Microplitis croceipes is a solitary endoparasitic wasp that oviposits in the hemocoel of Heleothis virescens larvae. Upon parasitization, the host larvaes physiology is altered; resulting in a compromised immune system and a decrease in the production of some vital proteins resulting in a terminal post-wandering prepupal state. Teratocytes, cells derived from the extraembryonic serosa of the parasitic wasp, mimic symptoms of parasitization when injected into host larvae, independent of other factors like polydnavirus and venom. Some of the inhibition of protein synthesis can be attributed to proteins secreted by the teratocytes (teratocyte secretory proteins or TSP). A fraction of TSP between 330 kDa inhibits protein synthesis in vivo, in the in vitro fat body and testes assays, and in the rabbit reticulocyte lysate and wheat germ extract assays. This fraction, however, has no effect on nucleic acid synthesis. Its effect on protein synthesis is dose dependent and exposure time sensitive. A 13.9 kDa protein isolated from TSP and expressed in a baculovirus system seems primarily responsible for the inhibition. Although TSP14 production was low, it did bind to the cell surface, enter the cell, and inhibit protein synthesis as the 330 kDa factor did.

This thesis addresses different questions regarding the rate, efficiency, and accuracy of peptide bond formation with natural as well as unnatural amino acids: Which step is rate-limiting during peptide bond formation? How does the accuracy vary with different transfer RNAs (tRNAs) and codons and how is it relevant to the living cells? Does proofreading selection of codon reading occur in a single- or multi-step manner as theoretically suggested? How does the E. coli translation system discriminate unnatural amino acids? Based on that, how to improve the incorporation efficiencies of unnatural amino acids? Based on the study on pH dependence of peptide bond formation, we show that the rate of the chemistry of peptidyl transfer to aminoacyl-tRNA (AA-tRNA) Gly-tRNAGly or Pro-tRNAPro limits the rate of peptide bond formation at physiological pH 7.5, and this could possibly be true for peptidyl transfer to all natural AA-tRNAs at physiological condition. By studying the efficiency-accuracy trade-off for codon reading by seven AA-tRNA containing ternary complexes, we observe a large variation on the accuracy of initial codon selection and identify several error hot-spots. The maximal accuracy varied 400-fold from 200 to 84000 depending on the tRNA identity, the type and position of the mismatches. We also propose a proofreading mechanism that contains two irreversible steps in sequence. This could be highly relevant to the living cells in relation to maintaining both high accuracy and high efficiency in protein synthesis. Finally, we show that peptide bond formation with small and large non-N-alkylated L- unnatural amino acids proceed at rates similar to those with natural amino acids Phe and Ala on the ribosome. Interestingly, the large side chain of the bulky unnatural amino acid only weakens its binding for elongation factor Tu (EF-Tu) but not slows down peptidyl transfer on the ribosome. Our results also suggest that the efficiency of unnatural amino acid incorporation could be improved in general by increasing EF-Tu concentration, lowering the reaction temperature and / or using tRNA bodies with optimal affinities for EF-Tu in the translation system.

Kadmis yra plačiai paplitęs aplinkos teršalas. Šis sunkusis metalas yra pavojingas žmonių sveikatai. Tuo tarpu cinkas ir selenas yra būtini elementai žmonėms ir gyvūnams. Todėl šio darbo tikslas buvo įvertinti kadmio, cinko ir selenito jonų ūmų poveikį baltymų sintezės sistemos ir jos komponentų bei ląstelių antioksidacinės sistemos aktyvumams laboratorinių pelių kepenyse. Tikslui pasiekti iškelti šie uždaviniai: įvertinti ūmų kadmio jonų poveikį baltymų sintezės sistemai pelių kepenyse, transliacijos sistemos komponentų – tRNR bei aminoacil-tRNR sintetazės – aktyvumams, metalotioneino koncentracijai pelių kepenyse, redukuoto glutationo koncentracijai ir lipidų peroksidacijai pelių kepenyse bei eritrocituose ir įvertinti cinko ir selenito jonų įtaką pelių baltymų sintezės ir antioksidacinėms sistemoms, esant ūmiam kadmio jonų poveikiui. Tyrimo rezultatai parodė, kad kadmio jonų poveikis baltymų sintezės procesui pelių kepenyse susijęs su tiesioginiu poveikiu šiame procese dalyvaujančių komponentų – tRNR bei aminoacil-tRNR sintetazės – aktyvumų pokyčiais. Be to, kadmio jonai keitė ląstelių antioksidacinės sistemos komponentų (redukuoto glutationo ir metalotioneino) koncentracijas ir aktyvino lipidų peroksidaciją pelių kepenyse. Cinko ir selenito jonai apsaugojo transliacijos ir antioksidacinę sistemas nuo toksinio kadmio jonų poveikio. / Cadmium is a widespread environmental pollutant. This heavy metal forms a serious hazard to the public health. Zinc and selenium are essential elements for humans and animals. So, the aim of present study was to evaluate the acute effects of cadmium, zinc, and selenite ions on the effectiveness of protein synthesis system and its components in the liver of laboratory mice as well as on the activity of cellular antioxidant system. The objectives of the study were to evaluate the effects of cadmium ions on the rate of liver protein synthesis, on the activities of components of translation machinery – tRNA and aminoacyl-tRNA synthetase, on the content of metallothionein in the liver, on the content of reduced glutathione and lipid peroxidation in the liver and red blood cells; to evaluate the influence of zinc and selenite ions on the protein synthesis system and antioxidative system of mice affected by cadmium ions. The obtained results showed, that the toxicity of cadmium ions on hepatic protein synthesis is related to the changes in activities of the key components of protein synthesizing system – tRNAs and aminoacyl-tRNA synthetases. Moreover, cadmium ions altered the content of antioxidant components (reduced glutathione, metallothionein) and activated liver lipid peroxidation. Mice pre-treatment with zinc and selenite ions prevented both translational machinery and antioxidative system against cadmium-induced disturbances.

