Impact of oxidative stress on the function,abundance, and turnover of the Arabidopsis 80S cytosolic ribosome

Abiotic stress in plants causes accumulation of reactive oxygen species (ROS) leading to the need for new protein synthesis to defend against ROS and to replace existing proteins that are damaged by oxidation. Functional plant ribosomes are critical for these activities, however we know little about the impact of oxidative stress on plant ribosome abundance, turnover, and function. Using Arabidopsis cell culture as a model system, we induced oxidative stress using 1 µm of H₂O₂ or 5 µm menadione to more than halve cell growth rate and limit total protein content. We show that ribosome content on a total cell protein basis decreased in oxidatively stressed cells. However, overall protein synthesis rates on a ribosome abundance basis showed the resident ribosomes retained their function in oxidatively stressed cells. ¹⁵N progressive labelling was used to calculate the rate of ribosome synthesis and degradation to track the fate of 62 r‐proteins. The degradation rates and the synthesis rates of most r‐proteins slowed following oxidative stress leading to an ageing population of ribosomes in stressed cells. However, there were exceptions to this trend; r‐protein RPS14C doubled its degradation rate in both oxidative treatments. Overall, we show that ribosome abundance decreases and their age increases with oxidative stress in line with loss of cell growth rate and total cellular protein amount, but ribosome function of the ageing ribosomes appeared to be maintained concomittently with differences in the turnover rate and abundance of specific ribosomal proteins. Data are available via ProteomeXchange with identifier PXD012840.     

Degradation of short-lived proteins is decreased by centrifugation

We have examined the effects of enucleation and of inhibitors of mRNA synthesis (actinomycin D and cordycepin) on protein turnover of HeLa cells. Enucleation markedly inhibited the rate of protein degradation for short-lived proteins. However, cells centrifuged in the absence of cytochalasin B at the speed required to obtain cytoplasts showed protein degradation rates identical to those of cytoplasts, while inhibitors of mRNA synthesis did not affect the process. Although enucleation may affect degradation of specific proteins, these results suggest that centrifugation is largely responsible for the inhibition of protein degradation in cytoplasts.     

Replacement perfusion of cultured eucaryotic cells: A method for the accurate measurement of the rates of growth,protein synthesis,and protein turnover

The fractional rates of protein synthesis (k_s) and degradation (k_p) were studied in the myeloma cell line SP2/0-AG14 grown at different rates (k_g). Cells in spinner flask suspension cultures were maintained at constant cellular density for prolonged periods by replacement perfusion of labeling medium at a rate equivalent to the rate of growth. Total protein synthesis was calculated from the specific-radioactivity of labeled L-leucine in the precursor (medium) and cellular protein. Fractional synthesis rates determined by approach to equilibrium labeling were the same as those determined by equilibrium-pulse labeling kinetics and pulse-chase kinetics. The rate of protein degradation was determined from the established relationship k_g = k_s – k_p. Protein synthesis rates remained constant over a threefold range in the rate of cell growth. At relatively slow growth rates (k_g = 0.017/hr) turnover represented a major fraction of total synthesis (k_p = 0.032/hr = 0.65ks). At rapid growth rates (k_g = 0.058/hr) the value of k_p was less than 0.005/hr. No major difference was observed between the k_s determined for individual cellular proteins (separated by SDS-polyacrylamide (7.5%) gel electro-phoresis) from rapid- and slow-growing cultures. Thus, with an invariable k_s, any change in growth rate is due to an inverse change in the rate of turnover. Since turnover is the balance between synthesis and degradation and since synthesis is unchanging then changes in the growth rate of SP2/0-AG14 should be due to changes in the rate of protein degradation. Experiments were therefore performed to determine the origin of the degradative machinery, ie, cytosolic or lysosomal; autolysis of prelabeled cellular protein (in vitro) was observed only at acidic pH (4.2) and WUS totally inhibited by addition of lcupcptin (10 μM) and pepstatin (2 μM), the specific inhibitors of lysosomal cathepsins B (L) and D, respectively. Since growth rate appears to be regulated by the alterations in the rate of protein degradation and degradation (in vitro) in SP2/0-AG14 appearsto be lysosomal, then one should be able to alter the rate of cellular growth by interfering with rate of lysosomal proteolysis. Indeed, when the lysosomotropic amine NH ₄Cl (10 mM) is added to cells growing with a k_g of 0.018/hr ± 0.001 (k_s = 0.050/hr ± 0.002) the growth rate increased to 0.051/hr ± 0.002 without change in the rate of protein synthesis (k_s = 0.049/hr ± 0.003). It is suggested from our data that the cellular growth rate of SP2/0-AG14 is regulated by the lysosomal apparatus; whether this regulation is itself regulated by either a specific compartmentalization of the lysosomal proteinases and/or their substrates or by endogenous protease inhibitors, should prove to be an exciting area for future investigation.     

