Synthesis Rates Of Individual Proteins Research Articles

Understanding the effects of nutrition and post-exercise nutrition on skeletal muscle protein turnover: Insights from stable isotope studies

Understanding the effects of nutrition and post-exercise nutrition on skeletal muscle protein turnover: Insights from stable isotope studies

Fractional Synthesis Rates of Individual Proteins in Rat Soleus and Plantaris Muscles.

Differences in the protein composition of fast- and slow-twitch muscle may be maintained by different rates of protein turnover. We investigated protein turnover rates in slow-twitch soleus and fast-twitch plantaris of male Wistar rats (body weight 412 ± 69 g). Animals were assigned to four groups (n = 3, in each), including a control group (0 d) and three groups that received deuterium oxide (D2O) for either 10 days, 20 days or 30 days. D2O administration was initiated by an intraperitoneal injection of 20 μL of 99% D2O-saline per g body weight, and maintained by provision of 4% (v/v) D2O in the drinking water available ad libitum. Soluble proteins from harvested muscles were analysed by liquid chromatography–tandem mass spectrometry and identified against the SwissProt database. The enrichment of D2O and rate constant (k) of protein synthesis was calculated from the abundance of peptide mass isotopomers. The fractional synthesis rate (FSR) of 44 proteins in soleus and 34 proteins in plantaris spanned from 0.58%/day (CO1A1: Collagen alpha-1 chain) to 5.40%/day NDRG2 (N-myc downstream-regulated gene 2 protein). Eight out of 18 proteins identified in both muscles had a different FSR in soleus than in plantaris (p < 0.05).

Rates of Synthesis of Individual Proteins in Muscles of Old Mice: A Heavy Water Approach.

Age‐associated loss of muscle mass and function is a major factor in the development of frailty, but the mechanisms responsible for this loss are unknown. Unquestionably, muscle atrophy is the consequence of changes in the balance between protein synthesis and breakdown. However, the nature and extent of these changes in old age is unclear, with some studies reporting reduced basal muscle protein synthetic rate in the aged compared with younger people and others suggesting no difference. Whole organism heavy water ([2H2]O) dynamic labelling of proteins, followed by LC‐MSMS, provides a significant improvement in analysis of protein turnover, allowing the rates of synthesis and degradation of both slow and fast turnover muscle proteins to be accurately modelled. The aim of this study was to use a [2H2]O labelling approach to determine the synthesis rates of individual proteins in the gastrocnemius muscles of adult and old mice.Ten adult and ten old male mice were primed with an intraperitoneal bolus injection of saline, prepared with 99% [2H2]O and subsequently, provided with 8% (v/v) [2H2]O in the drinking water, for up to 60 days. The sampling interval was distributed to obtain sufficient data points to cover both high and low turnover proteins. Muscle proteins were extracted and digested using standard procedures for analysis on a high‐resolution mass spectrometer (Thermo Q‐Exactive) and the raw data files were processed to allow protein identification using Crux‐tide ( McIlwain et al, 2014). The software RIAna (: https://github.com/Molecular‐Proteomics/riana) was then used to track time dependent incorporation of [2H] into tryptic peptides, yielding synthesis rates of proteins to be determined.Muscle mass was significantly reduced in the old mice and preliminary data demonstrated little change in the rate of synthesis of a large proportion of identified muscle proteins between adult and old mice. Several individual proteins showed a marked decrease in protein synthesis rates in muscles of old compared with adult mice, particularly structural proteins. Other proteins, particularly redox‐responsive proteins demonstrated an increased synthesis in muscles of old mice. Proteomic analysis of unlabelled muscle protein pools and RNASeq data will be integrated with the turnover data to further interpret these findings.This study was supported by the University of Liverpool, MRC and NIA (AG051442).

Determining synthesis rates of individual proteins in zebrafish (Danio rerio) with low levels of a stable isotope labelled amino acid.

