Protein turnover in exponential and stationary phase Vibrio cells

Vibrio strain 14 supports phage alpha 3a growth in standing stationary phase cells but not in shaking (aerated) stationary phase cells. In exponential cells, protein was turned over at 1.8% h-1, and the rate was increased by starvation or inhibition of protein synthesis. In shaking stationary phase cells the rate of protein turnover was low (1.0% h-1) for proteins synthesised during growth but high (20% h-1) for recently synthesised proteins. In contrast recently synthesised proteins in standing stationary phase cells were stable over 60 min and proteins synthesised during growth were turned over at 2.9% h-1. ppGpp and pppGpp were detected in exponential cells, but were not detected in stationary phase cells.     

Proteasome-dependent turnover of protein disulfide isomerase in oxidatively stressed cells.

Generalized increases in protein oxidation and protein degradation in response to mild oxidative stress have been widely reported, but only a few individual proteins have actually been shown to undergo selective, oxidation-induced proteolysis. Our goal was to find such proteins in Clone 9 liver cells exposed to hydrogen peroxide. Using metabolic radiolabeling of intracellular proteins with [35S]cysteine/methionine, and analysis by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), we found at least three labeled proteins ("A," "B," and "C") whose levels were decreased significantly more than the generalized protein loss after mild oxidative stress. "Protein C" was excised from 2-D PAGE and subjected to N-terminal amino acid microsequencing. "Protein C" was identified as Protein Disulfide Isomerase or PDI (E.C. 5.3.4.1), and this identity was reconfirmed by Western blotting with a C-terminal anti-PDI monoclonal antibody. A combination of quantitative radiometry and Western blotting in 2-D PAGE revealed that PDI was selectively degraded and then new PDI was synthesized, following H2O2 exposure. PDI degradation was blocked by inhibitors of the proteasome, and by cell treatment with proteasome C2 subunit antisense oligonucleotides, indicating that the proteasome was largely responsible for oxidation-induced PDI degradation.     

The role of alternative splicing and C-terminal amino acids in thromboxane receptor stabilization

The thromboxane receptor has two alternatively spliced isoforms, alpha and beta, which differ only in sequences within the cytoplasmic C-terminal domain. Oxidative stress induced by H(2)O(2) in a COS-7 cell model results in stabilization of the thromboxane receptor beta isoform by translocation from the endoplasmic reticulum to the Golgi complex, which in turn results in protection of the receptor from degradation. We now report that both the alpha and beta thromboxane receptor isoforms respond identically to oxidative stress. Further, mutagenesis studies indicate that replacing the normal C-terminus with a nonsense sequence also does not alter stabilization behaviour ruling out a role for the distinct C-termini in this process. Further mutagenesis implicates a cluster of arginine residues within the C-terminal domain as involved in oxidative stress-induced stabilization. These data identify a region of the thromboxane receptor that is responsible for responding to oxidative challenge and open the possibility of identification of the molecular machinery underpinning this response.     

Protein oxidation and loss of protease activity may lead to cataract formation in the aged lens

Over 95% of the dry mass of the eye lens consists of specialized proteins called crystallins. Aged lenses are subject to cataract formation, in which damage, cross-linking, and precipitation of crystallins contribute to a loss of lens clarity. Cataract is one of the major causes of blindness, and it is estimated that over 50,000,000 people suffer from this disability. Damage to lens crystallins appears to be largely attributable to the effects of UV radiation and/or various active oxygen species (oxygen radicals, ¹O₂, H₂O₂, etc.). Photooxidative damage to lens crystallins is normally retarded by a series of antioxidant enzymes and compounds. Crystallins which experience mild oxidative damage are rapidly degraded by a system of lenticular proteases. However, extensive oxidation and cross-linking severely decrease proteolytic susceptibility of lens crystallins. Thus, in the young lens the combination of antioxidants and proteases serves to prevent crystallin damage and precipitation in cataract formation. The aged lens, however, exhibits diminished antioxidant capacity and decreased proteolytic capabilities. The loss of proteolytic activity may actually be partially attributable to oxidative damage which proteases (like any other protein)_can sustain. We propose that the rate of crystallin damage increases as antioxidant capacity declines with age. The lower protease activity of aged lens cells may be insufficient to cope with such rates of crystallin damage, and denatured crystallins may begin to accumulate. As the concentration of oxidatively denatured crystallins rises, cross-linking reactions may produce insoluble aggregates which are refractive to protease digestion. Such a scheme could explain many events which are known to contribute to cataract formation, as well as several which have appeared to be unrelated. This hypothesis is also open to experimental verification and intervention.     

