Peroxin Pex21p Interacts with C-terminal Noncatalytic Domain of Yeast Seryl-tRNA Synthetase and Forms a Specific Ternary Complex with tRNA\u3csup\u3eSer\u3c/sup\u3e

The seryl‐tRNA synthetase from Saccharomyces cerevisiae interacts with the peroxisome biogenesis‐related factor Pex21p. Several deletion mutants of seryl‐tRNA synthetase were constructed and inspected for their ability to interact with Pex21p in a yeast two‐hybrid assay, allowing mapping of the synthetase domain required for complex assembly. Deletion of the 13 C‐terminal amino acids abolished Pex21p binding to seryl‐tRNA synthetase. The catalytic parameters of purified truncated seryl‐tRNA synthetase, determined in the serylation reaction, were found to be almost identical to those of the native enzyme. In vivo loss of interaction with Pex21p was confirmed in vitro by coaffinity purification. These data indicate that the C‐terminally appended domain of yeast seryl‐tRNA synthetase does not participate in substrate binding, but instead is required for association with Pex21p. We further determined that Pex21p does not directly bind tRNA, and nor does it possess a tRNA‐binding motif, but it instead participates in the formation of a specific ternary complex with seryl‐tRNA synthetase and tRNASer, strengthening the interaction of seryl‐tRNA synthetase with its cognate tRNASer

Modeling Strong Physically Unclonable Functions with Metaheuristics

Evolutionary algorithms have been successfully applied to attack Physically Unclonable Functions (PUFs). CMA-ES is recognized as the most powerful option for a type of attack called the reliability attack. In this paper, we take a step back and systematically evaluate several metaheuristics for the challenge-response pair-based attack on strong PUFs. Our results confirm that CMA-ES has the best performance, but we note several other algorithms with similar performance while having smaller computational costs

Seril-tRNA-sintetaze iz metanogenih arheja: supresija bakterijskih amber mutacija i heterologna toksičnost

Methanogenic archaea possess unusual seryl-tRNA synthetases (SerRS), evolutionarily distinct from the SerRSs found in other archaea, eucaryotes and bacteria. Our recent X-ray structural analysis of Methanosarcina barkeri SerRS revealed an idiosyncratic N-terminal domain and catalytic zinc ion in the active site. To shed further light on substrate discrimination by methanogenic-type SerRS, we set up to explore in vivo the interaction of methanogenic-type SerRSs with their cognate tRNAs in Escherichia coli or Saccharomyces cerevisiae. The expression of various methanogenic-type SerRSs was toxic for E. coli, resulting in the synthesis of erroneous proteins, as revealed by β-galactosidase stability assay. Although SerRSs from methanogenic archaea recognize tRNAsSer from all three domains of life in vitro, the toxicity presumably precluded the complementation of endogenous SerRS function in both, E. coli and S. cerevisiae. However, despite the observed toxicity, coexpression of methanogenic-type SerRS with its cognate tRNA suppressed bacterial amber mutation.Metanogene arheje imaju neobične seril-tRNA-sintetaze (SerRS), evolucijski udaljene od SerRS koje se mogu naći u drugih arheja, eukariota i bakterija. Naša nedavna analiza kristalne strukture SerRS iz metanogene arheje Methanosarina barkeri otkrila je karakterističnu N-terminalnu domenu i katalitički ion cinka na aktivnom mjestu. Da bi se rasvijetlio način na koji metanogeni tip SerRS diskriminira supstrate, autori su istraživali in vivo interakciju metanogenog tipa SerRS s pripadnim molekulama tRNA u bakteriji Escherichia coli ili kvascu Saccharomyces cerevisiae. Ekspresija raznih SerRS metanogenoga tipa bila je toksična za bakteriju E. coli, te je rezultirala sintezom proteina s greškama u aminokiselinskom sastavu, što se vidi iz testa stabilnosti β-galaktozidaze. Iako je SerRS iz metanogenih arheja mogla prepoznati tRNASer iz sve tri domene života in vitro, vjerojatno je toksičnost zasjenila komplementaciju funkcije endogene SerRS u bakteriji E. coli i kvascu S. cerevisiae. Međutim, unatoč toksičnosti, koekspresija metanogenoga tipa SerRS s pripadnom tRNA suprimirala je bakterijsku amber mutaciju

