Rate Of Net Protein Synthesis Research Articles

Altered proteostasis in aging and heat shock response in C. elegans revealed by analysis of the global and de novo synthesized proteome

Protein misfolding and aggregation as a consequence of impaired protein homeostasis (proteostasis) not only characterizes numerous age-related diseases but also the aging process itself. Functionally related to the aging process are, among others, ribosomal proteins, suggesting an intimate link between proteostasis and aging. We determined by iTRAQ quantitative proteomic analysis in C. elegans how the proteome changes with age and in response to heat shock. Levels of ribosomal proteins and mitochondrial chaperones were decreased in aged animals, supporting the notion that proteostasis is altered during aging. Mitochondrial enzymes of the tricarboxylic acid cycle and the electron transport chain were also reduced, consistent with an age-associated energy impairment. Moreover, we observed an age-associated decline in the heat shock response. In order to determine how protein synthesis is altered in aging and in response to heat shock, we complemented our global analysis by determining the de novo proteome. For that, we established a novel method that enables both the visualization and identification of de novo synthesized proteins, by incorporating the non-canonical methionine analogue, azidohomoalanine (AHA), into the nascent polypeptides, followed by reacting the azide group of AHA by ‘click chemistry’ with an alkyne-labeled tag. Our analysis of AHA-tagged peptides demonstrated that the decreased abundance of, for example, ribosomal proteins in aged animals is not solely due to degradation but also reflects a relative decrease in their synthesis. Interestingly, although the net rate of protein synthesis is reduced in aged animals, our analyses indicate that the synthesis of certain proteins such as the vitellogenins increases with age.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-014-1558-7) contains supplementary material, which is available to authorized users.

Calcineurin is required for translational control of protein synthesis in rat pancreatic acini.

CCK increases the rate of net protein synthesis in rat pancreatic acini by activating initiation and elongation factors required for translation. The immunosuppressant FK506 inhibits the Ca(2+)-calmodulin-dependent phosphatase calcineurin in pancreatic acinar cells and blocks pancreatic growth induced by chronic CCK treatment. To test a requirement for calcineurin in the activation of the translational machinery stimulated by CCK, we evaluated the effects of FK506 on protein synthesis and on regulatory initiation and elongation factors in rat pancreatic acini in vitro. CCK acutely increased protein synthesis in acini from normal rats with a maximum increase at 100 pM CCK to 170 +/- 11% of control. The immunosuppressant FK506 dose-dependently inhibited CCK-stimulated protein synthesis over the same concentration range that blocked calcineurin activity, as assessed by dephosphorylation of the calcineurin substrate calcium-regulated heat-stable protein of 24 kDa. Another immunosuppressant, cyclosporin A, inhibited protein synthesis, but its effects appeared more complex. FK506 also inhibited protein synthesis stimulated by bombesin and carbachol. FK506 did not significantly affect the activity of the initiation factor-2B, or the phosphorylation of the initiation factor-2alpha, ribosomal protein protein S6, or the mRNA cap binding protein eukaryotic initiation factor (eIF) 4E. Instead, blockade of calcineurin with FK506 reduced the phosphorylation of the eIF4E binding protein, reduced the formation of the eIF4F complex, and increased the phosphorylation of eukaryotic elongation factor 2. From these results, we conclude that calcineurin activity is required for protein synthesis, and this action may be related to an effect on the formation of the mRNA cap binding complex and the elongation processes.

In Vivo Pulse Labeling of Proteins in Attached Leaves with Radioactive Amino Acids

The pulse labeling of proteins in attached leaves by the incorporation of radioactive amino acids can be a valuable approach to study a wide range of topics pertaning to plant gene expression. Transient changes in the rate of net protein synthesis can be measured since pulse labeling permits discrimination between proteins actively synthesized during the labeling interval from those accumulated at earlier times. Specific procedures for a rapid and convenient method of in vivo pulse labeling are outlined which we believe could be broadly useful in plant biological research. A particularly important technical feature of the protocol is electroblotting of radioactive proteins from the sodium dodecyl sulfate acrylamide resolving gel matrix to polyvinylidene di-fluoride transfer membrane. This step permits a single polyacrylamide slab gel to be used for autoradiography, immunoblot analysis, and protein staining, thereby greatly facilitating comparison among these complementary techniques and resulting in a significant savings in time and radioactive sample. Central considerations concerning the quantitation and interpretation of in vivo pulse labeling data are discussed.

