Compartmentalisation and localisation of the translation initiation factor (eIF) 4F complex in normally growing fibroblasts

Previous observations of association of mRNAs and ribosomes with subcellular structures highlight the importance of localised translation. However, little is known regarding associations between eukaryotic translation initiation factors and cellular structures within the cytoplasm of normally growing cells. We have used detergent-based cellular fractionation coupled with immunofluorescence microscopy to investigate the subcellular localisation in NIH3T3 fibroblasts of the initiation factors involved in recruitment of mRNA for translation, focussing on eIF4E, the mRNA cap-binding protein, the scaffold protein eIF4GI and poly(A) binding protein (PABP). We find that these proteins exist mainly in a soluble cytosolic pool, with only a subfraction tightly associated with cellular structures. However, this "associated" fraction was enriched in active "eIF4F" complexes (eIF4E.eIF4G.eIF4A.PABP). Immunofluorescence analysis reveals both a diffuse and a perinuclear distribution of eIF4G, with the perinuclear staining pattern similar to that of the endoplasmic reticulum. eIF4E also shows both a diffuse staining pattern and a tighter perinuclear stain, partly coincident with vimentin intermediate filaments. All three proteins localise to the lamellipodia of migrating cells in close proximity to ribosomes, microtubules, microfilaments and focal adhesions, with eIF4G and eIF4E at the periphery showing a similar staining pattern to the focal adhesion protein vinculin

Cysteinyl-tRNA Deacylation Can Be Uncoupled from Protein Synthesis

Aminoacyl-tRNA synthetases (ARSs) are critical components of protein translation, providing ribosomes with aminoacyl-tRNAs. In return, ribosomes release uncharged tRNAs as ARS substrates. Here, we show that tRNA deacylation can be uncoupled from protein synthesis in an amino acid specific manner. While tRNAs coupled to radiolabeled Met, Leu Lys, or Ser are stable in cells following translation inhibition with arsenite, radiolabeled Cys is released from tRNA at a high rate. We discuss possible translation independent functions for tRNACys

Structural basis of yeast aminoacyl-tRNA synthetase complex formation revealed by crystal structures of two binary sub-complexes

The yeast aminoacyl-tRNA synthetase (aaRS) complex is formed by the methionyl- and glutamyl-tRNA synthetases (MetRS and GluRS, respectively) and the tRNA aminoacylation cofactor Arc1p. It is considered an evolutionary intermediate between prokaryotic aaRS and the multi- aaRS complex found in higher eukaryotes. While a wealth of structural information is available on the enzymatic domains of single aaRS, insight into complex formation between eukaryotic aaRS and associated protein cofactors is missing. Here we report crystal structures of the binary complexes between the interacting domains of Arc1p and MetRS as well as those of Arc1p and GluRS at resolutions of 2.2 and 2.05 Å, respectively. The data provide a complete structural model for ternary complex formation between the interacting domains of MetRS, GluRS and Arc1p. The structures reveal that all three domains adopt a glutathione S-transferase (GST)-like fold and that simultaneous interaction of Arc1p with GluRS and MetRS is mediated by the use of a novel interface in addition to a classical GST dimerization interaction. The results demonstrate a novel role for this fold as a heteromerization domain specific to eukaryotic aaRS, associated proteins and protein translation elongation factors

Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA

We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution through quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). The localization of fl-tRNA to active translation sites was confirmed through its co-localization with cellular factors and its dynamic alterations upon inhibition of protein synthesis. Moreover, fluorescence resonance energy transfer (FRET) signals, generated when fl-tRNAs, separately labeled as a FRET pair occupy adjacent sites on the ribosome, quantitatively reflect levels of protein synthesis in defined cellular regions. In addition, FRET signals enable detection of intra-populational variability in protein synthesis activity. We demonstrate that FtTM allows quantitative comparison of protein synthesis between different cell types, monitoring effects of antibiotics and stress agents, and characterization of changes in spatial compartmentalization of protein synthesis upon viral infection

The emerging role of exosome and microvesicle- (EMV-) based cancer therapeutics and immunotherapy

