Tissue-specific Protein Synthesis Research Articles

Trabecular, but not cortical, bone tissue protein synthesis rates are lower in the femoral head when compared to the proximal femur following an intracapsular hip fracture.

All musculoskeletal tissues are in a constant state of turnover, with a dynamic equilibrium between tissue protein synthesis and breakdown rates. The synthesis of protein allows musculoskeletal tissues to heal following injury. Yet, impaired tissue healing is observed following certain injuries, such as geriatric hip fractures. It is assumed that the regenerative properties of femoral head bone tissue are compromised following an intracapsular hip fracture and therefore hip replacement surgery is normally performed. However, the actual impact on in vivo bone protein synthesis rates has never been determined. In the present study, 10 patients (age: 79±10 y, BMI: 24±4kg/m2) with an acute (<24h) intracapsular hip fracture received a primed continuous intravenous infusion of L-[ring-13C6]-phenylalanine before and throughout their hip replacement surgery. Trabecular and cortical bone tissue from both the femoral head and proximal femur were sampled during surgery to assess protein synthesis rates of affected (femoral head) and unaffected (proximal femur) bone tissue, respectively. In addition, tissue samples of gluteus maximus muscle, synovium, ligamentum teres, and femoral head cartilage were collected. Tissue-specific protein synthesis rates were assessed by measuring L-[ring-13C6]-phenylalanine incorporation in tissue protein. Femoral head trabecular bone protein synthesis rates (0.056 [0.024-0.086] %/h) were lower when compared to proximal femur trabecular bone protein synthesis rates (0.081 [0.056-0.118] %/h; P=0.043). Cortical bone protein synthesis rates did not differ between the femoral head and proximal femur (0.041 [0.021-0.078] and 0.045 [0.028-0.073] %/h, respectively; P>0.05). Skeletal muscle, synovium, ligamentum teres, and femoral head cartilage protein synthesis rates averaged 0.080 [0.048-0.089], 0.093 [0.051-0.130], 0.121 [0.110-0.167], and 0.023 [0.015-0.039] %/h, respectively. In contrast to the general assumption that the femoral head is avital after an intracapsular displaced hip fracture in the elderly, our data show that bone protein synthesis is still ongoing in femoral head bone tissue during the early stages following an intracapsular hip fracture in older patients. Nonetheless, trabecular bone protein synthesis rates are lower in the femoral head when compared to the proximal femur in older patients following an acute intracapsular hip fracture. Trial register no: NL9036.

Using protein-per-mRNA differences among human tissues in codon optimization

BackgroundCodon usage and nucleotide composition of coding sequences have profound effects on protein expression. However, while it is recognized that different tissues have distinct tRNA profiles and codon usages in their transcriptomes, the effect of tissue-specific codon optimality on protein synthesis remains elusive.ResultsWe leverage existing state-of-the-art transcriptomics and proteomics datasets from the GTEx project and the Human Protein Atlas to compute the protein-to-mRNA ratios of 36 human tissues. Using this as a proxy of translational efficiency, we build a machine learning model that identifies codons enriched or depleted in specific tissues. We detect two clusters of tissues with an opposite pattern of codon preferences. We then use these identified patterns for the development of CUSTOM, a codon optimizer algorithm which suggests a synonymous codon design in order to optimize protein production in a tissue-specific manner. In human cell-line models, we provide evidence that codon optimization should take into account particularities of the translational machinery of the tissues in which the target proteins are expressed and that our approach can design genes with tissue-optimized expression profiles.ConclusionsWe provide proof-of-concept evidence that codon preferences exist in tissue-specific protein synthesis and demonstrate its application to synthetic gene design. We show that CUSTOM can be of benefit in biological and biotechnological applications, such as in the design of tissue-targeted therapies and vaccines.