Sunkieji metalai yra vieni toksiškiausių ir žalingiausių aplinkos veiksnių. Organizme jie skatina aktyviųjų deguonies formų susidarymą, dėl to pažeidžiamos ląstelės membranos, nukleorūgščių ir baltymų struktūros. Šiame darbe ištyrime sunkiųjų metalų Pb ir Ni toksinį poveikį kepenų antioksidacinei apsaugos sistemai, baltymų sintezės (transliacijos) sistemai bei antioksidanto Zn apsauginį veikimą prieš toksinį Pb ir Ni poveikį. Įvertinome kompleksinį šių metalų jonų poveikį oksidacinio streso rodikliui – redukuoto glutationo koncentracijai, lipidų peroksidacijai, bendro baltymo, apsauginio baltymo metalotioneino, bei hemo sintezei. Nustatėme, kad Zn jonai geba apsaugoti ląstelę nuo toksinio Pb ir Ni jonų poveikio, tačiau jų apsauginis poveikis yra nevienareikšmiškas. Parodėme, kad Zn jonai apsaugo ląsteles nuo Pb jonų sukeltų oksidacinių pažeidimų ankstyvoje intoksikavimo stadijoje. Tačiau, šio metalo apsauginį poveikį prieš Ni jonų toksiškumą nustatėme tik po dviejų savaičių. Šis darbas leidžia detaliau suprasti Zn pajėgumą apsaugoti organizmą nuo toksinio Pb ir Ni poveikio. / Heavy metals are among the most toxic and harmful environmental factors. In the body, they promote the formation of active oxygen species, resulting the damage to the cell membranes, nucleic acid and protein structure. This study was conducted to evaluate the toxic effects of Pb and Ni on antioxidant defence and protein synthesis (translation) systems in liver cells and to determine the possible protective effect of Zn against Pb and Ni toxicity. We established the complex effect of these metal ions on the content of an oxidative stress marker reduced glutathione, lipid peroxidation as well as on total protein, metallothionein and heme synthesis. It was determined that Zn ions are able to pro¬tect cells from the toxic effects of Pb and Ni ions exposure, but the protective effect was ambiguous. The Zn ions protected cells from Pb ions induced oxidative damage in the early stages of intoxication. The protective effect against Ni ions toxicity, on the contrary, was observed only after two weeks of intoxication. This study provides more detailed understanding of Zn ability to pro¬tect the organism from toxic effects of Pb and Ni.