Growth rate correlates negatively with protein turnover in Arabidopsis accessions

Previous studies with Arabidopsis accessions revealed that biomass correlates negatively to dusk starch content and total protein, and positively to the maximum activities of enzymes in photosynthesis. We hypothesized that large accessions have lower ribosome abundance and lower rates of protein synthesis, and that this is compensated by lower rates of protein degradation. This would increase growth efficiency and allow more investment in photosynthetic machinery. We analysed ribosome abundance and polysome loading in 19 accessions, modelled the rates of protein synthesis and compared them with the observed rate of growth. Large accessions contained less ribosomes than small accessions, due mainly to cytosolic ribosome abundance falling at night in large accessions. The modelled rates of protein synthesis resembled those required for growth in large accessions, but were up to 30% in excess in small accessions. We then employed ¹³CO₂ pulse‐chase labelling to measure the rates of protein synthesis and degradation in 13 accessions. Small accessions had a slightly higher rate of protein synthesis and much higher rates of protein degradation than large accessions. Protein turnover was negligible in large accessions but equivalent to up to 30% of synthesised protein day^?1 in small accessions. We discuss to what extent the decrease in growth in small accessions can be quantitatively explained by known costs of protein turnover and what factors may lead to the altered diurnal dynamics and increase of ribosome abundance in small accessions, and propose that there is a trade‐off between protein turnover and maximisation of growth rate.     

A quantitative spatial proteomics analysis of proteome turnover in human cells

Measuring the properties of endogenous cell proteins, such as expression level, subcellular localization, and turnover rates, on a whole proteome level remains a major challenge in the postgenome era. Quantitative methods for measuring mRNA expression do not reliably predict corresponding protein levels and provide little or no information on other protein properties. Here we describe a combined pulse-labeling, spatial proteomics and data analysis strategy to characterize the expression, localization, synthesis, degradation, and turnover rates of endogenously expressed, untagged human proteins in different subcellular compartments. Using quantitative mass spectrometry and stable isotope labeling with amino acids in cell culture, a total of 80,098 peptides from 8,041 HeLa proteins were quantified, and their spatial distribution between the cytoplasm, nucleus and nucleolus determined and visualized using specialized software tools developed in PepTracker. Using information from ion intensities and rates of change in isotope ratios, protein abundance levels and protein synthesis, degradation and turnover rates were calculated for the whole cell and for the respective cytoplasmic, nuclear, and nucleolar compartments. Expression levels of endogenous HeLa proteins varied by up to seven orders of magnitude. The average turnover rate for HeLa proteins was ~20 h. Turnover rate did not correlate with either molecular weight or net charge, but did correlate with abundance, with highly abundant proteins showing longer than average half-lives. Fast turnover proteins had overall a higher frequency of PEST motifs than slow turnover proteins but no general correlation was observed between amino or carboxyl terminal amino acid identities and turnover rates. A subset of proteins was identified that exist in pools with different turnover rates depending on their subcellular localization. This strongly correlated with subunits of large, multiprotein complexes, suggesting a general mechanism whereby their assembly is controlled in a different subcellular location to their main site of function.     

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-¹⁴C 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-¹⁴C 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.     

Increased bone turnover is associated with protein and energy metabolism in adolescents with sickle cell anemia

Contribution of bone turnover to the hypercatabolic state observed in sickle cell anemia is unknown. We examined the association between markers of bone turnover and basal rates of whole body protein turnover and energy expenditure in 28 adolescents with homozygous sickle cell anemia (HbSS) and in 26 matched controls with normal phenotype (HbAA). Whole body protein breakdown and synthesis were measured using a stable isotope of [15N]glycine, resting energy expenditure was measured by whole room indirect calorimetry, and the rate of pyridinoline cross-link (PYD) excretion in urine and fasting serum levels of the type I procollagen carboxy-terminal propeptide (PICP) were measured with commercial kits. Urinary PYD and serum PICP were significantly elevated in HbSS patients. The increase in procollagen synthesis, indicated by high levels of PICP, was significantly correlated with increased whole body protein synthesis. The increase in type I collagen degradation, indicated by high PYD excretion, was significantly correlated with increased protein breakdown. We conclude that increased rates of bone turnover contribute to the increased rates of protein turnover and energy expenditure observed in adolescents with homozygous sickle cell anemia.     

