Protein Synthesis Factors Research Articles

Diosgenin stimulates osteogenic activity by increasing bone matrix protein synthesis and bone-specific transcription factor Runx2 in osteoblastic MC3T3-E1 cells

Diosgenin, a steroid saponin extracted from the root of wild yam ( Dioscorea villossa) is claimed to have osteogenic property. However, detailed studies providing evidence to this claim have not been fully undertaken. In this study, we investigated the effect of diosgenin on the osteogenesis of murine MC3T3-E1 osteoblastic cells. Cells were cultured with varying levels of diosgenin (0–10 μM) within 25 days of bone formation period. Diosgenin was found to stimulate proliferation within the range of 0.01–5 μM using MTT assay. The medium and cellular levels of Type 1 collagen and alkaline phosphatase (ALP), both of which are major bone matrix proteins, increased within the low range of diosgenin concentration (>0–3 μM), and this pattern was further confirmed by collagen and ALP staining of the extracellular matrix (ECM). The cellular protein expression of ALP and collagen Type 1 was also increased at 0.1–1 μM diosgenin treatment as analyzed by Western blot. Calcium deposition within the ECM also showed the same pattern as assessed by Alizarin Red S and Von Kossa staining. Bone-specific transcription factor runt-related transcription factor 2 (Runx2) and Runx2-regulated osteopontin protein expressions were induced at low concentration (0.1–1 μM) and again decreased with high diosgenin concentrations. Based on our findings, our study suggests that diosgenin can enhance bone formation by stimulating the synthesis and secretion of Type 1 collagen and ALP and bone marker proteins Runx2 and osteopontin expression. The increased levels of these marker proteins, in turn, can increase the formation of calcium deposits within the ECM thereby increasing bone formation.

Direct integration of cell‐free‐synthesized connexin‐43 into liposomes and hemichannel formation

Proteoliposomes were directly prepared by synthesizing membrane proteins with the use of minimal protein synthesis factors isolated from Escherichia coli (the PURE system) in the presence of liposomes. Connexin-43 (Cx43), which is a water-insoluble integral membrane protein that forms a hexameric complex in membranes, was cotranslationally integrated with an essentially uniform orientation in liposomes. The addition of liposomes following protein expression (post-translational presence of liposomes) did not lead to the integration of Cx43 into the liposome membranes. The amount of integrated Cx43 increased as the liposome concentration increased. The presence of liposomes did not influence the total amount of synthesized Cx43. The Cx43 integrated into the liposome membranes formed open membrane pores. These results indicate that the liposomes act in a chaperone-like manner by preventing Cx43 from aggregating in solution, because of integration into the bilayer, and also by functionalization of the integrated Cx43 in the membrane. This is the first report that cell-free-synthesized water-insoluble membrane protein is directly integrated with a uniform orientation as a functional oligomer into liposome membranes. This simple proteoliposome preparation procedure should be a valuable approach for structural and functional studies of membrane proteins.

Mutations at the accommodation gate of the ribosome impair RF2-dependent translation termination

During protein synthesis, aminoacyl-tRNA (aa-tRNA) and release factors 1 and 2 (RF1 and RF2) have to bind at the catalytic center of the ribosome on the 50S subunit where they take part in peptide bond formation or peptidyl-tRNA hydrolysis, respectively. Computer simulations of aa-tRNA movement into the catalytic site (accommodation) suggested that three nucleotides of 23S rRNA, U2492, C2556, and C2573, form a "gate" at which aa-tRNA movement into the A site is retarded. Here we examined the role of nucleotides C2573 of 23S rRNA, a part of the putative accommodation gate, and of the neighboring A2572 for aa-tRNA binding followed by peptide bond formation and for the RF2-dependent peptide release. Mutations at the two positions did not affect aa-tRNA accommodation, peptide bond formation, or the fidelity of aa-tRNA selection, but impaired RF2-catalyzed peptide release. The data suggest that the ribosome is a robust machine that allows rapid aa-tRNA accommodation despite the defects at the accommodation gate. In comparison, peptide release by RF2 appears more sensitive to these mutations, due to slower accommodation of the factor or effects on RF2 positioning in the A site.

