Regulation of breakdown of canavanyl proteins in Escherichia coli by growth conditions in Ion+ and Ion− cells

In vivo rates of proteolysis of canavanyl proteins were compared in lon+ and lon- Escherichia coli strains following growth in a variety of media. Both lon+ and lon- cells grown rapidly in complex media possessed higher levels of constitutive degradative activity than when cultured in minimal media. Pre-growth of lon+ cells in the presence of canavanine induced proteolytic activity following growth in minimal media as did stress agents such as heat, alcohol and puromycin: the lon mutant did not show the increased activity following canavanine treatment. The results suggest the presence of a proteolytic activity which selectively degrades aberrant proteins which does not involve protease La, the product of the lon gene, and which furthermore is regulated in part by growth conditions independently of the stress response.     

Effects of canavanine on the secretion of plasma proteins by Hep G2 cells

Many secretory proteins contain an amino-terminal propeptide extension which is removed prior to secretion. The point of cleavage is usually marked by a basic pair of amino acids containing arginine. Canavanine, an analogue of arginine, is incorporated into protein and has been shown to inhibit the proteolytic processing of several of these prosecretory proteins. The addition of 3 mM canavanine to Hep G2 cells incubated with L-[35S]methionine inhibited the secretion of 11 plasma proteins studied. Of the secretory proteins studied only albumin is thought to contain a propeptide, which is marked by a pair of arginine residues at its point of proteolytic processing. Canavanine had varying effects on the secretion of plasma proteins; ranging from a 43-53% inhibition of secretion of alpha 1 antitrypsin and alpha 1 anti-chrymotrypsin to nearly abolishing (93% inhibition) secretion of transferrin. Canavanine also caused most of the proteins studied to migrate slower on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Two of the canavanine-treated proteins (albumin and transferrin) which underwent marked changes in electrophoretic mobility were more sensitive than untreated proteins to proteolysis by Staphylococcus Aureus V8 proteinase. The slower electrophoretic migration and the greater sensitivity to proteolysis of these proteins may be attributed to marked structural changes caused by the incorporation of canavanine. This suggests that the inhibition of plasma protein secretion by canavanine is not only due to an inhibition of the processing of proteins but may be caused by structural distortions of the secretory proteins.     

Autostimulation of dihydrostreptomycin uptake in Bacillus subtilis

In Bacillus subtilis it was shown that the membrane potential (delta psi) has to reach a threshold value of -180 to -190 mV for efficient uptake of dihydrostreptomycin to occur. The magnitude of delta psi is raised above this threshold, and dihydrostreptomycin uptake greatly enhanced, not only by dihydrostreptomycin itself (autostimulation) and by other miscoding aminoglycoside antibiotics, but also by puromycin, bacitracin and N,N'-dicyclohexylcarbodiimide. Stimulation of uptake by dihydrostreptomycin or puromycin was dependent on a specific interference with ongoing protein synthesis. Thus, chloramphenicol prevented the stimulating effect of puromycin by lowering the magnitude of delta psi. Although normally severely antagonizing streptomycin accumulation, K+, as well as spermidine and putrescine, which are known to stabilize ribosomes, consequently enhanced autostimulation of dihydrostreptomycin uptake in a K+-retention mutant with impaired protein synthesis. It is suggested that miscoding aminoglycosides and puromycin both enhance dihydrostreptomycin uptake by increasing delta psi due to ion fluxes, which are themselves caused by a dramatic stimulation of intracellular proteolysis of faulty proteins.     

Structural aberrations in T-even bacteriophage. VII. In vitro analysis of the canavanine-mediated inhibition of proteolytic cleavage.

Canavanine arrests a critical function in head morphogenesis and the potential for forming giant T-even phage particles termed lollipops is induced. Formation of the particles requires the addition of arginine and the restoration of normal functions. We now report on an investigation into the effects of canavanine on both the T4-induced proteolytic activity and on the substrate proteins. Using an in vitro cleavage assay we have shown that the gene 21-dependent proteolytic activity from canavanine-treated extracts is markedly inhibited, whereas the substrate proteins retain a high susceptibility for cleavage. The proteolytic activity in extracts treated with canavanine followed by arginine is readily detectable, and proteins previously synthesized in the presence of canavanine can be cleaved. Protein synthesis is apparently required for the appearance of the proteolytic activity after the canavanine-arginine treatment. Mixing experiments suggest the requirement for a component of the gene 21-dependent proteolytic activity that is not coded for by gene 21.     

