Properties of antithrombin-thrombin complex formed in the presence and in the absence of heparin.

Protein degradation by diploid human-embryo lung fibroblasts (MRC5 cells) in monolayer culture was studied. 1. Varying the labelling period of proteins was found to alter the half-lives of labelled abnormal canavanine-containing proteins to an extent very similar to that obtained with normal proteins. 2. By manipulating the times of labelling it was possible to generate a species of abnormal protein with a greater half-life than that of a species of normal protein. A comparison of the lysosomal involvement in their degradation as determined both by inhibition by methylamine, a lysosomotropic agent, and by the degree of increase in protein degradation in step-down conditions, indicated that the degree of lysosomal involvement was not entirely dependent upon the half-life of the protein, but that abnormal proteins are preferentially degraded non-lysosomally. 3. The microtubule inhibitors colchicine and vinblastine were found to stimulate statistically basal protein degradation of normal long-labelled protein, whereas they had less effect upon the basal degradation of the other species of proteins studied and very little effect upon step-down degradation of all proteins studied. The stimulation in protein degradation found did not seem to involve the acid proteinases of lysosomes.     

Ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutant ts85

We have shown that covalent conjugation of ubiquitin to proteins is temperature-sensitive in the mouse cell cycle mutant ts85 due to a specifically thermolabile ubiquitin-activating enzyme (accompanying paper). We show here that degradation of short-lived proteins is also temperature sensitive in ts85 , in contrast to wild-type and revertant cells. While more than 70% of the prelabeled abnormal proteins (containing amino acid analogs) or puromycyl peptides are degraded within 4 hr at the permissive temperature in both ts85 and wild-type cells, less than 15% are degraded in ts85 cells at the nonpermissive temperature. Degradation of abnormal proteins and puromycyl peptides in both ts85 cells and wild-type cells is nonlysosomal and ATP-dependent. Immunochemical analysis shows a strong and specific reduction in the levels of in vivo labeled ubiquitin-protein conjugates at the nonpermissive temperature in ts85 cells. Degradation of normal, short-lived proteins is also specifically temperature sensitive in ts85 . We suggest that the contribution of ubiquitin-independent pathways to the degradation of short-lived proteins in this higher eucaryotic cell is no more than 10%, and possibly less.     

Turnover of abnormal proteins in Bacillus megaterium and Saccharomyces cerevisiae: differences between in vivo and in vitro degradation

Degradation of abnormal proteins in Bacillus megaterium and Saccharomyces cerevisiae in vivo was compared with that in cell-free extracts. Protein degradation in vivo, when the cells were labelled with ¹⁴C-leucine during growth in the presence of ethionine, was affected by the concentration of the analogue used. Proteins synthesized in the presence of 0.2–1 mM ethionine were degraded most rapidly in both organisms. The proteolytic enzyme system of yeast degraded the analogue-containing proteins in vitro faster than the normal proteins. This holds also for proteins synthesized in the presence of 5 mM ethionine, whose degradation in vivo was impaired. The proteolytic system of B. megaterium, on the other hand, was unable in vitro to differentiate between normal and abnormal proteins. Denatured proteins underwent preferential degradation over normal and ethionine-containing proteins.Participant in the UNESCO Postgraduate Course On Modern Problems in Biology and Microbial Technology.     

Abnormal protein metabolism in skin fibroblasts in vitro from patients with Duchenne muscular dystrophy

Rates of protein turnover have been measured on a statistical basis in Duchenne muscular dystrophy and normal skin fibroblasts populations in vitro. At comparable numbers of cumulative population doublings, protein synthesis was significantly reduced by about 24% in DMD fibroblasts as compared to normal fibroblasts (p less than 0.01, N = 12). Degradation of short lived proteins was significantly enhanced by about 60% (p less than 0.05, N = 18), and the degradation of long lived proteins was significantly increased by about 28% (p less than 0.05, N = 18) in DMD fibroblast populations in vitro. The enhanced degradation of long lived proteins in DMD fibroblasts can be reduced to basal levels of degradation by the use of the protease inhibitors leupeptin and Ep475 (p less than 0.05, N = 9).     

Rapid, ATP-dependent degradation of a truncated D1 protein in the chloroplast.