Lysine Dipeptide Enhances Gut Structure and Whole-Body Protein Synthesis in Neonatal Piglets with Intestinal Atrophy

BackgroundParenteral nutrition (PN) is often a necessity for preterm infants; however, prolonged PN leads to gut atrophy, weakened gut barrier function, and a higher risk of intestinal infections. Peptide transporter-1 (PepT1) is a di- or tripeptide transporter in the gut and, unlike other nutrient transporters, its activity is preserved with the onset of intestinal atrophy from PN. As such, enteral amino acids in the form of dipeptides may be more bioavailable than free amino acids when atrophy is present. ObjectivesIn Yucatan miniature piglets with PN-induced intestinal atrophy, we sought to determine the structural and functional effects of enteral refeeding with lysine as a dipeptide, compared to free L-lysine. MethodsPiglets aged 7-8 days were PN-fed for 4 days to induce intestinal atrophy, then were refed with enteral diets with equimolar lysine supplied as lysyl-lysine (Lys-Lys; n = 7), free lysine (n = 7), or Lys-Lys with glycyl-sarcosine (n = 6; to determine whether competitive inhibition of Lys-Lys uptake would abolish PepT1-mediated effects). The diets provided lysine at 75% of the requirement and were gastrically delivered for a total of 18 hours. Whole-body and tissue-specific protein synthesis, as well as indices for gut structure and barrier function, were measured. ResultsThe villus height, mucosal weight, and free lysine concentration were higher in the Lys-Lys group compared to the other 2 groups (P < 0.05). Lysyl-lysine led to greater whole-body protein synthesis compared to free lysine (P < 0.05). Mucosal myeloperoxidase activity was lower in the Lys-Lys group (P < 0.05), suggesting less inflammation. The inclusion of glycyl-sarcosine with Lys-Lys abolished the dipeptide effects on whole-body and tissue-specific protein synthesis (P < 0.05), suggesting that improved lysine availability was mediated by PepT1. ConclusionsImproved intestinal structure and whole-body protein synthesis suggests that feeding strategies designed to exploit PepT1 may help to avoid adverse effects when enteral nutrition is reintroduced into the compromised guts of neonatal piglets.

537 Animal Growth and Development—A Scorecard and Recalibration

Abstract The ASAS Public Policy Committee (PPC) provides updates of Grand Challenges (GCs; www.asas.org/about/public-policy/asas-grand-challenges) to clearly articulate research priorities, to provide science-based information for shaping public policy, and to enhance future funding for research and education programs in animal sciences (AS). In this nexus symposium for 2021, PPC examines previous stated priorities and provides a progress report card and offers additional perspectives and recommendations for research needed to address some of the GCs continuing to face animal agriculture. Among the GC topics is growth and development, a rather broad field of inquiry focused on improving the overall growth efficiency of meat producing animals. The genesis of this discipline and its popularity grew mainly in response to heightened efforts by pharmaceutical companies to identify, develop and adopt novel new growth promotants. This included a myriad of work on the highly heralded technologies involving estrogenic and androgenic implants, somatotropin, and beta-adrenergic agonists. Because the potential application of these technologies was so broad, many disciplines within the animal sciences became involved in the process of creating knowledge around these drivers of productivity. In the process, our understanding of how tissues grow in response to these compounds, under a myriad of other conditions, and our fundamental understanding of the molecular and cellular mechanisms regulating growth and development, expanded significantly. Areas of significant expansion included but were not restricted to: satellite cell biology and myogenesis, whole body and tissue-specific protein synthesis and degradation, growth factor biology, adipogenesis, and repartitioning of nutrients throughout the body. In our quest to increase productivity and product quality, coupled with advances in scientific techniques, long-existing and emerging genetic mutations with desirable traits were studied and mechanisms undergirding their biology began to develop. Applying the most innovative tools for the detailed manipulation of cellular processes, great strides were made during this time. However, this eclectic area of investigation is perhaps more important than ever given the inevitable replacement of growth promotant technologies with new and emerging genomic technologies. Many biological research challenges lie ahead such as applications of gene editing, RNA control, and epigenetic regulation through fetal programming. This presentation will review some of the significant advances made in the growth and development area and explore where significant gains may be possible in the future.

