Vanadate inhibits the ATP-dependent degradation of proteins in reticulocytes without affecting ubiquitin conjugation

Reticulocytes contain a nonlysosomal, ATP-dependent system for degrading abnormal proteins and normal proteins during cell maturation. Vanadate, which inhibits several ATPases including the ATP-dependent proteases in Escherichia coli and liver mitochondria, also markedly reduced the ATP-dependent degradation of proteins in reticulocyte extracts. At low concentrations (K1 = 50 microM), vanadate inhibited the ATP-dependent hydrolysis of [3H]methylcasein and denatured 125I-labeled bovine serum albumin, but it did not reduce the low amount of proteolysis seen in the absence of ATP. This inhibition by vanadate was rapid in onset, reversed by dialysis, and was not mimicked by molybdate. Vanadate inhibits proteolysis at an ATP-stimulated step which is independent of the ATP requirement for ubiquitin conjugation to protein substrates. When the amino groups on casein and bovine serum albumin were covalently modified so as to prevent their conjugation to ubiquitin, the derivatized proteins were still degraded by an ATP-stimulated process that was inhibited by vanadate. In addition, vanadate did not reduce the ATP-dependent conjugation of 125I-ubiquitin to endogenous reticulocyte proteins, although it markedly inhibited their degradation. In intact reticulocytes vanadate also inhibited the degradation of endogenous proteins and of abnormal proteins containing amino acid analogs. This effect was rapid and reversible; however, vanadate also reduced protein synthesis and eventually lowered ATP levels in the intact cells. Vanadate (10 mM) has also been reported to decrease intralysosomal proteolysis in hepatocytes. However, in liver extracts this effect on lysosomal proteases required high concentrations of vanadate (K1 = 500 microM) and was also observed with molybdate, unlike the inhibition of ATP-dependent proteolysis in reticulocytes.     

ATP-dependent degradation of 125I-bovine serum albumin by rabbit reticulocytes does not need repression of an endogenous inhibitor

The ubiquitin-dependent proteolysis of 125I-bovine serum albumin in rabbit reticulocytes has been investigated. Using various reticulocyte fractions (reticulocyte protease, inhibitor-free protease, "ubiquitin" and "inhibitor") in the presence or absence of ATP, we found that the repression of an endogenous inhibitor, as suggested by others for alpha-casein proteolysis, is unlikely for bovine serum albumin. Therefore, differences exist in the ATP-dependent proteolytic pathway of rabbit reticulocytes depending on the substrate. Fractionation of the reticulocyte ATP-dependent proteolytic system revealed at least two proteolytic and two inhibitory fractions involved in the proteolysis of bovine serum albumin.     

Transfer RNA is required for conjugation of ubiquitin to selective substrates of the ubiquitin- and ATP-dependent proteolytic system

Degradation of intracellular proteins via the ubiquitin- and ATP-dependent proteolytic pathway involves several steps. In the initial event, ubiquitin, an abundant 76-residue polypeptide is covalently linked to the protein substrate in an ATP-requiring reaction. Proteins marked by ubiquitin are selectively proteolyzed in a reaction that also requires ATP. Ubiquitin conjugation to proteins appears also to be involved in regulation of cell cycle and cell division, and probably in the regulation of gene expression at the level of chromatin structure. We have previously shown (Ciechanover, A., Wolin, S. L., Steitz, J. A., and Lodish, H. F. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1341-1345) that transfer RNA is an essential component of the ubiquitin pathway. Ribonucleases strongly and specifically inhibited the degradation of 125I-labeled bovine serum albumin, while tRNA purified from reticulocyte extract could restore the proteolytic activity. Specifically, pure tRNAHis isolated by immunoprecipitation with human autoimmune serum could restore the proteolytic activity. Here we demonstrate that tRNA is required for conjugation of ubiquitin to some but not all proteolytic substrates of the ubiquitin mediated pathway. Conjugation of 125I-labeled ubiquitin to reduced carboxymethylated bovine serum albumin, alpha-lactalbumin, and soybean trypsin inhibitor was strongly and specifically inhibited by ribonucleases. Consequently, the ATP-dependent degradation of these substrates in the cell-free ubiquitin-dependent reticulocyte system was inhibited as well. Addition of tRNA to the ribonuclease inhibited system (following inhibition of the ribonuclease) restored both the conjugation activity and the ubiquitin- and ATP-dependent degradation of these substrates. Conjugation of ubiquitin to some endogenous reticulocyte proteins was also inhibited by ribonucleases and could be restored by the addition of tRNA. In striking contrast, the conjugation of radiolabeled ubiquitin to lysozyme, oxidized RNase A, alpha-casein, and beta-lactoglobulin was not affected by the ribonuclease treatment, and the degradation of these substrates was significantly accelerated by the ribonucleases. These findings indicate that there are at least two distinct ubiquitin conjugation systems. One requires tRNA, and the other is tRNA independent. These pathways, however, must share some common component(s) of the system, since the inhibition of one system accelerates the other. The possible function of tRNA in the selective conjugation reaction and the possible role of the two distinct ubiquitin marking mechanisms are discussed.     