Prolonged selenium-deficient diet in MsrA knockout mice causes enhanced oxidative modification to proteins and affects the levels of antioxidant enzymes in a tissue-specific manner

The methionine sulfoxide reductase (Msr) system (comprised of MsrA and MsrB) is responsible for reducing methionine sulfoxide (MetO) to methionine. One major form of MsrB is a selenoprotein. Following prolonged selenium deficient diet (SD), through F2 generation, the MsrA - / -mice exhibited higher protein-MetO and carbonyl levels relative to their wild-type (WT) control in most organs. More specifically, the SD diet caused alteration in the expression and/or activities of certain antioxidants as follows: lowering the specific activity of MsrB in the MsrA - / -cerebellum in comparison to WT mice; lowering the activities of glutathione peroxidase (Gpx) and thioredoxin reductase (Trr) especially in brains of MsrA - / -mice; elevation of the cellular levels of selenoprotein P (SelP) in most tissues of the MsrA - / -relative to WT. Unexpectedly, the expression and activity of glucose-6-phosphate dehydrogenase (G6PD) were mainly elevated in lungs and hearts of MsrA - / -mice. Moreover, the body weight of the MsrA - / -mice lagged behind the WT mice body weight up to 120 days of the SD diet. In summary, it is suggested that the lack of the MsrA gene in conjunction with prolonged SD diet causes decreased antioxidant capability and enhanced protein oxidation.     

Prolonged selenium deficient diet in MsrA knockout mice causes enhanced oxidative modification to proteins and affects the levels of antioxidant enzymes in a tissue-specific manner

The methionine sulfoxide reductase (Msr) system (comprised of MsrA and MsrB) is responsible for reducing methionine sulfoxide (MetO) to methionine. One major form of MsrB is a selenoprotein. Following prolonged selenium deficient diet (SD), through F2 generation, the MsrA ? / ?mice exhibited higher protein–MetO and carbonyl levels relative to their wild-type (WT) control in most organs. More specifically, the SD diet caused alteration in the expression and/or activities of certain antioxidants as follows: lowering the specific activity of MsrB in the MsrA ? / ?cerebellum in comparison to WT mice; lowering the activities of glutathione peroxidase (Gpx) and thioredoxin reductase (Trr) especially in brains of MsrA ? / ?mice; elevation of the cellular levels of selenoprotein P (SelP) in most tissues of the MsrA ? / ?relative to WT. Unexpectedly, the expression and activity of glucose-6-phosphate dehydrogenase (G6PD) were mainly elevated in lungs and hearts of MsrA ? / ?mice. Moreover, the body weight of the MsrA ? / ?mice lagged behind the WT mice body weight up to 120 days of the SD diet. In summary, it is suggested that the lack of the MsrA gene in conjunction with prolonged SD diet causes decreased antioxidant capability and enhanced protein oxidation.     

Protein turnover in the attached leaves of non-stressed and stressed barley seedlings

Protein turnover was examined, using tritiated water, in various 2-cm regions of 7-11-d-old, first leaves of barley (Hordeum vulgare). Differences were found between the regions in their protein turnover and their responses to stress. The rate constant for degradation for total protein was the same throughout the leaf and the average half-life (t_1/2) of protein=approx. 220 h. Only in the older regions did a 24-h pulse of³H₂O preferentially label protein with a t_1/2 (90 h) considerably shorter than the t_1/2 for total protein. Soluble protein was degraded faster than insoluble protein and contained an appreciable short-lived protein component observable by short-pulse labelling. The rate of protein synthesis was greatest in the cells of the youngest region and declined as each region aged. The mean rate of protein synthesis over the 4-d period was 4 and 7 nmol h^-1 of amino-N with respect to the regions 1–3 and 7–9 cm from the leaf tip. Seedlings, stressed by adding polyethylene glycol (2.0 MPa) to the roots, showed a marked loss of protein from the older leaf regions with only small losses in the younger regions. Amino acids accumulated in the younger region continuously whereas in the older region little accumulation occurred until day 3 of stress when proline levels increased. Protein synthesis was decreased by between 30% and 50% in all leaf regions. In the region 1–3 cm from the leaf tip, the rate of protein degradation of total protein was enhanced and equalled the rate of degradation of 24-h-pulse-labelled protein which was not itself significantly affected by stress (t_1/2=approx. 90 h). In the region 3–5 cm, the degradation of both 4-d and 24-h-labelled protein was enhanced by stress to rates similar to those found in the region 1–3 cm. This was largely through increases in the degradation of the insoluble protein, but the degradation of soluble protein was also raised. Protein degradation in the region 7–9 cm was not affected by stress.Abbreviations t_1/2 average half-life - PEG polyethylene glycol     