Effect of microtubular or translational inhibitors on general cell protein degradation. Evidence for a dual catabolic pathway.

Rat embryo fibroblasts were grown in Eagle's minimal essential medium with 10% serum and cell proteins prelabelled with L-[1-(14)C]leucine, followed by a 24h chase. When transferred to medium deprived of serum these cells showed a 2--3-fold increase in the production of trichloroacetic acid-soluble radioactivity during a 4h observation period. The microtubular poisons vinblastine, vincristine and colchicine partially inhibited this induced proteolysis, but had no effect on the proteolytic rate of cells maintained in medium with 10% serum. A similar discriminating effect on induced proteolysis was observed with cycloheximide, puromycin and insulin. The inhibitory effects of cycloheximide and vinblastine were not additive. These data support the hypothesis that, in addition to the basal turnover of cell proteins, a second mechanism of protein degradation involving cytoplasmic autophagy can be activated by nutritional step-down and is selectively inhibited by agents that interfere with microtubular function and protein synthesis.     

Cultured animal cells exposed to amino acid analogues or puromycin rapidly synthesize several polypeptides

Four major acidic polypeptides, with molecular weights of 88, 72, 71, and 23 thousand, and minor polypeptides with molecular weights of 110, 50, 38, and 30 thousand rapidly accumulated in cultured chick embryo (CE) cells which were exposed for three hours to the arginine analogue canavanine. P₁₁₀, P₈₈, P_71,72, and P₂₃ had unique peptide maps. Evidence of a 27,000 dalton precursor to P₂₃ was obtained. The analogue-stimulated proteins were not related to another set of inducible avian polypeptides known as the glucose-regulated proteins. In mammalian cells, the rate of accumulation of several polypeptides, which were similar in size to the avian proteins, sharply increased after canavanine treatment. Proteins with the same electrophoretic mobilities, isoelectric points, and peptide maps as the analogue-stimulated proteins were expressed at low levels in untreated cultures. To determine the time courses of the canavanine-mediated increases in protein accumulation and the recovery of protein metabolism after analogue treatment, radioactively labeled proteins were extracted from CE cells and analyzed on SDS-polyacrylamide gels. In cultures exposed to canavanine, the rates of accumulation of P₈₈ and P_71,72 increased from basal to new plateau levels in about 1.5 hours, while P₂₃ required about 2.5 hours. When added with the analogue, actinomycin D and cordycepin blocked the increases in protein accumulation. These inhibitors also blocked the rapid decline in the rates of accumulation of the enhanced proteins which occurred after removal of canavanine. Studies of the metabolic stability of the enhanced proteins indicated that the changes in their accumulation were caused by alterations in their rates of synthesis. Thus, the analogue-mediated response fulfilled several of the criteria for inducible eucaryotic gene expression. The amino acid analogue p-fluorophenylalanine and the chain-terminating analogue of amino acyl-tRNA puromycin stimulated the synthesis of the same set of proteins induced by canavanine. The enhanced synthesis of these proteins appeared to be a cellular response to either the presence or catabolism of abnormal proteins and puromycyl peptides.     

Changes in intracellular proteolysis in Escherichia coli during prolonged growth with a low concentration of dihydrostreptomycin

Abstract Escherichia coli was cultured with a low concentration of dihydrostreptomycin (2.5 μg/ml.). Growth was similar to untreated controls for 10 h after which a slow decline in growth rate occurred; growth ceased after 20 h. Intracellular catabolism of pulse-labelled protein synthesised at various points during the antibiotic treatment increased during the first 10 h, but during the second 10 h proteolysis progressively declined to almost control values. The production of an aberrant proteolytic system is one possible explanation.     

