High levels of accuracy in transcription, aminoacylation of tRNA, and mRNA translation are essential for all life forms. However, high accuracy also necessarily means large energy dissipation and slow kinetics. Therefore, in vivo there is a fine tuned balance between rate and accuracy of key chemical reactions. We have shown that in our optimized in vitro bacterial protein synthesis system we have in vivo compatible rate and accuracy of ribosomal protein elongation. Our measurements of the temperature and the pH dependence of peptide bond formation with native substrates also suggest that the chemical step of peptidyl transfer, rather than tRNA accommodation, limits the rate of peptide bond formation. This work has made it possible to study ribosomal peptidyl transfer with native substrates. Furthermore, we have developed a general theoretical model for the rate-accuracy trade-off in enzymatic reactions. When considering this trade-off for protein synthesis in the context of the living bacterial cell, where cognate aa-tRNAs compete for ribosome binding with an excess of non-cognate aa-tRNAs, the model predicts an accuracy optimum where the inhibitory effect of non-cognate substrate binding and the efficiency loss due to high discard rate of cognate aa-tRNAs are minimized. However, these results also show that commonly used biochemical systems for protein synthesis studies operate at exceptionally suboptimal conditions. This makes it difficult, if not impossible, to relate the biochemical data to protein synthesis in the living cell. To validate our theoretical model we developed a method, based on variation of the concentration of Mg2+ ions in the buffer, to study the rate-accuracy trade-off of bacterial protein synthesis in vitro. We found a linear trade-off between rate and accuracy of tRNA selection on the ribosome, from which we could estimate the maximal accuracy. Exploiting this method for a complete set of single-mismatch readings by one tRNA species, we found simple patterns of genetic code reading, where the accuracy was highest for the second and lowest for the third codon position. The results bridge the gap between in vivo and in vitro protein synthesis and allow calibration of our test tube conditions to those of the living cell.

Insulin as well as glucagon are important hormones in maintaining glucose homeostasis and regulating the metabolism in the body. Insulin receptors (IR) are transmembrane receptors that promote a signal transduction when activated by insulin. This can for example cause an increased influx of glucose into the cell performed by so called glucose transporters (GLUTs). These membrane proteins facilitate the transport of glucose from the blood into the cells, so the cell always has a constant supply of energy. Peroxisome proliferator-activated receptors (PPAR) are nuclear fatty acid receptors. They are activated by lipids and regulate fatty acid transcription. PPARδ/β is located in skeletal muscle and can promote fatty acid catabolism as well as cause a switch in fuel preference from glucose to fatty acids. It has been suggested that ligands for PPARδ could act as insulin sensitizers. The PPARγ coactivator-1α can increase mitochondrial content in skeletal muscle if over expressed. The same is true for endurance exercise. Hormones released from adipose tissue can cause hyperphagia and obesity if over- or under expressed. They can also work in the opposite way by decreasing appetite with weight loss as an effect. Impaired signalling or dysfunctional receptor can cause insulin resistance, obesity and diabetes. Lipolysis occurs in adipose tissues and is conducted by three enzymes, namely adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and monoglyceride lipase (MGL). There are some factors that can increase lipolysis such as caffeine, a low glycemic index, high protein intake and training. The enzyme PEPCK is involved in the gluconeogensis in the liver and kidney cortex, and also in the glyceroneogenesis in the liver, as well as in brown and white adipose tissue. When overexpressed in skeletal muscle the enzyme increases the muscle activity. The overexpression of the enzyme did promote the β-oxidation as energy source for the muscles during exercise, instead of muscle glycogen as fuel. The processes of protein synthesis and breakdown are together called protein turnover. Muscle grows when synthesis is greater than breakdown, and withers if breakdown exceeds the level of synthesis. Acute effects of training is catabolic, but long time exercise causes however an increased protein synthesis. Leucine, an essential amino acid, has an important role in the initiation phase of translation. Glutamine is probably important in the regulation of muscle protein synthesis and breakdown. Together with glutamate, aspartate and asparagine, these are responsible for the amino acid metabolism that occurs in the muscles. Protein synthesis reaches its maximum in the recovery phase after intense training.