Uncoupling ubiquitin-protein conjugation from ubiquitin-dependent proteolysis by use of beta, gamma-nonhydrolyzable ATP analogues.

Pathways of ubiquitin-dependent protein degradation have in common two requirements for ATP. Ubiquitin activation by the enzyme E1 is accompanied by ATP hydrolysis to yield AMP and PPi, and during conjugate breakdown, the ubiquitin-dependent protease hydrolyzes ATP to ADP and Pi. We show here that either of two beta, gamma-nonhydrolyzable ATP analogues, 5'-adenylyl imidodiphosphate or 5'-adenylyl methylenediphosphate, can support ubiquitin-protein conjugation. With the ubiquitin-dependent protease, however, neither analogue could substitute for ATP. Thus, the substitution of a beta, gamma-nonhydrolyzable analogue for ATP offers a simple method to uncouple ubiquitin conjugation from proteolysis in crude systems. On the basis of pyrophosphate exchange kinetics, E1 has apparent Km and Vmax values that are similar for ATP and the analogues, but substrate inhibition by 5'-adenylyl methylenediphosphate made use of the beta, gamma-imido analogue preferable. In one application, beta, gamma-imido-ATP was used in combination with ubiquitin aldehyde (an inhibitor of ubiquitin-protein isopeptidases) to establish that several unfolded RNase A derivatives are recognized equally as ubiquitination substrates. This result extends an earlier study [Dunten, R. L., & Cohen, R. E. (1989) J. Biol. Chem. 264, 16739-16747] to show that conjugate yields, upon which relative ubiquitination rates were based, were not influenced by differential ubiquitin-dependent proteolysis. In a second application, ATP and beta, gamma-imido-ATP were compared in a pulse-chase experiment to investigate the contributions of ATP-dependent proteolysis and isopeptidase activities to conjugate stability.     

Effects of ATP, vanadate, and molybdate on cathepsin D-catalyzed proteolysis

The effects of ATP, vanadate, and molybdate on cathepsin D-catalyzed hydrolysis of proteins and peptides were examined. Hydrolysis of bovine serum albumin, hemoglobin, parathyroid hormone, and a synthetic octapeptide was activated by ATP. Degradation of the protein substrates all had similar ATP concentration dependence, but the magnitude of the activation varied. Kinetic constants for ATP activation were obtained with a synthetic substrate. ATP increased kcat from 0.4 to 2 s-1 but did not change KM. Kact for ATP was 800 microM. Studies with pepstatin-Sepharose confirm that ATP does not alter the substrate binding site on cathepsin D. Pepsin, a homologous aspartate protease, was not activated by ATP. It was also found that vanadate and molybdate inhibit cathepsin D-catalyzed proteolysis. However, this inhibition was dramatically dependent on substrate concentration and was eliminated at high substrate. Hydrolysis of the synthetic peptide was not inhibited at concentrations of molybdate below 50 microM, and above this concentration the peptide precipitated. Protein substrates were also found to precipitate in the presence of molybdate. The ATP dependence of the enzyme was not altered by molybdate or vanadate. These results suggest that inhibition by vanadate and molybdate is related to interactions with the substrate rather than with cathepsin D. It is concluded that ATP activation of cathepsin D may play a physiological role in regulation of proteolysis in lysosomes, but that vanadate and molybdate inhibition of lysosomal proteolysis does not establish ATP dependence.     

