Yeast has evolved to minimize protein resource cost for synthesizing amino acids.

Proteins, as essential biomolecules, account for a large fraction of cell mass, and thus the synthesis of the complete set of proteins (i.e., the proteome) represents a substantial part of the cellular resource budget. Therefore, cells might be under selective pressures to optimize the resource costs for protein synthesis, particularly the biosynthesis of the 20 proteinogenic amino acids. Previous studies showed that less energetically costly amino acids are more abundant in the proteomes of bacteria that survive under energy-limited conditions, but the energy cost of synthesizing amino acids was reported to be weakly associated with the amino acid usage in Saccharomyces cerevisiae Here we present a modeling framework to estimate the protein cost of synthesizing each amino acid (i.e., the protein mass required for supporting one unit of amino acid biosynthetic flux) and the glucose cost (i.e., the glucose consumed per amino acid synthesized). We show that the logarithms of the relative abundances of amino acids in S. cerevisiae's proteome correlate well with the protein costs of synthesizing amino acids (Pearson's r = -0.89), which is better than that with the glucose costs (Pearson's r = -0.5). Therefore, we demonstrate that S. cerevisiae tends to minimize protein resource, rather than glucose or energy, for synthesizing amino acids.

A flow-cytometry-based assessment of global protein synthesis in human senescent cells.

Senescent cells constantly experience stressful conditions and restrain their protein translation to cope with it. Here, we present a detailed protocol to measure the rate of global protein synthesis using L-azidohomoalanine (L-AHA)-based click chemistry in human senescent fibroblasts. We optimized several aspects of the procedure, including senescence induction, a flow cytometry analysis of senescent cells, and the duration of L-AHA incorporation. This protocol uses senescent human fibroblasts but can be applied to other types of cells or circumstances. For complete details on the use and execution of this protocol, please refer to Lee et al. (2021).

Thermodynamic Cost, Speed, Fluctuations, and Error Reduction of Biological Copy Machines.

Due to large fluctuations in cellular environments, transfer of information in biological processes without regulation is error-prone. The mechanistic details of error-reducing mechanisms in biological copying processes have been a subject of active research; however, how error reduction of a process is balanced with its thermodynamic cost and dynamical properties remain largely unexplored. Here, we study the error reducing strategies in light of the recently discovered thermodynamic uncertainty relation (TUR) that sets a physical bound to the cost-precision trade-off for dissipative processes. We found that the two representative copying processes, DNA replication by the exonuclease-deficient T7 DNA polymerase and mRNA translation by the E. coli ribosome, reduce the error rates to biologically acceptable levels while also optimizing the processes close to the physical limit dictated by TUR.

Gene length as a regulator for ribosome recruitment and protein synthesis: theoretical insights.

Protein synthesis rates are determined, at the translational level, by properties of the transcript's sequence. The efficiency of an mRNA can be tuned by varying the ribosome binding sites controlling the recruitment of the ribosomes, or the codon usage establishing the speed of protein elongation. In this work we propose transcript length as a further key determinant of translation efficiency. Based on a physical model that considers the kinetics of ribosomes advancing on the mRNA and diffusing in its surrounding, as well as mRNA circularisation and ribosome drop-off, we explain how the transcript length may play a central role in establishing ribosome recruitment and the overall translation rate of an mRNA. According to our results, the proximity of the 3' end to the ribosomal recruitment site of the mRNA could induce a feedback in the translation process that would favour the recycling of ribosomes. We also demonstrate how this process may be involved in shaping the experimental ribosome density-gene length dependence. Finally, we argue that cells could exploit this mechanism to adjust and balance the usage of its ribosomal resources.

The Cost of Protein Production.

The economy of protein production is central to cell physiology, being intimately linked with cell division rate and cell size. Attempts to model cellular physiology are limited by the scarcity of experimental data defining the molecular processes limiting protein expression. Here, we distinguish the relative contribution of gene transcription and protein translation to the slower proliferation of budding yeast producing excess levels of unneeded proteins. In contrast to widely held assumptions, rapidly growing cells are not universally limited by ribosome content. Rather, transcription dominates cost under some conditions (e.g., low phosphate), translation in others (e.g., low nitrogen), and both in other conditions (e.g., rich media). Furthermore, cells adapted to enforced protein production by becoming larger and increasing their endogenous protein levels, suggesting limited competition for common resources. We propose that rapidly growing cells do not exhaust their resources to maximize growth but maintain sufficient reserves to accommodate changing requirements.

