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The process which uses the cyclin-dependent kinase inhibitor Sic1 and its ubiquitination by the cullin-RING ubiquitin ligase SCF and the ubiquitin-conjugating enzyme Cdc34 is discussed. The results showed that Sic1 ubiquitination is separated into two steps which are attachment of the first ubiquitin which is a rate limiting, followed by rapid elongation of a K48-linked ubiquitin chain.

A subunit of RNA polymarase II, Rpb4p, is required for the decay of a class of mRNAs whose products are involved in protein synthesis. Rpb4p promotes the deadenylation process of specific mRNAs and recruits Pat1/Lsm1-7 to mRNAs, thus stimulating their decapping and further decay.

Study is conducted to show that, Sgo 1, a protein required for protecting centromeric cohesins from removal during meiosis I, localizes to cohesin-associated regions at the centromere and the 50-kb region surrounding it. It is shown that the 50-kb Sgo 1-binding domain is the chromosomal region where cohesions are protected from removal during meiosis I.

Recently synthesized proteins are sorted at the trans-Golgi network into specialized routes for exocytosis. Surprisingly little is known about the underlying molecular machinery. Here, we present a visual screen to search for proteins involved in cargo sorting and vesicle formation. We expressed a GFP-tagged plasma membrane protein in the yeast deletion library and identified mutants with altered marker localization. This screen revealed a requirement of several enzymes regulating the synthesis of sphingolipids and ergosterol in the correct and efficient delivery of the marker protein to the cell surface. Additionally, we identified mutants regulating the actin cytoskeleton (Rvs161p and Vrplp), known membrane traffic regulators (Kesl p and Chs5p), and several unknown genes. This visual screening method can now be used for different cargo proteins to search in a genome-wide fashion for machinery involved in post-Golgi sorting. exocytosis | lipid rafts | Saccharomyces cerevisiae | sorting | Golgi

Many newly synthesized proteins have to become unfolded during translocation across biological membranes. We have analyzed the effects of various stabilization/destabilization mutations in the lg-like module of the muscle protein titin upon its import from the N terminus or C terminus into mitochondria. The effects of mutations on the import of the titin module from the C terminus correlate well with those on forced mechanical unfolding in atomic-force microscopy (AFM) measurements. On the other hand, as long as turnover of the mitochondrial Hsp70 system is not rate-limiting for the import, import of the titin module from the N terminus is sensitive to mutations in the N-terminal region but not the ones in the C-terminal region that affect resistance to global unfolding in AFM experiments. We propose that the mitochondrial-import system can catalyze precursor-unfolding by reducing the stability of unfolding intermediates. Mitochondria | titin | Hsp70 | mechanical stability

Subcellular compartments have unique protein compositions, yet protein synthesis only occurs in the cytosol and in mitochondria and chloroplasts. How do proteins get where they need to go? The first steps are targeting to an organelle and efficient translocation across its limiting membrane. Given that most transport systems are exquisitely substrate specific, how are diverse protein sequences recognized for translocation? Are they translocated as linear polypeptide chains or after folding? During translocation, how are diverse amino acyl side chains accommodated? What are the proteins and the lipid environment that catalyze transport and couple it to energy? How is translocation coordinated with protein synthesis and folding, and how are partially translocated transmembrane proteins released into the lipid bilayer? We review here the marked progress of the past 35 years and salient questions for future work., Emerging technologies have enabled progress in understanding protein translocation. In vitro protein synthesis led to the discovery that the mRNAs for secreted proteins are attached to membranes, whereas cytosolic proteins [...]

Muscle protein synthesis requires energy and amino acids to proceed and can be stimulated by insulin under certain circumstances. We hypothesized that short-term provision of insulin and nutritional energy would stimulate muscle protein synthesis in healthy subjects only if amino acid availability did not decrease. Using stable isotope techniques, we compared the effects on muscle phenylalanine kinetics across the leg of an amino acid-lowering, high-energy (HE, n = 6, 162 [+ or -] 20 kcal/h) hyperglycemic hyperlipidemic hyperinsulinemic clamp with systemic insulin infusion to a low-energy (LE, n = 6, 35 [+ or -] 3 kcal/h, P < 0.05 vs. HE) euglycemic hyperinsulinemic clamp with local insulin infusion in the femoral artery. Basal blood phenylalanine concentrations and phenylalanine net balance, muscle protein breakdown, and synthesis (nmol * [min.sup.-1] * 100 g leg [muscle.sup.-1]) were not different between groups. During insulin infusion, femoral insulinemia increased to a similar extent between groups and blood phenylalanine concentration decreased 27 [+ or -] 3% in the HE group but only 9 [+ or -] 2% in the LE group (P < 0.01 HE vs. LE). Phenylalanine net balance increased in both groups, but the change was greater (P < 0.05) in the LE group. Muscle protein breakdown decreased in the HE group (58 [+ or -] 12 to 35 [+ or -] 7 nmol * [min.sup.-1] * 100 g leg [muscle.sup.-1]) and did not change in the LE group. Muscle protein synthesis was unchanged in the HE group (39 [+ or -] 6 to 30 [+ or -] 7 nmol * [min.sup.-1] 100 g leg [muscle.sup.-1]) and increased (P < 0.05) in the LE group (41 [+ or -] 9 to 114 [+ or -] 26 nmol * [min.sup.-1] * 100 g leg [muscle.sup.-1]). We conclude that amino acid availability is an important factor in the regulation of muscle protein synthesis in response to insulin, as decreased blood amino acid concentrations override the positive effect of insulin on muscle protein synthesis even if excess energy is provided.