Response of Intracellular Proteolysis to Alteration of Bacterial Protein and the Implications in Metabolic Regulation

An assessment has been made of the extent to which the breakdown of microbial cellular proteins is regulated by their metabolic state or function. For this purpose, a number of agents and conditions that alter the synthesis, structure, or utility of cellular protein were examined for the effect on their lability. In Escherichia coli, 5-fluorouracil, p-fluorophenylalanine, norleucine, canavanine, thienylalanine, and puromycin, which engender nonfunctional cellular protein en masse, and ultraviolet irradiation increase the breakdown rate of proteins synthesized in their presence as much as two- to threefold without altering the general capacity for proteolysis. The effects are complicated by, but experimentally distinguishable from, secondary changes in proteolysis that accompany growth inhibition. In contrast, no potentiation of proteolysis is elicited by the presence of suppressor genes, by the administration of heat, or by the biosynthetic alterations attending large changes in the conditions of cultivation or by those attending bacteriophage infection. Thus, although mass perturbations in protein conformation are catabolically distinguishable, the more individual and limited conformational modifications that might occur in disuse do not appear to be the primary determinants of the protein turnover rate. In Bacillus subtilis, turnover synthesis of protein during starvation is as susceptible to treatment with actinomycin D as that during growth. Treatment alters neither the rate of intracellular proteolysis nor the catabolic pattern of the modicum of proteins that are still synthesized. It is concluded that there is no correlation between metabolic stability of protein and the stability of its messenger ribonucleic acid.     

Kinetics of synthesis of nitrogenase in batch and continuous culture of Anabaena flos-aquae

Summary Nitrogenase of Anabaena flos-aquae was inactivated by oxygen and recovery of activity was measured in batch and iron, phosphate and urea-limited continuous cultures. In batch culture, canavanine, chloramphenicol, methylamine, proflavine, puromycin and urea inhibited the recovery process. The rate of recovery of nitrogenase activity in continuous cultures was dependent on light intensity, concentration of urea, ammonium salts and nitrate, and independent of growth rate. Oxygen and urea caused an inactivation of nitrogenase in continuous cultures.     

Infulence of hormones and medium composition on the degradation of phosphoenolpyruvate carboxykinase (GTP) and total protein in Reuber H35 cells.

Reuber H35 cells were pulse-labeled with radioactive leucine and the influence of hormones, serum, and amino acids on protein degradation was investigated during a subsequent chase period. Radioactive, immunoprecipitable phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) had a half-life of 5 to 6 hours which was not influenced by either N6, O2-dibutyryl adenosine 3':5'-monophosphate, dexamethasone, or insulin. The rate of phosphoenolpyruvate carboxykinase degradation was the same under steady state conditions as during the approach to a new steady state following hormonal induction or deinduction of the enzyme. Therefore, hormonal regulation of enzyme activity in vivo is the result of changes in the rate of enzyme synthesis. The rate of proteolysis for total cell proteins was increased under nutritional step-down conditions produced by the removal of serum or amino acids, or both, from the medium. This effect was completely prevented by insulin. Cycloheximide and puromycin, but not actinomycin D or cordycepin, inhibited protein degradation under step-down conditions but did not further decrease the basal rate of proteolysis measured in the presence of either insulin or serum plus amino acids. There was a good correlation between changes in proteolysis produced by serum and amino acids and changes in the degradation rate of phosphoenolpyruvate carboxykinase. Also, inhibition of proteolysis with cycloheximide and puromycin was accompanied by a decrease in the degradation rate for enzyme antigen. It is suggested that nutritional step-down leads either to the synthesis or activation of a proteolytic system.     

Protein degradation during sporulation ofBacillus megaterium: Effect of actinomycin D

The first acceleration of protein degradation in cells ofBacillus megaterium was found at the stage 0–I of sporulation, the second one at the stage II–III, where the sporulation process became irreversible. These accelerations were reduced by actinomycin D inhibiting RNA and protein syntheses by more than 95%. In the presence of the antibiotic, only 8% of prelabeled proteins were degraded. Actinomycin D did not lower either the concentration of ATP or the proteolytic activity in the homogenate prepared from sporulating cells. This indicates that the inhibition of protein catabolism by actinomycin D was not owing to the absence of ATP or proteolytic enzymes. Actinomycin probably inhibited an unknown step preceding the proteolytic attack of the protein molecules during sporulation, because it had no significant effect on proteolysis during vegetative growth.     