EEF1A: Thinking Outside the Ribosome

Eukaryotic translation elongation factor 1A (eEF1A) is one of the most abundant protein synthesis factors. eEF1A is responsible for the delivery of all aminoacyl-tRNAs to the ribosome, aside from initiator and selenocysteine tRNAs. In addition to its roles in polypeptide chain elongation, unique cellular and viral activities have been attributed to eEF1A in eukaryotes from yeast to plants and mammals. From preliminary, speculative associations to well characterized biochemical and biological interactions, it is clear that eEF1A, of all the translation factors, has been ascribed the most functions outside of protein synthesis. A mechanistic understanding of these non-canonical functions of eEF1A will shed light on many important biological questions, including viral-host interaction, subcellular organization, and the integration of key cellular pathways.

Microtubule-Dependent mRNA Transport in Fungi

The localization and local translation of mRNAs constitute an important mechanism to promote the correct subcellular targeting of proteins. mRNA localization is mediated by the active transport of mRNPs, large assemblies consisting of mRNAs and associated factors such as RNA-binding proteins. Molecular motors move mRNPs along the actin or microtubule cytoskeleton for short-distance or long-distance trafficking, respectively. In filamentous fungi, microtubule-based long-distance transport of vesicles, which are involved in membrane and cell wall expansion, supports efficient hyphal growth. Recently, we discovered that the microtubule-mediated transport of mRNAs is essential for the fast polar growth of infectious filaments in the corn pathogen Ustilago maydis. Combining in vivo UV cross-linking and RNA live imaging revealed that the RNA-binding protein Rrm4, which constitutes an integral part of the mRNP transport machinery, mediates the transport of distinct mRNAs encoding polarity factors, protein synthesis factors, and mitochondrial proteins. Moreover, our results indicate that microtubule-dependent mRNA transport is evolutionarily conserved from fungi to higher eukaryotes. This raises the exciting possibility of U. maydis as a model system to uncover basic concepts of long-distance mRNA transport.

The increase in Akt phosphorylation following feeding is independent of age in growing and mature Thoroughbred mares

The activation of the initiation factors for protein synthesis in skeletal muscle has been shown to increase following consumption of a protein‐containing meal due to increases in both insulin (INS) and amino acid (AA) concentrations. In neonates, INS sensitivity has been shown to decrease with age, reducing the downstream signalling of Akt and resulting in lower skeletal muscle protein synthesis. The objective of this study was to determine the effects of an 18h period of feed witholding, both with (postprandial; PP) and without (post‐absorptive; PA) the re‐feeding of a high protein meal, on the activation of Akt in gluteal muscle of yearling, 2y old, and mature Thoroughbred mares (N=18). Blood samples were taken throughout the experimental period to measure plasma AA via HPLC. A gluteal muscle biopsy was taken after the last blood sample (t = 90 min) to measure the phosphorylation (P) of Akt at Ser473 and Thr308 using Western blotting techniques. For all horses, indispensible AA were ~ 25‐ 110% higher in the PP versus the PA period (P< 0.0001). There was a significant increase in Akt P‐Ser473 (P< 0.0001) and a trend for higher AktP‐Thr308 (P= 0.09) in the PP relative to PA state. Age did affect on the Akt phosphorylation (P > 0.05). Consumption of a high protein meal, following feed withholding, appeared to activate Akt in adolescent and mature horses. Additional research is necessary to determine whether the increase in Akt phosphorylation corresponded to an increase in the phosphorylation of downstream translation initiation factors. Funded by the Department of Animal and Food Sciences.