Is Supplemental Leucine Intake Associated with Enhanced Post Exercise Muscle Protein Synthesis and Attenuated Muscle Protein Breakdown? Knight AD, Benardot D, Thompson W, and Henes ST Introduction: The role of individual amino acids on protein synthesis and their impact on physical performance is of high importance to athletes and to those studying the science of sports nutrition. Leucine, one of three branched-chain amino acids, is a frequently researched amino acid because of its potential stimulatory effect on muscle protein synthesis (MPS) following exercise in humans. Purpose: Although there have been many studies conducted on leucine’s muscle stimulatory effect, questions remain as to the efficacy and feasibility of leucine as an MPS catalyst. Contributing to these questions are the widely varied dosing and timing strategies that different researchers have employed. It is the purpose of this thesis, therefore, to assess the differences in study protocols and shed light on the potential effectiveness on leucine as a MPS stimulator. Central to this issue is whether supplemental leucine intake is associated with enhanced post exercise MPS and, if so, what associated factors, including timing and level of intake, are most likely to influence this effect. Methods: A comprehensive review of the literature on leucine and its effect on MPS was performed. Studies were organized into similar topics, with an assessment and summary of effect produced for each topic area. A general conclusion was made that was based on the summary of each topic area. Results: Leucine is involved in protein metabolism regulation through its role in stimulating the mammalian target of rapamycin (mTOR) signaling cascade and by indicating energy and amino acid availability. It functions to initiate MPS and decrease muscle protein breakdown by downregulating the ubiquitin-proteasome system, lysosomal activity, and/or increasing circulating insulin. Conclusions: Supplementation with the amino acid leucine effectively enhances MPS and attenuates muscle protein degradation in humans following bouts of physical exertion. Leucine intake in amounts greater than that found in ~20g whole protein saturates MPS and increases leucine oxidation. For this reason, an upper limit of leucine intake should be established. While leucine successfully increases MPS, it remains unclear whether this translates to enhanced physical performance, an area that requires more studies to be conducted.

The GTPase elongation factor G (EF-G) is an important player in the complex process of protein synthesis by bacterial ribosomes. Although extensively studied much remains to be learned about this fascinating protein. In the elongation phase, after incorporation of each amino acid into the growing peptide chain, EF-G translocates the ribosome along the mRNA template. In the recycling phase, when the synthesis of a protein has been completed, EF-G, together with ribosome recycling factor (RRF), splits the ribosome into its subunits. We developed the first in vitro assay for measuring the average time of a complete translocation step at any position along the mRNA. Inside the open reading frame, at saturating EF-G concentration and low magnesium ion concentration, translocation rates were fast and compatible with elongation rates observed in vivo. We also determined the complete kinetic mechanism for EF-G- and RRF-dependent splitting of the post-termination ribosome. We showed that splitting occurs only when RRF binds before EF-G and that the rate and GTP consumption of the reaction varies greatly with the factor concentrations. The antibiotic fusidic acid (FA) inhibits bacterial protein synthesis by binding to EF-G when the factor is ribosome bound, during translocation and ribosome recycling. We developed experimental methods and a theoretical framework for analyzing the effect of tight-binding inhibitors like FA on protein synthesis. We found that FA targets three different states during each elongation cycle and that it binds to EF-G on the post-termination ribosome both in the presence and absence of RRF. The stalling time of an FA-inhibited ribosome is about hundred-fold longer than the time of an uninhibited elongation cycle and therefore each binding event has a large impact on the protein synthesis rate and may induce queuing of ribosomes on the mRNA. Although ribosomes in the elongation and the recycling phases are targeted with similar efficiency, we showed that the main effect of FA in vivo is on elongation. Our results may serve as a basis for modelling of EF-G function and FA inhibition inside the living cell and for structure determination of mechanistically important intermediate states in translocation and ribosome recycling.