Dietary Methyl Donors Contribute to Whole-Body Protein Turnover and Protein Synthesis in Skeletal Muscle and the Jejunum in Neonatal Piglets

Background: The neonatal methionine requirement must consider not only the high demand for rapid tissue protein expansion but also the demands as the precursor for a suite of critical transmethylation reactions. However, methionine metabolism is inherently complex because upon transferring its methyl group during transmethylation, methionine can be reformed by the dietary methyl donors choline (via betaine) and folate.Objective: We sought to determine whether dietary methyl donors contribute to methionine availability for protein synthesis in neonatal piglets.Methods: Yucatan miniature piglets aged 4–8 d were fed a diet that provided 38 μg folate/(kg·d), 60 mg choline/(kg·d), and 238 mg betaine/(kg·d) [methyl-sufficient (MS); n = 8] or a diet devoid of these methyl precursors [methyl-deficient (MD); n = 8]. After 5 d, dietary methionine was reduced from 0.30 to 0.20 g/(kg·d) in both groups. On day 6, piglets received a constant [1-13C]phenylalanine infusion to measure whole-body protein kinetics, and on day 8 they received a constant [3H-methyl]methionine infusion to measure tissue-specific protein synthesis in skeletal muscle, the liver, and the jejunum.Results: Whole-body phenylalanine flux, protein synthesis, and protein breakdown were 13%, 12%, and 22% lower, respectively, in the MD group than in the MS group (P < 0.05). Reduced whole-body protein synthesis in the MD piglets was attributed to 50% lower protein synthesis in skeletal muscle and the jejunum than in the MS piglets (P < 0.05). Furthermore, methionine availability in skeletal muscle was halved in piglets fed the MD diet (P < 0.05), and the specific radioactivity of methionine was doubled in the jejunum of MD piglets (P < 0.05), suggesting lower intestinal remethylation. Liver protein synthesis did not significantly differ between the groups, but secreted proteins were not measured.Conclusions: Dietary methyl donors can affect whole-body and tissue-specific protein synthesis in neonatal piglets and should be considered when determining the methionine requirement.

Leucine and Protein Metabolism in Obese Zucker Rats

Branched-chain amino acids (BCAAs) are circulating nutrient signals for protein accretion, however, they increase in obesity and elevations appear to be prognostic of diabetes. To understand the mechanisms whereby obesity affects BCAAs and protein metabolism, we employed metabolomics and measured rates of [1-14C]-leucine metabolism, tissue-specific protein synthesis and branched-chain keto-acid (BCKA) dehydrogenase complex (BCKDC) activities. Male obese Zucker rats (11-weeks old) had increased body weight (BW, 53%), liver (107%) and fat (∼300%), but lower plantaris and gastrocnemius masses (−21–24%). Plasma BCAAs and BCKAs were elevated 45–69% and ∼100%, respectively, in obese rats. Processes facilitating these rises appeared to include increased dietary intake (23%), leucine (Leu) turnover and proteolysis [35% per g fat free mass (FFM), urinary markers of proteolysis: 3-methylhistidine (183%) and 4-hydroxyproline (766%)] and decreased BCKDC per g kidney, heart, gastrocnemius and liver (−47–66%). A process disposing of circulating BCAAs, protein synthesis, was increased 23–29% by obesity in whole-body (FFM corrected), gastrocnemius and liver. Despite the observed decreases in BCKDC activities per gm tissue, rates of whole-body Leu oxidation in obese rats were 22% and 59% higher normalized to BW and FFM, respectively. Consistently, urinary concentrations of eight BCAA catabolism-derived acylcarnitines were also elevated. The unexpected increase in BCAA oxidation may be due to a substrate effect in liver. Supporting this idea, BCKAs were elevated more in liver (193–418%) than plasma or muscle, and per g losses of hepatic BCKDC activities were completely offset by increased liver mass, in contrast to other tissues. In summary, our results indicate that plasma BCKAs may represent a more sensitive metabolic signature for obesity than BCAAs. Processes supporting elevated BCAA]BCKAs in the obese Zucker rat include increased dietary intake, Leu and protein turnover along with impaired BCKDC activity. Elevated BCAAs/BCKAs may contribute to observed elevations in protein synthesis and BCAA oxidation.