Yeast AEP3p Is an Accessory Factor in Initiation of Mitochondrial Translation

Initiation of protein synthesis in mitochondria and chloroplasts normally uses a formylated initiator methionyl-tRNA (fMet-tRNA(f)(Met)). However, mitochondrial protein synthesis in Saccharomyces cerevisiae can initiate with nonformylated Met-tRNA(f)(Met), as demonstrated in yeast mutants in which the nuclear gene encoding mitochondrial methionyl-tRNA formyltransferase (FMT1) has been deleted. The role of formylation of the initiator tRNA is not known, but in vitro formylation increases binding of Met-tRNA(f)(Met) to translation initiation factor 2 (IF2). We hypothesize the existence of an accessory factor that assists mitochondrial IF2 (mIF2) in utilizing unformylated Met-tRNA(f)(Met). This accessory factor might be unnecessary when formylated Met-tRNA(f)(Met) is present but becomes essential when only the unformylated species are available. Using a synthetic petite genetic screen in yeast, we identified a mutation in the AEP3 gene that caused a synthetic respiratory-defective phenotype together with Delta fmt1. The same aep3 mutation also caused a synthetic respiratory defect in cells lacking formylated Met-tRNA(f)(Met) due to loss of the MIS1 gene that encodes the mitochondrial C(1)-tetrahydrofolate synthase. The AEP3 gene encodes a peripheral mitochondrial inner membrane protein that stabilizes mitochondrially encoded ATP6/8 mRNA. Here we show that the AEP3 protein (Aep3p) physically interacts with yeast mIF2 both in vitro and in vivo and promotes the binding of unformylated initiator tRNA to yeast mIF2. We propose that Aep3p functions as an accessory initiation factor in mitochondrial protein synthesis.

Roles of the translationally controlled tumour protein (TCTP) and the double-stranded RNA-dependent protein kinase, PKR, in cellular stress responses

Translationally controlled tumour protein (TCTP) is a highly conserved protein present in all eukaryotic organisms. Various cellular functions and molecular interactions have been ascribed to this protein, many related to its growth-promoting and antiapoptotic properties. TCTP levels are highly regulated in response to various cellular stimuli and stresses. We have shown recently that the double-stranded RNA-dependent protein kinase, PKR, is involved in translational regulation of TCTP. Here we extend these studies by demonstrating that TCTP is downregulated in response to various proapoptotic treatments, in particular agents that induce Ca(++) stress, in a PKR-dependent manner. This regulation requires phosphorylation of protein synthesis factor eIF2alpha. Since TCTP has been characterized as an antiapoptotic and Ca(++)-binding protein, we asked whether it is involved in protecting cells from Ca(++)-stress-induced apoptosis. Overexpression of TCTP partially protects cells against thapsigargin-induced apoptosis, as measured using caspase-3 activation assays, a nuclear fragmentation assay, using fluorescence-activated cell sorting analysis, and time-lapse video microscopy. TCTP also protects cells against the proapoptotic effects of tunicamycin and etoposide, but not against those of arsenite. Our results imply that cellular TCTP levels influence sensitivity to apoptosis and that PKR may exert its proapoptotic effects at least in part through downregulation of TCTP via eIF2alpha phosphorylation.

Differential expression of soybean SLTI100 gene encoding translation elongation factor 1A by abiotic stresses

Abstract The translation elongation factor 1A, eEF1A, catalyzes the binding of aminoacyl-tRNA to the A-site of the ribosome by a GTP-dependent mechanism. By subtractive suppression hybridization technique, we have isolated a soybean low-temperature inducible gene, SLTI100 encoding translation elongation factor 1A. Multiple sequence align-ments and phylogenic analysis showed that SLTI100 and other eEF1As originated from diverse organisms are highly conserved. RNA expression of SLTI100 was specifically induced by low temperature, high salt, ABA, or drought stress. Based on the subcellular localization of the corre-sponding gene product fused to GFP, we were able to con-firm that SLTI100-GFP was restricted to the nucleus and cytoplasm. We propose that soybean eEF1A may play an important role in translational regulation during abiotic stress responses in plants. Introduction In the translational elongation process, eukaryote transla-tional elongation factor 1 (eEF1) is a protein synthesis factor composed of four subunits: A, B α, Bβ and Bγ (Browing et al. 1990). Subunit eEF1A, also known as EF1α, binds ami-noacyl-tRNAs to the acceptor (A) site of the ribosome during the peptide chain elongation phase of protein syn-thesis. eEF1A (49,174 kDa) is an abundant protein repre-senting upto 5% of the soluble protein in wheat germ extracts (Browing et al. 1990) and is highly conserved in evolution (77% conservation of wheat EF1A with human EF1A) (Metz et al. 1992). In higher eukaryotes, eEF1A is typically encoded by a multigene family: in man there are 18 genes (Lund et al. 1996) while in