Pagrindinis tyrimo tikslas – įvertinti švino ir nikelio jonų įtaką baltymų ir metalotioneinų sintezei laboratorinių pelių kepenyse bei cinko apsauginį poveikį, veikiant švinui ir nikeliui. Tyrimams naudotos 4-6 savaičių nelinijinės baltosios laboratorinės pelės, sveriančios nuo 20 iki 25 gramų. Pelių laikymo sąlygos atitiko reikalavimus. Eksperimentams pelės buvo suskirstytos į 6 grupes: I – kontrolinė, II – gaunančios Pb(CH3COO)2 (dozė 10 mg Pb/kg), III – gaunančios ZnSO4 (1,56 mg Zn/kg), IV – ZnSO4 ir po 20 minučių Pb(CH3COO)2 (anksčiau nurodytos dozės), V – NiCl2 (1,12 mg Ni/kg) , VI – ZnSO4 ir po 20 minučių NiCl2 (anksčiau nurodytos dozės). Druskų tirpalai į pilvo ertmę švirkšti 14 dienų. Praėjus 14 dienų, pelės buvo anestezuotos, joms atlikta cervikalinė dislokacija. Tyrimams naudotos pelių kepenys. Baltymų koncentracijai nustatyti naudojome Beer/Lambert metodą, spektrofotometriškai matavome sugertį ties 260 ir 280 nm banga. Metalotioneinų koncentraciją nustatėme pagal Peixoto metodiką, spektrofotometriškai sugertį matavome ties 412 nm banga. Nustatėme, jog metalai skirtingai veikia bendrų baltymų ir metalotioneinų sintezę. Švinas neturėjo statistiškai reikšmingo poveikio bendrų baltymų sintezei, o nikelis skatino bendrą baltymų sintezę. Nikelis metalotioneinų sintezę taip pat aktyvino labiau nei švinas. Padidėjus metalotioneinų koncentracijai bei suaktyvėjus bendrų baltymų sintezei, užfiksuotas cinko jonų apsauginis poveikis. / The present study was conducted to investigate the effects of lead and nickel ions on total proteins and metallothioneins synthesis in mice liver and evaluate protective effects of zinc ions. Experiments were done on 4-6 weeks old white laboratory outbreed mice weighing 20-25 g. All experiments performed according to the Republic of Lithuania Law on the Care, Keeping and Use of animals (License of State Veterinary Service for working with laboratory animals No 0221). For experiment mice were sorted out into 6 groups: I group – control; II group – Pb(CH3COO)2 (10 mg Pb per kg of body mass), III group – ZnSO4 (1,56 mg Zn per kg of body mass), IV – ZnSO4 and after 20 minutes Pb(CH3COO)2 (the same doses), V – NiCl2 (1,12 mg Ni per kg of body mass) , VI – ZnSO4 and after 20 minutes NiCl2 (the same doses). Concentration of protein was determined by Lowry method. MTs were assayed in mice liver according to the method of Peixoto N. C. There is no statistically significant effect of lead on protein synthesis in mice liver. ZnSO4, injected 20 minutes before Pb(CH3COO)2, increased protein synthesis. After injection of NiCl2 solution, marked amino acid actuation to new synthesized protein has increased, but ZnSO4, injected 20 minutes before NiCl2, decreased protein synthesis. Obtained data showed, that in mice liver treated with NiCl2 and Pb(CH3COO)2 solutions, MTs content was increased. According to the data, ZnSO4 injected 20 minutes before NiCl2 or Pb(CH3COO)2, decreased MTs content... [to full text]

Ribosomal stalk proteins are known to play important role in protein synthesis. The ‘stalk’, an extended structure on the large subunit of the ribosome is composed mainly of two to three dimers of L12 and one L10 protein, which forms the base of the stalk. In E. coli, four copies of L12 molecules exist as dimer of dimers forming the pentameric L8 complex together with L10. This thesis is a collection of four interlinked studies on the structure, function and application of the ribosomal stalk protein L12. In the first study, we have mapped the interaction sites of the four major translation GTPase factors (IF2, EF-Tu, EF-G & RF3) on L12 molecule using heteronuclear NMR spectroscopy. Surprisingly, all these factors produced an overlapping interaction map spanning two α-helices on the C terminal domain of L12, thereby suggesting a general nature of the interaction between L12 and the GTPase factors. L12 is known to stimulate GTPase activity of the elongation factors EF-Tu and EF-G. Here, we have clarified the role of L12 in IF2 mediated initiation of protein synthesis. Our data suggest that rapid subunit association requires a specific interaction between the L12 protein on the 50S and IF2·GTP on the 30S preinitiation complex. We have also shown that L12 is not a GAP for IF2 and GTP hydrolysis triggers IF2 release from the 70S initiation complex. The next question we have addressed is why multiple copies of L12 dimer are needed on the ribosome. For this purpose, we created a pure E. coli strain JE105, where the terminal part of rplJ gene coding for the binding site of one L12 dimer on protein L10 was deleted in the chromosomal locus. Using ribosomes with single L12 dimer we have observed that the rate of the initiation and elongation involving IF2 and EF-G gets most compromised, which in turn decreases the growth rate of the bacteria.  This study also indicates that L12 can interact with different GTPase factors in a specialized manner. Lastly, we have developed an application making advantage of the multiple L12 dimers on the ribosome. By inserting a (His)6-tag at the C-terminus of the L12 protein we have created a novel E. coli strain (JE28), where all ribosomes are tetra-(His)6-tagged. Further, we have developed a single step method for purification of the active (His)6-tagged ribosomes from JE28.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.