Regulation of protein turnover versus growth state: ascites hepatoma as a model for studies both in the animal and in vitro

Cell protein turnover states as related to growth phase have been analyzed in a rat ascites hepatoma (Yoshida AH-130), which after transplantation entered a period of exponential growth, followed by a quasi-stationary state. Evaluation of AH-130 cell protein turnover in the animal (slow-turnover protein pool) was combined with rapid assays of proteolytic rates of cells transferred in vitro. Protein accumulation in the exponential phase reflected the balance between sustained synthetic rates and relatively low degradative rates. Cessation of growth resulted from convergent reduction of synthesis (from 3.10 to 1.49%/h) and enhancement of protein breakdown (from 0.61 to 1.43%/h). Endogenous proteolytic rates in vitro were very close to the above degradation rates. As shown by incubation with ammonia or other lysosomal inhibitors, the acidic vacuolar pathway for protein degradation, while totally suppressed in exponential tumor cells, was activated in cells from stationary tumors to such an extent that it fully accounted for the enhanced proteolysis. In contrast, energy metabolism inhibitors were effective on cells in either growth state, the residual ongoing proteolysis being similar in both cells. The possible contribution of cell death to activation of the acidic vacuolar proteolysis in stationary tumors is discussed.     

Activation of ERK by sodium tungstate induces protein synthesis and prevents protein degradation in rat L6 myotubes

The balance between the rates of protein synthesis and degradation in muscle is regulated by PI3K/Akt signaling. Here we addressed the effect of ERK activation by sodium tungstate on protein turnover in rat L6 myotubes. Phosphorylation of ERK by this compound increased protein synthesis by activating MTOR and prevented dexamethasone-induced protein degradation by blocking FoxO3a activity, but it did not alter Akt phosphorylation. Thus, activation of ERK by tungstate improves protein turnover in dexamethasone-treated cells. On the basis of our results, we propose that tungstate be considered an alternative to IGF-I and its analogs in the prevention of skeletal muscle atrophy.     

Role of lysosomes in protein turnover: Catch-up proteolysis after release from NH4Cl inhibition

Cultured rat embryo fibroblasts, when placed in media with 10% serum containing 20 mM NH₄Cl, show an inhibition of protein degradation and, concurrently, an accumulation of numerous, large vacuoles, partially filled with cellular debris. Cells placed in a serum-free media exhibit an enhanced degradation of cell protein, which is also inhibited by NH₄Cl. When these cells are removed from media containing NH₄Cl and placed in fresh media, the material accumulated in these vacuoles is rapidly and quantitatively released to the media in both an acid-soluble and acid-insoluble form. NH₄Cl inhibits rapidly and specifically the lysosomal proteolytic mechanism, and is without effect on the basal turnover mechanism. The lysosomal proteolytic mechanism accounts for approximately 25% of protein turnover, and, at least in low density cultures, can be stimulated to levels which account for more than half of the protein turnover in the cell. The major pathway for the degradation of fast turnover proteins appears to be separate from lysosomal mechanism.     

HnRNP core proteins: Synthesis,turnover and intracellular distribution

Our present data indicate that the M_r 34–40,000 polypeptides which are involved in the binding of a large fraction of hnRNA sequences, including mRNA, are for the most part metabolically stable species in mouse ascites tumor cells. An exception to this generalization is the smallest of 30S RNP core polypeptides, the M_r 34,000 protein, which has a relatively high turnover rate. The relationship of the various synthesis and degradation rates to the physiological state of mammalian cells remains to be determined, as does the pathway of assembly and disassembly of RNP substructures during re-utilization of the proteins and during their turnover. Immunofluorescent studies, which have confirmed the expected nucleoplasmic or euchromatic localization of the RNP core proteins, have also indicated that these species are stable during mitosis, at which time they are dispersed through the cell away from the condensed chromosomes. The proteins appear to relocate in the nucleus as soon as the nuclear envelope is reformed.     

Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope–labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study, we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [¹³C₆]lysine or [²H₂]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first-order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates.     

Tri-iodothyronine effects on Sertoli cell protein turnover in the prepubertal pig

The direct effect of Tri-iodothyronine (T₃: 0.1–100 nM) on protein turnover was studied using primary cultures of Sertoli cells isolated from immature piglet testis. The results demonstrate that T₃ significantly increases protein synthesis without altering the protein degradation rate. These data and previous ones, showing the presence of specific T₃ receptors in Sertoli cell nuclei, indicate that T₃ plays a fundamental role in the early regulation of porcine Sertoli cell growth and maturation.     