Feeding‐induced time course of changes in protein synthesis in neonatal pig skeletal muscle

Feeding increases protein synthesis by promoting translation initiation, and in the neonate, this increase is greatest in skeletal muscle. This study aimed to identify the feeding‐induced time course of the changes in protein synthesis and translation factor activation in muscle. Piglets (n=36; 5‐7 day old) enterally fed a complete meal were killed 0 (fasting), 30, 60, 90, 120, or 240 min post‐feeding. Skeletal muscle protein synthesis reached a maximum 30 min post‐feeding, was sustained through 120 min, and returned to baseline by 120 min. The relative proportion of polysomes vs. nonpolysomes increased at 30 min only. Protein kinase B phosphorylation was maximal 30 min after feeding and fell to baseline by 90 min. Phosphorylation of mammalian target of rapamycin, eukaryotic initiation factor (eIF) 4E binding protein (4EBP1), ribosomal protein S6, and eIF4G phosphorylation increased by 30 min, remained elevated to 120 min, and returned to baseline by 240 min. The association of 4EBP1▸eIF4E was reduced and eIF4E▸eIF4G increased 30 min post‐feeding, remained so to 120 min, and then returned to baseline. Feeding rapidly increased neonatal muscle protein synthesis by enhancing translation initiation and this increase was sustained for at least 120 min after a meal, but returned to baseline by 240 min. (Supported by NIH grants AR‐44474 and DK‐15658, and USDA/ARS 6250510000‐33).

Cover Picture: Proteomics 5/2009

AbstractIn this issue of Proteomics you will find the following highlighted articles:Heart (pump) broken?Hearts are pumps within pumps within channels and pumps. Calcium is pumped, potassium, sodium, amino acids, and electrons are all pumped, channeled or driven until, finally, blood is pumped. Failure of one or more pumps leads to a heart attack. This report from Zlatkovic et al. looks at the sub‐proteome associated with hypertensive failure of the K+ATP channel and associated cardiomyopathy that develops in KIR6.2 knock‐out mice. Out of >900 reproducible 2‐DE spots, 81 displayed significant over‐ or under‐expression, a number of which validated previously proposed interactions with the Kir6.2 channel. Two‐thirds were down‐regulations, including creatine kinase, adenylate kinase, and lactate dehydrogenase. A total of 114 proteins were ontologically mapped into the K+ATP‐dependent sub‐proteome and a role in hypertensive heart failure. Interaction mapping found >240 nodes and >1200 interactions/edges. A good foundation for future work.Zlatkovic, J. et al., Proteomics 2009, 9, 1314‐1325.The deeper you dig, the more you findA classical biochemist interested in protein‐protein interactions purifies his protein away from other proteins, seeking the highest “‐fold purification”. A proteomicist, on the other hand, looks for “consistent contamination” – i.e. association – of the protein of interest with other proteins. This requires high resolution separations and high accuracy concentration determinations. You can only work with species with concentrations above the detection limit (DL) for the detection method. 2‐DE MS has a DL of approximately 10−8 M, LC‐MS/MS is ∼10−10 M and saturating Cy5 dye method is ∼10−13 M. Archakov et al. report on an atomic force microscope technique that can yield a DL of 10−16 M when the target is irreversibly fixed to the bait to avoid the losses due to dissociation kinetics. At that level, over 1 000 000 different proteins can be seen in human plasma. How many biomarkers do you want? Math warning: more equations than figures.Archakov, A. et al., Proteomics 2009, 9, 1326‐1343.Unexplored territory: a catfish pathogen's proteomeAs genomic and proteomic tools become more powerful and cheaper per base or peptide, we can expect to see more papers like this one by Dumpala et al., focused on an organism of modest economic value. Each paper will, however, contribute a new niche with alternative adaptations for survival. In this case, we are introduced to Edwardsiella ictaluri, a Gram negative pathogen of farm‐raised channel catfish. Enteric septicemia of catfish is the most frequent disease of the commercially farmed catfish and appears in acute and chronic forms. For the work reported here, the bacteria were grown in culture, washed, lysed and separated by 2‐DE TOF/TOF or 2‐D LC‐MS/MS for peptide identification. The combined methods identified 788 unique proteins, including 73 ribosomal proteins, several protein synthesis factors, tRNA synthases and a number of other proteins that could be assigned by orthology to Escherichia coli or Edwardsiella tarda.Dumpala, P. R. et al., Proteomics 2009, 9, 1353‐1363.