Surveillance mechanisms that monitor protein synthesis can promote rapid elimination of misfolded nascent proteins. We showed that the translation elongation factor eEFIA and the proteasome subunit Rpt1 play a central role in the translocation of nascent-damaged proteins to the proteasome. We show here that multiubiquitinated proteins, and the ubiquitin-conjugating (E2) enzyme Ubc4, are rapidly detected in the proteasome following translational damage. However, Ubc4 levels in the proteasome were reduced significantly in a strain that expressed a mutant Rpt1 subunit. Ubc4 and Ubc5 are functionally redundant E2 enzymes that represent ideal candidates for ubiquitinating damaged nascent proteins because they lack significant substrate specificity, are required for the degradation of bulk, damaged proteins, and contribute to cellular stress-tolerance mechanisms. In agreement with this hypothesis, we determined that ubc4[DELTA] ubc5[DELTA] is exceedingly sensitive to protein translation inhibitors. Collectively, these studies suggest a specific role for Ubc4 and Ubc5 in the degradation of cotranslationally damaged proteins that are targeted to the proteasome.

The purpose of this study is to develop a dioxin detection method using a short peptide alternative to an immunoantibody. A full peptide library consisting of 2.5 million possible amino acid combinations was constructed by a solid-phase split synthesis approach using 19 natural amino acids. The peptide beads were subjected to a competitive binding assay between 2,3,7-trichlorodibenzo-p-dioxin and N-NBD-3-(3',4'-dichlorophenoxy)-1-propylamine (NBD-DCPPA) in a buffer containing 20% 1,4-dioxane. Two almost identical pentapeptides, FLDQI and FLDQV, that could bind dioxin were screened from the combinatorial library. NBD-DCPPA and the peptide synthesized on resin beads could be utilized to determine dioxin concentrations. The fluorescence intensity of the beads was measured using fluorescence microscopy to make a calibration curve for the dioxin concentrations. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TeCDD) could also detected in the presence of 30% 1,4-dioxane. To optimize the peptide sequence, a one-amino acid-substituted library was prepared using amino acids including nonnatural amino acids. The internal amino acids, LDQ, could not be substituted by any other amino acids. This result indicates that these three side chains are essential to recognize dioxins. The peptide C terminus substituted by phenylglycine showed a 10 times lower detection limit of 2,3,7,8-TeCDD of 150 pM (50 pg/mL) than the original sequence FLDQV. The cross reactivity of the dioxin binding peptides including the secondary derivatives was investigated. Some polycyclic aromatic hydrocarbons bound to the peptide beads, but nonchlorinated dibenzo-p-dioxin and PCB did not. From these results, we demonstrate the potential of short peptides as a practical sensor material targeting low molecular weight compounds such as dioxin.

Protein synthesis has long been known to be required for associative learning to consolidate into long-term memory. Here we demonstrate that PKC isozyme activation on days before training can induce the synthesis of proteins necessary and sufficient for subsequent long-term memory consolidation. Bryostatin (Bryo), a macrolide lactone with efficacy in subnanomolar concentrations and a potential therapeutic for Alzheimer's disease, is a potent activator of PKC, some of whose isozymes undergo prolonged activation after associative learning. Under normal conditions, two training events with paired visual and vestibular stimuli cause short-term memory of the mollusc Hermissenda that lasts [approximately equal to] 7 min. However, after 4-h exposures to Bryo (0.25 ng/ml) on two preceding days, the same two training events produced long-term conditioning that lasted >1 week and that was not blocked by anisomycin (1 [micro]g/ml). Anisomycin, however, eliminated long-term memory lasting at least 1 week after nine training events. Both the nine training events alone and two Bryo exposures plus two training event regimens caused comparably increased levels of the PKC [alpha]-isozyme substrate calexcitin in identified type B neurons and enhanced PKC activity in the membrane fractions. Furthermore, Bryo increased overall protein synthesis in cultured mammalian neurons by up to 60% for >3 days. The specific PKC antagonist Ro-32-0432 blocked much of this Bryo-induced protein synthesis as well as the Bryo-induced enhancement of the behavioral conditioning. Thus, Bryo-induced PKC activation produces those proteins necessary and sufficient for long-term memory on days in advance of the training events themselves. bryostatin | PKC isozymes

The role of topoisomerase IV (Topo IV) and of the structural maintenance of chromosomes (SMC) complex in chromosome compaction and in global protein synthesis was investigated. Lowering of the levels of Topo IV led to chromosome decondensation, while overproduction induced chromosome hyper-compaction, showing that Topo IV has an influence on the compaction of the whole chromosome, in a manner similar to that of the SMC protein, though different in mechanism. Increased synthesis of Topo IV in smc-deleted cells partially rescued the growth and condensation defect of the deletion, but not the segregation defect, revealing that the two systems interact at a genetic level. Two-dimensional gel investigations showed that global protein synthesis is highly aberrant in smc-deleted cells, and, to a different extent, also in cells lacking ScpA or ScpB, which form the SMC complex together with SMC protein. Overproduction of Topo IV partially rescued the defect in protein synthesis in smc mutant cells, indicating that Topo IV can restore the loss of negative super-coiling caused by the absence of SMC protein, but does not fully rescue the segregation defect. The data also show that the SMC protein has a dual function, in chromosome supercoiling and in active segregation.