Assessment of mTOR-Dependent Translational Regulation of Interferon Stimulated Genes.

Type-I interferon (IFN)-induced activation of the mammalian target of rapamycin (mTOR) signaling pathway has been implicated in translational control of mRNAs encoding interferon-stimulated genes (ISGs). However, mTOR-sensitive translatomes commonly include mRNAs with a 5' terminal oligopyrimidine tract (TOP), such as those encoding ribosomal proteins, but not ISGs. Because these translatomes were obtained under conditions when ISG expression is not induced, we examined the mTOR-sensitive translatome in human WISH cells stimulated with IFN ß. The mTOR inhibitor Torin1 resulted in a repression of global protein synthesis, including that of ISG products, and translation of all but 3 ISG mRNAs (TLR3, NT5C3A, and RNF19B) was not selectively more sensitive to mTOR inhibition. Detailed studies of NT5C3A revealed an IFN-induced change in transcription start site resulting in a switch from a non-TOP to a TOP-like transcript variant and mTOR sensitive translation. Thus, we show that, in the cell model used, translation of the vast majority of ISG mRNAs is not selectively sensitive to mTOR activity and describe an uncharacterized mechanism wherein the 5'-UTR of an mRNA is altered in response to a cytokine, resulting in a shift from mTOR-insensitive to mTOR-sensitive translation.

Dystropathology increases energy expenditure and protein turnover in the mdx mouse model of duchenne muscular dystrophy.

The skeletal muscles in Duchenne muscular dystrophy and the mdx mouse model lack functional dystrophin and undergo repeated bouts of necrosis, regeneration, and growth. These processes have a high metabolic cost. However, the consequences for whole body energy and protein metabolism, and on the dietary requirements for these macronutrients at different stages of the disease, are not well-understood. This study used juvenile (4- to 5- wk-old) and adult (12- to 14-wk-old) male dystrophic C57BL/10ScSn-mdx/J and age-matched C57BL/10ScSn/J control male mice to measure total and resting energy expenditure, food intake, spontaneous activity, body composition, whole body protein turnover, and muscle protein synthesis rates. In juvenile mdx mice that have extensive muscle damage, energy expenditure, muscle protein synthesis, and whole body protein turnover rates were higher than in age-matched controls. Adaptations in food intake and decreased activity were insufficient to meet the increased energy and protein needs of juvenile mdx mice and resulted in stunted growth. In (non-growing) adult mdx mice with less severe dystropathology, energy expenditure, muscle protein synthesis, and whole body protein turnover rates were also higher than in age-matched controls. Food intake was sufficient to meet their protein and energy needs, but insufficient to result in fat deposition. These data show that dystropathology impacts the protein and energy needs of mdx mice and that tailored dietary interventions are necessary to redress this imbalance. If not met, the resultant imbalance blunts growth, and may limit the benefits of therapies designed to protect and repair dystrophic muscles.

A traffic flow model for bio-polymerization processes.

Bio-polymerization processes like transcription and translation are central to proper function of a cell. The speed at which the bio-polymer grows is affected both by the number of pauses of elongation machinery, as well the number of bio-polymers due to crowding effects. In order to quantify these effects in fast transcribing ribosome genes, we rigorously show that a classical traffic flow model is the limit of a mean occupancy ODE model. We compare the simulation of this model to a stochastic model and evaluate the combined effect of the polymerase density and the existence of pauses on the instantaneous transcription rate of ribosomal genes.

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.

Nutrient signaling in protein homeostasis: an increase in quantity at the expense of quality.

The discovery that rapamycin extends the life span of diverse organisms has triggered many studies aimed at identifying the underlying molecular mechanisms. Mammalian target of rapamycin complex 1 (mTORC1) regulates cell growth and may regulate organismal aging by controlling mRNA translation. However, how inhibiting mTORC1 and decreasing protein synthesis can extend life span remains an unresolved issue. We showed that constitutively active mTORC1 signaling increased general protein synthesis but unexpectedly reduced the quality of newly synthesized polypeptides. We demonstrated that constitutively active mTORC1 decreased translation fidelity by increasing the speed of ribosomal elongation. Conversely, rapamycin treatment restored the quality of newly synthesized polypeptides mainly by slowing the rate of ribosomal elongation. We also found distinct roles for mTORC1 downstream targets in maintaining protein homeostasis. Loss of S6 kinases, but not 4E-BP family proteins, which are both involved in regulation of translation, attenuated the effects of rapamycin on the quality of newly translated proteins. Our results reveal a mechanistic connection between mTORC1 and protein quality, highlighting the central role of nutrient signaling in growth and aging.