The regulation of disassembly of alphavirus cores

Alphaviruses are used as model viruses for structure determination and for analysis of virus entry. They are used also as vectors for protein expression and gene therapy. Virus particles are assembled by budding, using preformed cores and a modified cellular membrane. During entry, alphaviruses release the viral core into the cytoplasm. Cores are disassembled during virus entry and accumulate in the cytoplasm during virus multiplication. The regulation of core disassembly is the subject of this review. A working model compatible with all experimental data is formulated. This model comprises the following steps: (1) The incoming core is present in the cytoplasm in a metastable state, primed for disassembly. A core structure containing the so-called linker region of the core protein in an exposed position susceptible to proteolytic cleavage on the core surface might represent the primed state. (2) The primed core allows access of cellular proteins to the viral genome RNA, e.g. initiation factors of protein synthesis. (3) In a following step, ribosomal 60S subunits bind to the complex and lead to core disassembly with a concomitant transfer of core protein or of core protein fragments to the 28S rRNA. The linker region may be involved in this transfer. (4) During the later stages of virus multiplication, cellular components involved in step (2) and/or in step (3) are inactivated. This inactivation might involve the binding of newly synthesised core protein to 28S rRNA. (5) Unprimed cores, e.g. core particles containing the linker region in an unexposed position, are assembled during virus multiplication. Priming of cores and inactivation of host-cell factors each represent a complete mechanism of regulation of core disassembly. Future experiments will show whether or not both processes are actually used. Since alphaviruses, e.g. Chikungunya virus, Ross River virus, Semliki Forest virus, and Sindbis virus, are human pathogens, these experiments are of practical relevance, since they might identify targets for antiviral chemotherapy.

The battle to keep or lose skeletal muscle with ageing

Ageing is associated with both a loss in muscle mass and a loss in skeletal muscle metabolic quality. These changes lead to sarcopenia and subsequent reduced daily function, and increase the risk for fall and fractures. Also, the metabolic deficits result in an increased risk for insulin resistance, obesity and type 2 diabetes. A major part – but not all – of these age-associated changes are related to a decreased level of physical activity, and thus this age-associated loss can be partly counteracted by increased physical activity. Resistance training in particular has been documented to have beneficial effects upon muscle mass and strength also in very old untrained people (Fiatarone et al. 1990; Welle et al. 1993; Sheffield-Moore et al. 2005). How much and how intense should elderly people perform strength training in order to counteract their age-associated loss in skeletal muscle mass remains a challenge, and only few studies have addressed this issue. In this issue of The Journal of Physiology, the Rennie group (Kumar et al. 2009) document that older compared to young individuals demonstrate a blunted rise in myofibrillar protein synthesis after exercise, and that this was associated with a lower response in the phosphorylation of two of the post-translational modification factors, p70 ribosomal S6 kinase (p70s6K) and eukaryotic initiation factor 4E binding protein 1 (4EBP1). The correlation between protein synthesis and regulatory factors adds to our present knowledge, but clearly still represents associations, and the statement that p70s6K predicts rate of myofibrillar synthesis rate in young but not in old is somewhat optimistic. It does, however, support the view that elderly demonstrate a certain ‘anabolic resistance’, a finding that earlier has been supported by the same research group in relation to nutritional intake, where elderly demonstrated less stimulation of myofibrillar protein synthesis than younger counterparts when challenged with increasing circulating levels of amino acids (Cuthbertson et al. 2005).

