Protein Synthesis Initiation Factor Research Articles

Structural Dynamics of Bacterial Translation Initiation Factor IF2

Bacterial translation initiation factor IF2 promotes ribosomal subunit association, recruitment, and binding of fMet-tRNA to the ribosomal P-site and initiation dipeptide formation. Here, we present the solution structures of GDP-bound and apo-IF2-G2 of Bacillus stearothermophilus and provide evidence that this isolated domain binds the 50 S ribosomal subunit and hydrolyzes GTP. Differences between the free and GDP-bound structures of IF2-G2 suggest that domain reorganization within the G2-G3-C1 regions underlies the different structural requirements of IF2 during the initiation process. However, these structural signals are unlikely forwarded from IF2-G2 to the C-terminal fMet-tRNA binding domain (IF2-C2) because the connected IF2-C1 and IF2-C2 modules show completely independent mobility, indicating that the bacterial interdomain connector lacks the rigidity that was found in the archaeal IF2 homolog aIF5B.

Abstract P180: Atrogin-1, a Muscle-Specific Ubiquitin Ligase, Drives Atrophic Remodeling of the Heart

The heart adapts to changes in load by remodeling both metabolically and structurally. During this process, cardiomyocytes break down unnecessary or damaged proteins and use the resulting amino acids for the synthesis of new proteins and/or energy provision. Protein degradation via the ubiquitin proteasome system is controlled by ubiquitin ligases, which determine the specific proteins to be degraded. Atrogin-1, a muscle specific ubiquitin ligase, is required for skeletal muscle atrophy, and over-expressing Atrogin-1 inhibits the development of cardiac hypertrophy. We now tested the hypothesis that Atrogin-1 is required for atrophic remodeling of the unloaded heart. Hearts from wild type (WT) and Atrogin-1 -/- mice (8-10 weeks old, n =8-12) were subjected to mechanical unloading by heterotopic transplantation. In WT hearts, seven days of unloading significantly reduced heart weight and myocyte cross-sectional area, while hearts lacking Atrogin-1 significantly hypertrophied (at least a 1.5-fold increase in heart weight, 2-fold increase in myocyte area). Conventional markers of atrophic remodeling, such as the reactivation of the fetal gene program (ANF, MHC isoform switch), were detected in both WT and Atrogin-1 -/- transplanted hearts. Proteasome activity and markers of autophagy were increased after unloading, although not significantly different between WT and Atrogin-1 -/- hearts. Pathways regulating protein synthesis were enhanced in the absence of Atrogin-1; there was an increase in activated Akt and its downstream pathway including mTOR, 4E-BP1, and p70 S6 kinase. Additionally, two known targets of Atrogin-1 involved in hypertrophy and protein synthesis, calcineurin and eukaryotic initiation factor 3f, were upregulated in unloaded Atrogin-1 deficient hearts. Consequently, “unloaded” cardiomyocytes lacking Atrogin-1 in vitro exhibit increased basal rates of protein synthesis. The results suggest that Atrogin-1 not only enhances protein degradation, but also keeps protein synthesis in check. Thus Atrogin-1 has a duel role in regulating cardiac mass.

The Alpha Subunit of Eukaryotic Initiation Factor 2B (eIF2B) Is Required for eIF2-Mediated Translational Suppression of Vesicular Stomatitis Virus

Eukaryotic translation initiation factor 2B (eIF2B) is a heteropentameric guanine nucleotide exchange factor that converts protein synthesis initiation factor 2 (eIF2) from a GDP-bound form to the active eIF2-GTP complex. Cellular stress can repress translation initiation by activating kinases capable of phosphorylating the alpha subunit of eIF2 (eIF2α), which sequesters eIF2B to prevent exchange activity. Previously, we demonstrated that tumor cells are sensitive to viral replication, possibly due to the occurrence of defects in eIF2B that overcome the inhibitory effects of eIF2α phosphorylation. To extend this analysis, we have investigated the importance of eIF2Bα function and report that this subunit can functionally substitute for its counterpart, GCN3, in yeast. In addition, a variant of mammalian eIF2Bα harboring a point mutation (T41A) was able overcome translational inhibition invoked by amino acid depravation, which activates Saccharomyces cerevisiae GCN2 to phosphorylate the yeast eIF2α homolog SUI2. Significantly, we also demonstrate that the loss of eIF2Bα, or the expression of the T41A variant in mammalian cells, is sufficient to neutralize the consequences of eIF2α phosphorylation and render normal cells susceptible to virus infection. Our data emphasize the importance of eIF2Bα in mediating the eIF2 kinase translation-inhibitory activity and may provide insight into the complex nature of viral oncolysis.