Arc mRNA docks precisely at the base of individual dendritic spines indicating the existence of a specialized microdomain for synapse-specific mRNA translation.

Arc (aka Arg 3.1) is induced by neural activity and learning experience. Arc mRNA is rapidly exported into dendrites where it localizes near activated synapses. By imaging green fluorescent protein (GFP)-tagged mRNA in living neurons in culture, we show that fusion transcripts containing the Arc 30'UTR (untranslated region) localize with remarkable precision in a microdomain at the base of dendritic spines. Transcripts with the Arc 30'UTR that encode a reporter protein rather than Arc show precise localization. Localization persists in the presence of translation inhibitors, indicating that localization does not require ongoing translation. Similarly, polyribosome complexes remained stably positioned at spine bases in brain tissue treated with the translation inhibitor (puromycin) that releases ribosomes from mRNA. Single particle tracking revealed that Arc mRNA particles positioned at spine bases exhibited highly constrained submicron movements. These observations imply the existence of a microdomain at the spine base where Arc mRNA docks in association with a previously unknown mRNA-binding structural element.

Hypercapnia induced shifts in gill energy budgets of Antarctic notothenioids.

Mechanisms responsive to hypercapnia (elevated CO(2) concentrations) and shaping branchial energy turnover were investigated in isolated perfused gills of two Antarctic Notothenioids (Gobionotothen gibberifrons, Notothenia coriiceps). Branchial oxygen consumption was measured under normo- versus hypercapnic conditions (10,000 ppm CO(2)) at high extracellular pH values. The fractional costs of ion regulation, protein and RNA synthesis in the energy budgets were determined using specific inhibitors. Overall gill energy turnover was maintained under pH compensated hypercapnia in both Antarctic species as well as in a temperate zoarcid (Zoarces viviparus). However, fractional energy consumption by the examined processes rose drastically in G. gibberifrons (100-180%), and to a lesser extent in N. coriiceps gills (7-56%). In conclusion, high CO(2) concentrations under conditions of compensated acidosis induce cost increments in epithelial processes, however, at maintained overall rates of branchial energy turnover.

Lack of adequate appreciation of physical exercise's complexities can pre-empt appropriate design and interpretation in scientific discovery.

Two major issues are presented. First, a challenge is made by us that a misunderstanding of physiology has led to incomplete or wrong functional designations of genes in some cases. Normal physiological processes are dynamic, integrated and periodic, and, therefore, it is difficult to define normal physiological function by looking at a single time point or single process in a non-stressed subject. The ability of the organism to successfully respond to homeostatic disruptions defines normal physiology. Genes were selected for survival and to appropriately respond to stresses, such as physical activity. Omitting gene functions by restricting them to non-stressful conditions could lead to less than optimal primary preventions, treatments and cures for diseases. Physical exercise, as a stressor, should be used to better demonstrate the complete functional classifications of some genes. Second, the challenge from others of an 'exercise pill' as a mimetic of natural physical activity will be shown to be lacking a scientific basis. The concept of an 'exercise pill'/'exercise mimetic' demonstrates an inadequate appreciation of the complexities in integrating cell, tissue, organ and systems during both acute disruptions in homeostasis by a single bout of exercise, and longer-term chronic adaptations to different types of exercise such as resistance and endurance. It is our opinion that those promoting drugs targeting a single or few molecules should not redefine the term 'exercise' and exercise concepts in an attempt to sensationalize findings. Additionally, the scientific criteria that the authors demand to be met to legitimately use the terms 'exercise pill' and 'exercise mimetic' are presented.

Regulatory control and the costs and benefits of biochemical noise.

