05-235 Sigma-AldrichAnti-EF1α Antibody, clone CBP-KK1

Mitochondrial protein import is essential for all eukaryotes and mediated by hetero-oligomeric protein translocases thought to be conserved within all eukaryotes. We have identified and analysed the function and architecture of the non-conventional outer membrane (OM) protein translocase in the early diverging eukaryote Trypanosoma brucei. It consists of six subunits that show no obvious homology to translocase components of other species. Two subunits are import receptors that have a unique topology and unique protein domains and thus evolved independently of the prototype receptors Tom20 and Tom70. Our study suggests that protein import receptors were recruited to the core of the OM translocase after the divergence of the major eukaryotic supergroups. Moreover, it links the evolutionary history of mitochondrial protein import receptors to the origin of the eukaryotic supergroups.

Lung cancer is the leading cause of cancer death worldwide, and brain metastasis is a major cause of morbidity and mortality in lung cancer. CDH2 (N-cadherin, a mesenchymal marker of the epithelial-mesenchymal transition) and ADAM9 (a type I transmembrane protein) are related to lung cancer brain metastasis; however, it is unclear how they interact to mediate this metastasis. Because microRNAs regulate many biological functions and disease processes (e.g., cancer) by down-regulating their target genes, microRNA microarrays were used to identify ADAM9-regulated miRNAs that target CDH2 in aggressive lung cancer cells. Luciferase assays and western blot analysis showed that CDH2 is a target gene of miR-218. MiR-218 was generated from pri-mir-218-1, which is located in SLIT2, in non-invasive lung adenocarcinoma cells, whereas its expression was inhibited in aggressive lung adenocarcinoma. The down-regulation of ADAM9 up-regulated SLIT2 and miR-218, thus down-regulating CDH2 expression. This study revealed that ADAM9 activates CDH2 through the release of miR-218 inhibition on CDH2 in lung adenocarcinoma.

TNF receptor-associated protein 1 (TRAP1), the main mitochondrial member of the heat shock protein (HSP) 90 family, is induced in most tumor types and is involved in the regulation of proteostasis in the mitochondria of tumor cells through the control of folding and stability of selective proteins, such as Cyclophilin D and Sorcin. Notably, we have recently demonstrated that TRAP1 also interacts with the regulatory protein particle TBP7 in the endoplasmic reticulum (ER), where it is involved in a further extra-mitochondrial quality control of nuclear-encoded mitochondrial proteins through the regulation of their ubiquitination/degradation. Here we show that TRAP1 is involved in the translational control of cancer cells through an attenuation of global protein synthesis, as evidenced by an inverse correlation between TRAP1 expression and ubiquitination/degradation of nascent stress-protective client proteins. This study demonstrates for the first time that TRAP1 is associated with ribosomes and with several translation factors in colon carcinoma cells and, remarkably, is found co-upregulated with some components of the translational apparatus (eIF4A, eIF4E, eEF1A and eEF1G) in human colorectal cancers, with potential new opportunities for therapeutic intervention in humans. Moreover, TRAP1 regulates the rate of protein synthesis through the eIF2α pathway either under basal conditions or under stress, favoring the activation of GCN2 and PERK kinases, with consequent phosphorylation of eIF2α and attenuation of cap-dependent translation. This enhances the synthesis of selective stress-responsive proteins, such as the transcription factor ATF4 and its downstream effectors BiP/Grp78, and the cystine antiporter system xCT, thereby providing protection against ER stress, oxidative damage and nutrient deprivation. Accordingly, TRAP1 silencing sensitizes cells to apoptosis induced by novel antitumoral drugs that inhibit cap-dependent translation, such as ribavirin or 4EGI-1, and reduces the ability of cells to migrate through the pores of transwell filters. These new findings target the TRAP1 network in the development of novel anti-cancer strategies.

Prion diseases are fatal, transmissible neurodegenerative diseases of the central nervous system. An abnormally protease-resistant and insoluble form (PrP(Sc)) of the normally soluble protease-sensitive host prion protein (PrP(C)) is the major component of the infectious prion. During the course of prion disease, PrP(Sc) accumulates primarily in the lymphoreticular and central nervous systems. Recent studies have shown that co-infection of prion-infected fibroblast cells with the Moloney murine leukemia virus (Mo-MuLV) strongly enhanced the release and spread of scrapie infectivity in cell culture, suggesting that retroviral coinfection might significantly influence prion spread and disease incubation times in vivo. We now show that another retrovirus, the murine leukemia virus Friend (F-MuLV), also enhanced the release and spread of scrapie infectivity in cell culture. However, peripheral co-infection of mice with both Friend virus and the mouse scrapie strain 22L did not alter scrapie disease incubation times, the levels of PrP(Sc) in the brain or spleen, or the distribution of pathological lesions in the brain. Thus, retroviral co-infection does not necessarily alter prion disease pathogenesis in vivo, most likely because of different cell-specific sites of replication for scrapie and F-MuLV.