Archaeal aminoacyl-tRNA synthetases interact with the ribosome to recycle tRNAs

Aminoacyl-tRNA synthetases (aaRS) are essential enzymes catalyzing the formation of aminoacyl-tRNAs, the immediate precursors for encoded peptides in ribosomal protein synthesis. Previous studies have suggested a link between tRNA aminoacylation and high-molecular-weight cellular complexes such as the cytoskeleton or ribosomes. However, the structural basis of these interactions and potential mechanistic implications are not well understood. To biochemically characterize these interactions we have used a system of two interacting archaeal aaRSs: an atypical methanogenic-type seryl-tRNA synthetase and an archaeal ArgRS. More specifically, we have shown by thermophoresis and surface plasmon resonance that these two aaRSs bind to the large ribosomal subunit with micromolar affinities. We have identified the L7/L12 stalk and the proteins located near the stalk base as the main sites for aaRS binding. Finally, we have performed a bioinformatics analysis of synonymous codons in the Methanothermobacter thermautotrophicus genome that supports a mechanism in which the deacylated tRNAs may be recharged by aaRSs bound to the ribosome and reused at the next occurrence of a codon encoding the same amino acid. These results suggest a mechanism of tRNA recycling in which aaRSs associate with the L7/L12 stalk region to recapture the tRNAs released from the preceding ribosome in polysome

Lipidomics Provides New Insight into Pathogenesis and Therapeutic Targets of the Ischemia-Reperfusion Injury

Lipids play an essential role in both tissue protection and damage. Tissue ischemia creates anaerobic conditions in which enzyme inactivation occurs, and reperfusion can initiate oxidative stress that leads to harmful changes in membrane lipids, the formation of aldehydes, and chain damage until cell death. The critical event in such a series of harmful events in the cell is the unwanted accumulation of fatty acids that leads to lipotoxicity. Lipid analysis provides additional insight into the pathogenesis of ischemia/reperfusion (I/R) disorders and reveals new targets for drug action. The profile of changes in the composition of fatty acids in the cell, as well as the time course of these changes, indicate both the mechanism of damage and new therapeutic possibilities. A therapeutic approach to reperfusion lipotoxicity involves attenuation of fatty acids overload, i.e., their transport to adipose tissue and/or inhibition of the adverse effects of fatty acids on cell damage and death. The latter option involves using PPAR agonists and drugs that modulate the transport of fatty acids via carnitine into the interior of the mitochondria or the redirection of long-chain fatty acids to peroxisomes

