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.     

Divergent effects of a dnaK mutation on abnormal protein degradation in Escherichia coli

Escherichia coli bacteria produce at least one 70 kD stress protein, the product of the dnaK gene. We have compared the rates of degradation of different types of abnormal proteins in null Ion E. coli with a partial deletion of the dnaK gene with the rates observed in null Ion dnaK+ cells. We have found that both canavanyl proteins and puromycyl polypeptides are degraded more slowly in the null dnaK mutants than in the dnaK+ strain. However, a temperature-sensitive mutant LacI protein is degraded more rapidly in the null dnaK strain. The stability of this temperature-sensitive LacI protein was also examined in detail under various other conditions.     

Limited degradation of oxidized calmodulin by proteasome: formation of peptides

Oxidized proteins are recognized and degraded preferentially by the proteasome. This is true for numerous proteins including calmodulin (CaM). The degradation of CaM was investigated in a human fibroblast cell line under conditions of oxidative stress. Low molecular CaM fragments or peptides were found under such conditions. In in vitro experiments it was investigated whether this CaM breakdown product formation is induced by protein oxidation or is due to a limited proteolysis-derived degradation by the 20S proteasome. Native unoxidized CaM was not degraded by 20S proteasome, oxidized CaM was degraded in a time- and H₂O₂ concentration-dependent manner. Peptides of similar molecular weight were detected in isolated calmodulin as in oxidatively stressed fibroblasts. The peptides were identified using isolated calmodulin. Therefore, in oxidatively stressed fibroblasts and in vitro CaM is forming oxidation-driven fragments and proteasomal cleavage peptides of approximately 30 amino acids which undergo a slow or no degradation.     

The clp proteases of Bacillus subtilis are directly involved in degradation of misfolded proteins

The presence of the heat stress response-related ATPases ClpC and ClpX or the peptidase ClpP in the cell is crucial for tolerance of many forms of stress in Bacillus subtilis. Assays for detection of defects in protein degradation suggest that ClpC, ClpP, and ClpX participate directly in overall proteolysis of misfolded proteins. Turnover rates for abnormal puromycyl peptides are significantly decreased in clpC, clpP, and clpX mutant cells. Electron-dense aggregates, most likely due to the accumulation of misfolded proteins, were noticed in studies of ultrathin cryosections in clpC and clpP mutant cells even under nonstress conditions. In contrast, in the wild type or clpX mutants such aggregates could only be observed after heat shock. This phenomenon supports the assumption that clpC and clpP mutants are deficient in the ability to solubilize or degrade damaged and aggregated proteins, the accumulation of which is toxic for the cell. By using immunogold labeling with antibodies raised against ClpC, ClpP, and ClpX, the Clp proteins were localized in these aggregates, showing that the Clp proteins act at this level in vivo.     

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.     

The degradation of bioactive peptides and proteins by dipeptidyl peptidase IV from human placenta

The degradation of several bioactive peptides and proteins by purified human dipeptidyl peptidase IV is reported. It was hitherto unknown that human gastrin-releasing peptide, human chorionic gonadotropin, human pancreatic polypeptide, sheep prolactin, aprotinin, corticotropin-like intermediate lobe peptide and (Tyr-)melanostatin are substrates of this peptidase. Kinetic constants were determined for the degradation of a number of other natural peptides, including substance P, the degradation of which has been described earlier in a qualitative manner. Generally, small peptides are degraded much more rapidly than proteins. However, the Km-values seem to be independent of the peptide chain length. The influence of the action of dipeptidyl peptidase IV on the biological function of peptides and proteins is discussed.     

Sequence- and species-dependence of proteasomal processivity

The proteasome is the degradation machine at the center of the ubiquitin-proteasome system and controls the concentrations of many proteins in eukaryotes. It is highly processive so that substrates are degraded completely into small peptides, avoiding the formation of potentially toxic fragments. Nonetheless, some proteins are incompletely degraded, indicating the existence of factors that influence proteasomal processivity. We have quantified proteasomal processivity and determined the underlying rates of substrate degradation and release. We find that processivity increases with species complexity over a 5-fold range between yeast and mammalian proteasome, and the effect is due to slower but more persistent degradation by proteasomes from more complex organisms. A sequence stretch that has been implicated in causing incomplete degradation, the glycine-rich region of the NFκB subunit p105, reduces the proteasome's ability to unfold its substrate, and polyglutamine repeats such as found in Huntington's disease reduce the processivity of the proteasome in a length-dependent manner.     