Protein Turnover in Brain of the Rat Fetus

Protein synthesis in vivo was studied in whole brain of rat fetuses using continuous intravenous infusion of L-[U-14C]tyrosine into unrestrained pregnant rats at 19 and 21 days gestation. Protein degradation (KD) was calculated by subtracting fractional growth rate of brain protein (KG) from the fractional synthesis rate (KS). KS was high at both gestational ages (0.42 +/- 0.03 days-1 at day 19, 0.47 +/- 0.029 days-1 at 21 days), comparable to values previously reported for newborn rat cerebral hemispheres, and threefold higher than is seen in adult animals. KD was similar at both 19 and 21 days gestation (0.19-0.24) and lower than that reported in neonatal rat brain using similar techniques. Protein accretion during the most rapid phase of brain growth (fetus) is accomplished by similar rates of protein synthesis, but decreased rates of degradation when compared with a slower growth phase (newborn). KD in the brain of the rapidly growing fetus is slightly higher than in adult cerebral hemispheres.     

Synthesis of novel heme-interacting acridone derivatives to prevent free heme-mediated protein oxidation and degradation

Heme is an important prosthetic molecule for various hemoproteins and serves important function in living aerobic organisms. But degradation of hemoprotein, for example, hemoglobin during different pathological conditions leads to the release of heme, which is very toxic as it induces oxidative stress and inflammation due to its pro-oxidant nature. Thus, synthesis of compound that will detoxify free heme by interacting with it would be fruitful for the management of heme-induced pathogenesis. Here, we report the synthesis of a novel natural product arborinine and some other acridone derivatives, which interact with free heme. These acridones in vitro block heme-mediated protein oxidation and degradation, markers for heme-induced oxidative stress.     

Protein turnover in a psychrotrophic bacterium

Protein breakdown in pulse-labelled and longlabelled cells of Arthrobacter S ₁-55, a psychrotrophic bacterium, has been assessed at different temperatures. The temperature at which the cells were grown and labelled affected the breakdown of pulsed-labelled but not long-labelled proteins. Inhibitors of ATP synthesis inhibited proteolysis. Miscoding antibiotics stimulated the production of rapidly degradable proteins.     

Metal-catalyzed oxidation induces carbonylation of peroxisomal proteins and loss of enzymatic activities

Peroxisomes are involved in oxidative metabolic reactions and have the capacity to generate large amounts of reactive oxygen species that could damage biomolecules including their own resident proteins. The purpose of this study was to determine whether peroxisomal proteins are susceptible to oxidation and whether oxidative damage affects their enzymatic activity. Peroxisomal proteins were subjected to metal-catalyzed oxidation (MCO) with CuCl(2)/ascorbate and derivatized with 2,4-dinitrophenylhydrazine which allowed for spectrophotometric quantification of carbonylation. Immunochemical detection of carbonylated peroxisomal proteins, resolved by gel electrophoresis and detected with anti-DNP antibodies, revealed five oxidatively modified proteins with the following molecular weights: 80, 66, 62, 55, and 50 kDa. The proteins at 66, 62, and 55 kDa were identified as malate synthase (MS), isocitrate lyase, and catalase (CAT), respectively. MS and CAT were estimated to contain 2-3 mol of carbonyl/mol of protein as a result of MCO. Enzymatic assays revealed varying degrees of activity loss. Isocitrate lyase and malate synthase showed significant loss of activity while catalase and malate dehydrogenase were less inhibited by carbonylation. Our findings show that peroxisomal proteins are vulnerable to MCO damage and thus may also be affected by in vivo exposure to reactive oxygen species.     