Točnost sinteze seril-tRNA

The high level of translational fidelity is ensured by various types of quality control mechanisms, which are adapted to prevent or correct naturally occurring mistakes. Accurate aminoacyl-tRNA synthesis is mostly dependent on the specificity of the aminoacyl-tRNA synthetases (aaRS), i.e. their ability to choose among competing structurally similar substrates. Our studies have revealed that accurate seryl-tRNA synthesis in yeast and plants is accomplished via tRNA-assisted optimization of amino acid binding to the active site of seryl-tRNA synthetase (SerRS). Based on our recent kinetic data, a mechanism is proposed by which transient protein : RNA complex activates the cognate amino acid more efficiently and more specifically than the apoenzyme alone. This may proceed via a tRNA induced conformational change in the enzyme’s active site. The influence of tRNASer, on the activation of serine by SerRS variants mutated in the active site, is much less pronounced. Although SerRS misactivates structurally similar threonine in vitro, the formation of such erroneous threonyl-adenylate is reduced in the presence of nonchargeable tRNASer analog. Thus, the sequence-specific tRNA : SerRS interactions enhance the accuracy of amino acid recognition. Another type of quality control mechanism in tRNA serylation is assumed to be based on the complex formation between SerRS and a nonsynthetase protein. Using in vivo interaction screen, yeast peroxin Pex21p was identified as SerRS interacting protein. This was confirmed by an in vitro binding assay. Kinetic experiments performed in the presence of Pex21p revealed that this peroxin acts as an activator of seryl-tRNA synthetase in the aminoacylation reaction.Točnost biosinteze proteina nadziru različiti kontrolni mehanizmi koji sprečavaju ili ispravljaju gre{ke u translaciji. Specifičnost aminoacil tRNA-sintetaza (aaRS) pri izboru i kovalentnom povezivanju pripadnih aminokiselina i tRNA ključna je u ovom procesu. Istraživanja u našem laboratoriju pokazala su da se specifičnost i učinkovitost sinteze seril-tRNA u kvascu i biljkama povećavaju tRNA-ovisnim prilagođavanjem veznog mjesta za serin u aktivnom mjestu seril tRNA-sintetaze (SerRS). Dakle, makromolekularni kompleksi tRNA i enzima imaju bolja katalitička svojstva od apoenzima. Naši rezultati kinetike pokazuju da se vezanjem tRNA bitno mijenja konformacija veznog mjesta za serin u enzimu divljeg tipa, dok je konformacijska promjena slabija kod enzima s mutacijama u aktivnom mjestu. Iako SerRS može aktivirati i serinu sličan treonin, stvaranje treonil adenilata smanjeno je u prisutnosti aminoacilacijski inaktivnog analoga tRNA. Time je pokazano da su interakcije između pripadne tRNA i SerRS bitne za točan izbor aminokiseline. Djelotvornost serilacije povećava se i interakcijom SerRS s nesintetaznim proteinom, peroksinom Pex21p. Ta neočekivana interakcija uočena je prvo in vivo, pretragom kvaščeve biblioteke u sustavu dvaju hibrida sa SerRS kao interakcijskim proteinom. Interakcija je potvrđena in vitro. Kinetički eksperimenti pokazali su da Pex21p djeluje kao aktivator SerRS, što ovu neobičnu interakciju čini biološki značajnom jer povećava učinkovitost aminoaciliranja

Genetic Algorithm to Evolve Ensembles of Rules for On-Line Scheduling on Single Machine with Variable Capacity

International Work-Conference on the Interplay Between Natural and Artificial Computation, IWINAC (8th . 2019. Almería, Spain

Time-Dependent Effects of Starvation on Pituitary, Hypothalamic and Serum Prolactin Levels in Rats: Comparison to the Galanin Expression Pattern

Given that both prolactin and galanin take part in the regulation of energy homeostasis and that galanin is localized within lactotrophs, this study was aimed at comparing the pituitary expression patterns of prolactin and galanin during different phases of metabolic response to starvation in adult Wistar male rats. Food was removed at the onset of the dark phase (6:00 pm) and the animals were deprived for 6, 12, 24 and 48 h. Each of the starved groups (n=6) was killed simultaneously with a group of ad libitum-fed rats (n=6), and the intrapituitary levels of prolactin and galanin were examined. Galanin expression in the hypothalamus and the circulating levels of prolactin were also assessed. Starvation induced a rise in the intrapituitary prolactin level (p LT 0.001), whereas the opposite trend was detected in the serum (p LT 0.05). The galanin pituitary level was initially increased (6, 12 h) (p LT 0.05), but as starvation progressed, it first reached (at 24 h) and ultimately fell below the level recorded in the ad libitum rats (at 48 h) (p LT 0.05). Both prolactin and galanin were elevated in the hypothalamus after 24- and 48-h starvation. The results show that the starvation-induced increase in the pituitary prolactin expression did not lead to the rise in prolactin circulating levels, but rather resulted in the elevation of the prolactin hypothalamic content. Furthermore, the results suggest that under the circumstances of disturbed energy homeostasis, galanin might be responsible for the augmented prolactin production, initially at the pituitary and subsequently at the hypothalamic level