Vegetarian Diets in Children and Adolescents

1. Meredith Renda, MD* 2. Philip Fischer, MD* 1. *Department of Pediatrics, Mayo Clinic, Rochester, Minn Vegetarianism is becoming more common among adults, with 1 in 40 adults currently choosing a vegetarian diet. Consequently, more children are raised as vegetarians. Vegetarianism is adopted for various reasons, including moral, religious, and health. Numerous studies have shown significant health benefits for individuals following this type of diet. Pediatricians should be well informed about vegetarianism and its role in our pediatric population. A first-time mother and her 2-month-old boy present to the clinic for a health supervision visit. The mother is breastfeeding, and her baby is growing appropriately. At the end of the visit, the mother mentions that she is a vegetarian and that she would like to raise her son in the same way. She would like to know what she should be doing as a breastfeeding mother and how she can make sure that her son has the appropriate nutrition in the future . The physician should begin by taking a dietary history from the mother to determine what type of vegetarianism she practices. He or she should explain to the mother that her infant's nutrition is based on her own dietary intake for as long as she continues to breastfeed. The pediatrician should counsel the mother on the importance of diet variety. If the mother is vegan, the pediatrician must take time to ensure that she is getting enough vitamin B12 and calcium in her diet. When she is ready to wean her infant, she should continue to focus on dietary variety. The mother should be cautioned that adult vegetarian diets that are high in bulk and low in calories are not always appropriate for the growing and developing child. Finally, the pediatrician should ensure that the mother has the appropriate resources to guide her in finding the best foods for her child's nutritional needs. …

The Isolation of the Vitamin B12 Coenzyme and the Role of the Vitamin in Methionine Synthesis

The invitation to contribute a “Reflections” article has brought back many memories as I recall the events early in my career that were most significant in determining the path I have taken for more than 50 years. I could reflect on several periods in my career, such as the early work on serotonin biosynthesis and metabolism or later studies on the role of GTP and elongation factors in protein synthesis, but the period when I worked on vitamin B12 in the late 1950s and early 1960s especially stands out in my mind, and I will focus mainly on these studies.

Regulation of the eIF2/eIF2B system during alcohol‐induced inhibition of hepatic protein synthesis in rats

Alcohol abuse damages hepatic structure and function. The effect of alcohol ingestion on protein synthesis and initiation factors eIF2 and eIF2B in rat liver was studied 1) after acute and chronic ethanol, 2) in males and females after chronic alcohol intake, and 3) after acute alcohol withdrawal. Protein synthesis was measured by incorporation of [3H]‐phenylalanine into liver protein and eIF2/eIF2B content and phosphorylation were determined by immunoblot. Protein synthesis was inhibited 37% after acute and 30% after chronic (>8 wk) ethanol and was associated with 65% increase in eIF2α phosphorylation in either case. eIF2 content decreased in rats fed alcohol for >12 weeks. Basal protein synthesis was 34% higher in females than in males and alcohol‐induced inhibition was greater in females (34%) than in males (19%). Withdrawal of alcohol reversed eIF2α phosphorylation but not the eIF2 deficit so hepatic protein synthesis was partially restored. Acute alcohol administration did not affect eIF2B content or eIF2Bε phosphorylation in male rats whereas chronic alcohol feeding revealed sex‐differential changes in both. eIF2B content in livers from males increased in the alcohol group but decreased in females; eIF2Bε phosphorylation decreased in males and increased in females. Changes in hepatic protein synthesis induced by alcohol consumption reflect underlying alterations in eIF2/eIF2B. NIH R21AA015349

Protein Synthesis and Neurotrophin-Dependent Structural Plasticity of Single Dendritic Spines

Long-term potentiation (LTP) at glutamatergic synapses is considered to underlie learning and memory and is associated with the enlargement of dendritic spines. Because the consolidation of memory and LTP require protein synthesis, it is important to clarify how protein synthesis affects spine enlargement. In rat brain slices, the repetitive pairing of postsynaptic spikes and two-photon uncaging of glutamate at single spines (a spike-timing protocol) produced both immediate and gradual phases of spine enlargement in CA1 pyramidal neurons. The gradual enlargement was strongly dependent on protein synthesis and brain-derived neurotrophic factor (BDNF) action, often associated with spine twitching, and was induced specifically at the spines that were immediately enlarged by the synaptic stimulation. Thus, this spike-timing protocol is an efficient trigger for BDNF secretion and induces protein synthesis-dependent long-term enlargement at the level of single spines.