Whey protein supplementation, containing large amount of leucine, has been a traditional intervention to maintain net protein balance in the past decades. It has been recognized that leucine alone is able to stimulate protein synthesis by activating mTOR and its related downstream pathway without affecting protein degradation, whereas its metabolite β-hydroxy-β-methylbutyrate (HMB) is known to attenuate protein degradation when provided chronically. However, the mechanism of HMB’s benefit remains unclear. To address how HMB regulates protein synthesis and degradation signaling pathways, we compared one dose of whey protein (187.5mg/kg), HMB (400mg/kg) or leucine (1.4g/kg) by oral gavage. Blood was collected at 0, 45 and 90 min for blood glucose and plasma insulin analysis. Red and white gastrocnemius muscle was taken separately 90 min after gavage. Blood glucose was reduced by leucine at 45 and 90 min post gavage. Plasma insulin was enhanced by leucine at 45 min and then decreased at 90 min post gavage, whereas HMB decreased plasma insulin through 90 min post gavege. Western blot analysis showed that HMB phosphorylated Akt in red gastronemius, and enhanced phosphorylation of mTOR in both types of muscles. Leucine phosphorylated mTOR, p70s6k and 4E-BP1 in both red and white gastronemius. Regarding protein degradation signals, phosphorylation of FOXO3A was enhanced by HMB, but not in the other treatment groups. Whey protein had no effect on those cellular signaling. Our results indicate that both HMB and leucine may stimulate protein synthesis through the mTOR pathway in red and white gastrocnemius muscles by different degrees with leucine more effective than HMB. HMB may have a greater effect than leucine on limiting protein degradation by phosphorylating Akt and FOXO3A in red and white gastrocnemius muscles. A combination of HMB and leucine, as a new interventional strategy, is predicted to maximize protein accretion by increasing protein synthesis as well as inhibiting protein degradation. / text

The human body consists of about 40 % skeletal muscles which control the body’s movement, ability to stand up, force generation, locomotion, heat production and are also the body’s protein reservoir. Muscle mass is controlled by the relationship between protein synthesis and protein degradation. Atrophy, a decrease in muscle mass, can be trigged by disuse, immobilization, inflammation and cancer. Hypertrophy, an increase in muscle mass, can occur after increased mechanical load, high usage and/or anabolic stimulation. The aim of this thesis was to investigate changes in expression and post translational modifications of some factors involved in the regulation of protein synthesis and protein degradation in 6-days denervated atrophic hind-limb muscles (anterior tibial and pooled gastrocnemius and soleus muscles) and in 6-days denervated hypertrophic hemidiaphragm muscle in mice. Protein expression and post translational modifications were studied semi-quantitatively using Western blots with whole muscle homogenates and separated nuclear and cytosolic fractions from both innervated and denervated muscles.  An increase in protein synthesis after denervation in both atrophic and hypertrophic muscles was suggested after studies of factors downstream of mTOR (paper I).  Other results suggest that FoxO1 and MuRF1 (paper II) participate in the tissue remodeling that occurs after denervation. A differential response of MK2 phosphorylation in denervated hypertrophic and atrophic muscles was confirmed (paper III). An increase in phosphorylation of the MK2 substrate Hsp 25 in all denervated muscles studied (paper III) indicates that other factors than MK2 are involved in regulating this phosphorylation. eIF4G phosphorylation at S1108 was investigated (paper IV) and a decrease was observed in atrophic muscle but an increase in hypertrophic muscle. The results in this thesis suggest that there are several factors that control protein degradation and protein synthesis in denervated atrophic and hypertrophic skeletal muscles. This is an intricate labyrinth with many different cell signaling factors, the function of which are still far from fully understood.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 4 uppsatser.