The zebrafish is a powerful model organism for the analysis of human cardiovascular development and disease. Understanding these processes at the protein level not only requires changes in protein concentration to be determined but also the rate at which these changes occur on a protein-by-protein basis. The ability to measure protein synthesis and degradation rates on a proteome-wide scale, using stable isotope labelling in conjunction with mass spectrometry is now a well-established experimental approach. With the advent of more selective and sensitive mass spectrometers, it is possible to accurately measure lower levels of stable isotope incorporation, even when sample is limited. In order to challenge the sensitivity of this approach, we successfully determined the synthesis rates of over 600 proteins from the cardiac muscle of the zebrafish using a diet where either 30% or 50% of the L-leucine was replaced with a stable isotope labelled analogue ([(2) H7 ]L-leucine]. It was possible to extract sufficient protein from individual zebrafish hearts to determine the incorporation rate of the label into hundreds of proteins simultaneously, with the two labelling regimens showing a good correlation of synthesis rates.

On the Rate of Synthesis of Individual Proteins within and between Different Striated Muscles of the Rat.

The turnover of muscle protein is responsive to different (patho)-physiological conditions but little is known about the rate of synthesis at the level of individual proteins or whether this varies between different muscles. We investigated the synthesis rate of eight proteins (actin, albumin, ATP synthase alpha, beta enolase, creatine kinase, myosin essential light chain, myosin regulatory light chain and tropomyosin) in the extensor digitorum longus, diaphragm, heart and soleus of male Wistar rats (352 ± 30 g body weight). Animals were assigned to four groups (n = 3, in each), including a control and groups that received deuterium oxide (2H2O) for 4 days, 7 days or 14 days. Deuterium labelling was initiated by an intraperitoneal injection of 10 μL/g body weight of 99.9% 2H2O-saline, and was maintained by administration of 5% (v/v) 2H2O in drinking water provided ad libitum. Homogenates of the isolated muscles were analysed by 2-dimensional gel electrophoresis and matrix-assisted laser desorption ionisation time of flight mass spectrometry. Proteins were identified against the SwissProt database using peptide mass fingerprinting. For each of the eight proteins investigated, the molar percent enrichment (MPE) of 2H and rate constant (k) of protein synthesis was calculated from the mass isotopomer distribution of peptides based on the amino acid sequence and predicted number of exchangeable C–H bonds. The average MPE (2.14% ± 0.2%) was as expected and was consistent across muscles harvested at different times (i.e., steady state enrichment was achieved). The synthesis rate of individual proteins differed markedly within each muscle and the rank-order of synthesis rates differed among the muscles studied. After 14 days the fraction of albumin synthesised (23% ± 5%) was significantly (p < 0.05) greater than for other muscle proteins. These data represent the first attempt to study the synthesis rates of individual proteins across a number of different striated muscles.

Assessment of Global Proteome Dynamics in Carp: A Model for Investigating Environmental Stress

Fish have to respond to a range of natural and man-made environmental stressors, which can lead to molecular changes within their tissues. Many studies focused on environmental stress in fish have examined the change in protein abundance or mRNA level. However, it is well-known that there is a disconnect between mRNA and protein expression. In order to bridge this gap, protein turnover must also be considered. We have developed an experimental strategy to determine the synthesis rates of individual proteins in the tissues of fish on a proteome-wide scale. This approach has been applied to the common carp ( Cyprinus carpio ), a key model species for investigating environmentally induced physiological plasticity. We have calculated the rates of protein synthesis for over a thousand individual proteins from the skeletal muscle and liver of carp. The median synthesis rate of proteins from liver was higher than that of skeletal muscle. The analysis further revealed that the same protein can have a different rate of synthesis depending on the tissue type. Our strategy permits a full investigation of proteome dynamics in fish and will have relevance to the fields of integrative biology and ecotoxicology.