Experiments in recent years have vividly demonstrated that gene expression can be highly stochastic. How protein concentration fluctuations affect the growth rate of a population of cells is, however, a wide-open question. We present a mathematical model that makes it possible to quantify the effect of protein concentration fluctuations on the growth rate of a population of genetically identical cells. The model predicts that the population's growth rate depends on how the growth rate of a single cell varies with protein concentration, the variance of the protein concentration fluctuations, and the correlation time of these fluctuations. The model also predicts that when the average concentration of a protein is close to the value that maximizes the growth rate, fluctuations in its concentration always reduce the growth rate. However, when the average protein concentration deviates sufficiently from the optimal level, fluctuations can enhance the growth rate of the population, even when the growth rate of a cell depends linearly on the protein concentration. The model also shows that the ensemble or population average of a quantity, such as the average protein expression level or its variance, is in general not equal to its time average as obtained from tracing a single cell and its descendants. We apply our model to perform a cost-benefit analysis of gene regulatory control. Our analysis predicts that the optimal expression level of a gene regulatory protein is determined by the trade-off between the cost of synthesizing the regulatory protein and the benefit of minimizing the fluctuations in the expression of its target gene. We discuss possible experiments that could test our predictions.

Energetic cost of synthesizing proteins in Antarctic limpet, Nacella concinna (Strebel, 1908), is not temperature dependent.

The energetic cost of protein synthesis is thought to account for a significant proportion of total metabolism. However, attempts to estimate the energetic cost of synthesizing proteins have resulted in surprisingly variable results, particularly for the small number of polar organisms studied, where cost estimates vary by two orders of magnitude. Much of this variability is probably the result of differing methodologies and experimental designs. Here we have used two different, carefully validated methods to measure the costs of protein synthesis in Antarctic limpets. One method, which utilized a specific protein synthesis inhibitor, was used to measure the cost of protein synthesis at two temperatures to test the hypothesis that the cost of protein synthesis varies with temperature. The cost of protein synthesis measured using the inhibitor cycloheximide was 13.95 +/- 0.77 micromol O2/mg protein, while correlation of absolute protein synthesis with oxygen consumption suggested the cost of protein synthesis was 19.58 micromol O2/mg protein. Water temperature did not alter the cost of protein synthesis in Nacella concinna (Student's t-test, P = 0.849, t = 0.19, df = 12). In a meta-analysis of literature values for the cost of protein synthesis there was no significant effect of temperature, but there was a significant relationship between the concentration of cycloheximide used to inhibit protein synthesis and the measured cost.

Cost of protein synthesis and energy allocation during development of antarctic sea urchin embryos and larvae.

Cold environments represent a substantial volume of the biosphere. To study developmental physiology in subzero seawater temperatures typically found in the Southern Ocean, rates and costs of protein synthesis were measured in embryos and larvae of Sterechinus neumayeri, the Antarctic sea urchin. Our analysis of the "cost of living" in extreme cold for this species shows (1) that cost of protein synthesis is strikingly low during development, at 0.41 +/- 0.05 J (mg protein synthesized)(-1) (n = 16); (2) that synthesis cost is fixed and independent of synthesis rate; and (3) that a low synthesis cost permits high rates of protein turnover at -1 degrees C, at rates comparable to those of temperate species of sea urchin embryos developing at 15 degrees C. With a low synthesis cost, even at the highest synthesis rates measured (gastrulae), the proportion of total metabolism accounted for by protein synthesis in the Antarctic sea urchin was 54%-a value similar to that of temperate sea urchin embryos. In the Antarctic sea urchin, up to 87% of metabolic rate can be accounted for by the combined energy costs of protein synthesis and the sodium pump. We conclude that, in Antarctic sea urchin embryos, high rates of protein synthesis can be supported in extreme-cold environments while still maintaining low rates of respiration.

Foreign protein degradation and instability in plants and plant tissue cultures.

Low production cost is a key factor driving the development of plants and plant tissue cultures for the synthesis of therapeutic and other foreign proteins. Because product yield and concentration exert a major influence on process economics, improving foreign protein accumulation is crucial for enhancing the commercial success of plant-based production systems. Strategies aimed at increasing transgene expression have been effective; however, a critical but poorly understood factor contributing to low foreign protein yield is post-synthesis and/or post-secretion instability and degradation. Loss of foreign protein as result of biological and physical processes such as proteolytic destruction and irreversible surface adsorption can occur in plants and plant culture systems. This review highlights the need to consider such mechanisms and outlines a range of remedial strategies aimed at minimizing foreign protein degradation and loss.