Decoding is the key step during protein synthesis that enables information transfer from RNA to protein, a process critical for the survival of all organisms. We have used large-scale (2.64 x [10.sup.6] atoms) all-atom simulations of the entire ribosome to understand a critical step of decoding. Although the decoding problem has been studied for more than four decades, the rate-limiting step of cognate tRNA selection has only recently been identified. This step, known as accommodation, involves the movement inside the ribosome of the aminoacyl-tRNA from the partially bound 'A/T' state to the fully bound 'A/A' state. Here, we show that a corridor of 20 universally conserved ribosomal RNA bases interacts with the tRNA during the accommodation movement. Surprisingly, the tRNA is impeded by the A-loop (23S helix 92), instead of enjoying a smooth transition to the A/A state. In particular, universally conserved 23S ribosomal RNA bases U2492, C2556, and C2573 act as a 3D gate, causing the acceptor stem to pause before allowing entrance into the peptidyl transferase center. Our simulations demonstrate that the flexibility of the acceptor stem of the tRNA, in addition to flexibility of the anticodon arm, is essential for tRNA selection. This study serves as a template for simulating conformational changes in large (>[10.sup.6] atoms) biological and artificial molecular machines. proofreading | protein synthesis | molecular dynamics simulations RNA | high-performance computing

A study is undertaken to provide a complete model of factor Va that can be used for the investigation and understanding of its physiological functions. The model represents the foundation for the establishment of a complete prothrombinase complex model, which might be successful in describing accurately the ternary protein-protein interaction and thus accounts for experimental observations.

Cerulenin is an antibiotic that inhibits fatty acid synthesis by covalent modification of the active thiol of the chain-elongation subtypes of [beta]-ketoacyl-acyl carrier protein synthase. It also inhibits other processes that utilize essential thiols. Cerulenin has been widely reported to block protein secretion at sub-MIC levels, an effect that has been postulated to represent interference with membrane function through interference with normal fatty acid synthesis. This study confirms the profound reduction in extracellular proteins caused by low concentrations of the antibiotic, and shows by Northern blot hybridization that this reduction is due to interference with transcription. By exchanging promoters between entB, a gene that is inhibited by cerulenin, and entA, a gene that is not, it was also shown that the antibiotic does not block secretion. Subinhibitory concentrations of cerulenin were also found to block transcriptional activation of at least two regulatory determinants, agr and sae, that function by signal transduction. Interference with the activation of these and other regulatory determinants probably accounts for much of the inhibitory effect on exoprotein production of sub-MIC concentrations of cerulenin.

The sterol regulatory element-binding protein (SREBP) family of transcription factors controls the biosynthesis of cholesterol and other lipids, and lipid synthesis is critical for cell growth and proliferation. We were, therefore, interested in the expression and activity of SREBPs during the cell cycle. We found that the expression of a number of SREBP-responsive promoter-reporter genes were induced in a SREBP-dependent manner in cells arrested in [G.sub.2]/M. In addition, the mature forms of SREBP1a and SREBP1c were hyperphosphorylated in mitotic cells, giving rise to a phosphoepitope recognized by the mitotic protein monoclonal-2 (MPM-2) antibody. In contrast, SREBP2 was not hyperphosphorylated in mitotic cells and was not recognized by the MPM-2 antibody. The MPM-2 epitope was mapped to the C terminus of mature SREBP1, and the mitosis-specific hyperphosphorylation of SREBP1 depended on this domain of the protein. The transcriptional and DNA-binding activity of SREBP1 was enhanced in cells arrested in [G.sub.2]/M, and these effects depended on the C-terminal domain of the protein. In part, these effects could be explained by our observation that mature SREBP1 was stabilized in [G.sub.2]/M. In agreement with these observations, we found that the synthesis of cholesterol was increased in [G.sub.2]/M-arrested cells. Thus, our results demonstrate that the activity of mature SREBP1 is regulated by phosphorylation during the cell cycle, suggesting that SREBP1 may provide a link between lipid synthesis, proliferation, and cell growth. cell cycle | cholesterol | phosphorylation | proliferation

In eukaryotes, tRNAs transcribed in the nucleus function in cytoplasmic protein synthesis. The Ran-GTP-binding exportin, Los1p/ Xpo-t, and additional pathway(s) mediate tRNA transport to the cytoplasm. Although tRNA movement was thought to be unidirectional, recent reports that yeast precursor tRNA splicing occurs in the cytoplasm, whereas fully spliced tRNAs can reside in the nucleus, require that either the precursor tRNA splicing machinery or mature tRNAs move from the cytoplasm to the nucleus. Our data argue against the first possibility and strongly support the second. Combining heterokaryon analysis with fluorescence in situ hybridization, we show that a foreign tRNA encoded by one nucleus can move from the cytoplasm to a second nucleus that does not encode the tRNA. We also discovered nuclear accumulation of endogenous cytoplasmic tRNAs in haploid yeast cells in response to nutritional deprivation. Nuclear accumulation of cytoplasmic tRNA requires Ran and the Mtr10/Kap111 member of the importin-[beta] family. Retrograde tRNA nuclear import may provide a novel mechanism to regulate gene expression in eukaryotes. amino acid deprivation | heterokaryon | Mtr10 | tRNA nuclear import | yeast