Identification of macrophage cell-surface binding sites for cationized bovine serum albumin.

Autoimmune diseases are characterized by the presence of autoantibodies often restricted to host proteins exhibiting charge rich domains. Charged polypeptides elicit strong immune responses, and cationized bovine serum albumin and other cationic proteins are significantly more immunogenic than their less charged counterparts. These phenomena may involve enhanced protein uptake by macrophages, resulting in greater processing and presentation of antigenic peptide-MHC complexes to T-cells. We compared macrophage cell-surface binding and uptake of native and cationized bovine serum albumin. Specific binding of [125I]cationized bovine serum albumin to THP-1 macrophages in vitro was 11-16 fold greater than for native albumin. Half-maximal inhibition of [125I]cationized albumin binding was observed at 10-7M ligand. The specificity of [125I]cationized bovine serum albumin binding and uptake was further studied in terms of competitive inhibition of proteolysis by proteins of varying charge content. Cationized bovine serum albumin, but not native albumin, inhibited proteolysis of [125I]cBSA. Calf thymus histones also inhibited cBSA degradation. High concentration of myelin basic protein was moderately effective at blocking cBSA degradation, while myoglobin and beta lactalbumin showed no inhibition. These results indicate that specific cell-surface binding sites which occur on macrophages may mediate selective uptake of certain proteins with highly charged domains including some autoantigens.     

Circular dichroic study of the conformational stability of sulfhydryl-blocked bovine serum albumin

1. The blockage of the single sulfhydryl-group of bovine serum albumin does not alter the secondary structure, although the alpha-helical structure is destabilized since lower concentrations of guanidine and of urea unfold the protein. 2. What happens to the previously helical structure depends upon the reagent used to block the sulfhydryl-group. Bovine serum albumin derivatized with 5,5'-dithiobis-(2-nitrobenzoic acid) and iodoacetate preferentially acquire the beta-structure in high concentrations of guanidine and urea, whereas iodoacetamide-derivatized bovine serum albumin acquires primarily the random coil structure. 3. Part of the helical structure is also lost in 5-6 mM sodium dodecyl sulfate; thionitrobenzoate-bovine serum albumin shows an increase in the random coil, whereas the two alkylated proteins display the increase both in beta-structure and random coil. 4. Carboxymethylation or carboxamidomethylation of fully reduced bovine serum albumin results in a drastic change in the secondary structure of the protein with a substantial decrease in alpha-helix and a corresponding increase in both beta-structure and random coil. These extensively alkylated proteins also display differences in denaturation profiles in solutions of guanidine and urea.     

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.     

Degradation of rat liver cytochromes P450 3A after their inactivation by 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine: characterization of the proteolytic system.

The suicide substrate 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4- dihydropyridine (DDEP) inactivates rat liver cytochrome P450 (P450) 3A isozymes through prosthetic heme alkylation of the apoprotein in a mechanism-based fashion, which marks them for rapid proteolysis. In this article, through the use of epitope-specific monoclonal antibodies, we show that both 3A1 and 3A2 isozymes are targeted for proteolysis. Furthermore, using intact rats, isolated rat hepatocytes, and rat liver subcellular fractions supplemented with ATP and MgCl2, as well as various proteolytic inhibitors as probes, we now report that the hepatic cytosolic ubiquitin-dependent proteolytic system rather than hepatic lysosomes is involved in the rapid degradation of DDEP-induced heme alkylated P450s 3A.     

Role of thiols, pH and cathepsin D in the lysosomal catabolism of serum albumin.