Leucine Supplementation of a Low-Protein Meal Increases Skeletal Muscle and Visceral Tissue Protein Synthesis in Neonatal Pigs by Stimulating mTOR-Dependent Translation Initiation ,

Protein synthesis and eukaryotic initiation factor (eIF) activation are increased in skeletal muscle of neonatal pigs parenterally infused with amino acids. Leucine appears to be the most effective single amino acid to trigger these effects. To examine the response to enteral leucine supplementation, overnight food-deprived 5-d-old pigs were gavage fed at 0 and 60 min a: 1) low-protein diet (LP); 2) LP supplemented with leucine (LP+L) to equal leucine in the high-protein diet (HP); or 3) HP diet. Diets were isocaloric and equal in lactose. Fractional protein synthesis rates and translation initiation control mechanisms were examined in skeletal muscles and visceral tissues 90 min after feeding. Protein synthesis rates in longissimus dorsi, gastrocnemius, and masseter muscles, heart, jejunum, kidney, and pancreas, but not liver, were greater in the LP+L group compared with the LP group and did not differ from the HP group. Feeding LP+L and HP diets compared with the LP diet increased phosphorylation of mammalian target of rapamycin (mTOR), 4E-binding protein 1, ribosomal protein S6 kinase-1, and eIF4G and formation of the active eIF4E·eIF4G complex in longissimus dorsi muscle. In all tissues except liver, activation of mTOR effectors increased in pigs fed LP+L and HP vs. LP diets. Our results suggest that leucine supplementation of a low-protein meal stimulates protein synthesis in muscle and most visceral tissues to a rate similar to that achieved by feeding a high-protein meal and this stimulation involves activation of mTOR downstream effectors.

Leucine and α-Ketoisocaproic Acid, but Not Norleucine, Stimulate Skeletal Muscle Protein Synthesis in Neonatal Pigs , ,

The branched-chain amino acid, leucine, acts as a nutrient signal to stimulate protein synthesis in skeletal muscle of young pigs. However, the chemical structure responsible for this effect has not been identified. We have shown that the other branched-chain amino acids, isoleucine and valine, are not able to stimulate protein synthesis when raised in plasma to levels within the postprandial range. In this study, we evaluated the effect of leucine, α-ketoisocaproic acid (KIC), and norleucine infusion (0 or 400 μmol·kg−1·h−1 for 60 min) on protein synthesis and activation of translation initiation factors in piglets. Infusion of leucine, KIC, and norleucine raised plasma levels of each compound compared with controls. KIC also increased (P < 0.01) and norleucine reduced (P < 0.02) plasma levels of leucine compared with controls. Administration of leucine and KIC resulted in greater (P < 0.006) phosphorylation of eukaryotic initiation factor (eIF) 4E binding protein-1 (4E-BP1) and eIF4G, lower (P < 0.04) abundance of the inactive 4E-BP1·eIF4E complex, and greater (P < 0.05) active eIF4G·eIF4E complex formation in skeletal muscle compared with controls. Protein synthesis in skeletal muscle was greater (P < 0.02) in leucine- and KIC-infused pigs than in those in the control group. Norleucine infusion did not affect muscle protein synthesis or translation initiation factor activation. In liver, neither protein synthesis nor activation of translation initiation factors was affected by treatment. These results suggest that the ability of leucine to act as a nutrient signal to stimulate skeletal muscle protein synthesis is specific for leucine and/or its metabolite, KIC.

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&lt; 0.0001). There was a significant increase in Akt P‐Ser473 (P&lt; 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 &gt; 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.

Tipping the balance: antagonism of PKR kinase and ADAR1 deaminase functions by virus gene products.