Hepatocyte growth factor activator inhibitor-1 (HAI-1) was initially identified as cognate inhibitor of matriptase, a membrane-bound serine protease. Paradoxically, HAI-1 is also required for matriptase activation, a process that requires sphingosine 1-phosphate (S1P)-mediated translocation of the protease to cell-cell junctions in human mammary epithelial cells. In the present study, we further explored how HAI-1 regulates this protease. First, we observed that after S1P treatment HAI-1 was cotranslocated with matriptase to cell-cell junctions and that the cellular ratio of HAI-1 to matriptase was maintained during this process. However, when this ratio was changed by cell treatment with HAI-1 small interfering RNA or anti-HAI-1 MAb M 19, spontaneous activation of matriptase occurred in the absence of SiP-induced translocation; S1P-induced matriptase activation was also enhanced. These results support a role for HAI-1 in protection of cell from uncontrolled matriptase activation. We next expressed matriptase, either alone or with HAI-1 in breast cancer cells that do not endogenously express either protein. A defect in matriptase trafficking to the cell surface occurred if wild-type matriptase was expressed in the absence of HAI-1; this defect appeared to result from matriptase toxicity to cells. Coexpression with matriptase of wild-type HAI-1, but not HAI-1 mutants altered in its Kunitz domain 1, corrected the trafficking defect. In contrast, catalytically defective matriptase mutants were normal in their trafficking in the absence of HAI-1. These results are also consistent with a role for HAI-1 to prevent inappropriate matriptase proteolytic activity during its protein synthesis and trafficking. Taken together, these results support multiple roles for HAI-1 to regulate matriptase, including its proper expression, intracellular trafficking, activation, and inhibition. protease-activated receptor-2; hepatocyte growth factor; urokinase; sphingosine 1-phosphate; Kunitz domain

Escherichia coli cells were constructed in which the dnaA gene was moved to a location opposite oriC on the circular chromosome. In these cells the dnaA gene was replicated with significant delay relative to the origin. Consequently, the period where the newly replicated and hemimethylated oriC was sequestered no longer coincided with the period where the dnaA gene promoter was sequestered. DnaA protein synthesis was therefore expected to continue during origin sequestration. Despite a normal length of the sequestration period in such cells, they had increased origin content and also displayed asynchrony of initiation. This indicated that reinitiation occasionally occurred at some origins within the same cell cycle. The extra initiations took place in spite of a reduction in total DnaA protein concentration to about half of the wild-type level. We propose that this more efficient utilization of DnaA protein results from an increased availability at the end of the origin sequestration period. Therefore, coordinated sequestration of oriC and dnaA is required for maintaining controlled once-per-cell-cycle initiation.

We previously reported that [sup.2][H.sub.2]O can be used to measure rates of protein synthesis during prolonged steady-state conditions (Previs SF, Fatica R, Chandramouli V, Alexander JC, Brunengraber H, and Landau BR. Am J Physiol Endocrinol Metab 286: E665-E672, 2004). The underlying premise of our method is that following the administration of [sup.2][H.sub.2]O, [sup.2]H atoms in body water rapidly equilibrate with free alanine before it is incorporated into newly synthesized proteins. We have now directly examined whether [sup.2][H.sub.2]O can be used to measure the influence of a single meal on protein synthesis. In addition, we have compared the use of [sup.2][H.sub.2]O for measuring rates of protein synthesis in vivo vs. in cell culture. Using a rat model, we observed rapid equilibration between [sup.2]H in body water and free alanine; therefore we were able to study the response of protein synthesis to a single meal. We observed that ~50% of the plasma albumin that is synthesized over the course of 24 h is made within ~5 h after eating (in rats trained to eat a complete 24-h ration of food in a single meal). Contrary to what we observed in vivo, feeding (the replenishment of cell culture medium) does influence the use of [sup.2][H.sub.2]O for in vitro studies. In particular, since there can be slow equilibration of [sup.2]H between water and alanine in the cell culture medium, special consideration must be made to avoid underestimating the rate of protein synthesis in vitro. albumin turnover; nutritional status; stable isotopes; gas chromatography-mass spectrometry

We aimed to determine whether there were differences in the extent and time course of skeletal muscle myofibrillar protein synthesis (MPS) and muscle collagen protein synthesis (CPS) in human skeletal muscle in an 8.5-h period after bouts of maximal muscle shortening (SC; average peak torque = 225 [+ or -] 7 N*m, means [+ or -] SE) or lengthening contractions (LC; average peak torque = 299 [ + or -] 18 N*m) with equivalent work performed in each mode. Eight healthy young men (21.9 [+ or -] 0.6 yr, body mass index 24.9 [+ or -] 1.3 kg/[m.sup.2]) performed 6 sets of 10 maximal unilateral LC of the knee extensors on an isokinetic dynamometer. With the contralateral leg, they then performed 6 sets of maximal unilateral SC with work matched to the total work performed during LC (10.9 [+ or -] 0.7 vs. 10.9 [+ or -] 0.8 kJ, P = 0.83). After exercise, the participants consumed small intermittent meals to provide 0.1 g*[kg.sup.-1] * [h.sup.-1] of protein and carbohydrate. Prior exercise elevated MPS above rest in both conditions, but there was a more rapid rise after LC (P < 0.01). The increases (P < 0.001) in CPS above rest were identical for both SC and LC and likely represent a remodeling of the myofibrillar basement membrane. Therefore, a more rapid rise in MPS after maximal LC could translate into greater protein accretion and muscle hypertrophy during chronic resistance training utilizing maximal LC. eccentric; concentric; resistance exercise; z-band streaming

Studies both in vivo and in vitro have shown that leucine at a very high dose can stimulate muscle protein synthesis, an effect that is enhanced in vivo by insulin secreted in response to the leucine dose. High leucine can also inhibit protein degradation in skeletal muscle, as well as in liver. In contrast, at normal physiological levels, increasing leucine concentration by infusion stimulates muscle protein synthesis by enhancing its sensitivity to insulin. It is concluded that the role of leucine in vivo is to provide a signal that amino acids are available, which in combination with the signal of energy availability from insulin, stimulates muscle protein synthesis. KEY WORDS: * leucine * isoleucine * valine * branched-chain amino acids * protein synthesis * insulin * muscle