Inactivation of a gene that is highly conserved in Gram-positive bacteria stimulates degradation of non-native proteins and concomitantly increases stress tolerance in Lactococcus lactis

Exposure of cells to elevated temperatures triggers the synthesis of chaperones and proteases including components of the conserved Clp protease complex. We demonstrated previously that the proteolytic subunit, ClpP, plays a major role in stress tolerance and in the degradation of non-native proteins in the Gram-positive bacterium Lactococcus lactis. Here, we used transposon mutagenesis to generate mutants in which the temperature- and puromycin-sensitive phenotype of a lactococcal clpP null mutant was partly alleviated. In all mutants obtained, the transposon was inserted in the L. lactis trmA gene. When analysing a clpP, trmA double mutant, we found that the expression normally induced from the clpP and dnaK promoters in the clpP mutant was reduced to wild-type level upon introduction of the trmA disruption. Additionally, the degradation of puromycyl-containing polypeptides was increased, suggesting that inactivation of trmA compensates for the absence of ClpP by stimulating an as yet unidentified protease that degrades misfolded proteins. When trmA was disrupted in wild-type cells, both stress tolerance and proteolysis of puromycyl peptides was enhanced above wild-type level. Based on our results, we propose that TrmA, which is well conserved in several Gram-positive bacteria, affects the degradation of non-native proteins and thereby controls stress tolerance.     

Insertional mutagenesis of the lon gene in Escherichia coli: lon is dispensable.

The lon gene of Escherichia coli codes for an ATP-dependent protease. Mutations in lon cause a defect in the intracellular degradation of abnormal and mutant proteins and lead to a number of phenotypic changes, such as UV sensitivity and overproduction of capsular polysaccharide. We have isolated lambda transducing phage carrying the lon gene and used the lon phage as a target for insertional mutagenesis by a defective transposon Tn10 to produce lon::delta 16 delta 17Tn10 derivatives. The delta 16 delta 17Tn10 (hereafter called delta Tn10) elements were inserted at sites throughout the lon gene and disrupted the coding region between 15 and 75% of the distance from the amino-terminal end. Radioactive labeling of proteins in vivo in cells infected with different lambda lon::delta Tn10 phage demonstrated that the insertions resulted in the synthesis of truncated Lon proteins. The lon::delta Tn10 mutations, when crossed from the phage into the bacterial chromosome, abolished the synthesis of intact Lon protein, as assayed by antibody on Western blots. An analysis of the protein-degradative ability of lon::delta Tn10 cells suggests that although the insertions in lon caused a reduction in ATP-dependent protein degradation, they did not completely eliminate such degradation either in vivo or in vitro. The lon::delta Tn10 mutations and a lon deletion retaining only the amino-terminal 25% of the gene did not affect the energy-dependent degradation of proteins during starvation and led to only a 40 to 60% reduction in the ATP-dependent degradation of canavanine-containing proteins and puromycyl peptides. Our data provide clear evidence that energy-dependent proteolytic enzymes other than Lon exist in E. coli.     

Degradation of peptides and proteins of different sizes by homogenates of human MRC5 lung fibroblasts. Aged cells have a decreased ability to degrade shortened proteins

The degradation of haemoglobin and haemoglobin-derived peptide fragments by homogenates of MRC5 fibroblasts has been investigated. Results show that the smaller fragments were degraded more rapidly than larger substrates at both pH 5.5 and pH 7.5. Only the smallest of the soluble cyanogen bromide peptides (Mr 3500) was degraded at pH 7.5. Degradation at pH 5.5 proceeded more rapidly than that at pH 7.5 for all substrates tested but was more marked with the larger substrates. Homogenates prepared from aged cells degraded puromycin peptides and, to a lesser extent, cyanogen bromide peptides at a slower rate, at pH 7.5, than those prepared from younger cells. We suggest that cytosolic degradation is less selective and at least one cytosolic proteolytic activity decreases as cells age.     