Rapid changes in protein synthesis after application of arachidonic acid to potato tuber tissue

Early changes in protein synthesis were investigated in potato ( Solanum tuberosum L.) cv. Kennebec alter application of arachidonic acid (AA), an elicitor of sesquiterpenoid phytoalexin accumulation and other responses associated with hypersensitive resistance in potato. In vivo labelling of disks from potato tubers with [ 3H]leucine showed that the rate of net protein synthesis in underlying tissues nearly doubled within 6 h of the application of AA. This is one of the earliest active responses to the elicitor known. Uptake of leucine was inhibited in the surface tissue during the first 9 h after treatment with AA but removal of the upper 1 mm of treated tissue immediately before application of leucine avoided the interference. Application of other polyunsaturated fatty acids to tuber disks also caused transient increases in protein synthesis of subsurface tissues. However, AA always induced the largest and most prolonged increases. These results clearly show that AA has marked effects on the protein metabolism of potato tubers soon after its application.

RATE AND EFFICIENCY OF GAIN, FROM WEANING TO SLAUGHTER, OF STEERS GIVEN HAY, HAY SUPPLEMENTED WITH RUMTNAL UNDEGRADABLE PROTEIN, OR CONCENTRATE

Holstein steers were given milk from birth until they reached a weight of 100 kg. Beginning at 60 kg, they received gradually increasing amounts of one of three different diets and these became their sole diet after milk was withdrawn. The three diets were: (a) 85% grass hay and 15% concentrate (group 1); (b) the same diet with enough rumen undegradable protein (formaldehyde-treated canola meal) added to raise the crude protein content by 10 g kg−1 (group 2); and (c) 85% concentrate and 15% grass hay (group 3). The amounts of the three diets offered were designed to provide 0.95 MJ of digestible energy per day per unit of liveweight0.75. Steers were slaughtered at about 500 kg liveweight and the right sides of their carcasses were dissected into separable muscle, adipose tissue, and bone. This information was used to derive estimates of the daily gains of protein, fat, and energy in the empty bodies and the carcasses of the steers. Empty liveweight gain of group 3 steers exceeded that of group 1 steers by 42% and they required only 58% as much extra-maintenance energy per unit of gain. Daily gains of carcass muscle, adipose tissue, bone, protein, fat, and energy, and of empty body protein, fat, and energy, were greater in steers in group 3 than in those in group 1. The extra gain of group 3 steers, compared with group 1 steers, was due largely to gain of extra body fat. The overall efficiency of energy gain was 50% greater in steers in group 3 than in those in group 1. Supplementing the forage diet (group 1) with rumen undegradable protein (group 2) increased daily empty liveweight gains by 13% and reduced the extra-maintenance energy requirement per unit weight gain by 12%. Daily gains of carcass muscle, bone, and protein, and of empty body protein, were greater in steers in group 2 than in those in group 1. The extra gain of group 2 steers, compared with group 1 steers, was due largely to gain of extra body protein and its associated water. There were no differences between steers in these two groups in efficiency of energy gain (energy gain per unit extra-maintenance energy intake). It was concluded that the higher energetic efficiency of empty body weight gain of steers in group 2, as opposed to those in group 1, was due to a higher rate of net protein synthesis as a consequence of the increased amounts of protein absorbed from the intestine, and that the higher energetic efficiency of weight gain of steers given the concentrate diet (group 3), as opposed to those given the forage diet (group 1), was due to a higher efficiency of conversion of the energy-yielding products of digestion into fat. Key words: Energetic efficiency, steers, protein, undegradable, hay, concentrate

Kinetic evidence for a critical rate of protein synthesis in the Saccharomyces cerevisiae yeast cell cycle.

The kinetics of cell cycle initiation were measured at pH 2.7 for cells that had been arrested at the "start" step of cell division with the polypeptide pheromone alpha-factor. Cell cycle initiation was induced by the removal of alpha-factor. The rate at which cells completed start was identical to the rate of subsequent bud emergence. After short times of prearrest with alpha-factor (e.g. 5.2 h), the kinetics of bud emergence were biphasic, indicative of two subpopulations of cells that differed by greater than 10-fold in their rates of cell cycle initiation. The subpopulation that exhibited a slow rate of cell cycle initiation is comprised of cells that resided in G1 prior to start at the time of removal of alpha-factor, whereas the subpopulation that initiated the cell cycle rapidly is comprised of cells that had reached and become blocked at start. A critical concentration of cycloheximide was found to reintroduce slow budding cells into a population of 100% fast budding cells, suggesting that the two subpopulations differ with respect to attainment of a critical rate of protein synthesis that is necessary for the performance of start. Cycloheximide and an increase in the time of prearrest with alpha-factor had opposite effects on both the partitioning of cells between the two subpopulations and the net rate of protein synthesis per cell, consistent with this conclusion. Cell cycle initiation by the subpopulation of fast budding cells required protein synthesis even though the critical rate of protein synthesis had been achieved during arrest. It is concluded that alpha-factor inhibits the synthesis of and/or inactivates specific proteins that are required for the performance of start, but alpha-factor does not prevent attainment of the critical rate of protein synthesis.