The protein kinase regulated by RNA (PKR) and the adenosine deaminase acting on RNA (ADAR1) are interferon-inducible enzymes that play important roles in biologic processes including the antiviral actions of interferons, signal transduction, and apoptosis. PKR catalyzes the RNA-dependent phosphorylation of protein synthesis initiation factor eIF-2 alpha, thereby leading to altered translational patterns in interferon-treated and virus-infected cells. PKR also modulates signal transduction responses, including the induction of interferon. ADAR1 catalyzes the deamination of adenosine (A) to generate inosine (I) in RNAs with double-stranded character. Because I is recognized as G instead of A, A-to-I editing by ADAR1 can lead to genetic recoding and altered RNA structures. The importance of PKR and ADAR1 in innate antiviral immunity is illustrated by a number of viruses that encode either RNA or protein viral gene products that antagonize PKR and ADAR1 enzymatic activity, localization, or stability.

Acute Effects of Enteral Leucine Supplementation of a Low Protein Diet on Muscle Protein Synthesis in Neonatal Pigs

Protein synthesis and eukaryotic initiation factor (eIF) activation are increased in skeletal muscle of neonatal pigs parenterally infused with insulin and amino acids (AA), particularly leucine. We hypothesized that enteral Leu supplementation of a low protein diets in neonatal pigs will acutely increase muscle protein synthesis to a rate similar to that achieved by feeding a high protein (HP) diet. Fasted 5‐d‐old piglets (n=12) were gavage fed at 0 and 60 min either: 1) low protein diet (LP), 2) low protein diet supplemented with Leu (LP+L) to equal HP diet, or 3) HP diet. Diets were isocaloric and lactose was equal. Fractional protein synthesis rates and translational initiation control mechanisms were examined 90 min after feeding. Muscle protein synthesis increased in the LP+L group compared to the LP group alone and did not differ from the HP group. LP+L and HP increased the phosphorylation of S6K1, eIF4G, and 4E binding protein‐1 (4E‐BP1), decreased inactive 4E‐BP1▸eIF4E complex abundance, and increased active eIF4E▸eIF4G complex formation in muscle. There were no difference between groups in PKB phosphorylation. Our results suggest that low protein diets supplemented with leucine stimulates muscle protein synthesis to a rate similar to that achieved by high protein diets and this stimulation involves activation of mTOR downstream effectors. (Supported by Ajinomoto Amino Acid Research Program and USDA 58‐6250‐6‐001).

Effect of circulating insulin concentration on whole body and muscle protein metabolism in mature thoroughbred mares

The role of insulin (INS) in carbohydrate metabolism and the development of certain metabolic diseases has been well studied in horsse; however, there has been minimal research with regards to the role that INS plays in equine protein metabolism. The objective of this study was to determine the effect of circulating INS concentration on whole‐body protein metabolism and the activation of translation initiation factors in muscle. Mature thoroughbred mares (n=8) underwent euglycemic clamp procedures at each of 4 different rates of INS infusion: 0, 1.2, 3 and 6 mU/kg/min. Blood samples were taken during the clamp and at the end of the clamp a biopsy from the gluteal muscle was taken. Western blotting was used to examine the phosphorylation of protein synthesis initiation factors Akt, rpS6 and 4E‐BP1 in the gluteal muscle, and HPLC was used to determine plasma and muscle free amino acid concentrations. INS infusion resulted in a decrease (P &lt; 0.05) in the plasma concentration of all indispensable amino acids, with greater decreases seen with increasing rates of INS infusion. Muscle free valine, leucine and isoleucine concentrations were also lower (P &lt; 0.05) following INS infusion, in a dose dependent manner. Akt phosphorylation was maximized at 6 mU/kg/min INS, while rpS6 and 4E‐BP1 phosphorylation was maximized at 3 mU/kg/min INS. INS infusion appeared to promote both whole‐body amino acid utilization and activation of translation initiation factors in muscle of mature horses. Future research should focus on the effects of altered INS sensitivity on equine protein metabolism. Funded by estate of the late Paul Mellon.

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.

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.

Loss of Protein Kinase PKR Expression in Human HeLa Cells Complements the Vaccinia Virus E3L Deletion Mutant Phenotype by Restoration of Viral Protein Synthesis