Since the in vitro study of Buse and Reid in 1975 showing a stimulatory effect of leucine upon rat muscle protein synthesis and reduction in proteolysis, a similar effect has been sought in humans. In 1978, Sherwin demonstrated in humans an improvement in N balance with infusion of leucine in obese subjects fasting to lose weight. A variety of subsequent studies have been performed in humans where leucine alone or the BCAAs have been administered in varying amounts and durations, and the effect upon protein metabolism has been measured. Measurements of changes in muscle amino acid metabolism were made by arteriovenous difference measurements and by biopsies. An anabolic effect of leucine and the branched-chain amino acids (BCAAs) on reduction of muscle protein breakdown was found in these studies, with no measured effect upon muscle protein synthesis. Later studies using stable isotope tracers to define both whole-body protein turnover and leg or arm protein metabolism have similarly concluded that leucine administration specifically induces a reduction in protein breakdown without increasing protein synthesis. This anabolic effect, produced through a reduction of protein breakdown in vivo in humans by leucine is contrary to in vitro studies of rat muscle where stimulation of protein synthesis, has been demonstrated by leucine. Likewise an increase in protein synthesis has also been demonstrated by insulin in rat muscle that is not seen in humans. Of the various studies administering BCAAs or leucine to humans for varying periods of time and amount, the results have been consistent. In addition, no untoward effects have been reported in any of these studies from infusion of the BCAAs at upward 3 times basal flux or 6 times normal dietary intake during the fed portion of the day. KEY WORDS: * protein synthesis * protein breakdown * leucine

Amino acids (AAs), especially BCAAs, play pivotal roles in hormonal secretion and action as well as in intracellular signaling. There is emerging data showing that BCAAs regulate gene transcription and translation. Signaling proteins such as the mammalian target of rapamycin act as sensors of BCAAs, especially leucine, to modulate anabolic action. AAs stimulate protein synthesis and inhibit protein breakdown in skeletal muscle and liver. The specific role of BCAAs in regulating synthesis and breakdown of individual protein or proteins with common function or functions remains to be defined. Future studies should also focus on potential adverse effects of BCAAs on insulin sensitivity, renal function, and tumor growth. It also remains to be determined whether potential adverse effects of BCAA supplementation is similar in people of different age groups. KEY WORDS: * branched-chain amino acids * signaling * hormonal secretion * hormonal action * protein synthesis * protein breakdown

The branched-chain amino acids (BCAAs) are required for protein synthesis and neurotransmitter synthesis. The branched-chain [alpha]-ketoacid dehydrogenase complex (BCKDC) is the most important regulatory enzyme in the catabolic pathways of the BCAAs. Activity of the complex is controlled by covalent modification with phosphorylation of its branched-chain [alpha]-ketoacid dehydrogenase subunits by a specific kinase [branched-chain kinase (BDK)] causing inactivation and dephosphorylation by a specific phosphatase [branched-chain phosphatase (BDP)] causing activation. Tight control of BCKDC activity is important for conserving as well as disposing of BCAAs. Phosphorylation of the complex occurs when there is a need to conserve BCAAs for protein synthesis; dephosphorylation occurs when BCAAs are present in excess. The relative activities of BDK and BDP set the activity state of BCKDC. BDK activity is regulated by [alpha]-ketoisocaproate inhibition and altered level of expression. Less is known about BDP but a novel mitochondrial phosphatase was identified recently that may contribute to the regulation of BCKDC. Reduced capacity to oxidize BCAAs, as in maple syrup urine disease, results in excess BCAAs in the blood and profound neurological dysfunction and brain damage. In contrast, loss of control of BCAA oxidation results in growth impairment and epileptic-like seizures. These findings emphasize the importance of control of BCAA catabolism for normal neurological function. It is proposed that the safe upper limit of dietary BCAA intake could be established with a BCAA tolerance test and clamp protocol. KEY WORDS: * leucine * branched-chain amino acids * branched-chain keto acids * branched-chain [alpha]-ketoacid dehydrogenase * branched-chain [alpha]-ketoacid dehydrogenase kinase

The high rate of muscle protein accretion in neonates is sustained by the marked increase in muscle protein synthesis in response to feeding. Little is known about the role of proteolysis in the regulation of protein accretion in response to feeding during the neonatal period. To determine the feeding-induced response of protein breakdown at the whole-body level and in the hindlimb of neonates, 10- and 28-d-old piglets that had been food deprived overnight were infused (7 h) with [1-[sup.13]C]phenylalanine and [ring-[sup.2][H.sub.4]]tyrosine during an initial food deprivation period (3 h), followed by a feeding period (4 h). During feeding, endogenous flux of phenylalanine decreased (P < 0.01) in both the whole body and the hindlimb. Feeding reduced (P < 0.01) whole-body proteolysis but increased hindlimb proteolysis (P = 0.04), suggesting that tissues other than the hindlimb are involved in the reduction in whole-body proteolysis during feeding. Overnight food deprivation resulted in a net mobilization of phenylalanine from whole-body proteins (P < 0.01) but not hindlimb proteins. These responses were unaffected by age. The results suggest that the hindlimb requires a continuous supply of free amino acids to sustain the high rate of muscle protein turnover in neonates and that adaptive mechanisms provide free amino acids to sustain skeletal muscle protein accretion in early postnatal life when the amino acid supply is limited. KEY WORDS: * phenylalanine hydroxylation * phenylalanine kinetics * neonate * skeletal muscle * stable isotopes