Assessment of cardiac proteome dynamics with heavy water: slower protein synthesis rates in interfibrillar than subsarcolemmal mitochondria

Traditional proteomics provides static assessment of protein content, but not synthetic rates. Recently, proteome dynamics with heavy water ((2)H2O) was introduced, where (2)H labels amino acids that are incorporated into proteins, and the synthesis rate of individual proteins is calculated using mass isotopomer distribution analysis. We refine this approach with a novel algorithm and rigorous selection criteria that improve the accuracy and precision of the calculation of synthesis rates and use it to measure protein kinetics in spatially distinct cardiac mitochondrial subpopulations. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated from adult rats, which were given (2)H2O in the drinking water for up to 60 days. Plasma (2)H2O and myocardial (2)H-enrichment of amino acids were stable throughout the experimental protocol. Multiple tryptic peptides were identified from 28 proteins in both SSM and IFM and showed a time-dependent increase in heavy mass isotopomers that was consistent within a given protein. Mitochondrial protein synthesis was relatively slow (average half-life of 30 days, 2.4% per day). Although the synthesis rates for individual proteins were correlated between IFM and SSM (R(2) = 0.84; P < 0.0001), values in IFM were 15% less than SSM (P < 0.001). In conclusion, administration of (2)H2O results in stable enrichment of the cardiac precursor amino acid pool, with the use of refined analytical and computational methods coupled with cell fractionation one can measure synthesis rates for cardiac proteins in subcellular compartments in vivo, and protein synthesis is slower in mitochondria located among the myofibrils than in the subsarcolemmal region.

Concomitant determination of absolute values of cellular protein amounts, synthesis rates, and turnover rates by quantitative proteome profiling.

Two-dimensional gel electrophoresis of protein fractions isolated from (35)S-radiolabeled cells provides qualitative information on intracellular amounts, (35)S incorporation rates, protein modifications, and subcellular localizations of up to thousands of individual proteins. In this study we extended proteome profiling to provide quantitative data on synthesis rates of individual proteins. We combined fluorescence detection of radiolabeled proteins with SYPRO ruby(TM) staining and subsequent autoradiography of the same gels, thereby quantifying protein amounts and (35)S incorporation. To calibrate calculation of absolute synthesis rates, we determined the amount and autoradiograph intensity of radiolabeled haptoglobin secreted by interleukin-6 pretreated HepG2 cells. This allowed us to obtain a standard calibration value for (35)S incorporation per autoradiograph intensity unit. This value was used to measure protein synthesis rates during time course experiments of heat-shocked U937 cells. We measured the increasing amounts of hsp70 and calculated it by integration of the determined hsp70 synthesis rates over time. Similar results were obtained by both methods, validating our standardization procedure. Based on the assumption that the synthesis rate of proteins in a steady state of cell metabolism would essentially compensate protein degradation, we calculated biological half-lives of proteins from protein amounts and synthesis rates determined from two-dimensional gels. Calculated protein half-lives were found close to those determined by pulse-chase experiments, thus validating this new method. In conclusion, we devised a method to assess quantitative proteome profiles covering determination of individual amounts, synthesis, and turnover rates of proteins.

Tracers to investigate protein and amino acid metabolism in human subjects.