Attempts were made to assess the role of thiols and to determine the cathepsins involved in the degradation of serum albumin in mouse liver and kidney lysosomes. Unlike cysteine or beta-mercaptoethanol, reduced glutathione (GSH) did not stimulate the degradation of formaldehyde-treated albumin in liver lysosomes, suggesting that the tripeptide did not penetrate the membrane. However, GSH was a much more effective stimulant of proteolysis in kidney lysosomes than was cysteine at low concentrations, and the effect was saturable at 1-2 mM concentrations. Thiols did not stimulate proteolysis in lysosomes when the disulphide bonds of albumin were reduced and alkylated, suggesting that the stimulatory effects were solely due to disulphide-bond reduction in protein substrates. Results obtained with thiols and iodoacetamide suggested that albumins denatured by disulphide-bond reduction and alkylation, disulphide-bond reduction without alkylation, or by treatment with 8 M-urea, were all degraded primarily by cathepsin D in lysosomes, but formaldehyde-denatured albumin was attacked by thiol proteinases. These findings correlated well with studies on the degradation of these proteins by rat liver lysosome (tritosome) extracts. Studies with the proteinase inhibitors leupeptin and pepstatin and the stimulatory effects of thiols in these extracts suggested that formaldehyde-denatured albumin was degraded primarily by the thiol proteinases, but that native albumin or albumins denatured by disulphide-bond reduction or by treatment with 8 M-urea were attacked by cathepsin D. Denaturation of serum albumin by any of the methods used caused a shift in the pH optimum of albumin catabolism by tritosome extracts or by purified cathepsin D from approx. 3-4 to 5-6. These results were discussed in terms of a possible mechanism for the catabolic aspect of serum albumin turnover.     

Role of ubiquitin conformations in the specificity of protein degradation: iodinated derivatives with altered conformations and activities

Three iodinated derivatives of ubiquitin have been synthesized and these derivatives have been characterized in the ubiquitin-dependent protein degradation system. With chloramine-T as the oxidant, a derivative containing monoiodotyrosine is formed in the presence of 1 M KI and a derivative containing diiodotyrosine is produced in the presence of 1 mM KI. These derivatives exhibit phenolate ionizations at pH 9.2 and 7.9 with absorbance maxima at 305 and 314 nm, respectively. In addition to modification of the tyrosine residue, these conditions lead to the oxidation of the single methionine residue and iodination of the single histidine residue [M.J. Cox, R. Shapira, and K.D. Wilkinson (1986) Anal. Biochem. 154, 345-352]. Iodination of ubiquitin under these conditions renders the protein sensitive to hydrolysis by trypsin and results in an enhanced susceptibility to alcohol-induced helix formation. When the derivatives are tested in the ATP: pyrophosphate exchange reaction catalyzed by the ubiquitin adenylating enzyme, they are found to exhibit activity comparable to the native protein. When these derivatives are tested for the ability to act as a cofactor in the ubiquitin-dependent protein degradation system, they are both found to support a rate of protein degradation that is twice that of native ubiquitin. At high concentrations of derivatives, the rate of protein degradation is inhibited, while the steady state level of conjugates increases. Thus, the free derivatives inhibit the protease portion of the reaction, but are fully active in the activation and conjugation portions of the reaction. With iodine as the modification reagent, monoiodination of tyrosine is the predominant reaction. This derivative exhibits activity similar to native ubiquitin. Thus, it appears that modification of the histidine residue is responsible for the increased activity of the more highly iodinated derivatives. The enzymes of the system must recognize different portions of the ubiquitin structure, or different conformations of ubiquitin that are affected by the iodination of the histidine residue. These results suggest a conformational change of the ubiquitin molecule may be important in determining the rate and specificity of proteolysis.     

Degradation of oxidatively denatured proteins in Escherichia coli

When exposed to oxidative stress, by oxygen radicals or H2O2, E. coli exhibited decreased growth, decreased protein synthesis, and dose-dependent increases in protein degradation. The quinone menadione induced proteolysis when cells were incubated in air, but was not effective when cells were incubated without oxygen. Anaerobically grown cells also exhibited significantly lower proteolytic capacity than did cells that were grown aerobically. Xanthine plus xanthine oxidase (which generate O2- and H2O2) caused a stimulation of proteolysis which was inhibitable by catalase, but not by superoxide dismutase: Indicating that H2O2 was responsible for the increased protein degradation. Indeed, H2O2 alone was effective in inducing increased intracellular proteolysis. Two-dimensional polyacrylamide gel electrophoresis of [3H]leucine labeled E. coli revealed greater than 50% decreases in the concentrations of 10-15 cell proteins following H2O2 or menadione exposure, while several other proteins were less severely affected. To test for the presence of soluble proteases, we prepared cell-free extracts of E. coli and incubated them with radio-labeled protein substrates. E. coli extracts degraded casein and globin polypeptides at rapid rates but showed little activity with native proteins such as superoxide dismutase, hemoglobin, bovine serum albumin, or catalase. When these same proteins were denatured by exposure to oxygen radicals or H2O2, however, they became excellent substrates for degradation in E. coli extracts. Studies with albumin revealed correlations greater than 0.95 between the degree of oxidative denaturation and proteolytic susceptibility. Pretreatment of E. coli with menadione or H2O2 did not increase the proteolytic capacity of cell extracts; indicating that neither protease activation, nor protease induction were required.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein substrates activate the ATP-dependent protease La by promoting nucleotide binding and release of bound ADP