Study of protein targets for covalent modification by the antitumoral and anti‐inflammatory prostaglandin PGA1: focus on vimentin

Prostaglandins with cyclopentenone structure (cyPG) display potent antiproliferative actions that have elicited their study as potential anticancer agents. Several natural and synthetic analogs of the cyPG prostaglandin A(1) (PGA(1)) have proven antitumoral efficacy in cancer cell lines and animal models. In addition, PGA(1) has been used as an inhibitor of transcription factor NF-kappaB-mediated processes, including inflammatory gene expression and viral replication. An important determinant for these effects is the ability of cyPG to form Michael adducts with free thiol groups. The chemical nature of this interaction implies that PGA(1) could covalently modify cysteine residues in a large number of cellular proteins potentially involved in its beneficial effects. However, only a few targets of PGA(1) have been identified. In previous work, we have observed that a biotinylated analog of PGA(1) that retains the cyclopentenone moiety (PGA(1)-B) binds to multiple targets in fibroblasts. Here, we have addressed the identification of these targets through a proteomic approach. Cell fractionation followed by avidin affinity chromatography yielded a fraction enriched in proteins modified by PGA(1)-B. Analysis of this fraction by SDS-PAGE and LC-MS/MS allowed the identification of the chaperone Hsp90, elongation and initiation factors for protein synthesis and cytoskeletal proteins including actin, tubulin and vimentin. Furthermore, we have characterized the modification of vimentin both in vitro and in intact cells. Our observations indicate that cysteine 328 is the main site for PGA(1) addition. These results may contribute to a better understanding of the mechanism of action of PGA(1) and the potential of cyPG-based therapeutic strategies.

Elongation factors in protein synthesis.

Recent discoveries of elongation factor-related proteins have considerably complicated the simple textbook scheme of the peptide chain elongation cycle. During growth and differentiation the cycle may be regulated not only by factor modification but also factor replacement. In addition, rare tRNAs may have their own rare factor proteins. A special case is the acquisition of resistance by bacteria to elongation factor-directed antibiotics. Pertinent data from the literature and our own work with Escherichia coli and Streptomyces are discussed. The GTP-binding domain of EF-Tu has been studied extensively, but little molecular detail is available on the interactions with its other ligands or effectors, or on the way they are affected by the GTPase switch signal. A growing number of EF-Tu mutants obtained by ourselves and others are helping us in testing current ideas. We have found a synergistic effect between EF-Tu and EF-G in their uncoupled GTPase reactions on empty ribosomes. Only the EF-G reaction is perturbed by fluoroaluminates.

Persistence of Long-Term Memory Storage Requires a Late Protein Synthesis- and BDNF- Dependent Phase in the Hippocampus

Persistence is the most characteristic attribute of long-term memory (LTM). To understand LTM, we must understand how memory traces persist over time despite the short-lived nature and rapid turnover of their molecular substrates. It is widely accepted that LTM formation is dependent upon hippocampal de novo protein synthesis and Brain-Derived Neurotrophic Factor (BDNF) signaling during or early after acquisition. Here we show that 12 hr after acquisition of a one-trial associative learning task, there is a novel protein synthesis and BDNF-dependent phase in the rat hippocampus that is critical for the persistence of LTM storage. Our findings indicate that a delayed stabilization phase is specifically required for maintenance, but not formation, of the memory trace. We propose that memory formation and memory persistence share some of the same molecular mechanisms and that recurrent rounds of consolidation-like events take place in the hippocampus for maintenance of the memory trace.