Proline Supplementation to Parenteral Nutrition Results in Greater Rates of Protein Synthesis in the Muscle, Skin, and Small Intestine in Neonatal Yucatan Miniature Piglets1–3

Proline and arginine are each indispensable during parenteral feeding due to limited interconversion by an atrophied gut. Commercial amino acid parenteral products designed for neonates contain proline concentrations that differ by almost 4-fold. To assess the adequacy of the lowest concentration of proline provided in commercial total parenteral nutrition (TPN) products, we compared rates of tissue-specific protein synthesis and nitrogen balance in neonatal piglets provided TPN at 2 different proline concentrations. Yucatan miniature piglets (9–11 d old, n = 12) were randomized to complete isonitrogenous TPN diets with low proline (LP; L-proline as 3% of amino acids) or proline supplemented (PS; 9%). After 7 d of receiving TPN, rates of protein synthesis in liver, gastrocnemius muscle, jejunal mucosa, and skin were determined by the flooding dose technique and tissue free amino acids were measured. Nitrogen balance was assessed during the last 3 d. The LP TPN resulted in lower free proline concentrations in plasma, muscle, and skin (P < 0.05) and lower rates of protein synthesis in the jejunum (by 25%; P = 0.02), muscle (by 45%; P = 0.015), and skin (by 60%; P = 0.01); there was no difference in liver. Nitrogen retention was 20% lower in the LP group (P = 0.01). In conclusion, muscle and skin protein synthesis was profoundly sensitive to parenteral proline supply and the reduced protein synthesis in the intestine could affect intestinal integrity. Low-proline TPN solutions that are currently in wide use in neonatal care may result in impaired tissue growth.

Carbon Turnover in Tissues of a Passerine Bird: Allometry, Isotopic Clocks, and Phenotypic Flexibility in Organ Size

Stable isotopes are an important tool for physiological and behavioral ecologists, although their usefulness depends on a thorough understanding of the dynamics of isotope incorporation into tissue(s) over time. In contrast to hair, claws, and feathers, most animal tissues continuously incorporate carbon (and other elements), and so carbon isotope values may change over time, depending on resource use and tissue-specific metabolic rates. Here we report the carbon turnover rate for 12 tissues from a passerine bird, the zebra finch (Taeniopygia guttata). We measured average carbon retention time (tau) for 8 d for small intestine; 10-13 d for gizzard, kidney, liver, pancreas, and proventriculus; 17-21 d for heart, brain, blood, and flight muscle; and 26-28 d for leg muscle and skin. We used these data, along with the few other published estimates, to confirm that the fractional rate of isotopic turnover for red blood cells, whole blood, liver, and leg muscle scales with body mass to approximately the -1/4 power. Our data also support several key assumptions of the "isotopic-clock" model, which uses differences in isotope value between tissues, along with estimates of turnover rate of these tissues, to predict time elapsed since a diet shift. Finally, we show that between-tissues differences in turnover rate largely, but not entirely, explain the extent of phenotypic flexibility in organs of garden warblers during their long-distance flight across the Sahara Desert during spring. More studies that measure tissue-specific protein synthesis, metabolic rate, and elemental turnover in many tissues from a variety of animals are needed.

Diet-induced obesity alters protein synthesis: tissue-specific effects in fasted versus fed mice

The influence of obesity on protein dynamics is not clearly understood. We have designed experiments to test the hypothesis that obesity impairs the stimulation of tissue-specific protein synthesis after nutrient ingestion. C57BL/6J mice were randomized into 2 groups: group 1 (control, n = 16) was fed a low-fat, high-carbohydrate diet, whereas group 2 (experimental, n = 16) was fed a high-fat, low-carbohydrate diet ad libitum for 9 weeks. On the experiment day, all mice were fasted for 6 hours and given an intraperitoneal injection of 2H 2O. They were then randomized into 2 subgroups and either given a sham saline gavage or a liquid-meal challenge. Rates of protein synthesis were determined via the incorporation of [ 2H]alanine (5 hours postchallenge) into total gastrocnemius muscle protein, total liver protein, and plasma albumin. High-fat feeding led to an increase in total body fat ( P < .001) and epididymal fat pad weights ( P < .001) and elevated fasting plasma glucose levels ( P < .01). Diet-induced obesity ( a) did not affect basal rates of skeletal muscle protein synthesis, but did impair the activation of skeletal muscle protein synthesis in response to nutrient ingestion ( P < .05), and ( b) slightly reduced basal rates of synthesis of total hepatic proteins and plasma albumin ( P = .10), but did not affect the synthesis of either in response to the meal challenge. In conclusion, there are alterations in tissue-specific protein metabolism in the C57BL/6J mouse model of diet-induced obesity. This model may prove to be helpful in future studies that explore the mechanisms that account for altered protein dynamics in obesity.