The implications of gene heterozygosity for protein folding and protein turnover

The offspring of closely related parents often suffer from inbreeding depression, sometimes resulting in a slower growth rate for inbred offspring relative to non-inbred offspring. Previous research has shown that some of the slower growth rate of inbred organisms can be attributed to the inbred organisms’ increased levels of protein turnover. This paper attempts to show that the higher levels of protein turnover among inbred organisms can be attributed to accumulations of misfolded and aggregated proteins that require degradation by the inbred organisms’ protein quality control systems. The accumulation of misfolded and aggregated proteins within inbred organisms are the result of more negative free energies of folding for proteins encoded at homozygous gene loci and higher concentrations of potentially aggregating non-native protein species within the cell. The theory presented here makes several quantitative predictions that suggest a connection between protein misfolding/aggregation and polyploidy that can be tested by future research.     

Microbial oxidation of methane,ammonium and carbon monoxide,and turnover of nitrous oxide and nitric oxide in soils

The effect of soil microbial processes on production and/or consumption of atmospheric trace gases was studied in four different soils which were preincubated in the presence of elevated concentrations of CH₄, NH ₄ ⁺ or CO, to simulate the growth of the resident populations of methanotrophic, nitrifying, or carboxydotrophic bacteria, respectively. Oxidation of CH₄, both at atmospheric (1.8 ppmv) and at elevated (3500 ppmv) CH₄ mixing ratios, was stimulated after preincubation with CH₄, but not with NH ₄ ⁺ or CO, indicating that CH₄ was oxidized by methanotrophic, but not by nitrifying or carboxydotrophic bacteria. However, the oxidation of CH₄ was partially inhibited by addition of NH ₄ ⁺ and CO. Analogously, oxidation of NH ₄ ⁺ was partially inhibited by addition of CH₄. Oxidation of CO at elevated mixing ratios (2300 ppmv) was stimulated after preincubation with CO, indicating oxidation by carboxydotrophs, but was also stimulated at a small extent after preincubation with CH₄, suggesting the involvement of methanotrophs. At atmospheric CO mixing ratios (0.13 ppmv), on the other hand, oxidation of CO was stimulated after preincubation with NH ₄ ⁺ , indicating that the activity was due to nitrifiers. NO uptake was stimulated in soils preincubated with CH₄, indicating the involvement of methanotrophs. However, production of N₂O was only stimulated, if CH₄ was added as a substrate. The results indicate that especially the methanotrophic and nitrifying populations in soil not only oxidize their specific substrates, but are also involved in the metabolism of other compounds.     

Protein oxidation in aging: endoplasmic reticulum as a target

Summary. Oxidatively modified proteins have been shown to correlate with the age of an organism or its tissues. An increase in tissue-susceptibility to experimentally induced protein oxidation not only depends on tissue type and age, but also on the maximum lifespan potential of the species. A general, although tissue dependent, decline in anti-oxidative defenses during aging may very well be responsible for this difference in vulnerability. In addition, the level of protein modifications also depends on the nature and the subcellular localization of the proteins involved. Damage to the endoplasmic reticulum (ER), and its subsequent impaired functionality may be involved in the process of aging. This is suggested by; (1) an upregulation of ER stress-response chaperones, (2) a preferential oxidation of ER-resident proteins and, (3) a disturbance of calcium homeostasis. Therefore, this review will focus on the putative involvement of the oxidized endoplasmic reticulum in the process of aging.     

Metal-catalyzed oxidation of human serum albumin does not alter the interactive binding to the two principal drug binding sites