This document is the Accepted Manuscript version of the following article: Colin Moore, Uchini Kosgodage, Sigrun Lange, and Jameel M. Inal, ‘The emerging role of exosome and microvesicle- (EMV-) based cancer therapeutics and immunotherapy’, International Journal of Cancer, Vol. 141 (3): 428-436, August 2017. DOI: https://doi.org/10.1002/ijc.30672. © 2017 UICC. This manuscript version may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.There is an urgent need to develop new combination therapies beyond existing surgery, radio- and chemo-therapy, perhaps initially combining chemotherapy with the targeting specificities of immunotherapy. For this, strategies to limit inflammation and immunosuppression and evasion in the tumour microenvironment are also needed. To devise effective new immunotherapies we must first understand tumour immunology, including the roles of T cells, macrophages, myeloid suppressor cells and of exosomes and microvesicles (EMVs) in promoting angiogenesis, tumour growth, drug resistance and metastasis. One promising cancer immunotherapy discussed uses cationic liposomes carrying tumour RNA (RNA-lipoplexes) to provoke a strong anti-viral-like (cytotoxic CD8+ ) anti-tumour immune response. Mesenchymal stem cell-derived EMVs, with their capacity to migrate towards inflammatory areas including solid tumours, have also been used. As tumour EMVs clearly exacerbate the tumour microenvironment, another therapy option could involve EMV removal. Affinity-based methods to deplete EMVs, including an immunodepletion, antibody-based affinity substrate, are therefore considered. Finally EMV and exosome-mimetic nanovesicles (NVs) delivery of siRNA or chemotherapeutic drugs that target tumours using peptide ligands for cognate receptors on the tumour cells are discussed. We also touch upon the reversal of drug efflux in EMVs from cancer cells which can sensitize cells to chemotherapy. The use of immunotherapy in combination with the advent of EMVs provides potent therapies to various cancers.Peer reviewe

Synonymous Codon Ordering: A Subtle but Prevalent Strategy of Bacteria to Improve Translational Efficiency

Background: In yeast coding sequences, once a particular codon has been used, subsequent occurrence of the same amino acid tends to use codons sharing the same tRNA. Such a phenomenon of co-tRNA codons pairing bias (CTCPB) is also found in some other eukaryotes but it is not known whether it occurs in prokaryotes. Methodology/Principal Findings: In this study, we focused on a total of 773 bacterial genomes to investigate their synonymous codon pairing preferences. After calculating the actual frequencies of synonymous codon pairs and comparing them with their expected values, we detected an obvious pairing bias towards identical codon pairs. This seems consistent with the previously reported CTCPB phenomenon, since identical codons are certainly read by the same tRNA. However, among co-tRNA but non-identical codon pairs, only 22 were often found overrepresented, suggesting that many co-tRNA codons actually do not preferentially pair together in prokaryotes. Therefore, the previously reported co-tRNA codons pairing rule needs to be more rigorously defined. The affinity differences between a tRNA anticodon and its readable codons should be taken into account. Moreover, both within-gene-shuffling tests and phylogenetic analyses support the idea that translational selection played an important role in shaping the observed synonymous codon pairing pattern in prokaryotes. Conclusions: Overall, a high level of synonymous codon pairing bias was detected in 73 % investigated bacterial species