The E3L proteins encoded by vaccinia virus bind double-stranded RNA and mediate interferon resistance, promote virus growth, and impair virus-mediated apoptosis. Among the cellular proteins implicated as targets of E3L is the protein kinase regulated by RNA (PKR). To test in human cells the role of PKR in conferring the E3L mutant phenotype, HeLa cells stably deficient in PKR generated by an RNA interference-silencing strategy were compared to parental and control knockdown cells following infection with either an E3L deletion mutant (DeltaE3L) or wild-type (WT) virus. The growth yields of WT virus were comparable in PKR-sufficient and -deficient cells. By contrast, the single-cycle yield of DeltaE3L virus was increased by nearly 2 log(10) in PKR-deficient cells over the impaired growth in PKR-sufficient cells. Furthermore, virus-induced apoptosis characteristic of the DeltaE3L mutant in PKR-sufficient cells was effectively abolished in PKR-deficient HeLa cells. The viral protein synthesis pattern was altered in DeltaE3L-infected PKR-sufficient cells, characterized by an inhibition of late viral protein expression, whereas in PKR-deficient cells, late protein accumulation was restored. Phosphorylation of both PKR and the alpha subunit of protein synthesis initiation factor 2 (eIF-2alpha) was elevated severalfold in DeltaE3L-infected PKR-sufficient, but not PKR-deficient, cells. WT virus did not significantly increase PKR or eIF-2alpha phosphorylation in either PKR-sufficient or -deficient cells, both of which supported efficient WT viral protein production. Finally, apoptosis induced by infection of PKR-sufficient HeLa cells with DeltaE3L virus was blocked by a caspase antagonist, but mutant virus growth was not rescued, suggesting that translation inhibition rather than apoptosis activation is a principal factor limiting virus growth.

On the discovery of interferon-inducible, double-stranded RNA activated enzymes: The 2′–5′oligoadenylate synthetases and the protein kinase PKR

The demonstration that double-stranded (ds) RNA inhibits protein synthesis in cell-free systems prepared from interferon-treated cells, lead to the discovery of the two interferon-induced, dsRNA-dependent enzymes: the serine/threonine protein kinase that is referred to as PKR and the 2′,5′-oligoadenylate synthetase (2′,5′-OAS), which converts ATP to 2′,5′-linked oligoadenylates with the unusual 2′-5′ instead of 3′-5′ phosphodiesterase bond. We raised monoclonal and polyclonal antibodies against human PKR and the two larger forms of the 2′,5′-OAS. Such specific antibodies proved to be indispensable for the detailed characterization of these enzyme and the cloning of cDNAs corresponding to the human PKR and the 69–71 and 100 kDa forms of the 2′,5′-OAS. When activated by dsRNA, PKR becomes autophosphorylated and catalyzes phosphorylation of the protein synthesis initiation factor eIF2, whereas the 2′-5′OAS forms 2′,5′-oligoadenylates that activate the latent endoribonuclease, the RNAse L. By inhibiting initiation of protein synthesis or by degrading RNA, these enzymes play key roles in two independent pathways that regulate overall protein synthesis and the mechanism of the antiviral action of interferon. In addition, these enzymes are now shown to regulate other cellular events, such as gene induction, normal control of cell growth, differentiation and apoptosis.

Involvement of ERKs, RSK2 and PKR in UVA-induced signal transduction toward phosphorylation of eIF2α (Ser51)

Double-stranded RNA-dependent protein kinase R (PKR) has been implicated in anti-viral (antitumor) and apoptotic responses. PKR is activated by extracellular stresses and phosphorylates the alpha subunit of protein synthesis initiation factor eIF2, thereby inhibiting protein synthesis and impeding virus multiplication. Phosphorylation of eIF2alpha in mammalian cells has been shown to be increased after ultraviolet (UV) stress and to be required for UV-induced repression of protein translation. UVA is an important etiological factor in skin carcinogenesis and we observed that UVA induced phosphorylation of PKR (Thr(451)) and eIF2alpha (Ser(51)) in mouse skin epidermal JB6 Cl41 cells. The induction was suppressed by the MEK1 inhibitor, PD 98059. UVA stimulation of PKR and eIF2alpha phosphorylation was also inhibited by a dominant-negative mutant (DNM) of ERK2- or RSK2-deficient cells (RSK2(-)). An inhibitor of p38, SB 202190 or a DNM of p38alpha kinase (DNM-p38alpha) suppressed UVA-induced phosphorylation of eIF2alpha (Ser(51)) but had no effect on phosphorylation of PKR (Thr(451)). Our data indicated that phosphorylation of PKR at Thr(451) is mediated through ERK2 and RSK2, but not through p38 kinase, and is involved in the regulation of Ser(51) phosphorylation of eIF2alpha in UVA-irradiated JB6 cells. In vitro and in vivo kinase assays indicated that phosphorylation of eIF2alpha at Ser(51) occurred indirectly through ERK2, RSK2 or p38 kinase in the cellular response to UVA. These data may lead to the use of these signaling molecules as targets to develop more effective chemopreventive agents with fewer side effects to control UV-induced skin cancer.