The D1 protein constitutes one of the reaction center subunits of photosystem II and turns over rapidly due to photooxidative damage. Here, we studied the degradation of a truncated D1 protein. A plasmid with a precise deletion in the reading frame of the psbA gene encoding D1 was introduced into the chloroplast of Chlamydomonas reinhardtii. A homoplasmic mutant containing the desired gene was able to synthesize the truncated form of the polypeptide, but could not accumulate significant levels of it. As a consequence, other central photosystem II subunits did not assemble within the thylakoid membrane. In vivo pulse-chase experiments showed that the abnormal D1 protein is rapidly degraded in the light. Degradation was delayed in the light in the presence of an uncoupler, or when cells were incubated in the dark. Pulse-chase experiments performed in vitro indicate that an ATP and metal-dependent protease is responsible for the breakdown process. The paper describes the first in vivo and in vitro functional test for ATP-dependent degradation of a defect polypeptide in chloroplasts. The possible involvement of proteases similar to those removing abnormal proteins in prokaryotic organisms is discussed on the basis of proteases recently identified in chloroplasts.     

Role of proteasomes in the degradation of short-lived proteins in human fibroblasts under various growth conditions

Degradation of proteins in the cells occurs by proteasomes, lysosomes and other cytosolic and organellar proteases. It is believed that proteasomes constitute the major proteolytic pathway under most conditions, especially when degrading abnormal and other short-lived proteins. However, no systematic analysis of their role in the overall degradation of truly short-lived cell proteins has been carried out. Here, the degradation of short-labelled proteins was examined in human fibroblasts by release of trichloroacetic acid-soluble radioactivity. The kinetics of degradation was decomposed into two, corresponding to short- and long-lived proteins, and the effect of proteasomal and lysosomal inhibitors on their degradation, under various growth conditions, was separately investigated. From the degradation kinetics of proteins labelled for various pulse times it can be estimated that about 30% of newly synthesised proteins are degraded with a half-life of approximately 1h. These rapidly degraded proteins should mostly include defective ribosomal products. Deprivation of serum and confluent conditions increased the degradation of the pool of long-lived proteins in fibroblasts without affecting, or affecting to a lesser extent, the degradation of the pool of short-lived proteins. Inhibitors of proteasomes and of lysosomes prevented more than 80% of the degradation of short-lived proteins. It is concluded that, although proteasomes are responsible of about 40-60% of the degradation of short-lived proteins in normal human fibroblasts, lysosomes have also an important participation in the degradation of these proteins. Moreover, in confluent fibroblasts under serum deprivation, lysosomal pathways become even more important than proteasomes in the degradation of short-lived proteins.     

Effect of hypophysectomy on the rates of protein synthesis and degradation in rat liver.

The effect of hypophysectomy on the protein metabolism of the liver in vivo was studied. Fractional rates of protein synthesis and degradation were determined in the livers of normal and hypophysectomized rats. Synthesis was measured after the injection of massive amounts of radioactive leucine. Degradation was estimated either as the balance between synthesis and accumulation of stable liver proteins or from the disappearance of radioactivity from the proteins previously labelled by the injection of NaH14CO3. The results indicate that: (1) hypophysectomy diminishes the capacity of the liver to synthesize proteins in vivo, mainly of those that are exported as plasma proteins; (2) livers of both normal and hypophysectomized rats show identical protein-degradation rates, whereas plasma proteins are degraded slowly after hypophysectomy.     

Collagen degradation in I-cells is normal

There is evidence that lysosomal proteases mediate the intracellular degradation of structurally abnormal collagen. I-Cell disease (Mucolipidosis II) is characterized by marked deficiency of many lysosomal hydrolases, including the collagenolytic enzyme cathepsin B. The experiments reported here tested the hypothesis that degradation of abnormal collagen would be severely impaired in I-cells. Skin fibroblasts from 3 patients with I-cell disease were incubated with and without cis-hydroxyproline, a proline analog that causes structural abnormalities in collagen, and [14C]proline. The amount of [14C]hydroxyproline in a low molecular weight fraction relative to total [14C]hydroxyproline was used as a measure of intracellular collagen degradation. Levels of degradation were significantly higher in I-cells exposed to cis-hydroxyproline than in cells incubated without the analog. Similar data were obtained for normal human fetal lung fibroblasts incubated under the same conditions. Degradation of [125I]-epidermal growth factor was used to assess the functionality of the lysosomal pathway for protein degradation, and it was much lower in I-cells than in normal cells. It can be concluded that a completely functional complement of lysosomal enzymes is not necessary for structurally abnormal collagen to be degraded intracellularly; the data suggest that a nonlysosomal pathway exists.     

Degradation of abnormal proteins in peptidase-deficient mutants of Salmonella typhimurium.