Mechanisms underlying geroprotective effects of peptides.

We review the role of peptides in aging and the mechanisms underlying the geroprotective effect of peptide preparations. Geroprotective properties of peptides are associated with their influence on systems maintaining homeostasis in the body and regulation of mechanisms underlying aging. Peptides normalize synthesis of tissue-specific proteins and regulate expression of genes responsible for proliferation and differentiation of cells. Thus, peptides maintain normal physiological functions and decelerate aging.

Modulation of milk protein synthesis through alteration of the cytoskeleton in mouse mammary epithelial cells cultured on a reconstituted basement membrane

Recent studies indicate that the cytoskeleton may be involved in modulating tissue-specific gene expression in mammalian cells. We have studied the role of the cytoskeleton in regulating milk protein synthesis and secretion by primary mouse mammary epithelial cells cultured on a reconstituted basement membrane that promotes differentiation. After 8 days in culture, cells were treated with cytochalasin D (CD) (0.5-1 micrograms/ml) to alter actin filaments or acrylamide (Ac) (5 mM) to alter intermediate filaments (cytokeratins). CD inhibited synthesis of most proteins in a concentration-dependent manner, with beta-casein being the first affected. In contrast, Ac increased protein synthesis and secretion by 17-31% after a 12 hr treatment. Polyacrylamide gel electrophoresis of total secreted proteins indicates that synthetic rates of most proteins were increased equally by Ac treatment. This increase is apparently controlled at the level of translation, because control and Ac-treated cells contained the same amount of poly-A+ RNA, and neither CD nor Ac altered mRNA levels for beta-casein. There was also no indication that either CD or Ac can induce the expression of milk proteins in quiescent cells cultured on a plastic substratum. In conjunction with the biochemical studies, changes in cytoskeletal morphology caused by the drug treatments were analyzed by immunofluorescence microscopy. As has been observed in other cell types, low concentrations of CD caused cells to round up by disrupting actin filaments. Ac treatment slightly decreased the intensity of actin staining, but no changes in microfilament organization were observed. Ac-treated cells showed slight disorganization of the cytokeratin filaments, with some peripheral interfibrillar bundling, but the cytokeratin network did not collapse and no retraction of cell extensions or breakdown of cell-cell contacts was observed. These results confirm previous reports that the actin cytoskeleton may play a role in regulating tissue-specific protein synthesis. How Ac stimulates protein synthesis is unknown, but it is unlikely that this effect is directly mediated through intermediate filaments.

Epidermal growth factor inhibits thyrotropin-mediated synthesis of tissue-specific proteins in cultured ovine thyroid cells

Primary cultures of ovine thyroid cells were induced to differentiate by addition of thyrotropin (TSH). This was demonstrated as an accumulation of 2 thyroid-specific proteins, thyroglobulin and thyroid peroxidase, using immunofluorescent staining methods and immunoprecipitation of biosynthetically labeled cultures. As an additional measure of differentiation, cells exhibited a morphological response to TSH and regained the ability to incorporate radioactive iodide. Epidermal growth factor (EGF) markedly inhibited differentiation when added together with TSH. Thyroglobulin synthesis was reduced to low levels and peroxidase synthesis was reduced to levels that were undetectable by the methods used. Morphological changes in response to TSH were also diminished by EGF. The antagonistic interaction between TSH and EGF in regulating differentiation in cultured thyroid cells may reflect the type of control that exists in vivo.

DNA synthesis in Rana catesbeiana tadpole liver during spontaneous and triiodothyronine-induced metamorphosis

The incorporation of 3H-thymidine into the DNA of Rana catesbeiana tadpole liver was studied in vivo during spontaneous and 3,5,3′-triiodothyronine (T 3)-induced metamorphosis. Some early macromolecular responses occurring in the liver of T 3-treated tadpoles were elucidated by an improved technique for the fractionation of tadpole liver organelles. There is a transitory stimulation in the rate of 3H-thymidine and 14C-lysine incorporation into nuclear DNA and protein occurring 2–4 hours after T 3 administration. An enhanced rate of 3H-thymidine incorporation into liver mitochondrial DNA began 1 hr after hormone treatment of the tadpole and persisted for 3 days; thereafter the rate of incorporation declined to the control level. An increased rate of 3H-thymidine incorporation into tadpole liver nuclear DNA occurs 4 days after T 3 treatment and between stages XVII and XX in spontaneous metamorphosis. An increase of the thymine components in the acid-soluble pool of the liver coincides with the increased rate of 3H-thymidine incorporation. In both spontaneous and T 3-induced metamorphosis, the synthesis of tissue-specific proteins is preceded by, or coincides with, the enhanced nuclear DNA biosynthesis.