Οι ανησυχητικές διαστάσεις που έχει λάβει τα τελευταία χρόνια η περιβαλλοντική ρύπανση και ιδιαίτερα η ρύπανση του θαλάσσιου περιβάλλοντος, καθιστούν επιτακτική την ανάγκη εύρεσης και εγκαθίδρυσης νέων βιοδεικτών που μπορούν να συμβάλουν δραστικά στην έγκαιρη αναγνώριση της κατάστασης της υγείας του θαλάσσιου οικοσυστήματος και συνεπώς στη λήψη βελτιωτικών μέτρων για την αποκατάστασή του. Κυρίαρχο ρόλο ανάμεσα στους θαλάσσιους ρύπους κατέχουν τα βαρέα μέταλλα, τα οποία λόγω της ικανότητάς τους να επάγουν ή να προκαλούν οξειδωτικό στρες μπορούν να προκαλέσουν σημαντικές, ακόμη και θανατογόνες βλάβες στους θαλάσσιους οργανισμούς. Η έκθεση σε μέταλλα προκαλεί την απορρύθμιση πολλών κυτταρικών διαδικασιών και κυρίως αυτών που διεξάγονται από μακρομοριακά σύμπλοκα, όπως η μεταφραστική μηχανή, τα οποία είναι ιδιαίτερα ευαίσθητα σε συνθήκες stress. Οι αλλαγές που προκαλούνται από το κυτταρικό stress στη διαδικασία της μετάφρασης καλύπτουν ένα ευρύ φάσμα αποκρίσεων, που μπορεί να περιλαμβάνει μείωση της ολικής μετάφρασης ή αύξηση της μετάφρασης ειδικών mRNAs. Ένας αποτελεσματικός τρόπος εκτίμησης των μεταφραστικών αποκρίσεων ενός οργανισμού στο περιβαλλοντικό stress είναι ο προσδιορισμός του μεταφραστικά ενεργού ριβοσωματικού κλάσματος των πολυσωμάτων. Επίσης, καθώς ρυθμιστικά γεγονότα μπορεί να συμβούν σε οποιοδήποτε βήμα της μετάφρασης, έγινε μία σειρά πειραμάτων, που αφορούν στον έλεγχο της μεταφραστικής λειτουργίας του μυδιού Mytilus galloprovincialis, οργανισμού που χρησιμοποιείται συχνά ως βιομάρτυρας, σε εργαστηριακές συνθήκες, υπό την επίδραση τριών μετάλλων, του υδραργύρου, του χαλκού και του καδμίου. Για την εκτίμηση της κατάστασης της υγείας των εκτεθειμένων μυδιών έγινε προσδιορισμός ορισμένων κλασικών, ευρέως χρησιμοποιούμενων βιοδεικτών, καθώς και βιοδεικτών οξειδωτικού στρες. Ο έλεγχος των μεταφραστικών αποκρίσεων των εκτεθειμένων μυδιών περιλάμβανε εκτίμηση του ποσοστού πολυσωμάτων και προσδιορισμό της επίδρασης των μετάλλων τόσο στο προπαρασκευαστικό στάδιο της μετάφρασης, δηλαδή την αμινοακυλίωση των υποστρωμάτων, όσο και στα τρία κύρια στάδια της μετάφρασης, δηλαδή την έναρξη, την επιμήκυνση και τον τερματισμό. Τα δεδομένα της μελέτης αυτής αποκαλύπτουν ότι τόσο ο υδράργυρος, όσο και ο χαλκός προκαλούν το ίδιο πρότυπο αλλαγών στη μεταφραστική λειτουργία των εκτεθειμένων μυδιών, οδηγώντας σε απορρύθμιση όλων των σταδίων και των ενδιάμεσων βημάτων της πρωτεϊνοσύνθεσης. Οι διαταραχές αυτές οφείλονται στο οξειδωτικό στρες που προκαλείται από τα μέταλλα αυτά και στην παράλληλη αδυναμία του συστήματος αντιοξειδωτικής άμυνας των κυττάρων να το αντιμετωπίσει. Η στατιστική επεξεργασία των αποτελεσμάτων επιβεβαιώνει την αρνητική επίδραση του οξειδωτικού στρες που προκαλείται από τα μέταλλα στη μετάφραση, με μία στατιστικά σημαντική συσχέτιση το ποσοστού πολυσωμάτων με τους βιοδείκτες οξειδωτικού στρες. Από την άλλη, τα αποτελέσματα της έκθεσης των μυδιών στο κάδμιο διαφοροποιούνται σημαντικά. Αρχικά, η έκθεση των μυδιών στο μέταλλο αυτό προκαλεί μία έντονη διαταραχή στην πρωτεϊνοσυνθετική διαδικασία, οφειλόμενη στην πρόκληση οξειδωτικού στρες. Με την πάροδο όμως το χρόνου, τα κύτταρα καταφέρνουν να διεγείρουν τους αντιοξειδωτικούς μηχανισμούς, που σε αυτή την περίπτωση αντιπροσωπεύονται κυρίως από τις μεταλλοθειονίνες, με αποτέλεσμα να καταφέρνουν να ανταπεξέλθουν στη δύσκολες συνθήκες και να αναχαιτίσουν την αρνητική επίδραση του καδμίου, γεγονός που οδηγεί σε ανάκαμψη της μεταφραστικής ικανότητας των μυδιών αυτών, στο τέλος της περιόδου έκθεσης. Το διφασικό προφίλ της επίδρασης του καδμίου αντανακλάται στην έλειψη συσχέτισης μεταξύ του ποσοστού πολυσωμάτων των εκτεθειμένων στο κάδμιο μυδιών και των βιοδεικτών οξειδωτικού στρες αλλά και των λοιπών βιοδεικτών. / The alarming levels of the environmental and especially the marine pollution in recent years constitute an urgent need of finding and establishing new biomarkers which may contribute to the early detection of the health status of the marine ecosystem leading to ameliorating measures towards its restoration. Heavy metals, which are capable of causing severe or even leathal defects to marine organisms, through their ability to induce or produce oxidative stress, posess a leading role among the marine pollutants. Exporure to heavy metals may cause the deregulation of many cellular processeses, mainly of those that are carried out by macromolecular complexes, such as the translational machinery, which are particularly sensitive to stress conditions. The alterations induced by the cellular stress in the translation may cover a broad range of responses, including a decline of the global translation or an increase of the translation of certain mRNAs. An effective way to reveal translational responses of an organism to environmetal stress is the evaluation of the translationally active ribosomal fraction of polysomes. Moreover, considering the fact that regulatory events may occur at any step of the translational process, a set of experiments was carried out, concerning the examination of the translational function of the mussel Mytilus galloprovindialis, which