The degradation of abnormal proteins produced as a result of incorporation of the arginine analog L-canavanine or generated by exposure to puromycin was studied in wild-type and multiply peptidase-deficient strains of Salmonella typhimurium. Both types of abnormal protein were rapidly degraded during growth of Pep+ strains of this organism. Peptidase--deficient mutants (lacking peptidases N, A, B, and D) could also degrade these abnormal proteins, although the rate of production of trichloroacetic acid-soluble degradation products was slower in the mutant strain than in a strain carrying a normal complement of peptidases. Analysis of these trichloroacetic acid-soluble degradation products of ion-exchange chromatography showed that free amino acid was the major breakdown product produced by the wild-type strain. The acid-soluble degradation product produced by the mutant strain, however, was a complex mixture that contained a variety of small peptides as well as free amino acids. These results indicate that the same group of peptidases shown previously to function in the degradation of exogenously supplied peptides and in protein turnover during carbon starvation also lie on the pathway by which abnormal proteins are degraded.     

Cytokinin inhibits the proteasome-mediated degradation of carbonylated proteins in Arabidopsis leaves

Under normal conditions, plants contain numerous carbonylated proteins, which are thought to be indicative of oxidative stress damage. Conditions that promote formation of reactive oxygen species (ROS) enhance protein carbonylation, and protein degradation is required to reverse the damage. However, it is not clear how the degradation of carbonylated proteins is controlled in planta. In this report, we show that detached Arabidopsis leaves rapidly and selectively degrade carbonylated proteins when kept in the dark. The loss of carbonylated proteins corresponded to a loss of soluble protein and accumulation of free amino acids. Degradation of carbonylated proteins and the loss of soluble protein was blocked by MG132 but not 3-methyladenine, suggesting that the 26S proteasome pathway rather than the autophagic pathway was involved. Consistent with this, rpn10 and rpn12 mutants, which are defective in proteasome function, had increased (rather than decreased) levels of carbonylated proteins when detached in the dark. Feeding metabolites (amino acids and sucrose) to detached leaves of wild-type Arabidopsis in the dark had little or no effect on the loss of carbonylated proteins, whereas providing soybean xylem sap via the transpiration stream effectively prevented degradation. The effect of xylem sap was mimicked by feeding 10 muM kinetin. We postulate that disruption of cytokinin flux to detached leaves triggers the selective degradation of carbonylated proteins via the proteasome pathway. The results may have implications for the control of protein mobilization in response to changes in N availability.     

Biosynthetic incorporation of oxidized amino acids into proteins and their cellular proteolysis

We demonstrate that oxidized amino acids can be incorporated into proteins by protein synthesis. The level of incorporation into protein was dependent on the concentration of oxidized amino acid supplied to the cells. At low levels of incorporation, the oxidized amino acids examined increased the degradation rate of the cell proteins. Degradation of certain proteins containing high levels of DOPA (but not ortho or meta tyrosine) was decreased to below the basal degradation rates suggesting that DOPA may contribute to proteins becoming resistant to proteolysis. Changes in the degradation rates of the oxidized amino acid-containing proteins was shown to have no impact on the degradation rates of native proteins, indicating that the activity of the degradative machinery was not affected. We demonstrate that oxidized proteins are selectively degraded by the proteasomes and provide evidence to suggest that the proteasomes and the endosomal-lysosomal systems may act in sequence as well as in parallel. The incorporation approach, unlike cell studies in which an exogenous oxidant is used, allows the degradation rates of the oxidatively modified proteins to be selectively measured, offering a greater sensitivity as well as greatly reducing toxicity to the cell and avoiding oxidative modification of other cell components.     

Senescence-associated degradation of chloroplast proteins inside and outside the organelle

Leaf proteins, and in particular the photosynthetic proteins of plastids, are extensively degraded during senescence. Although this involves massive amounts of protein, the mechanisms responsible for chloroplast protein degradation are largely unknown. Degradation within the plastid itself is supported by the observation that chloroplasts contain active proteases, and that chloroplasts isolated from senescing leaves can cleave Rubisco to release partially digested fragments. It is less clear whether chloroplasts can complete Rubisco degradation. Chloroplastic proteases are likely involved in the breakdown of the D1 and LHCII proteins of photosystem II. Small s enescence- a ssociated v acuoles (SAVs) with high-proteolytic activity develop in senescing leaf cells, and there is evidence that SAVs contain chloroplast proteins. Thus, an extra-plastidic pathway involving SAVs might participate in the degradation of some chloroplast proteins. Plastidic and extra-plastidic pathways might cooperate in the degradation of chloroplast proteins, or they might represent alternative, redundant pathways for photosynthetic protein degradation.     