Limited data suggest that the growth of low-birth-weight infants is enhanced by feeding a high-protein diet; however, the mechanisms involved in the effect have not been delineated. To identify these mechanisms, 34 pigs were fed from 2 to 7 d of age [60 g dry matter/(kg body weight * d)] isocaloric milk diets that contained levels of dietary protein that were marginal, adequate, and in excess of the piglets protein requirement (21, 33, and 45% of dry matter, respectively). Dietary protein replaced lactose and fat on an isocaloric basis. Fractional protein synthesis rates, various biomarkers of translational regulation, and plasma glucose and insulin levels were measured in overnight food-deprived and fed pigs. Mean daily weight gain of pigs fed the 33 and 45% protein diets was greater than that of pigs fed the 21% protein diet (P < 0.01). Plasma glucose (P = 0.07) and insulin (P < 0.01) levels decreased as dietary protein increased 60 min after feeding. Protein synthesis rates in longissimus dorsi, gastrocnemius, masseter, heart, liver, kidney, jejunum, and pancreas were greater in the fed than in the food-deprived state (P < 0.01). Protein synthesis in skeletal muscle did not change with protein intake in the fed state, but decreased quadratically (P < 0.01) with increasing dietary protein in the food-deprived state. Protein kinase B, ribosomal protein S6 kinase 1(S6K1), and eukaryotic initiation factor (elF) 4E binding protein-1 (4E-BP1) were more phosphorylated, and assembly of the inactive eukaryotic initiation factor 4E * 4E-BP1 complex in muscle and liver was reduced in the fed state (P < 0.001) and were not consistently affected by dietary protein level. The results suggest that feeding stimulates protein synthesis, and this is modulated by the activation of initiation factors that regulate mRNA binding to the ribosomal complex. However, the provision of a high-protein diet that exceeds the protein requirement does not further enhance protein synthesis or translation initiation factor activation. KEY WORDS: * pigs * dietary protein * S6K1 * 4E-binding protein 1 * protein synthesis

Formylglycine (FGly)-generating enzyme (FGE) is a single-domain monomer with a surprising paucity of secondary structure and adopts a unique fold based on the crystal structure. FGE is localized in the endoplasmic reticulum (ER) and interacts with and modifies the unfolded form of newly synthesized sulfatases.

Fluorescence approaches are used to measure the dissociation constant for the Ssp DnaE split intein interaction and to determine the on and off rates of fragment association. The information obtained from the results is used to develop a tandem trans-splicing system based on native and engineered split inteins.

Although aminoacyl-tRNA synthetases (ARSs) are essential for protein synthesis, they also function as regulators and signaling molecules in diverse biological processes. Here, we screened 11 different human ARSs to identify the enzyme that is secreted as a signaling molecule. Among them, we found that lysyl-tRNA synthetase (KRS) was secreted from intact human cells, and its secretion was induced by TNF-[alpha]. The secreted KRS bound to macrophages and peripheral blood mononuclear cells to enhance the TNF-[alpha] production and their migration. The mitogen-activated protein kinases, extracellular signal-regulated kinase and p38 mitogen-activated protein kinase, and G[alpha]i were determined to be involved in the signal transduction triggered by KRS. All of these activities demonstrate that human KRS may work as a previously uncharacterized signaling molecule, inducing immune response through the activation of monocyte/macrophages. aminoacyl-tRNA synthetase | cytokine | TNF-[alpha] | immune response | cell migration

The bacterial parD toxin-antitoxin system of plasmid R1 encodes two proteins, the Kid toxin and its cognate antitoxin, Kis. Kid cleaves RNA and inhibits protein synthesis and cell growth in Escherichia coli. Here, we show that Kid promotes RNA degradation and inhibition of protein synthesis in rabbit reticulocyte lysates. These new activities of the Kid toxin were counteracted by the Kis antitoxin and were not displayed by the KidR85W variant, which is nontoxic in E. coli. Moreover, while Kid cleaved single- and double-stranded RNA with a preference for UAA or UAC triplets, KidR85W maintained this sequence preference but hardly cleaved double-stranded RNA. Kid was formerly shown to inhibit DNA replication of the ColE1 plasmid. Here we provide in vitro evidence that Kid cleaves the ColE1 RNA II primer, which is required for the initiation of ColE1 replication. In contrast, KidR85W did not affect the stability of RNA II, nor did it inhibit the in vitro replication of ColE1. Thus, the endoribonuclease and the cytotoxic and DNA replication-inhibitory activities of Kid seem tightly correlated. We propose that the spectrum of action of this toxin extends beyond the sole inhibition of protein synthesis to control a broad range of RNA-regulated cellular processes.

Murine L6 and human rhabdomyosarcoma cells were cultured standardized in low (0.28 mM) and normal (9 mM) amino acid (AA) concentrations to reevaluate by independent methods to what extent AA activate initiation of protein synthesis. Methods used were incorporation of radioactive AA into proteins, distribution analysis of RNA in density gradient, and Western blots on initiation factors of translation of proteins in cultured cells as well as in vivo (gastrocnemius, C57B1 mice) during starvation/refeeding. Incorporation rate of AA gave incorrect results in a variety of conditions, where phenylalanine stimulated the incorporation rate of phenylalanine into proteins, but not of tyrosine, and tyrosine stimulated incorporation of tyrosine but not of phenylalanine. Similar problems were observed when [[sup.35]S]methionine was used for labeling of fractionated cellular proteins. However, the methods entirely independent of labeled AA incorporation indicated that essential AA activate initiation of translation, whereas nonessential AA did not. Branched-chain AA and glutamine, in combination with some other AA, also stimulated initiation of translation. Starvation/refeeding in vitro agreed qualitatively with results in vivo evaluated by initiation factors. Insulin at physiological concentrations (100 [micro]M/ml) did not stimulate global protein synthesis at low or normal AA concentrations but did so at supraphysiological levels (3 mU/ml), confirmed by independent methods. Our results reemphasize that labeled AA should he used with caution for quantification of protein synthesis, since the precursor pool(s) for protein synthesis is not in complete equilibrium with surrounding AA. 'Flooding' tracee experiments did not overcome this problem. amino acids; insulin; initiation of translation; protein synthesis; muscle cells