Three tracer methods have been used to measure protein synthesis, protein breakdown and protein oxidation at whole-body level. The method using L-[1-(13)C]leucine is considered the method of reference. These methods have contributed greatly to the existing knowledge on whole-body protein turnover and its regulation by feeding, fasting, hormones and disease. How exercise and ingestion of mixed protein-containing meals affect whole-body protein metabolism is still open to debate, as there are discrepancies in results obtained with different tracers. The contribution of whole-body methods to the future gain of knowledge is expected to be limited due to the fact that most physiological disturbances have been investigated extensively, and due to the lack of information on the relative contribution of various tissues and proteins to whole-body changes. Tracer amino acid-incorporation methods are most suited to investigate these latter aspects of protein metabolism. These methods have shown that some tissues (liver and gut) have much higher turnover rates and deposit much more protein than others (muscle). Massive differences also exist between the fractional synthesis rates of individual proteins. The incorporation methods have been properly validated, although minor disagreements remain on the identity of the true precursor pool (the enrichment of which should be used in the calculations). Arterio-venous organ balance studies have shown that little protein is deposited in skeletal muscle following a protein-containing meal, while much more protein is deposited in liver and gut. The amount deposited in the feeding period in each of these tissues is released again during overnight fasting. The addition of tracers to organ balance studies allows the simultaneous estimation of protein synthesis and protein breakdown, and provides information on whether changes in net protein balance are caused primarily by a change in protein synthesis or in protein breakdown. In the case of a small arterio-venous difference in a tissue with a high blood flow, estimates of protein synthesis and breakdown become very uncertain, limiting the value of using the tracer. An additional measurement of the intracellular free amino acid pool enrichment allows a correction for amino acid recycling and quantification of the inward and outward transmembrane transport. However, in order to obtain reliable estimates of the intramuscular amino acid enrichment and, therefore, of muscle protein synthesis and breakdown in this so-called three-pool model, the muscle should be freeze-dried and the resulting fibres should be freed from connective tissue and small blood clots under a dissection microscope. Even when optimal precautions are taken, the calculations in these tracer balance methods use multiple variables and, therefore, are bound to lead to more variability in estimates of protein synthesis than the tracer amino acid incorporation methods. In the future, most studies should focus on the measurement of protein synthesis and breakdown in specific proteins in order to understand the mechanisms behind tissue adaptation in response to various stimuli (feeding, fasting, exercise, trauma, sepsis, disuse and disease). The tracer laboratories, therefore, should improve the methodology to allow the measurement of low tracer amino acid enrichments in small amounts of protein.

Mass Spectrometric Methods for Determination of [13C]Leucine Enrichment in Human Muscle Protein

Two modified GC-based isotope ratio MS (IRMS) techniques, for measurement of [13C]leucine enrichment in muscle protein, were compared with a conventional dual inlet technique. Of these three, two involved HPLC purification of leucine and liberation of carbon dioxide (CO2) using the ninhydrin reaction. In the conventional technique the CO2was introduced into the MS by a dual inlet system following cryogenic concentration. In the second method (ninhydrin/GC/IRMS) the CO2was purified by on-line GC. In the third technique, GC/combustion/IRMS, derivatized amino acids are separated by GC and analyzed after combustion. A primed continuous infusion ofL-[1-13C]leucine was given intravenously to human subjects and needle quadriceps muscle biopsies were taken to measure [13C]leucine enrichment in muscle protein. All three methods demonstrated excellent correlation (r> 0.999). Differences in the measurement of [13C]leucine enrichment in muscle protein were <6%. The ninhydrin techniques require microgram quantities of leucine, with the conventional technique requiring twice the amount as the ninhydrin/GC/IRMS method. The GC/combustion/IRMS technique requires only nanogram quantities of leucine with similar precision enabling measurement of synthesis rates of individual proteins from biopsy samples. We have measured the isotopic enrichment of myosin heavy chain and mixed muscle protein in human subjects using the GC/combustion/IRMS technique.

Somatotropin Enhances the Rate of Amino Acid Deposition but Has Minimal Impact on Amino Acid Balance in Growing Pigs1

This experiment was conducted to establish the influence of porcine somatotropin on tissue distribution and deposition rates of amino acids in growing pigs. Barrows were treated daily with buffer or porcine somatotropin (100 µmg/kg body wt) when they weighed between 30 and 64 kg (eight pigs/group). Pigs were restrictively fed so that the average food intake was 1.86 kg/d. The corn-soybean meal-skim milk-based diet contained 18% crude protein and 1.2% lysine, and was designed to meet muscle amino acid ratio profile with respect to lysine as the first limiting amino acid for growth. Tissue levels of eighteen amino acids were determined on lyophilized samples that were appropriately hydrolyzed and analyzed by HPLC. The concentrations (mg/g dry wt) of all amino acids were greater in carcass, skin, head and empty body of porcine somatotropin-treated pigs; amino acid concentration in viscera was not influenced by porcine somatotropin. However, when the concentration of each amino acid was expressed on a per unit protein basis, the amino acid profiles of control and porcine somatotropin-treated pigs were quite similar. The average deposition rate of each amino acid was increased ∼67% by porcine somatotropin. When the deposition of each amino acid was calculated in relation to lysine, however, the pattern of amino acid utilization for growth was similar for control and porcine somatotropin-treated pigs; exceptions were arginine, glycine and tryptophan. The ratio of indispensable to dispensable amino acids that were deposited was also similar for control and porcine somatotropin-treated pigs. These data indicate that the synthesis rate of individual proteins can be influenced by porcine somatotropin, but the balance of amino acids remains largely unaffected, suggesting that the changes in protein and amino acid metabolism elicited by porcine somatotropin are consistent with normal growth processes.