Molecular Cloning, Mapping, and Expression Analysis of the EIF4A2 Gene in Pig

A full-length cDNA clone encoding the eukaryotic translation initiation factor 4A, isoform 2 (EIF4A2), was cloned from the fetal skeletal cDNA library from the pig (Sus scrofa). EIF4A2 is a highly conserved gene for one of the protein-synthesis initiation factors involved in the binding of mRNA to the ribosome. Based on this cDNA sequence, the deduced protein of 407 amino acids contains the characteristic motifs shared by the DEAD-box supergene family. The genomic nucleotide sequence of this gene was determined and a single nucleotide polymorphism located in the 5' untranslated region was genotyped. The porcine EIF4A2 was expressed in all tissues examined but in variable amounts. The EIF4A2 expression level in muscle was upregulated through embryonic and neonatal development until adult, suggesting that porcine EIF4A2 was possibly involved in translation regulation of other muscle-related genes in muscle formation and development. In addition, we mapped porcine EIF4A2 to q4.1 of SSC13, in agreement with comparative mapping data.

How Initiation Factors Maximize the Accuracy of tRNA Selection in Initiation of Bacterial Protein Synthesis

During initiation of bacterial protein synthesis, messenger RNA and fMet-tRNAfMet bind to the 30S ribosomal subunit together with initiation factors IF1, IF2, and IF3. Docking of the 30S preinitiation complex to the 50S ribosomal subunit results in a peptidyl-transfer competent 70S ribosome. Initiation with an elongator tRNA may lead to frameshift and an aberrant N-terminal sequence in the nascent protein. We show how the occurrence of initiation errors is minimized by (1) recognition of the formyl group by the synergistic action of IF2 and IF1, (2) uniform destabilization of the binding of all tRNAs to the 30S subunit by IF3, and (3) an optimal distance between the Shine-Dalgarno sequence and the initiator codon. We suggest why IF1 is essential for E. coli, discuss the role of the G-C base pairs in the anticodon stem of some tRNAs, and clarify gene expression changes with varying IF3 concentration in the living cell.

Irreversible aggregation of protein synthesis machinery after focal brain ischemia

Focal brain ischemia leads to a slow type of neuronal death in the penumbra that starts several hours after ischemia and continues to mature for days. During this maturation period, blood flow, cellular ATP and ionic homeostasis are gradually recovered in the penumbral region. In striking contrast, protein synthesis is irreversibly inhibited. This study used a rat focal brain ischemia model to investigate whether or not irreversible translational inhibition is due to abnormal aggregation of translational complex components, i.e. the ribosomes and their associated nascent polypeptides, protein synthesis initiation factors and co-translational chaperones. Under electron microscopy, most rosette-shaped polyribosomes were relatively evenly distributed in the cytoplasm of sham-operated control neurons, but clumped into large abnormal aggregates in penumbral neurons subjected to 2 h of focal ischemia followed by 4 h of reperfusion. The abnormal ribosomal protein aggregation lasted until the onset of delayed neuronal death at 24-48 h of reperfusion after ischemia. Biochemical study further suggested that translational complex components, including small ribosomal subunit protein 6 (S6), large subunit protein 28 (L28), eukaryotic initiation factors 2alpha, 4E and 3eta, and co-translational chaperone heat-shock cognate protein 70 (HSC70) and co-chaperone Hdj1, were all irreversibly clumped into large abnormal protein aggregates after ischemia. Translational complex components were also highly ubiquitinated. This study clearly demonstrates that focal ischemia leads to irreversible aggregation of protein synthesis machinery that contributes to neuronal death after focal brain ischemia.