Proteolytic degradation of insulin and glucagon.

The degradation of insulin and glucagon by a highly purified enzyme isolated from rat skeletal muscle was investigated. A sensitive assay for proteolytic degradation of insulin and glucagon using fluorescamine to detect an increase in primary amine groups was established. As measured by an increase in fluorescamine reactive materials, insulin was rapidly degraded by this highly purified enzyme without requiring initial disulfide cleavage. Associated with the increase in fluorescamine reactive materials was a decrease in immunoassayable insulinmglucagon wal also proteolytically degraded by this enzyme but a number of other peptides and proteins including proinsulin, and A and B chains of insulin were not degraded. Thus, we have demonstrated that insulin (and glucagon) can be proteolytically degraded by an enzyme isolated from an insulin sensitive tissue, skeletal muscle. Proteolytic degradation by this enzyme requires the intact insulin molecule rather than separate A and B chains.     

Peptide sequences that target proteins for enhanced degradation during serum withdrawal

Fibroblasts increase the catabolism of certain intracellular proteins in response to serum withdrawal, and these proteins contain specific peptide regions that may be required for their increased degradation. We show that the increased degradation of microinjected ribonuclease A during serum withdrawal can be blocked by co-injection of a pentapeptide corresponding to residues 7-11 of ribonuclease A, Lys-Phe-Glu-Arg-Gln. Furthermore, similar peptide sequences appear to play a widespread role in targeting proteins for enhanced degradation. Affinity-purified antibodies raised against the pentapeptide are able to precipitate 20-35% of radiolabeled cytosolic proteins from fibroblasts. Such proteins are preferentially degraded when cells are deprived of serum while nonimmunoprecipitable proteins are degraded at the same rate in the presence and absence of serum. Immunoreactive cytosolic proteins also exist in rat liver and kidney, and these proteins are depleted when protein degradation rates are enhanced due to starvation. Several types of evidence suggest that the peptides recognized in cellular proteins are similar to Lys-Phe-Glu-Arg-Gln but are not this exact sequence. Analyses of amino acid sequences for four proteins whose degradative rates are enhanced in response to serum withdrawal and for four proteins that are degraded in a serum-independent manner indicate two possible peptide motifs related to Lys-Phe-Glu-Arg-Gln that may target cellular proteins for enhanced degradation. These results, combined with previous studies (McElligott, M. A., Miao, P., and Dice, J. F. (1985) J. Biol. Chem. 260, 11986-11993), suggest that these peptide regions target specific proteins to a lysosomal pathway of degradation during serum withdrawal.     

An enzyme related to the high molecular weight multicatalytic proteinase, macropain, participates in a ubiquitin-mediated, ATP-stimulated proteolytic pathway in soluble extracts of BHK 21/C13 fibroblasts

Soluble, cell-free extracts of BHK 21/C13 fibroblasts degraded a variety of exogenous proteins to acid-soluble peptides at pH 8.0. ATP stimulated the rates of proteolysis. Both the absolute rate of proteolysis and the magnitude of the ATP effect depended on the specific substrate. For example, casein was degraded approximately 10-fold faster than lysozyme, but lysozyme degradation was more highly stimulated by ATP than was casein degradation. Ubiquitin enhanced the ATP-stimulated proteolysis of each substrate in both postmicrosomal extracts and DEAE-cellulose fractionated extracts. In each extract, ubiquitin enhanced the ATP-stimulated degradation of lysozyme to a greater degree than that of casein. These results suggested that lysozyme was degraded by a pathway that was more dependent upon ubiquitin than was casein. Further evidence for this conclusion was obtained in studies using substrates whose amino groups were blocked by extensive methylation or carbamoylation. The high molecular weight proteinase, macropain, appears to be involved in the ATP-stimulated degradation of both substrates. Specific immunoprecipitation of macropain with polyclonal antibodies resulted in the inhibition of ATP-stimulated proteinase activity both in the absence and presence of ubiquitin. These results indicate that macropain plays a role in both ubiquitin-mediated and ubiquitin-independent ATP-stimulated proteolysis in BHK cell extracts.     