REPAIR OF PHOTOINHIBITORY DAMAGE IN ANACYSTIS NIDULANS 625 (SYNECHOCOCCUS 6301): RELATION TO CATALYTIC CAPACITY FOR, AND ENERGY SUPPLY TO, PROTEIN SYNTHESIS, AND IMPLICATIONS FOR μmax AND THE EFFICIENCY OF LIGHT‐LIMITED GROWTH

SummaryPreviously published data on photoinhibition in Anacystis nidulans (Richt.) Drouet 625 (=Synechococcus Naeg. 6301) in the presence (gross rate) and absence (net rate) of an inhibitor of protein synthesis, and on the rate of repair of photoinhibitory damage, are analysed. The analysis makes assumptions about the relationship between changes in the efficiency of photosynthesis and changes in the number of undamaged photoreaction two reaction centres, the number of photoreaction centres of photoreaction two per unit cell volume, and the Mr of the photodamageable polypeptide associated with photoreaction two. With this background, the rate of protein synthesis (mol amino acid incorporated (m3 cell volume)−1 s−1) which is required to just balance the rate of protein destruction in gross photoinhibition, or to support the rate of protein synthesis in repair, is computed. The conclusions drawn from this analysis are:(1) Repair of (gross) photodamage which is known to be submaximal requires rates of protein synthesis up to 1/10 of the rate of net protein synthesis at the μmax of Anacystis nidulans, and about 1/50 of the maximum possible capacity for protein synthesis deduced from the RNA content of Anacystis nidulans and the RNA‐based protein synthesis capacity of Escherichia coli.(2) The rates of protein synthesis computed in (1) use up to 1/5 of the ATP production rate from oxidative phosphorylation in darkness and up to 1/10 of the ATP production rate (including photophosphorylation) at an incident photon flux density of 5 μmol photon (400 to 700nm)m−2 s−1(3) Cells subject to photoinhibitory damage, and its repair, have an additional requirement for protein synthesis and ATP generation over cells not subject to photoinhibition. These additional requirements may limit both μmax and the efficiency of light‐limited growth.

Heat Stress Effects on Protein Synthesis and Exosmosis of Cell Solutes in Three Turfgrass Species1

AbstractKentucky bluegrass (Poa pratensisL.), annual bluegrass (Poaannua L.), and perennial ryegrass (Lolium perenneL.) differ in their heat tolerance as determined by plant dry weight 2 weeks after exposure to heat stress. The status of the plants at that time depends on the degree of injury that occurs during the stress treatment and the repair or tolerance of that injury. The purpose of this research was to evaluate species differences in the degree of initial injury due to heat stress. The incorporation of radio‐labeled leucine as an indicator of the net rate of protein synthesis and the efflux of cell solutes from plant tissue sections into distilled water were evaluated immediately after turfgrass shoots or tissue sections were exposed to temperatures in the range of 43 to 51 °C for 30 min. Net protein synthesis in the grasses was very heat labile. Incorporation of radiolabeled leucine declined an average of 69% in plants previously heated at 43 °C compared to plants held at 27 °C. Efflux of cell solutes from the tissue sections did not increase in plants that had been heated to 43, 45, 47, or 49 °C compared to plants held at 25 °C. No significant differences were found between the grasses for the parameters tested. Either disruption of some other physiological process or differential repair or tolerance of the heat stress injury accounted for the heat tolerance rankings of the grasses. Also, the results of this study indicate that indirect rather than direct heat injury is responsible for the behavior of the grasses stressed at the temperatures and exposure periods used in this study.

Protein turnover, nitrogen balance and rehabilitation.