Growth and cytodifferentiation of embryonic chick lens epithelial cells in vitro

Cytodifferentiation, mitotic activity, tritiated thymidine binding and cell migration were studied in 297 6-day embryonic chick lens epithelia explanted into defined medium alone (UM) or into defined medium supplemented with serum (SM). Lens epithelial cells explanted into UM alone did not undergo significant cytodifferentiation nor cellular proliferation. Cells explanted directly into SM rapidly differentiated into short prospective lens fibers and failed to show significant proliferative activity. If the lens epithelial cells were precultured for 12 to 24 h in UM before transfer to SM, the cells rapidly lost their capacity to differentiate under these experimental conditions. As the ability to form short fibers was lost, the explanted cells showed greatly enhanced mitotic and migratory activity when stimulated by the addition of serum containing medium. The possibility is discussed that the rapidly lost capacity of lens epithelial cells to form fibers is due to the loss or inactivation of a cell component or components involved in the synthesis of tissue-specific proteins, and, being unable to differentiate under these experimental conditions, the cells are stimulated to proliferate and to migrate by the addition of serum.

Molecular aspects of lens cell differentiation.

I have presented a series of observations on macromolecular interactions which occur during the terminal stages of lens cell differentiation. These are summarized in Fig. 2. Other cell types that undergo similar changes are the erythrocyte and skin cells (epidermis) during the process of keratinization. These other cells are also involved in the synthesis of highly specific proteins, and there are indications that molecular alterations similar to those described for the lens may also occur in these cells (26). Thus, elucidation of a specific series of macromolecular initeractions such as those described may provide a basis for the biochemical definition of the terminal stages of cellular differentiation. Differentiation of the reticulocyte, for example, involves inactivation of the nucleus, stabilization of mRNA, and possibly a ribosomal breakdown such as I have described here (26). Furthermore, elucidation of the mechanisms of reactions involving the initiation of tissue-specific protein synthesis and suLbsequent nuclear inactivation, stabilization of mRNA, and breakdown of the ribosomes may provide a basis for defining the mechanisms of terminal cellular differentiation. The lens cell has reached its highest form of cellular differentiation when it has formed the fiber cell. With respect to the mechanism of lens fiber cell formation, we would like to know whether specific biochemical changes such as gamma-crystallin synthesis are intiniately linked to fiber cell formation-that is, whether gamma-crystallins are required to bring about the formation of a fiber cell. The potential for synthesizing gamma-crystallins is inherent in the genome of the cell. This part of the genome is nonfunctional in the epithelial cell. Can these genes be activated without bringing about a simultaneous cellular elongation, nuclear inactivation and loss of cellular replication, stabilization of mRNA, and breakdown of the ribosomes? The degree of coupling or uncoupling of tissue-specific-protein synthesis to morphogenesis is an important part of the mechanism of cellular differentiation. We feel that we have now reached the stage where we can begin to answer these questions.

Structural Organization and Embryonic Differentiation

Publisher Summary In the development of a living organism complexity arises in the form of cellular differentiation. Cellular differentiation involves biochemical events which bring about synthesis of tissue-specific proteins, such as cellular proteins or enzymic complements characterizing the cell types. The differential activity of the genes, which is responsible for changing the character of different parts of the embryo, is itself a result of a series of interactions between the genetic material and the cytoplasmic environment. Thus primary proteins which may be formed interacts with the genes; this interaction leads to further differentiation of that region. This means, from the time of the initial interactions, there occur a need for restriction of certain substances or biosynthetic activities to particular regions of the embryo. This chapter focuses on structural organization and embryonic differentiation, and discusses the structural changes during neural tissue differentiation, structural changes during differentiation of lens, and structural changes during differentiation of other tissues.