To make more practical the total chemical synthesis of proteins by the ligation of unprotected peptide building blocks, we have developed a method to facilitate the isolation and handling of intermediate products. The synthetic technique makes use of a [His.sub.6] tag at the C terminus of the target polypeptide chain, introduced during the synthesis of the C-terminal peptide segment building block. The presence of a [His.sub.6] tag enables the isolation of peptide or protein products directly from ligation reaction mixtures by Ni-NTA affinity column purification. This simple approach enables facile buffer exchange to alternate reaction conditions and is compatible with direct analytical control by protein MS of the multiple ligation steps involved in protein synthesis. We used syntheses of crambin and a modular tetratricopeptide repeat protein of 17 kDa as models to examine the utility of this affinity purification approach. The results show that [His.sub.6] tag-assisted chemical protein synthesis is a useful method that substantially reduces handling losses and provides for rapid chemical protein syntheses. affinity purification | native chemical ligation

The phosphorylated signal transduction protein [P.sub.II] ([P.sub.II]-P) in the cyanobacterium Synechocystis sp. strain PCC 6803 is dephosphorylated by PphA, a protein phosphatase of the 2C family (PP2C). In this study, the physiological conditions of [P.sub.II]-P dephosphorylation were investigated with respect to the in vivo specificity of [P.sub.II]-P towards PphA and the cellular abundance of PphA in cells growing under different nitrogen regimes. Furthermore, the consequences of impaired [P.sub.II]-P dephosphorylation with respect to short-term inhibition of glutamine synthetase (GS) were studied. With a contribution of approximately 15% of total [Mn.sup.2+]-dependent p-nitrophenyl phosphate hydrolysis activity, PphA has only a minor impact on the total PP2C activity in Synechocystis extracts. Nevertheless, residual [P.sub.II]-P dephosphorylation in PphA-deficient cells could only be observed after prolonged incubation in the presence of ammonium. The abundance of PphA correlates with the phosphorylation state of [P.sub.II] under nitrogen-replete conditions and is specifically enhanced by nitrite. Regulation of pphA expression operates at the post-transcriptional level. In the presence of nitrate/nitrite, PphA is present in molar excess over [P.sub.II]-P, enabling the cells to rapidly dephosphorylate [P.sub.II]-P in response to changing environmental conditions. A PphA-deficient mutant is not impaired in short-term inhibition of GS activity following ammonium treatment. Down-regulation of GS occurs by induction of gif genes (encoding GS inactivating factors 7 and 17), which is controlled by NtcA-mediated gene repression. Thus, impaired [P.sub.II]-P dephosphorylation does not affect ammonium-prompted inactivation of NtcA.

The cytosolic pathway for Gln-tRNA synthesis into the mitochondrion, which is caused due to the Saccharomyces cerevisiae, is studied. Mitochondrial protein synthesis is carried out normally by mitochondrial enzymes and organelle-encoded tRNAs that are different from their cytoplasmic counterparts and found that the presence of cytoplasmic glutaminyl-tRNA synthetase (GlnRS) in the organelle and supports its involvement in mitochondrial Gln-tRNA synthesis.

An essential spliceosomal protein Sf3b1 is identified that physically interacts with the Class II Polycomb group (PcG) proteins and generated Sf3b1 knockout mice and the mammalian Polycomb-mediated repression of homeotic (Hox) genes is studied. The findings suggest that for a true PcG- mediated repression of Hox genes, the interaction of spliceosomal (Sf3b1)-PcG protein is very important.

A detailed description of current knowledge about the structure, function, and interactions of the individual components involved in bacterial translation initiation is presented. An overview of mechanisms of regulation of the translation initiation event is provided.

How growth hormone (GH) stimulates protein synthesis is unknown. Phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathways balance anabolic and catabolic activities in response to nutrients and growth factor signaling. As a test of GH signaling, immunoassays of two downstream translation regulatory proteins were compared in ad libitum-fed 2-month-old normal and Ames (Prop1df) dwarf mice. Phosphorylation of the p70 and p85 isoforms of S6 kinase 1 in liver and the p70 isoform in gastrocnemius muscle were significantly decreased in dwarfs. Messenger RNA (mRNA) Cap-binding demonstrated significantly higher levels of translation repressor 4E-BP1/eukaryotic initiation factor 4E (eIF4E) (coprecipitates) from dwarf livers, but not muscle. Consistent with these binding data, significantly less phosphorylation of 4E-BP1 was documented in dwarf liver. These data suggest a link between GH signaling and translation control in a model of extended longevity.