Certain stress conditions can induce cleavage of tRNAs around the anticodon loop via the use of the ribonuclease angiogenin. The cellular factors that regulate tRNA cleavage are not well known. In this study we used normal and eIF2α phosphorylation-deficient mouse embryonic fibroblasts and applied a microarray-based methodology to identify and compare tRNA cleavage patterns in response to hypertonic stress, oxidative stress (arsenite), and treatment with recombinant angiogenin. In all three scenarios mouse embryonic fibroblasts deficient in eIF2α phosphorylation showed a higher accumulation of tRNA fragments including those derived from initiator-tRNA(Met). We have shown that tRNA cleavage is regulated by the availability of angiogenin, its substrate (tRNA), the levels of the angiogenin inhibitor RNH1, and the rates of protein synthesis. These conclusions are supported by the following findings: (i) exogenous treatment with angiogenin or knockdown of RNH1 increased tRNA cleavage; (ii) tRNA fragment accumulation was higher during oxidative stress than hypertonic stress, in agreement with a dramatic decrease of RNH1 levels during oxidative stress; and (iii) a positive correlation was observed between angiogenin-mediated tRNA cleavage and global protein synthesis rates. Identification of the stress-specific tRNA cleavage mechanisms and patterns will provide insights into the role of tRNA fragments in signaling pathways and stress-related disorders.

Finding genetic polymorphisms and mutations linked to addictive behavior can provide important targets for pharmaceutical and therapeutic interventions. Forward genetic approaches in model organisms such as zebrafish provide a potentially powerful avenue for finding new target genes. In order to validate this use of zebrafish, the molecular nature of its reward system must be characterized. We have previously reported the use of cocaine-induced conditioned place preference (CPP) as a reliable method for screening mutagenized fish for defects in the reward pathway. Here we test if CPP in zebrafish involves the dopaminergic system by co-treating fish with cocaine and dopaminergic antagonists. Sulpiride, a potent D2 receptor (DR2) antagonist, blocked cocaine-induced CPP, while the D1 receptor (DR1) antagonist SCH23390 had no effect. Acute cocaine exposure also induced a rise in the expression of tyrosine hydroxylase (TH), an important enzyme in dopamine synthesis, and a significant decrease in the expression of elongation factor 1α (EF1α), a housekeeping gene that regulates protein synthesis. Cocaine selectively increased the ratio of TH/EF1α in the telencephalon, but not in other brain regions. The cocaine-induced change in TH/EF1α was blocked by co-treatment with sulpiride, but not SCH23390, correlating closely with the action of these drugs on the CPP behavioral response. Immunohistochemical analysis revealed that the drop in EF1α was selective for the dorsal nucleus of the ventral telencephalic area (Vd), a region believed to be the teleost equivalent of the striatum. Examination of TH mRNA and EF1α transcripts suggests that regulation of expression is post-transcriptional, but this requires further examination. These results highlight important similarities and differences between zebrafish and more traditional mammalian model organisms.

Postsynaptic density (PSD) is a protein supramolecule lying underneath the postsynaptic membrane of excitatory synapses and has been implicated to play important roles in synaptic structure and function in mammalian central nervous system. Here, PSDs were isolated from two distinct regions of porcine brain, cerebral cortex and cerebellum. SDS-PAGE and Western blotting analyses indicated that cerebral and cerebellar PSDs consisted of a similar set of proteins with noticeable differences in the abundance of various proteins between these samples. Subsequently, protein localization in these PSDs was analyzed by using the Nano-Depth-Tagging method. This method involved the use of three synthetic reagents, as agarose beads whose surface was covalently linked with a fluorescent, photoactivable, and cleavable chemical crosslinker by spacers of varied lengths. After its application was verified by using a synthetic complex consisting of four layers of different proteins, the Nano-Depth-Tagging method was used here to yield information concerning the depth distribution of various proteins in the PSD. The results indicated that in both cerebral and cerebellar PSDs, glutamate receptors, actin, and actin binding proteins resided in the peripheral regions within ∼ 10 nm deep from the surface and that scaffold proteins, tubulin subunits, microtubule-binding proteins, and membrane cytoskeleton proteins found in mammalian erythrocytes resided in the interiors deeper than 10 nm from the surface in the PSD. Finally, by using the immunoabsorption method, binding partner proteins of two proteins residing in the interiors, PSD-95 and α-tubulin, and those of two proteins residing in the peripheral regions, elongation factor-1α and calcium, calmodulin-dependent protein kinase II α subunit, of cerebral and cerebellar PSDs were identified. Overall, the results indicate a striking similarity in protein organization between the PSDs isolated from porcine cerebral cortex and cerebellum. A model of the molecular structure of the PSD has also been proposed here.