The peroxin Pex14p is involved in LC3-dependent degradation of mammalian peroxisomes

As a step toward understanding the homeostasis of peroxisomes in mammalian cells, we investigated a degradation system of peroxisomes in Chinese hamster ovary (CHO)-K1 cells in response to the nutrient-starvation. Peroxisomal proteins were degraded apparently in a preferential manner as compared to cytosolic proteins, when CHO-K1 cells were starved in Hank's solution and then re-cultured in a normal medium. We verified whether microtubule-associated protein I light chain 3 (LC3), an essential factor for autophagy, was involved in the degradation of peroxisomal proteins. In the LC3-knocked-down CHO-K1 cells, the specific degradation of peroxisomal proteins was no longer observed and proteins including peroxisomal and cytosolic proteins were rather non-selectively degraded under the starvation condition. The starvation-dependent non-selective protein degradation was inhibited with proteasome inhibitors, MG132 and Epoxomicin. The integral membrane peroxin, Pex14p interacted with membrane-bound LC3-II, the modified form of LC3, via microtubules under the starvation condition. Taken together, these results suggest that peroxisomal proteins are degraded by two degradation systems involving autophagy and proteasomes depending on various cell-culture conditions, and that Pex14p plays a pivotal role as a prerequisite factor for the degradation of peroxisomal proteins by autophagy with the aid of microtubules.     

Species variation in ATP-dependent protein degradation: protease profiles differ between mycobacteria and protease functions differ between Mycobacterium smegmatis and Escherichia coli

We report here that the existence of the potentially broad substrate specificity protease Lon (also called La), is evolutionarily discontinuous within the order Actinomycetales. Lon homologues were identified in the fast-growing species Mycobacterium smegmatis, and the slow-growing species Micobacterium avium and Mycobacterium intracellulare. However, Lon homologues were not detected in the slow-growing species Mycobacterium tuberculosis, Mycobacterium bovis, or Mycobacterium leprae; or in the non-mycobacterial Actinomycetale Corynebacterium glutamica. To characterize the function of the Lon protease within the Actinomycetales, a viable M. smegmatis Deltalon strain was constructed, demonstrating that lon is not essential under certain conditions. Surprisingly, lon was also dispensable in M. smegmatis cells already lacking intact 20S proteasome alpha- and beta-subunit genes (called prcA and prcB, respectively). Creation of the later double deletion strain (prcBA::kan Deltalon) necessitated use of a novel gene deletion strategy that does not require an antibiotic resistance marker. The M. smegmatis prcBA::kan Deltalon double mutants displayed wild type (wt) growth rates and wt stress tolerances. In addition, the M. smegmatis prcBA::kan Deltalon double mutants degraded at wt rates the broad spectrum of truncated proteins induced by treating cells with puromycin. This later result was in sharp contrast to those in Escherichia coli, where either lon or hslUV single mutants are strongly impaired in their degradation of puromycyl peptides (hslV is a prcB homologue). Overall these data suggested that mycobacterial species contain additional ATP-dependent proteases that have broad substrate specificity. Consistent with this suggestion, M. smegmatis and M. tuberculosis each contain at least one homologue of ClpP, the proteolytic subunit common to the ClpAP and ClpXP proteases.     

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.     

Alterations of protein degradation and 2-D protein pattern in muscle cells of MDX and DMD origin.

Intracellular protein turnover of MDX, DMD and normal muscle was determined by [35S]methionine pulse-chase experiments and subsequent high resolution 2-D gel electrophoresis. In MDX myotubes intracellular degradation of short-lived and long-lived proteins was markedly increased by a factor of 1,4-2,1. In wildtype the rate of degradation of short-lived proteins was approximately 2.6%/h, whereas in MDX these proteins were degraded by 5.7%/h. Long-lived proteins were degraded in wildtype at a rate of 1.8%/h, and in MDX at a rate of 2.5%/h. Furthermore, we have described a 51.000 Da protein with an IEP of 5.1 (p51/5.1), whose net content is highly and specifically reduced in cultured MDX and DMD muscle cells as well as in isolated MDX muscle fibers. Treatment with calcium-channel blockers Dantrolene and Verapamil inhibited the degradation of p51/5.1 in MDX myotubes by more than 90% in contrast to controls.     

