological function of CpBV15 β as an immunosuppressive agent. The effect of CpBV15 β on cellular immunity was analyzed by assessing hemocyte-spreading behavior. Parasitization by C. plutellae caused altered behavior of hemocytes of P. xylostella, in which the hemocytes were not able to attach and spread on glass slides. CpBV15 β was expressed in Sf9 cells using a baculovirus expression system and purified from the culture media. When hemocytes of nonparasitized P. xylostella were incubated with purified CpBV15 β protein, spreading behavior was impaired in a dose-dependent manner at low micro-molar range. This inhibitory effect of CpBV15 β could also be demonstrated on hemocytes of a non-natural host, Spodoptera exigua. CpBV15 β protein significantly inhibited F-actin growth of hemocytes in response to an insect cytokine. Similarly, cycloheximide, a eukaryotic translation inhibitor, strongly inhibited the spreading behavior and F-actin growth of P. xylostella hemocytes. Under in vitro condition, hemocytes of nonparasitized P. xylostella released proteins into the surrounding medium. Upon incubation of hemocytes with either CpBV15 β or cycloheximide, their ability to release protein molecules was markedly inhibited. This study suggests that CpBV15 β suppresses hemocyte behavior by inhibiting protein translation.

Inhibitory properties of nucleic acid-binding ligands on protein synthesis

The use of small molecule inhibitors in the study of cellular processes is a powerful approach to understanding gene function. During the course of a high throughput screen for novel inhibitors of eukaryotic translation, we identified a number of nucleic acid binding ligands that showed activity in our assay. When tested on a panel of mRNA transcripts displaying different modes of translation initiation, these ligands showed a range of biological activities – with some inhibiting both cap-dependent and internal initiation and others preferentially blocking internal initiation. We used this information to identify a novel threading intercalator that inhibits Hepatitis C virus internal initiation.