How do microbes adapt to challenges from the environment? In this thesis, two distinct cases were examined through structural and biochemical methods. In the first, we followed a real-time protein evolution of HisA to a novel function. The second case was fusidic acid (FA) resistance mediated by the protein FusB in Staphylococcus aureus. In the first study, the aim was to understand how mutants of HisA from the histidine biosynthetic pathway could evolve a novel TrpF activity and further evolve to generalist or specialist enzymes. We solved the crystal structure of wild type Salmonella enterica HisA in its apo-state and the structures of the mutants D7N and D7N/D176A in complex with the substrate ProFAR. These two distinct complex structures showed us the coupled conformational changes of HisA and ProFAR before catalysis. We also solved crystal structures of ten mutants, some in complex with substrate or product. The structures indicate that bi-functional mutants adopt distinct loop conformations linked to the two functions and that mutations in specialist enzymes favor one of the conformations. We also observed biphasic relationships in which small changes in the activities of low-performance enzymes had large effects on fitness, until a threshold, above which large changes in enzyme performance had little effect on fitness. Fusidic acid blocks protein translation by locking elongation factor G (EF-G) to the ribosome after GTP hydrolysis in elongation and recycling of bacterial protein synthesis. To understand the rescue mechanism, we solved the crystal structure of FusB at 1.6Å resolution. The structure showed that FusB is a two-domain protein and C-terminal domain contains a treble clef zinc finger. Using hybrid constructs between S. aureus EF-G that binds to FusB, and E. coli EF-G that does not, the binding determinants were located to domain IV of EF-G. This was further supported by small-angle X-ray scattering studies of the FusB·EF-G complex. Using single-molecule methods, we observed FusB frequently binding to the ribosome and rescue of FA-inhibited elongation by effects on the non-rotated state ribosome. Ribosome binding of FusB was confirmed by isothermal titration calorimetry.

The main aim of this thesis was to increase our understanding of the metabolic responses associated with short-term high-fat overfeeding. To this end, four separate studies are described in this thesis; each of which involved the provision of a high-fat, high-energy diet to young, healthy, lean individuals. The first of these experimental chapters (Chapter 2) determined the effects of a 7-day, high-fat (65%), high-energy (+50%) diet on postprandial metabolic and endocrine responses to a mixed meal challenge. This chapter demonstrates that 7-days of overfeeding impaired glycaemic control in our subject cohort but did not influence the response of selected gut hormones (acylated ghrelin, GLP-1 and GIP). In a mechanistic follow up study utilising stable isotope tracer methodology we then demonstrate that overfeeding-induced impairments in glycaemic control are attributable to subtle alterations in plasma glucose flux, rather than the overt tissue-specific adaptations (e.g. increased EGP, or reduced glucose disposal) that have previously been reported (Chapter 3). In an attempt to delineate the time-course of diet-induced impairments in glycaemic control, we then investigated the effects of 1-day of overfeeding (+80% energy with 73% of total energy coming as fat) (Chapter 4). Results demonstrate that a single day of overfeeding elicits responses which are comparable to 7-days of high-fat overfeeding; highlighting the rapidity with which excessive high-fat food intake can negatively influence glucose metabolism. In chapter 5 we utilised stable isotope tracer and muscle biopsy techniques to demonstrate that 7-days of high-fat overfeeding impairs glycaemic control but does not influence the fed-state mixed muscle protein fractional synthesis rate (FSR). In conclusion, the findings of this thesis demonstrate that while short-term high-fat overfeeding negatively influences whole-body glucose metabolism, skeletal muscle protein metabolism appears to be relatively unaffected in young, lean, healthy humans.

During cellular stress, the regulation of protein synthesis is a key adaptive mechanism used by cells to survive. In response to various stresses, heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), an RNA binding protein principally found within the nucleus, is phosphorylated and consequently accumulates in the cytoplasm. Among other roles, cytoplasmic hnRNP A1 functions as an auxiliary translation factor for internal ribosome entry site (IRES)-mediated translation of specific mRNA, including the anti-apoptotic protein B-cell lymphoma-extra large (Bcl-xL). To identify which kinases control the cytoplasmic accumulation of hnRNP A1, an RNAi-based kinome-wide screen was performed in hypertonically stressed U2OS cells, from which AMPK-related kinase 5 (ARK5) was identified as a potential regulator of hnRNP A1’s localization. Here we show that ARK5 directly phosphorylates hnRNP A1 and that the inhibition of ARK5 expression blocks the stress induced cytoplasmic accumulation of hnRNP A1, modulates expression of Bcl-xL protein and increases cell viability. Our data points to a novel role for ARK5 and provides further insight into the mechanisms regulating cellular stress response.