The 1987 Borden Award lecture. Protein metabolism in premature human infants

Our studies have focused on the regulation of whole body and skeletal muscle protein metabolism in premature infants. Net deposition of protein is the result of a positive balance between protein synthesis and breakdown. To measure protein metabolism we have employed end-product studies with [15N]glycine and 13[C]leucine. Myofibrillar protein degradation was estimated by measuring the excretion of N-t-methylhistidine in urine. Energy expenditure and substrate utilization were also measured. Premature infants have high rates of protein synthesis (12 g.kg-1.d-1), twice those measured in children and four times those found in adults. Intrauterine malnourished babies have increased rates of protein turnover. Very low birth weight infants (less than 1500 g) have higher myofibrillar protein turnover than larger babies. Intravenous feeding decreases whole body protein turnover, and we estimate visceral protein synthesis to be approximately 4 g.kg-1.d-1. Suboptimal energy intake worsens nitrogen utilization by reducing the reutilization of endogenous amino acids for protein synthesis. We have also examined the effects of varying the source of nonprotein energy (i.e., glucose only versus glucose plus lipid) at requirement levels and have shown there is no effect on protein metabolism. Recent improvements in technology have opened the way to detailed study of individual amino acid metabolism in neonates in the future.     

Systems-wide proteomic analysis in mammalian cells reveals conserved, functional protein turnover

The turnover of each protein in the mammalian proteome is a functionally important characteristic. Here, we employed high-resolution mass spectrometry to quantify protein dynamics in nondividing mammalian cells. The ratio of externally supplied versus endogenous amino acids to de novo protein synthesis was about 17:1. Using subsaturating SILAC labeling, we obtained accurate turnover rates of 4106 proteins in HeLa and 3528 proteins in C2C12 cells. Comparison of these human and mouse cell lines revealed a highly significant turnover correlation of protein orthologs and thus high species conservation. Functionally, we observed statistically significant trends for the turnover of phosphoproteins and gene ontology categories that showed extensive covariation between mouse and human. Likewise, the members of some protein complexes, such as the proteasome, have highly similar turnover rates. The high species conservation and the low complex variances thus imply great regulatory fine-tuning of protein turnover.     

Abnormality in Translational Regulation of Catalase Expression in Disorders of Peroxisomal Biogenesis

Abstract: Peroxisomal disorders are a newly described group of inherited neurological diseases. In disorders of peroxisomal biogenesis, e.g., Zellweger syndrome, owing to the lack of peroxisomes, catalase, a peroxisomal enzyme, is found to be present in the cytoplasm instead. We observed higher catalase activity (7.59 ± 0.41 mU/mg of protein) in cultured skin fibroblasts from Zellweger patients than in control fibroblasts (4.45 ± 0.29 mU/mg of protein). Moreover, we also found that the majority of the catalase in Zellweger cells was present in the inactive form. The specific activities following reactivation in Zellweger and control cells were 12.1 and 4.9 mU/mg of protein, respectively. To understand the molecular basis of higher levels of catalase in Zellweger than control cells, we examined the rate of synthesis and turnover of catalase and levels of catalase mRNA and protein levels in Zellweger cells as compared with control cells. The initial rates of synthesis of catalase in Zellweger (1.68 ± 0.15 mU/mg of protein) and control (1.51 ± 0.14 mU/mg of protein) cells were similar. The rates of turnover of catalase in Zellweger (t_1/2 = 47 ± 8 h) and control (t_1/2 = 49 ± 7 h) were also similar. Consistent with the enzyme activity, the levels of catalase protein were higher in Zellweger cells as compared with control cells. On the other hand, there was no difference in the level of catalase mRNA between control and Zellweger cells. Although the rate of synthesis in Zellweger and control cells were initially similar, it was down-regulated to a lower level at ～72 h of culture in control fibroblasts as compared with Zellweger cells, which continued to synthesize catalase at the same rate up to 5 days in culture. The presence of similar levels of mRNA in control and Zellweger cells and continued synthesis of catalase in Zellweger cells at a higher level as compared with control cells suggest a loss of regulation at the translational level.     