Protein deposition in Iberian pigs is low and the reasons for this are unknown. We investigated differences in protein synthesis rate in tissues of 30 Iberian and Landrace gilts fed 2 diets with adequate amino acid composition containing 160 or 120 g crude protein (CP)/kg, or a lysine-deficient diet (containing 120 or 160 g CP/kg for Iberian and Landrace pigs, respectively). Pigs were infused with a flooding dose of phenylalanine (15% as [[sup.2][H.sub.5]]-phenylalanine). Blood samples were taken from 12 to 40 min after the start of infusion, and samples from longissimus dorsi (ld), biceps femoris (bf), and semimembranosus (sm) muscles, liver, and duodenum were taken after slaughter. Body weights (BW) were 22.9 [+ or -] 0.37 and 27.1 [+ or -] 0.64 kg for Iberian and Landrace pigs, respectively. Iberian pigs fed the adequate diets had higher muscle fractional protein synthesis rates (FSR, %/d) than Landrace pigs. The FSR were 7.9 [+ or -] 0.34 vs. 6.3 [+ or -] 0.29%/d; 8.3 [+ or -] 0.36 vs. 6.3 [+ or -] 0.21%/d, and 7.7 [+ or -] 0.23 vs. 6.4 [+ or -] 0.36%/d for ld, bf, and sm muscles in Iberian and Landrace breeds, respectively (P < 0.01). However, muscles were between 20 and 32% smaller in the Iberian pigs (P < 0.01). Dietary protein level did not affect muscle FSR or size in either breed. Lysine deficiency reduced muscle FSR (46-49%, P < 0.001). Visceral tissues had greater relative weights in Iberian pigs (P < 0.001) with no breed differences in FSR. These findings might explain the low efficiency of protein and energy utilization by Iberian pigs compared with conventional pig breeds. KEY WORDS: * protein synthesis * muscle * viscera * Iberian pigs * lysine deficiency

We determined whether the steady-state levels of intestinal mucins are more sensitive than total proteins to dietary threonine intake. For 14 d, male Sprague-Dawley rats (158 [+ or -] 1 g. n = 32) were fed isonitrogenous diets (12.5% protein) containing 30% (group 30), 60% (group 60), 100% (control group), or 150% (group 150) of the theoretical threonine requirement for growth. All groups were pair-fed to the mean intake of group 30. The mucin and mucosal protein fractional synthesis rates (FSR) did not differ from controls in group 60. By contrast, the mucin FSR was significantly lower in the duodenum, ileum, and colon of group 30 compared with group 100, whereas the corresponding mucosal protein FSR did not differ. Because mucin mRNA levels did not differ between these 2 groups, mucin production in group 30 likely was impaired at the translational level. Our results clearly indicate that restriction of dietary threonine significantly and specifically impairs intestinal mucin synthesis. In clinical situations associated with increased threonine utilization, threonine availability may limit intestinal mucin synthesis and consequently reduce gut barrier function. KEY WORDS: * mucins * threonine * protein synthesis * intestine * rats

Oral administration of a single bolus of leucine in an amount equivalent to the daily intake (1.35 g/kg body wt) enhances skeletal muscle protein synthesis in food-deprived rats. To elucidate whether smaller amounts of leucine can also stimulate protein synthesis, rats were administered the amino acid at oncentrations ranging from 0.068 to 1.35 g/kg body wt by oral gavage. Thirty minutes following the administration of doses of leucine as low as 0.135 g/kg body wt, skeletal muscle protein synthesis was significantly greater than control values. The increase in protein synthesis was associated with changes in the regulation of biomarkers of mRNA translation initiation as evidenced by upregulated phosphorylation of the translational repressor, eukaryotic initiation factor (elF)4E-binding protein 1 (4E-BP1), the association of elF4G with the mRNA cap binding protein elF4E, and the phosphorylation of the 70-kDa ribosomal protein S6 kinase. Alterations in the phosphorylation of elF4G, as well as the association of 4E-BP1 with elF4E, were observed following leucine administration; however, these changes appeared to be biphasic with maximal changes occurring when circulating insulin concentrations were elevated. Thus it appears that leucine administration affects mRNA translation and skeletal muscle protein synthesis through modulation of multiple biomarkers of mRNA translation. The ability of small doses of leucine to stimulate skeletal muscle protein synthesis suggests that future research on the regulation of skeletal muscle protein synthesis by orally administered leucine will be feasible in humans. KEY WORDS: * translation initiation * gastrocnemius muscle * mTOR

This article reviews theories linked to endogenous bioelectric currents and the role they may play in wound repair with further appraisal of in vitro and in vivo research related to [...]

Cell death is an intrinsic part of metazoan development and mammalian immune regulation. Whereas the molecular events orchestrating apoptosis have been characterized extensively, little is known about the biochemistry of necrotic cell death. Here, we show that, in contrast to apoptosis, the induction of necrosis does not lead to the shut down of protein synthesis. The rapid drop in protein synthesis observed in apoptosis correlates with caspase-dependent breakdown of eukaryotic translation initiation factor (elF) 4G, activation of the double-stranded RNA-activated protein kinase PKR, and phosphorylation of its substrate elF2-[alpha]. In necrosis induced by tumor necrosis factor, double-stranded RNA, or viral infection, de novo protein synthesis persists and 28S ribosomal RNA fragmentation, elF2-[alpha] phosphorylation, and proteolytic activation of PKR are absent. Collectively, these results show that, in contrast to apoptotic cells, necrotic dying cells retain the opportunity to synthesize proteins.

As demonstrated with alr2835 (hepA) and alr2834 (hepC) mutants, heterocysts of Anabaena sp. strain PCC 7120, a filamentous cyanobacterium, must have an envelope polysaccharide layer (the He[p.sup.+] phenotype) to fix dinitrogen in an oxygen-containing milieu (the Fo[x.sup.+] phenotype). Transpositions presumptively responsible for a Fo[x.sup.-] phenotype were localized in open reading frames (ORFs) near hepA and hepC. A mutation in each of nine of these ORFs was complemented by a clone bearing only that single, intact ORF. Heterocysts of the nine mutants were found to lack an envelope polysaccharide layer. Complementation of mutations in air2832 and alr2840 may have resulted from recombination. However, alr2825, alr2827, alr2831, alr2833, alr2837, air2839, and alr2841, like hepA and hepC, are required for a He[p.sup.+] Fo[x.sup.+] phenotype.