Hydrogen storage in metastable MgyTi(1-y) thin films

Stebint pasaulio mokslinių institucijų ir pramonės įmonių aktyvumą vandenilio saugojimo srityje yra akivaizdu, kad vandenilis tapo potencialiu energijos nešėju, kuris jau artimiausioje ateityje gali pakeisti egzistuojančius energijos nešėjus (naftos produktus, gamtines dujas, anglį ir t. t.). Viena iš pagrindinių problemų, norint saugiai panaudoti vandenilį išlieka jo saugojimas. Dabartiniu metu vandenilio saugojimą bandoma realizuoti naudojant vandenilį suspaudžiant slėgiminiuose induose, suskystinant žemose temperatūrose, panaudojant metalų ir jų lydinių hidridus ir anglies nanostruktūras. Naudojant slėgiminius indus ir vandenilio suskystinimą žemose temperatūrose sunaudojama labai daug energijos (iki 30%). Šie metodai taip pat turi problemų susijusių su saugumo standartizavimu. Vieni iš perspektyviausių ir saugiausių metodų – vandenilio saugojimas anglies nanosturktūrose ir metalų ir jų lydinių hidridai. Analizuojant literatūros duomenis apie anglies nanostruktūrų tyrimus susiduriama su rezultatų atsikartojamumo problema, skirtingose laboratorijose. Tiriant tas pačias anglies nanostruktūras saugomas vandenilio kiekis svyruoja nuo 20 iki 1 masės procentų. Vienas iš perspektyviausių metodų saugoti ir transportuoti vandenilį – metalų hidridai. Šio darbo pagrindinis tikslas – išanalizuoti MgH2, naudojamo vandenilio saugojime, destabilizaciją, įvedant kontroliuojamus kiekius priemaišinių atomų į metalo hidridą ir taip sumažinant dekompozicijos temperatūrą.One of the most important problems for the efficient commercialization of hydrogen energetic is the development of reliable hydrogen storage elements. At present time, the hydrogen storage is performed in high pressure vessels at low temperature and using metal and alloy hydrides (carbon nanostructures). However, hydrogen conservation in high pressure vessels at low temperature requires about 30 % of conserved energy for the liquefaction. These methods have problems to the standardization of safety. Very perspective method for hydrogen storage is recently suggested in carbon nanostructures and metal and alloys hydrides. However, the existing information about the use of carbon nanostructures is much dispersed and not reproducible. The international committee on the hydrogen energy makes emphasis on the use of the metal hydrides as the most perspective method for the solution of hydrogen storage problem. The aim of this work is to analyse possibility to destabilise MgH2 used for hydrogen storage with controllable amounts of impurities (Ti) and as a result decrease decomposition temperature.Vytauto Didžiojo universiteta

Electric pulse-induced precipitation of biological macromolecules in electroporation

We found that electric discharge through solution of biological macromolecules (DNA, RNA and proteins) causes precipitation of significant portions of these macromolecules. This precipitation is a consequence of the interaction of biological macromolecules with the metal ions solubilized from the anode plate by the electric pulse, and occurs in both absence and presence of the cells in poration medium. Precipitated fractions of macromolecules sediments at the centrifugation speed used to pellet eukaryotic cells and does not dissolve when washed with buffer. Our data indicate a complication of the direct evaluation of electroporation efficiency based on the assumption that electroporated biological macromolecules which remain associated with the cells after several washes, are successfully electroinjected into the cytoplasm of cells

A channeled tRNA cycle during mammalian protein synthesis.

In earlier studies it was shown that the mammalian translation system is highly organized in vivo and that the intermediates in the process, aminoacyl-tRNAs, are channeled--i.e., they are directly transferred from the aminoacyl-tRNA synthetases to the elongation factor to the ribosomes without dissociating into the cellular fluid. Here, we examine whether spent tRNAs leaving the ribosome enter the fluid phase or are transferred directly to their cognate aminoacyl-tRNA synthetases to complete a channeled tRNA cycle. Using a permeabilized CHO cell system that closely mimics living cells, we find that there is no leakage of endogenous tRNA during many cycles of translation, and protein synthesis remains linear during this period, even though free aminoacyl-tRNA is known to rapidly equilibrate between the inside and outside of these cells. We also find that exogenous tRNA and periodate-oxidized tRNA have no effect on protein synthesis in this system, indicating that they do not enter the translation machinery, despite the fact that exogenous tRNA rapidly distributes throughout the cells. Furthermore, most of the cellular aminoacyl-tRNA synthetases function only with endogenous tRNAs, although a portion can use exogenous tRNA molecules. However, aminoacylation of these exogenous tRNAs is strongly inhibited by oxidized tRNA; this inhibitor has no effect on endogenous aminoacylation. On the basis of these and the earlier observations, we conclude that endogenous tRNA is never free of the protein synthetic machinery at any stage of the translation process and, consequently, that there is a channeled tRNA cycle during protein synthesis in mammalian cells