Assessment of individual protein turnover in three muscle types of rainbow trout

Protein synthesis rate was measured in three different muscle types which are ranked as follows: white skeletal muscle < red skeletal muscle < heart. The protein synthesis rates of mitochondrial and nuclear fractions were similar or slightly higher (white muscle) than those of whole tissue protein. The myofibrillar fraction accounted for 64%, 60% and 50% of the total protein in white muscle, red muscle and heart. The fractional rate of protein synthesis in the myofibrillar fraction is similar or slightly lower (red muscle) than that of whole muscle protein. Protein synthesis rates of individual proteins, myosin and actin, were similar to that of the myofibrillar fraction.

Synthesis of Proteins in Escherichia coli is Limited by the Concentration of Free Ribosomes : Expression from Reporter Genes does not always Reflect Functional mRNA Levels

Induction of β-galactosidase from high copy-number plasmids was found to reduce the synthesis of other cellular proteins in Escherichia coli. The reduction depends on the protein in question and on the induction level of the β-galactosidase. It could be observed transiently within one minute after induction and in some cases also during steady-state induction. Our interpretation is that the concentration of the free ribosomal subunits decreases after induction, leading to an increased competition among the individual ribosome binding sites for ribosomes. The immediate reduction in the synthesis rate of individual proteins after induction of β-galactosidase was used as an assay to measure in vivo the efficiency of a ribosome binding site. These efficiencies were compared to the calculated affinities between the ribosome binding site of specific mRNA species and the 3′ end of 16S RNA. For several mRNAs with similar Shine-Dalgarno sequences, the sensitivity to competition differed twofold. Our results show, that both transiently during induction of lacZ and also at very high steady-state expression levels, the expression from reporter genes, including the lacZ gene itself, does not reflect the levels of the mRNAs in a simple way.

In vitro synthesis and release of proteins by fat body and ovarian tissue of Leucophaea maderae during the sexual cycle

Fat body, ovaries without their surrounding connective tissue, and ovarian connective tissue of the ovoviviparous cockroach Leucophaea maderae were incubated in vitro. Incorporation of 14C-labelled amino acids into proteins by all three tissues was investigated quantitatively and qualitatively during oöcyte maturation (corpora allata active), immediately after ovulation, and during the gestation period (corpora allata inactive). Proteins in the tissues and in the incubation media were processed separately. The qualitative analysis involving disk electrophoretic separation of Ringer soluble tissue proteins or of proteins in the incubation media and subsequent autoradiography of the dried gels showed that all three tissues synthesize the same 26 proteins in all stages of the sexual cycle. An additional fraction is produced by the ovarian connective tissue only. All three tissues synthesize proteins at a higher rate during oöcyte maturation than during gestation. Tissue specificity was observed in the quantitative relations of the synthesized proteins and in the influence of hormones (juvenile hormone and neurosecretory factors) on the synthesis rates of individual proteins. New evidence supports the findings of Scheurer that the synthesis of some proteins is stimulated by juvenile hormone whereas others are influenced by neurosecretory factors. The synthesis of the female protein (22) is stimulated by juvenile hormone in different degrees in the three tissues during oöcyte maturation. At the same time juvenile hormone leads to an increased rate of release of this protein by fat body, but not by the two other tissues. In contrast to earlier findings the female specific protein was shown to be synthesized in small amounts by all three tissues also during gestation, when the corpora allata are supposedly inactive.