A Novel Pulse-Chase SILAC Strategy Measures Changes in Protein Decay and Synthesis Rates Induced by Perturbation of Proteostasis with an Hsp90 Inhibitor

Standard proteomics methods allow the relative quantitation of levels of thousands of proteins in two or more samples. While such methods are invaluable for defining the variations in protein concentrations which follow the perturbation of a biological system, they do not offer information on the mechanisms underlying such changes. Expanding on previous work [1], we developed a pulse-chase (pc) variant of SILAC (stable isotope labeling by amino acids in cell culture). pcSILAC can quantitate in one experiment and for two conditions the relative levels of proteins newly synthesized in a given time as well as the relative levels of remaining preexisting proteins. We validated the method studying the drug-mediated inhibition of the Hsp90 molecular chaperone, which is known to lead to increased synthesis of stress response proteins as well as the increased decay of Hsp90 “clients”. We showed that pcSILAC can give information on changes in global cellular proteostasis induced by treatment with the inhibitor, which are normally not captured by standard relative quantitation techniques. Furthermore, we have developed a mathematical model and computational framework that uses pcSILAC data to determine degradation constants k_d and synthesis rates V_s for proteins in both control and drug-treated cells. The results show that Hsp90 inhibition induced a generalized slowdown of protein synthesis and an increase in protein decay. Treatment with the inhibitor also resulted in widespread protein-specific changes in relative synthesis rates, together with variations in protein decay rates. The latter were more restricted to individual proteins or protein families than the variations in synthesis. Our results establish pcSILAC as a viable workflow for the mechanistic dissection of changes in the proteome which follow perturbations. Data are available via ProteomeXchange with identifier PXD000538.     

Regulation of protein turnover by L-glutamine in porcine intestinal epithelial cells

L-Glutamine (Gln) plays an important role in sustaining the intestinal mucosal mass of humans and animals. However, the underlying mechanisms are largely unknown. This study tested the hypothesis that Gln regulates protein turnover in intestinal epithelial cells. Intestinal porcine epithelial cells (IPEC-1) were cultured for 3 h (short-term study) or 96 h (long-term study) in Gln-free Dulbecco's modified Eagle-F12 Ham medium containing 0, 0.5 or 2.0 mM Gln. To determine effects of ammonia (a metabolite of Gln, i.e., 0.18 mM ammonia produced from 2 mM Gln in 3 h) on protein turnover, additional experiments were conducted in which medium contained 0.5 mM Gln and 0, 0.2, 0.5 or 2.0 mM NH(4)Cl. Variables of analysis included cell growth, protein synthesis, proteolysis and mammalian target of rapamycin (mTOR) signaling. IPEC-1 cell growth increased with extracellular Gln concentrations. Compared with 0 mM Gln, the addition of 0.5 and 2 mM Gln to medium stimulated protein synthesis and inhibited protein degradation in those cells in both the short- and long-term studies. Ammonia (0.05 to 2.0 mM) did not affect protein synthesis, although higher levels of ammonia (0.5 and 2.0 mM) reduced protein degradation in IPEC-1 cells. Consistent with the data on protein turnover, 0.5 and 2 mM Gln increased abundance of phosphorylated eIF4E-binding protein-1 and phosphorylated S6 kinase-1 proteins. Collectively, these results demonstrate that physiological levels of Gln regulate protein turnover independent of ammonia production in intestinal cells through the mTOR signaling pathway.     

Effect of vitamin B6 supplementation on degradation rates of short-lived proteins in human neutrophils

Metabolic pathways are controlled primarily by protein degradation rates. Degradation rates, in turn, are controlled by changes in physiologic condition or nutrient supply. Vitamin B(6) is associated with a greater variety of reactions than most other vitamins. Moreover, the vitamin B(6) needs of the elderly tend to be higher than those of young adults. Neutrophils seem to be appropriate cells for assessing protein turnover as affected by macronutrients and micronutrients. Thus, we assumed that vitamin B(6) supplementation, particularly in an elderly population, would change the turnover rates of the neutrophil proteins. Protein synthesis was measured after 30 minutes of (35)S-Met incorporation followed by a 30-minute washout incubation; degradation was measured after an additional 5-hour incubation. Following protein separation, radioactive images of short-lived proteins were electronically separated into bands. Vitamin B(6) supplementation significantly increased the synthesis of most neutrophil protein bands. There was a significant decrease of 25 to 66% in the degradation rates of 235 protein bands. We even detected by statistical evaluation a 20% decrease in the degradation rates of distinct protein bands. Activation coefficients of erythrocyte aspartate aminotransferase (AC-AST) decreased markedly. There was a significant positive correlation between the decrease in AC-AST and protein degradation. The N-end rule proposes that pyridoxal 5'-phosphate decreases degradation rates of short-lived proteins by binding to lysyl residues. A biochemical model of the mechanism of cellular protein turnover, as affected by nutritional intervention, in human neutrophils is demonstrated.