is commonly used as a bioindicator, in laboratory conditions, under the influence of three metals, mercury, copper and cadmium. In order to evaluate the health condition of the exposed mussels, a battery of standard biomarkers was applied, including biomarkers of oxidative stress. The determination of the translational responses of the treated mussels included measurement of the polysome content and assessment of the metal effect on the preparative stage of protein synthesis, the aminacylation of the substrates, as well as on the three main stages of translation, that is the initiation, the elongation and the termination. The data of the study reveal that mercury as well as copper cause the same patern of alterations in the translational function of the exposed mussels, leading to a deregulation of every stage and intermediate step of protein synthesis. These perturbations are a consequence of the metal induced oxidative stress, followed by a failure of the antioxidant defense system to confront it. Statistical analysis of the results confirms the negative effect of the metals on the translation, with a statistically important correlation of the polysome content with the oxidative stress biomarkers. On the other hand, the results of the cadmium exposed mussels follow a different pattern. First, the exposure of mussels to the metal causes a severe perturbation of the translational process, due to the oxidative stress induction. Finally, cells stimulate antioxidant mechanisms, in this case represented by metallotheioneins, managing to cope with the difficult conditions and to block the negative effect of cadmium, which leads to a restoration of the translational capacity of the mussels, at the end of the exposure period.The two-phase profile of the cadmium effect is reflected on the absence of statistical correlation between the polysomal content and biomarkers of oxidative stress, as well as other biomarkers.

Reading of messenger RNA (mRNA) by aminoacyl-tRNAs (aa-tRNAs) on the ribosomes in the bacterial cell occurs with high accuracy. It follows from the physical chemistry of enzymatic reactions that there must be a trade-off between rate and accuracy of initial tRNA selection in protein synthesis: when the current accuracy, the A-value, approaches its maximal possible value, the d-value, the kinetic efficiency of the reaction approaches zero. We have used an in vitro system for mRNA translation with purified E. coli components to estimate the d- and A-values by which aa-tRNAs discriminate between their cognate and near cognate codons displayed in the ribosomal A site. In the case of tRNALys, we verified the prediction of a linear trade-off between kinetic efficiency of cognate codon reading and the accuracy of codon selection. These experiments have been extended to a larger set of tRNAs, including tRNAPhe, tRNAGlu, tRNAHis, tRNACys, tRNAAsp and tRNATyr, and linear efficiency-accuracy trade-off was observed in all cases. Similar to tRNALys, tRNAPhe discriminated with higher accuracy against a particular mismatch in the second than in the first codon position. Remarkably high d-values were observed for tRNAGlu discrimination against a C-C mismatch in the first codon position (70 000) and for tRNAPhe discrimination against an A-G mismatch in the second codon position (79 000). At the same time, we have found a remarkably small d-value (200) for tRNAGlu misreading G in the middle position of the codon (U-G mismatch). Aminoglycoside antibiotics induce large codon reading errors by tRNAs. We have studied the mechanism of aminoglycoside action and found that the drug stabilized aminoacyl-tRNA in a codon selective in relation to a codon non-selective state. This greatly enhanced the probability of near cognate aminoacyl-tRNAs to successfully transcend the initial selection step of the translating ribosome. We showed that Mg2+ ions, in contrast, favour codon non-selective states and thus induce errors in a principally different way than aminoglycosides.  We also designed experiments to estimate the overall accuracy of peptide bond formation with, including initial selection accuracy and proofreading of tRNAs after GTP hydrolysis on EF-Tu. Our experiments have now made it possible to calibrate the accuracy of tRNA selection in the test tube to that in the living cells. We will now also be able to investigate the degree to which the accuracy of tRNA selection has been optimized for maximal fitness.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第13792号 / 工博第2896号 / 新制||工||1427(附属図書館) / 26008 / UT51-2008-C708 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 今中 忠行, 教授 青山 安宏, 教授 濵地 格 / 学位規則第4条第1項該当