Not many studies have been done on protein turnover during recovery from malnutrition. Some relevant information can, however, be obtained from measurements on normal growing animals, since rehabilitation and normal growth have in common a rapid rate of net protein synthesis. The key question is the extent to which net gain in protein results from an increase in synthesis or a decrease in breakdown or both. Different studies have used different methods, and all methods for measuring protein turnover have some disadvantages and sources of error. It is important to bear this in mind in evaluating the results. Consequently, part of this paper will be devoted to questions of methodology. Whole body protein turnover has been measured in children recovering from severe malnutrition. During the phase of rapid catch-up growth the rate of protein synthesis is increased. As might be expected, it increases linearly with the rate of weight gain. At the same time there is a smaller increase in the rate of protein breakdown. The resultant of these two processes is that, over and above the basal rate of protein synthesis, 1.4 grams of protein have to be synthesized for 1 gram to be laid down. Very similar results have been obtained in rapidly growing young pigs. Experimental studies on muscle growth in general confirm the conclusion that, at least in muscle, rapid growth is associated with rapid rates of protein breakdown as well as of synthesis. This has been shown in muscles of young growing rats, as well as in muscles in which hypertrophy has been induced by stretch or other stimuli. In contrast, the evidence suggests that rapid growth involves a fall in the rate of protein degradation. The magnitude of the nitrogen balance under any conditions is determined by the difference between synthesis and breakdown. In the absence of any storage of amino acids, this must be the same as the difference between intake and excretion (S - B = I - E). A question of great interest is whether, at a given intake, the extent of N balance is determined primarily by regulation of synthesis and breakdown or by regulation of amino acid oxidation. Clearly, a reduction in amino acid degradation is equivalent to an increase in amino acid intake. An interesting subject for future research is the extent to which the amino acid degrading enzymes adapt to the requirements imposed by growth and rehabilitation.

Rates of protein synthesis through the cell cycle of Saccharomyces cerevisiae

Exponentially growing cells of Saccharomyces cerevisiae were fractionated by centrifugation in isotonic, self-generated gradients of Percoll. Rapidly growing cells, μ = 0.5 ḡ × h −1, with nearly equal length of the daughter and the parental cell cycle were fractionated according to a cell cycle-related density variation. In these cells the net rate of protein synthesis varies nearly 2-fold during the cell cycle. Subsequent separations according to cell size revealed that the highest rate is observed during G2 period. Slow-growing cells, μ = 0.2 ḡ × h −1, were fractionated on shallow Percoll gradients in a bimodal fashion, primarily as a dense daughter fraction and a composite light fraction. Thereby a marked high rate of protein synthesis in large unbudded daughter cells was revealed. Separations according to cell size revealed a cell cycle-related separation of budded cells, and the highest rate is observed, as before, in the G2 period. Irrespective of the growth rate a non-exponential increase of cell protein is thereby observed through the cell cycle of budding yeast. Septation and cell separation coincide with a low degree of ribosome exploitation.

Protein synthesis by cultured jejunal mucosa from control subjects and patients with coeliac disease.

Jejunal biopsies from patients with coeliac disease and from controls were cultured in vitro for 24 hours with 14C-labelled leucine. The net rate of protein synthesis was found to be linear over 24 hours for mucosa from control subjects and patients with coeliac disease. Protein synthesis by mucosa from untreated coeliac patients was significantly greater than by control mucosa. Protein synthesis by treated gluten-sensitive coeliac mucosa was significantly less than that by untreated coeliac mucosa and did not differ from control mucosa. Protein synthesis by treated non-responsive coeliac mucosa was significantly less than untreated coeliac mucosa but greater than control mucosa. The differences in protein synthesis could not be accounted for by differences in the size of the enterocyte leucine pool. Analytical subcellular fractionation of cultured jejunal mucosa showed that most of the protein synthesised in vitro was found in the cytosol and endoplasmic reticulum-brush border fractions of the enterocyte.

Metabolism and metabolic effects of ketoacids

Administration of any of the three branched-chain amino or ketoacids necessarily yields the respective aminated or deaminated compound because the ubiquitous enzyme, branched-chain amino acid transaminase, catalyzes the reversible transfer of amino groups between the three amino acids and their ketoacids. Branched-chain amino acid transaminase activity increases by unknown mechanisms in certain physiological and nutritional conditions and also in the presence of an excess of alpha-ketoisocaproate. This compound directly stimulates the enzyme to increase catalytic efficiency. The effect is rapid in onset and specific for the keto analogue of leucine. Irreversible branched-chain ketoacid degradation is initiated by a mitochondrial dehydrogenase enzyme which (like branched-chain amino acid transaminase) reacts with all three ketoacids and is important in regulating the pool size and proportions of branched-chain amino acids. In rats; hepatic dehydrogenase activity is induced by feeding branched-chain ketoacids. In muscle, branched-chain ketoacids or their degradation products affect both energy supply and the rate of net protein synthesis. An improved understanding of ketoacid metabolism will lead to more effective use of these compounds in treating patients with diseases of nitrogen retention.