It is well known that various physiological factors such as pH, endogenous substances or post-translational modifications can affect the conformational state of human serum albumin (HSA). In a previous study, we reported that both pH- and long chain fatty acid-induced conformational changes can alter the interactive binding of ligands to the two principal binding sites of HSA, namely, site I and site II. In the present study, the effect of metal-catalyzed oxidation (MCO) caused by ascorbate/oxygen/trace metals on HSA structure and the interactive binding between dansyl-L-asparagine (DNSA; a site I ligand) and ibuprofen (a site II ligand) at pH 6.5 was investigated. MCO was accompanied by a time-dependent increase in carbonyl content in HSA, suggesting that the HSA was being oxidized. In addition, The MCO of HSA was accompanied by a change in net charge to a more negative charge and a decrease in thermal stability. SDS-PAGE patterns and α-helical contents of the oxidized HSAs were similar to those of native HSA, indicating that the HSA had not been extensively structurally modified by MCO. MCO also caused a selective decrease in ibuprofen binding. In spite of the changes in the HSA structure and ligand that bind to site II, no change in the interactive binding between DNSA and ibuprofen was observed. These data indicated that amino acid residues in site II are preferentially oxidized by MCO, whereas the spatial relationship between sites I and II (e.g. the distance between sites), the flexibility or space of each binding site are not altered. The present findings provide insights into the structural characteristics of oxidized HSA, and drug binding and drug-drug interactions on oxidized HSA.     

Ferritin oxidation and proteasomal degradation: protection by antioxidants

The accumulation of oxidatively damaged proteins is a well-known hallmark of aging and several neurodegenerative diseases including Alzheimer's, Parkinson's and Huntigton's diseases. These highly oxidized protein aggregates are in general not degradable by the main intracellular proteolytic machinery, the proteasomal system. One possible strategy to reduce the accumulation of such oxidized protein aggregates is the prevention of the formation of oxidized protein derivatives or to reduce the protein oxidation to a degree that can be handled by the proteasome. To do so an antioxidative strategy might be successful. Therefore, we undertook the present study to test whether antioxidants are able to prevent the protein oxidation and to influence the proteasomal degradation of moderate oxidized proteins. As a model protein we choose ferritin. H₂O₂ induced a concentration dependent increase of protein oxidation accompanied by an increased proteolytic susceptibility. This increase of proteolytic susceptibility is limited to moderate hydrogen peroxide concentrations, whereas higher concentrations are accompanied by protein aggregate formation.

Protective effects of the vitamin E derivative Trolox, the pyridoindole derivative Stobadine and of the standardized extracts of flavonoids from bark of Pinus Pinaster Pycnogenol^® and from leaves of Ginkgo biloba (EGb 761) were studied on moderate damaged ferritin.     

Ferritin oxidation and proteasomal degradation: Protection by antioxidants

The accumulation of oxidatively damaged proteins is a well-known hallmark of aging and several neurodegenerative diseases including Alzheimer's, Parkinson's and Huntigton's diseases. These highly oxidized protein aggregates are in general not degradable by the main intracellular proteolytic machinery, the proteasomal system. One possible strategy to reduce the accumulation of such oxidized protein aggregates is the prevention of the formation of oxidized protein derivatives or to reduce the protein oxidation to a degree that can be handled by the proteasome. To do so an antioxidative strategy might be successful. Therefore, we undertook the present study to test whether antioxidants are able to prevent the protein oxidation and to influence the proteasomal degradation of moderate oxidized proteins. As a model protein we choose ferritin. H₂O₂ induced a concentration dependent increase of protein oxidation accompanied by an increased proteolytic susceptibility. This increase of proteolytic susceptibility is limited to moderate hydrogen peroxide concentrations, whereas higher concentrations are accompanied by protein aggregate formation.

Protective effects of the vitamin E derivative Trolox, the pyridoindole derivative Stobadine and of the standardized extracts of flavonoids from bark of Pinus Pinaster Pycnogenol^® and from leaves of Ginkgo biloba (EGb 761) were studied on moderate damaged ferritin.     

Metal-catalyzed oxidation and cleavage of octopus glutathione transferase by the CU(II)-ascorbate system