Protein degradation in skin fibroblasts from patients with Duchenne muscular dystrophy.

The rates of degradation of [3H]leucine-labelled proteins have been measured in cultures of skin fibroblasts obtained from normal controls (five subjects) and patients with Duchenne muscular dystrophy (six subjects). Cultures were incubated with [3H]leucine (10 microCi/ml) for 60 min to label "short-lived" proteins, and with [3H]leucine (5 microCi/ml) for 60 h to label "long-lived" proteins. Optimal wash procedures were devised for removal of [3H]leucine from the extracellular space and from cell pools before beginning degradation measurements. Re-utilization of [3H]leucine released from degraded labelled proteins was prevented by supplementing the medium with 4mM-leucine. Rates of degradation did not depend on the growth state of the cells or on cell age over the range used (passages eight-20). Degradation of long-lived proteins was approximately linear over a 24h period, at a rate of 1.0% per h. 30% of short-lived protein was degraded within 6h. No differences were observed between protein degradation in normal fibroblasts and in those from patients with Duchenne muscular dystrophy.     

Action spectrum of Drosophila cryptochrome

Cryptochromes are a highly conserved class of UV-A/blue light photoreceptors. In Drosophila, cryptochrome is required for the normal entrainment of circadian rhythms to light dark cycles. The photocycle and molecular mechanism of animal cryptochrome photoreception are presently unknown. Drosophila cryptochrome undergoes light-dependent degradation when heterologously expressed in Schneider-2 cells. We have generated Drosophila luciferase-cryptochrome fusion proteins to more precisely monitor light-dependent cryptochrome degradation. We found that the luciferase-cryptochrome fusion protein undergoes light-dependent degradation with luciferase activity declining approximately 50% within 5 min of light exposure and approximately 85% within 1 h of light exposure. Degradation is inhibited by MG-132, consistent with a proteasomal degradation mechanism. Irradiance-response curves yield an action spectrum similar to absorption spectra for prokaryotic and eukaryotic cryptochromes with highest sensitivity in the UV-A. A luciferase-cryptochrome fusion protein lacking the terminal 15 amino acids is stably expressed in the dark but demonstrates increased sensitivity to light-induced degradation. The conferral of light-dependent degradation on a heterologous protein by fusion to cryptochrome may be a useful tool for probing protein function in cell expression systems.     

The ubiquitin-mediated proteolytic pathway: enzymology and mechanisms of recognition of the proteolytic substrates

Degradation of proteins by the ubiquitin system involves two discrete steps. Initially, ubiquitin is covalently linked in an ATP-dependent mode to the protein substrate. The protein moiety of the conjugate is subsequently degraded by a specific protease into peptides and free amino acids with the release of free and reutilizable ubiquitin. The degradation process also requires energy. In this review we shall discuss the mechanisms involved in ubiquitin activation, selection of substrates for conjugation, and subsequent degradation of ubiquitin-conjugated proteins. In addition, we shall briefly summarize what is currently known of the role of the ubiquitin system in protein degradation in vitro and in vivo.     

Endocytosis of liposomes containing lysosomal proteins increases intracellular protein degradation in growing L-132 cells

We have used a new approach to test the possible participation of lysosomes in the degradation of long-lived proteins. Rat liver lysosomal proteins were introduced, via multilamellar liposomes, into L-132 cells. Viability and protein synthesis were not impaired by this treatment. The liposomal content was released into the lysosomes of the cultured cells, as revealed by ferritin uptake and electron microscopy. Degradation rates of long-lived proteins increased with the uptake of lysosomal proteases. However, the increased protein degradation of chloroquine and leupeptin, in contrast to the inhibition by these reagents of the increased protein degradation of cells 'starved' of serum (step-down conditions). This approach opens a new way of investigating the degradation of intracellular proteins in cultured cells.     

The ATP dependence of the degradation of short- and long-lived proteins in growing fibroblasts