Examination of intramolecular heterogeneity of plasma membrane protein degradation in canine renal tubular epithelial cells and in rat liver

We have examined the hypothesis that hydrophilic portions of membrane-bound proteins which lie on either side of the phospholipid bilayer may be degraded at a different rate than are the hydrophobic portions of membrane proteins which are within the bilayer. Plasma membrane fractions from cells of the Maden-Darby canine kidney cell line and rat liver were digested with papain and pronase to cleave a mixture of peptides which is enriched in hydrophilic amino acids. It is proposed that these peptides are derived from regions of membrane-bound proteins which lie outside the bilayer. The residual particulate protein is enriched in hydropholic amino acids and presumably contains the portion of membrane-bound proteins which are in direct contact with the bilayer. A double-isotope method was used to assess the relative degradation rates of these two protein fractions. There was no measurable difference in protein degradation rates between the two fractions and the initial plasma membranes. These results suggest that the intramolecular heterogeneity which results from insertion of membrane-bound proteins into a bilayer is not a factor in their degradation.     

The degradation of cartilage proteoglycans by tissue proteinases. Proteoglycan heterogeneity and the pathway of proteolytic degradation.

1. CaCl2-extracted proteoglycan from bovine nasal cartilage was degraded by four tissue proteinases till no further decrease in hydroynamic size was obtained. The proteoglycan and its final degradation products were then fractionated by Sepharose 2B chromatography. 2. The average size of the degradation products was least for cathepsin B and lysosomal elastase, and greatest for cathepsin D and cathepsin G. The latter two proteinases also produced degradation products that showed the widest range of sizes. 3. The structure of the degradation products ranged from peptides containing a single glycosaminoglycan chain to those containing twelve or more chains. Of the four proteinases, only cathepsin B produced peptides that contained a single chondroitin sulphate chain. 4. The proteoglycan was very heterogeneous with respect to size and chemical composition. Its behaviour on electrophoresis suggested that at least two genetically distinct core proteins might exist. 5. Irrespective of their structural variations, all proteoglycan molecules were able to interact with hyaluronic acid. In contrast, none of the degradation products were capable of this type of interaction. 6. A pathway for the proteolytic degradation of proteoglycans is postulated in which the sites of initial cleavage may be common to the majority of proteinases, whereas the production of the final clusters is dependent on the specificity of the proteinase. Only those proteinases of broadest specificity can produce single-chain chondroitin sulphate-peptides.     

Differential Degradation of Different Benzodiazepine Binding Proteins by Incubation of Membranes from Cerebellum or Hippocampus with Trypsin

When rat brain membranes were incubated with [3H]flunitrazepam in the presence of UV light, predominantly one protein (P51) was irreversibly labeled in cerebellum and at least two proteins (P51 and P55) were labeled in hippocampus. On digestion of membranes with increasing concentrations of trypsin up to 40% of radioactivity irreversibly bound to proteins was removed from the membranes. In addition, P51 was nearly completely degraded to a peptide with apparent molecular weight 39,000 and this peptide was further degraded to a peptide with apparent molecular weight 25,000. In contrast, protein P55 was only partially degraded by trypsin and yielded two proteolytic peptides with apparent molecular weights 42,000 and 45,000 which seemed to be rather stable against further attack by trypsin. Membranes treated with trypsin still had the capacity to bind [3H]-flunitrazepam reversibly with an affinity similar to that of membranes not previously treated with trypsin. When these membranes were irradiated with UV light, the same proteolytic peptides were detected as in membranes first photolabeled and then digested with trypsin. These results suggest a close association between reversible and irreversible benzodiazepine binding sites and indicate that membrane-associated proteins P51 and P55 are differentially protected against degradation by trypsin.