ATP-stimulated degradation of endogenous proteins in cell-free extracts of BHK 21/C13 fibroblasts. A key role for the proteinase, macropain, in the ubiquitin-dependent degradation of short-lived proteins.

Baby hamster kidney (BHK) 21/C13 cell proteins, labeled with [35S]methionine, [14C]leucine or [3H]leucine in intact cells, were degraded in soluble, cell-free extracts by an ATP-stimulated process. The stimulatory effect of ATP appeared to require ATP hydrolysis and was mediated to a large extent by ubiquitin. Although the cell extracts contained endogenous ubiquitin, supplementation with exogenous ubiquitin increased ATP-dependent proteolysis by up to 2-fold. Furthermore, antibodies against the E1 ubiquitin conjugating enzyme specifically inhibited both conjugation of [125I]ubiquitin to endogenous proteins and ATP/ubiquitin-dependent proteolysis. Addition of purified E1 to antibody-treated extracts restored conjugation and proteolysis. Proteins containing the amino acid analogues canavanine and azatryptophan were also degraded in vitro by an ATP/ubiquitin-dependent process but at a rate up to 2-fold faster than normal proteins. These results indicate that soluble, cell-free extracts of BHK cells can selectively degrade proteins whose rates of degradation are increased in intact cells. Treatment of cell-free extracts with antibodies against the high molecular weight proteinase, macropain, also greatly inhibited the ATP/ubiquitin-dependent degradation of endogenous proteins. Proteolysis was specifically restored when purified macropain L was added to the antibody-treated extracts. Treatment of cell extracts with both anti-macropain and anti-E1 antibodies reduced ATP/ubiquitin-dependent proteolysis to the same extent as treatment with either antibody alone. Furthermore, proteolysis could be restored to the double antibody treated extracts only after addition of both purified E1 and macropain. These results provide strong evidence for an important role for macropain in the ATP/ubiquitin-dependent degradation of endogenous proteins in BHK cell extracts.     

Metabolism of alpha-factor by a mating type cells of Saccharomyces cerevisiae.

When a mating type cells of Saccharomyces cerevisiae are exposed to the mating pheromone alpha-factor in liquid cultures, there is a time-dependent loss of alpha-factor activity from the culture fluid. This loss of biological activity can be directly correlated with the proteolysis of the pheromone by a mating type cells. The metabolism of alpha-factor by a mating type cells may be measured by using either in vitro 125I-labeled or in vivo 35S-labeled pheromone. Addition of chloroquine to growing cultures of a mating type cells at concentrations which cause no detectable alterations in cell growth produces a potentiation of alpha-factor mediated cell cycle arrest. This potentiation of alpha-factor activity is directly correlated with the inhibition of alpha-factor proteolysis. Thus, while proteolytic digestion of alpha-factor appears to be related to the mechanism whereby a mating type cells "detoxify" alpha-factor and recover from cell cycle arrest, proteolysis of the mating factor is not necessary for alpha-factor mediated cell cycle arrest.     

Protective antibodies inhibit reovirus internalization and uncoating by intracellular proteases.

We identified in vitro correlates of in vivo protection mediated by nonneutralizing antibodies specific for reovirus capsid proteins. We defined mechanisms of antibody action by analyzing monoclonal antibody (MAb) effects at sequential steps in reovirus serotype 3 strain Dearing (T3D) infection of L cells. Two types of experiments showed that protective MAbs specific for the outer capsid proteins sigma 3 or mu 1 inhibited T3D infection independent of effects on binding. First, MAbs which had no effect on T3D binding inhibited T3D growth. Second, MAb-coated T3D attached to L cells did not replicate as efficiently as T3D without bound antibody. We therefore defined sigma 3-specific MAb effects on postbinding steps in T3D infection. T3D coated with MAb sigma 3-10G10 exhibited prolonged sensitivity to growth inhibition by ammonium chloride. Since ammonium chloride inhibits endosomal acidification and proteolytic processing of the T3D capsid, this suggested that MAbs inhibit early steps in T3D infection. This was confirmed by direct demonstration that several sigma 3-specific MAbs inhibited proteolytic uncoating of virions by fibroblasts. We identified two mechanisms for antibody-mediated inhibition of virion uncoating: (i) inhibition of internalization of T3D-MAb complexes bound to the cell surface, and (ii) inhibition of intracellular proteolysis of the T3D capsid. Studies using a cell-free system confirmed that sigma 3-specific MAbs directly block proteolytic uncoating of the T3D virion. In addition, we found that sigma 3-specific MAbs block (and therefore define) two distinct steps in proteolytic uncoating of the reovirion. We conclude that antibodies which are protective in vivo inhibit postbinding events in reovirus infection of permissive cells. Protective antibodies act by inhibiting internalization and intracellular proteolytic uncoating of the virion. Analysis of postbinding mechanisms of MAb action may identify targets for vaccine development and antiviral therapy.     