Glutathione transferase (GST) from octopus hepatopancreas was rapidly inactivated by micromolar concentration of Cu(II) in the presence of ascorbate at neutral pH and 0°C. Omitting the metal ion or ascorbate, or replacing the Cu(II) with Fe(II) did not result in any inactivation. Glutathione or the conjugation product of glutathione and 1-chloro-2,4-dinitrobenzene offered complete protection of the enzyme from Cu(II)-induced inactivation. 1-Chloro-2,4-dinitrobenzene, however, did not provide any protection. The inactivation was time and Cu(II) concentration dependent. The dependence of inactivation rate on Cu(II) concentration displayed saturation kinetics, which suggests that the inactivation occurs in two steps with Cu(II) binding with the enzyme first (K_dCu = 260 μM), then the locally generated free radicals modify the essential amino acid residues in the active center, which results in enzyme inactivation. The Cu(II)-ascorbate system is, thus, an affinity reagent for the octopus GST. The enzyme inactivation was demonstrated to be followed by protein cleavage. Native octopus GST has a subunit M_r of 24,000. The inactivated enzyme was cleaved at the C-terminal domain (domain II) of the enzyme molecule and resulted in the formation of peptide fragment of M_r 15,300, which has the identical N-terminal amino acid sequence as the native enzyme. The other half of the peptide with M_r approximately 7700 was visible in the gels only after silver staining, which also revealed a minor cleavage site, also located at the domain II, to produce peptide fragments of M_r approximately 11,300 and 8300. The oxygen carrier molecule in the cephalopods' blood is the copper-containing hemocyanin, which during turnover will release Cu(II). Our results indicate that Cu(II) catalyzes a site-specific oxidation of the essential amino acid residues at the C-terminus of GST causing enzyme inactivation. The modified-enzyme is then affinity cleaved at the putative metal binding site. The ability of octopus GST to bind with free Cu(II) may have important biological implications to enable cephalopods to avoid copper-induced cellular toxicity.     

Protein turnover: measurement of proteome dynamics by whole animal metabolic labelling with stable isotope labelled amino acids

The measurement of protein turnover in tissues of intact animals is obtained by whole animal dynamic labelling studies, requiring dietary administration of precursor label. It is difficult to obtain full labelling of precursor amino acids in the diet and if partial labelling is used, calculation of the rate of turnover of each protein requires knowledge of the precursor relative isotope abundance (RIA). We describe an approach to dynamic labelling of proteins in the mouse with a commercial diet supplemented with a pure, deuterated essential amino acid. The pattern of isotopomer labelling can be used to recover the precursor RIA, and sampling of urinary secreted proteins can monitor the development of liver precursor RIA non-invasively. Time-series analysis of the labelling trajectories for individual proteins allows accurate determination of the first order rate constant for degradation. The acquisition of this parameter over multiple proteins permits turnover profiling of cellular proteins and comparisons of different tissues. The median rate of degradation of muscle protein is considerably lower than liver or kidney, with heart occupying an intermediate position.     

Chromium and human low-density lipoprotein oxidation

Chromium is a catalytic metal able to foster oxidant damage, albeit its capacity to induce human LDL oxidation is to date unkown. Thus, we have investigated whether trivalent and hexavalent chromium, namely Cr(III) and Cr(VI), can induce human LDL oxidation. Cr(III) as CrCl₃ is incapable of inducing LDL oxidation at pH 7.4 or 4.5. However, Cr(III), specifically at physiological pH of 7.4 and in the presence of phosphates, causes an absorbance increase at 234 resembling a spectrophotometric kinetics of LDL oxidation with a lag- and propagation-like phase. In this regard, it is conceivable that peculiar Cr(III) forms such as Cr(III) hydroxide and, especially, Cr(III) polynuclear hydroxocomplexes formed at pH 7.4 interact with phosphates generating species with an intrinsic absorbance at 234 nm, which increases over time resembling a spectrophotometric kinetics of LDL oxidation. Cr(VI), as K₂Cr₂O₇, can instead induce substantial human LDL oxidation at acidic pH such as 4.5, which is typical of the intracellular lysosomal compartment. LDL oxidation is related to binding of Cr(VI) to LDL particles with quenching of the LDL tryptophan fluorescence, and it is inhibited by the metal chelators EDTA and deferoxamine, as well as by the chain-breaking antioxidants butylated hydroxytoluene and probucol. Moreover, Cr(VI)-induced LDL oxidation is inhibited by mannitol conceivably by binding Cr(V) formed from LDL-dependent Cr(VI) reduction and not by scavenging hydroxyl radicals (OH); indeed, the OH scavengers sodium formate and ethanol are ineffective against Cr(VI)-induced LDL oxidation. Notably, heightened LDL lipid hydroperoxide levels and decreased LDL tryptophan fluorescence occur in Cr plating workers, indicating Cr-induced human LDL oxidation in vivo. The biochemical, pathophysiological and clinical implications of these novel findings on chromium and human LDL oxidation are discussed.