To characterize the system(s) responsible for degradation of short-lived and long-lived proteins in mammalian cells, we compared the concentrations of ATP required for the degradation of these classes of proteins in growing hamster fibroblasts. By treating CHEF-18 cells with increasing concentrations of dinitrophenol and 2-deoxyglucose, it was possible to reduce their steady-state ATP content by different amounts (up to 98%). These treatments caused a rapid decrease in the degradation of both short- and long-lived proteins. Removal of the inhibitors led to a prompt restoration of ATP and proteolysis. As ATP content fell below normal levels (about 3.1 mM), rates of proteolysis decreased in a graded biphasic fashion. Reduction in ATP by up to 90% (as may occur in anoxia or injury) decreased proteolysis up to 50%; and with further loss of ATP, protein breakdown fell more sharply. Degradation of both classes of proteins was inhibited by 80% when ATP levels were reduced by 98%. The levels of ATP required for the breakdown of short- and long-lived proteins were indistinguishable. Protein synthesis was much more sensitive to a decrease in ATP content than protein breakdown and fell by 50% when ATP was reduced by only 15%. Chloroquine, an inhibitor of lysosome function, did not reduce the degradation of either class of proteins in growing cells, but it did inhibit the enhanced degradation of long-lived proteins upon removal of serum (in accord with previous studies). Thus, in growing fibroblasts, an ATP-dependent nonlysosomal process appears responsible for the hydrolysis of both short- and long-lived proteins.     

Intracellular protein degradation in cultured bovine lens epithelial cells

Summary Although several proteases have been identified in homogenates of cultured epithelial cells of the eye lens and in lens tissues, there is little information regarding intracellular protein degradation in intact lens cells in vitro. Cultured lens cells may be useful in the study of intracellular protein degradation in the lens, a tissue with a wide range of protein half-lives. This is of interest because alterations in protein turnover in the lens have been implicated in cataract formation. This study examines intracellular protein degradation in cultured bovine lens epithelial cells (BLEC). Cell cultures were incubated with radiolabeled leucine to label intracellular proteins. Protein degradation was measured by monitoring the release of trichloroacetic-acid-soluble radioactivity into the culture medium. The average half-life of long-lived proteins (half-life >50 h) was typically about 57 h in serum-supplemented medium. Average rates of degradation of long-lived proteins increased by up to 73% when fetal bovine serum was withdrawn from the culture medium. Serum had no effect on the degradation of short-lived proteins (half-life <10 h). Degradation of long-lived proteins in the presence and absence of serum was further studied in cultured BLEC from population doubling level (PDL) 2 to 43. Average half-life of proteins in serum-supplemented medium was 52 to 58 h and did not vary significantly as a function of PDL. Degradation rates in serum-free medium increased approximately twofold up to PDL 7, but returned by PDL 25 to original levels, which were maintained through PDL 43. This work was supported in part by grants from U. S. Department of Agriculture contract 53-3K06-5-10, Massachusetts Lions Eye Research Fund, Inc., and the Daniel and Florence Guggenheim Foundation. D. A. E. is a recipient of a National Eye Institute postdoctoral fellowship.     

Involvement of the molecular chaperone Ydj1 in the ubiquitin-dependent degradation of short-lived and abnormal proteins in Saccharomyces cerevisiae.

In Escherichia coli and mitochondria, the molecular chaperone DnaJ is required not only for protein folding but also for selective degradation of certain abnormal polypeptides. Here we demonstrate that in the yeast cytosol, the homologous chaperone Ydj1 is also required for ubiquitin-dependent degradation of certain abnormal proteins. The temperature-sensitive ydj1-151 mutant showed a large defect in the overall breakdown of short-lived cell proteins and abnormal polypeptides containing amino acid analogs, especially at 38 degrees C. By contrast, the degradation of long-lived cell proteins, which is independent of ubiquitin, was not altered nor was cell growth affected. The inactivation of Ydj1 markedly reduced the rapid, ubiquitin-dependent breakdown of certain beta-galactosidase (beta-gal) fusion polypeptides. Although degradation of N-end rule substrates (arginine-beta-gal and leucine-beta-gal) and the B-type cyclin Clb5-beta-gal occurred normally, degradation of the abnormal polypeptide ubiquitin-proline-beta-gal (Ub-P-beta-gal) and that of the short-lived normal protein Gcn4 were inhibited. As a consequence of reduced degradation of Ub-P-beta-gal, the beta-gal activity was four to five times higher in temperature-sensitive ydj1-151 mutant cells than in wild-type cells; thus, the folding and assembly of this enzyme do not require Ydj1 function. In wild-type cells, but not in ydj1-151 mutant cells, this chaperone is associated with the short-lived substrate Ub-P-beta-gal and not with stable beta-gal constructs. Furthermore, in the ydj1-151 mutant, the ubiquitination of Ub-P-beta-gal was blocked and the total level of ubiquitinated protein in the cell was reduced. Thus, Ydj1 is essential for the ubiquitin-dependent degradation of certain proteins. This chaperone may facilitate the recognition of unfolded proteins or serve as a cofactor for certain ubiquitin-ligating enzymes.