The effect of endogenous proteases on the spectrin binding proteins of human erythrocytes

We have demonstrated that in human erythrocyte ghosts endogenous proteolytic activity is responsible for the digestion of the spectrin binding proteins (bands 2.1 to 2.6). The pH optimum, cofactor requirements and inhibitor sensitivity have been established. Our results indicate that proteolysis of bands 2.1 to 2.6 and the formation of 3′, a fragment containing an active spectrin binding site, can occur through two enzymatic pathways: a cascade of consecutive proteolytic cleavages of the spectrin binding proteins inhibited by phenylmethylsulfonyl fluoride or a Ca²⁺-stimulated, phenylmethylsulfonyl fluoride-insensitive, EDTA-inhibited cleavage of band 2.1 to band 2.3, followed by digestion to band 3′ by phenylmethylsulfonyl fluoride-inhibitable enzymes. These findings may provide the techniques necessary to prevent proteolysis of the spectrin binding proteins during purification and reconstitution experiments and provide insight into how they are formed in vivo.     

Investigation of the mechanism of protein turnover in HeLa S-3 cells by incubation at elevated temperatures

An apparent paradox relating to the degradation of endogenous proteins in HeLa S-3 cells occurs at 45 degrees C, at which their proteolysis is considerably enhanced in vitro but completely inhibited in vivo. No significant differences in rates of degradation of short-lived (nascent) and long-lived ('existing') proteins synthesised at 37 degrees C were found when chased at temperatures up to 43 degrees C, but at 45 degrees C degradation of both categories was reduced to zero in vivo. Synthesis of protein was suppressed at temperatures above 41 degrees C, being reduced by up to 60% at 43 degrees C. Proteolysis in vitro proceeded 1.6-1.7 times faster at 45 degrees C than at 37 degrees C and neutral pH. Evidence is presented for the involvement of the basal system; the findings both in vivo and in vitro do not seem to implicate the lysosomal system, no firm indication being obtained of its 'induction' at elevated temperatures. The results are discussed in terms of the arrest of intracellular circulation at elevated temperatures, thereby reducing the delivery rate of proteins as substrates of the intracellular basal proteolytic enzyme system to negligible levels (i.e., to the frequency of encounters due solely to the diffusion of protein molecules with the cytoplasm).     

Proteolytic digestion of bacterial inclusion body proteins during dynamic transition between soluble and insoluble forms.

Inclusion bodies formed by two closely related hybrid proteins, namely VP1LAC and LACVP1, have been compared during their building in Escherichia coli. Features of these proteins are determinant of aggregation rates and protein composition of the bodies, generating insoluble particles with distinguishable volume evolution. Interestingly, in LACVP1 and less perceptibly in VP1LAC bodies, an important fraction of the aggregated polypeptide is lost at a given stage of body construction. Stable degradation intermediates of the more fragile LACVP1 are concomitantly found embedded in the bodies. When recombinant protein synthesis is arrested in growing cells, the amount of aggregated protein drops while the amount of soluble protein undergoes a sudden rise before proteolysis. This indicates an architectural plasticity during the in vivo building of the studied inclusion bodies by a dynamic transition between soluble and insoluble forms of the recombinant proteins involved. During this transition, protease-sensitive polypeptides can suffer an efficient proteolytic attack and the resulting fragments further aggregate as inclusion body components.     