Glutamate, the major excitatory transmitter in the vertebrate brain, is involved in neuronal development and synaptic plasticity. Glutamatergic stimulation leads to differential gene expression patterns in neuronal and glial cells. A glutamate-dependent transcriptional control has been established for several genes. However, much less is known about the molecular events that modify the translational machinery upon exposure to this neurotransmitter. In a glial model of cerebellar cultured Bergmann cells, glutamate induces a biphasic effect on [(35)S]-methionine incorporation into proteins that suggests that the elongation phase of protein biosynthesis is the target for regulation. Indeed, after a 15min exposure to glutamate a transient increase in elongation factor 2 phosphorylation has been reported, an effect mediated through the activation of the elongation factor 2 kinase. In this contribution, we sought to characterize the phosphorylation status of the eukaryotic elongation factor 1A (eEF1A) and the ribosomal transit time under glutamate exposure. A dose-dependent increase in eEF1A phosphorylation was found after a 60min glutamate treatment; this phenomenon is Ca(2+)/CaM dependent, blocked with Src and phosphatidyl-inositol 3-kinase inhibitors and with rapamicyn. Concomitantly, the ribosomal transit time was increased with a 15min glutamate exposure. After 60 more minutes, the average time used by the ribosomes to complete a polypeptide chain had almost returned to its initial level. These results strongly suggest that glutamate exerts an exquisite time-dependent translational control in glial cells, a process that might be critical for glia-neuron interactions.

Overproduction of amyloid precursor protein (APP) and beta-amyloid likely contribute to neurodegeneration seen in Alzheimer's disease (AD). APP mRNA contains several, 3'-untranslated region (UTR), cis-acting regulatory elements. A 52 base element (52sce), immediately downstream from the stop codon, has been previously shown to complex with uncharacterized cytoplasmic proteins. In this study, we purify and identify six proteins that specifically bind to the 52sce, and show that these proteins interact with each other and with APP mRNA in intact human neuroblastoma cells. We also present evidence that at least one of these proteins, the DEAD-box helicase rck/p54, is involved in post-transcriptional regulation, as its overexpression in cultured cells results in elevated levels of APP mRNA and protein. These findings suggest a novel mechanism for post-transcriptional regulation of APP mRNA.

Gamendazole was recently identified as an orally active antispermatogenic compound with antifertility effects. The cellular mechanism(s) through which these effects occur and the molecular target(s) of gamendazole action are currently unknown. Gamendazole was recently designed as a potent orally active antispermatogenic male contraceptive agent. Here, we report the identification of binding targets and propose a testable mechanism of action for this antispermatogenic agent. Both HSP90AB1 (previously known as HSP90beta [heat shock 90-kDa protein 1, beta]) and EEF1A1 (previously known as eEF1A [eukaryotic translation elongation factor 1 alpha 1]) were identified as binding targets by biotinylated gamendazole (BT-GMZ) affinity purification from testis, Sertoli cells, and ID8 ovarian cancer cells; identification was confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and Western blot analysis. BT-GMZ bound to purified yeast HSP82 (homologue to mammalian HSP90AB1) and EEF1A1, but not to TEF3 or HBS1, and was competed by unlabeled gamendazole. However, gamendazole did not inhibit nucleotide binding by EEF1A1. Gamendazole binding to purified Saccharomyces cerevisiae HSP82 inhibited luciferase refolding and was not competed by the HSP90 drugs geldanamycin or novobiocin analogue, KU-1. Gamendazole elicited degradation of the HSP90-dependent client proteins AKT1 and ERBB2 and had an antiproliferative effect in MCF-7 cells without inducing HSP90. These data suggest that gamendazole may represent a new class of selective HSP90AB1 and EEF1A1 inhibitors. Testis gene microarray analysis from gamendazole-treated rats showed a marked, rapid increase in three interleukin 1 genes and Nfkbia (NF-kappaB inhibitor alpha) 4 h after oral administration. A spike in II1a transcription was confirmed by RT-PCR in primary Sertoli cells 60 min after exposure to 100 nM gamendazole, demonstrating that Sertoli cells are a target. AKT1, NFKB, and interleukin 1 are known regulators of the Sertoli cell-spermatid junctional complexes. A current model for gamendazole action posits that this pathway links interaction with HSP90AB1 and EEF1A1 to the loss of spermatids and resulting infertility.