A factor reducing protein synthesis from Ehrlich ascites cells

Ehrlich ascites cells show a decreasing rate of incorporation of labeled amino acids into the cold acid insoluble material when they are incubated in HBSS. This decreasing rate of incorporation is partly due to a dilution of the labeled amino acid by unlabeled amino acids escaping from the cells. It is demonstrated that also the net rate of protein synthesis is reduced under these conditions and this reduction occurs on a step following aminoacylation of transfer-RNA. When the medium is changed after 3–5 hr of incubation, the cells regain their original high rate of amino acid incorporation and the medium harvested after incubation with ascites cells reduces the rate of protein synthesis in freshly harvested cells. It is hypothesized that ascites cells under the incubation conditions release a factor into the medium which reversibly inhibits protein synthesis. The possible role of this factor in regulating the transformation of growing ascites cells into non-growing cells is discussed.

Characterization of disease resistance responses in certain gene-for-gene interactions between flax and Melampsora lini

Six gene-for-gene interactions between flax and Melampsora lini exhibited changes in the synthesis of soluble proteins 6 to 18 h after inoculation. The rates at which various-sized proteins were synthesized varied for each different gene-for-gene combination. The increased rate of synthesis detected in particular fractions from a column separation was reproducible and characteristic for certain gene combinations. In general, the net rate of protein synthesis increased in the four incompatible (resistant) interactions but did not increase in the two compatible (susceptible) interactions. It is concluded that this characteristic increase in protein synthesis is one of the first detectable expressions of disease resistance in the flax-flax rust interaction.

Protein turnover during maturation of mouse brain tissue.

The measurement of protein turnover involves the product of the rates of protein synthesis and degradation. It is the dynamic balance between these two components that determines the measured net rate of protein synthesis. The data reported here show that brain cells from newborn animals incorporate arginine-(14)C into acid-insoluble protein at a rate 10-fold greater than the rate for brain cells obtained from 15-day old animals. This difference in incorporation occurred even though the rate of arginine accumulation and the resulting pool size of radioactive precursor were similar for both ages. The measurement of protein turnover in brain cell suspensions prepared from 1-day old animals was shown to be complex and to exhibit a cyclic phenomenon in regard to arginine-(14)C incorporation into and release from protein. The variation in half-life calculations (0.5-3.5 hr) due to this cyclic phenomenon is discussed. Although puromycin was added in an attempt to amplify the rate of degradation by preventing the synthesis of new protein, it was found that degradation was inhibited as well, suggesting a relationship between protein synthesis and degradation.

Overall controls on the biosynthesis of ribosomes in growing bacteria

Under conditions of balanced growth, there is a relationship between λ, the growth rate constant and k r , the rate of synthesis of ribosomal RNA per unit amount of DNA. If k is the rate of protein synthesis per unit of ribosomal RNA and C is the ratio of DNA to protein; this relationship is k r = λ 2/ kC. It applies if proteins and ribosomes are stable and ribosomal precursors are present in small amounts. From direct estimates of k r from isotope incorporation rates, or indirectly from the relationship k r = ( r) λ/( d), it could be shown that as the growth rate is changed by a given factor through alteration of the nutritional environment, k r is altered by an even larger factor. These findings imply strong physiological control at the level of RNA synthesis. However, the change is not sufficiently large to make k r directly proportional to λ 2, which would be the case if C and k are constant, independent of the growth rate. Since C is, in experimental fact, essentially independent of growth rate, this is another way of saying that the rate of net protein synthesis per unit amount of ribosomal RNA is not constant. Although this would appear to cast doubt on the hypothesis of Maaløe and Kjeldgaard that protein synthesis by the ribosome has the same efficiency for balanced growth at any rate, there are several other alternative explanations for the “extra RNA” in slowly growing cells. Experimental studies from our laboratory to examine these possibilities are reviewed. Protein turnover, ribosome turnover, destruction of precursors to ribosomes can be excluded as the major raison d'être of the “extra” RNA for slowly growing chemostat cultures limited by the carbon source. Also, the presence of cells temporarily quiescent in protein synthesis cannot account quantitatively for this RNA. The “extra” RNA cannot be accounted for by assuming that the addition of an amino acid to a growing peptide chain takes longer in cells in a poor environment and therefore the synthesis of protein by a functioning ribosome is slower. However, experiments based on the theory presented here show that the “extra” RNA in the carbon-limited slow-growing cells is available in the cells in a form that can quickly function when the environment is enriched. It must be in the form of ribosomes and tRNA ready, or almost ready, to function, but not functioning at full efficiency in the poor environment.