Redox pacing of proteome turnover: influences of glutathione and ketonemia

In early starvation tissue protein degradation increases, however in later starvation proteolysis declines so as to pace gradual atrophy during synthetic failure. Secondary decline of proteolytic pathways under progressive nutritional desperation is unexplained. After several days of starvation tissue GSH is partly depleted and GSSG/GSH is increased, followed by onset of ketonemia from fat breakdown. Ketone bodies inexplicably delay net muscle protein loss. Recent studies identify a proteome subset of more than 200 proteins with reactive sulfhydryl sites as candidates for coordinate redox control of diverse cell functions. Ketones cause protein sulfhydryl oxidation and protein S-glutathionylation. Here, redox-responsive proteolytic pathways were bio-assayed by release of [3H]leucine from rat myocardium under non-recirculating perfusion. More than 75% of myocardial protein degradation was inhibited and defined by infusion of diamide (100 microM) under constant physiologic concentrations of complete amino acids. Diamide-inhibitable proteolysis includes all lysosomal and some extra-lysosomal proteolysis. Following diamide washout, the reversal of proteolytic inhibitory action was greatly enhanced by artificial repletion of GSH by supra-physiologic extra-cellular GSH (1mM) exposure. Therefore, GSH maintains much of constitutive protein degradation in a primary tissue bioassay. Physiologic acetoacetate infusion (5mM) inhibited redox-responsive protein degradation. Uniformly [3H]leucine labeled 3T3 cells exhibited similar redox-dependent and redox-independent subcomponents of protein degradation. Independent of ketones, steady state cathepsin B reaction rate ex vivo was graded in proportion to the GSH concentration without GSSG, and inversely proportional to the GSSG/GSH redox ratio with inhibitory threshold at 0.5% oxidized. Linkage of some cysteine protease reaction rates to the interplay between GSH-GSSG/GSH status and ketonemia is suggested among transcendent mechanisms coordinating and pacing proteome turnover under prolonged starvation. The possibility of pre-emptive, redox coordination of distinct proteolytic pathways is speculatively discussed.     

Subcellular distribution of abnormal proteins in rabbit reticulocytes. Effects of cellular maturation, phenylhydrazine and inhibitors of ATP synthesis

Inhibitors of ATP synthesis (cyanide, dinitrophenol, fluoride) inhibited proteolysis of pulse-labelled abnormal proteins in rabbit reticulocytes and caused an accumulation of the aberrant polypeptides in the cell debris fraction in a manner analogous to phenylhydrazine-induced Heinz bodies. When the reticulocytes were separated into age-groups by sedimentation through discontinuous gradients of bovine serum albumin, the ability of the cells to degrade puromycin peptides decreased with increasing cellular maturity and, in the more mature cells, up to 40% of the labelled abnormal polypeptide remained associated with the cell debris fraction at the end of the chase period. It is suggested that the association of the abnormal polypeptide with the cell debris fraction is a consequence of a maturation-induced loss, or an inhibitor-induced inactivation of the cellular proteolytic activity.     

Protein modification by phosphorylation during the process of nuclear membrane dissolution in puromycin-treated mouse oocytes.

The present study was undertaken to elucidate the mechanism of nuclear membrane dissolution (NMD) in puromycin-treated mouse oocytes. Treatment of germinal vesicle breakdown (GVBD) oocytes with puromycin (50 micrograms/ml) induced chromosome decondensation with formation of a polar body; these are designated nuclear membrane (NM) oocytes. After withdrawal of puromycin, NM oocytes underwent NMD (approximately 70%) during a 12-h culture period. Either dibutyryl cyclic AMP (dbcAMP, 25-100 micrograms/ml) or isobutylmethylxanthine (IBMX, 0.1-1.0 mM) inhibited the process of NMD in a dose-dependent manner, suggesting the involvement of cAMP in the process of NMD. To determine which protein(s) participated in the transition from interphase to metaphase II during NMD, NM oocytes were labeled with [35S]methionine, and one- and two-dimensional gel electrophoresis were performed. Although the synthesis of stage-specific proteins during NMD was not found, two specific proteins of Mr 27,000 and 46,000, which were synthesized at interphase following removal of puromycin, were modified during NMD. Phosphatase treatment and 32PO4-labeling experiments indicated that phosphorylation was responsible for these modifications, which were inhibited by either dbcAMP or IBMX. Therefore, it appears that phosphorylation of specific proteins may play an important role in the transition from interphase to metaphase II.