Effect of the leucine analogs, α‐ketoisocaproic acid (KIC) and norleucine, on muscle protein synthesis and translation initiation factor activation in neonatal pigs

Leucine acts as a nutrient signal to stimulate protein synthesis in skeletal muscle of neonatal pigs; however, the chemical structure responsible for this effect has not been identified. We have shown that isoleucine and valine are not able to stimulate protein synthesis when raised in plasma within the physiological postprandial range. The current study evaluated the effect of leucine, KIC and norleucine infusion (0 or 400 μmol·kg−1·h−1 for 60 min) on protein synthesis and activation of translation initiation factors. Infusion of leucine, KIC and norleucine raised plasma levels of each compound compared to controls. In addition, KIC increased (P < 0.01) and norleucine reduced (P < 0.02) plasma levels of leucine compared to controls. Administration of leucine and KIC increased the phosphorylation of eukaryotic initiation factor (eIF) 4E binding protein-1 (4E-BP1, P < 0.01) and decreased the inactive 4E BP1·eIF4E complex in longissimus dorsi muscle. Protein synthesis was increased (P < 0.02) in muscle with infusion of leucine and KIC. Norleucine infusion did not affect muscle protein synthesis or translation initiation factor activation. In liver, neither protein synthesis nor activation of translation initiation factors was affected by any treatment. Results suggest that the ability of leucine to act as a nutrient signal to stimulate protein synthesis is likely specific for leucine or its metabolites. (NIH AR 44474 and USDA 58-6250-6-001)

Dietary protein and lactose increase translation initiation factor activation and tissue protein synthesis in neonatal pigs

Protein synthesis and eukaryotic initiation factor (eIF) activation are increased in muscle and liver of pigs parenterally infused with amino acids and insulin. To examine the effects of enteral protein and carbohydrate on protein synthesis, pigs (n = 42, 1.7 kg body wt) were fed isocaloric milk diets containing three levels of protein (5, 15, and 25 g x kg body wt(-1) x day(-1)) and two levels of lactose (low = 11 and high = 23 g x kg body wt(-1) x day(-1)) from 1 to 6 days of age. On day 7, pigs were gavage fed after 4-h food deprivation, and tissue protein synthesis rates and biomarkers of mRNA translation were assessed. Piglet growth and protein synthesis rates in muscle and liver increased with dietary protein and plateaued at 15 g x kg body wt(-1) x day(-1) (P < 0.001). Growth tended to be greater in high-lactose-fed pigs (P = 0.07). Plasma insulin was lowest in pigs fed 5 g x kg body wt(-1) x day(-1) protein (P < 0.0001). Plasma branched-chain amino acids increased as protein intake increased (P < 0.0001). Muscle (P < 0.001) and liver (P < or = 0.001) ribosomal protein S6 kinase-1 and eIF4E-binding protein phosphorylation increased with protein intake and plateaued at 15 g x kg body wt(-1) x day(-1). The results indicate that growth and protein synthesis rates in neonatal pigs are influenced by dietary protein and lactose intake and might be mediated by plasma amino acids and insulin levels. However, feeding protein well above the piglet's requirement does not further stimulate the activation of translation initiation or protein synthesis in skeletal muscle and liver.

Modulation of Differentiation-related Gene 1 Expression by Cell Cycle Blocker Mimosine, Revealed by Proteomic Analysis

L-mimosine, a plant amino acid, can reversibly block mammalian cells at late G1 phase and has been found to affect translation of mRNAs of the cyclin-dependent kinase inhibitor p27, eIF3a (eIF3 p170), and ribonucleotide reductase M2. The effect of mimosine on the expression of these genes may be essential for the G1 phase arrest. To determine additional genes that may be early respondents to the mimosine treatment, we performed two-dimensional gel electrophoretic analysis of [35S]methionine-labeled cell lysates followed by identification of the altered protein spots by LC-tandem mass spectrometry. In this study, the synthesis of two protein spots (MIP42 and MIP17) was found to be enhanced by mimosine, whereas the formation of another protein spot (MSP17) was severely blocked following mimosine treatment. These protein spots, MIP42, MIP17, and MSP17, were identified to be differentiation-related gene 1 (Drg-1; also called RTP, cap43, rit42, Ndrg-1, and PROXY-1), deoxyhypusine-containing eIF5A intermediate, and mature hypusine-containing eIF5A, respectively. The effect of mimosine on eIF5A maturation was due to inhibition of deoxyhypusine hydroxylase, the enzyme catalyzing the final step of hypusine biosynthesis in eIF5A. The mimosine-induced expression of Drg-1 was mainly attributable to increased transcription likely by the c-Jun/AP-1 transcription factor. Because induction of Drg-1 is an early event after mimosine treatment and is observed before a notable reduction in the steady-state level of mature eIF5A, eIF5A does not appear to be involved in the modulation of Drg-1 expression.