The interaction of protein substrates with protease La from Escherichia coli enhances its ability to hydrolyze ATP and peptide bonds. These studies were undertaken to clarify how unfolded proteins allosterically stimulate this ATPase activity. The tetrameric protease can bind four molecules of ATP, which activates proteolysis, or four molecules of ADP, which inhibits enzymatic activity. Protein substrates stimulate binding of the nonhydrolyzable ATP analog [3H] adenyl-5'yl imidodiphosphate, although they do not increase the net binding of [3H]ATP or [3H]ADP. Once bound, ATP is quickly hydrolyzed to ADP, which remains noncovalently associated with protease La even through repeated gel filtrations. Exposure to protein substrates (e.g. denatured bovine serum albumin at 37 degrees C) induces the release of all the bound ADP from the enzyme. Nonhydrolyzable ATP analogs bound to the enzyme were not released by these substrates. Proteins that are not degraded (e.g. native bovine serum albumin) and oligopeptides that only bind to the catalytic site do not induce ADP release. Thus, polypeptide substrates have to interact with an allosteric site to induce this effect. The protein-induced ADP release is inhibited by high concentrations of Mg2+ and is highly temperature-dependent. Protein substrates promoted [3H]ATP binding in the presence of ADP and Mg2+ (i.e. ATP-ADP exchange) and reduced the ability of ADP to inhibit the enzyme's peptidase and ATPase activities. These results indicate that: 1) ADP release is a rate-limiting step in protease La function; 2) bound ADP molecules inhibit protein and ATP hydrolysis in vivo; 3) denatured proteins interact with the enzyme's regulatory site and promote ADP release, ATP binding, and their own hydrolysis.     

ATP serves two distinct roles in protein degradation in reticulocytes, one requiring and one independent of ubiquitin

Protein degradation in rabbit reticulocytes is a nonlysosomal process requiring ATP. Recently, appreciable evidence has been presented that ATP is required for the covalent binding of the polypeptide ubiquitin to epsilon-amino groups on protein substrates. To test whether linkage of ubiquitin to substrates is required for ATP-dependent proteolysis, the amino groups of 3H-methyl-casein and denatured 125I-bovine serum albumin (BSA) were completely (93-99%) blocked by methylation, acetylation, carbamylation, or succinylation. In each case, the proteins lacking amino groups were still degraded by an ATP-stimulated process, although these various treatments altered absolute rates of proteolysis and reduced the magnitude of the ATP stimulation (two- to fourfold) below that seen measured with the unmodified substrates. When ubiquitin was removed by ion exchange chromatography, ATP still stimulated breakdown of casein and carbamylated casein twofold. The addition of ubiquitin in the presence of ATP caused a further twofold increase in the hydrolysis of unmodified casein but did not affect the degradation of casein lacking amino groups. Thus ubiquitin conjugation to substrates appears important in the breakdown of certain substrates (especially of BSA), but this reaction is not essential for ATP- stimulated proteolysis. The ATP-activated step that is independent of ubiquitin probably is also involved in the degradation of unblocked proteins, since both processes require Mg++ and ATP hydrolysis and are inhibited by hemin but not by protoporphyrin IX. These results suggest that ATP has distinct roles at different steps in the degradative pathway.     

Characterization of ATP-dependent proteolysis in embryos of the brine shrimp, Artemia franciscana

Under anoxia, embryos of Artemia franciscana enter a state of quiescence. During this time protein synthesis is depressed, and continued degradation of proteins could jeopardize the ability to recover from quiescence upon return to favorable conditions. In this study, we developed an assay for monitoring ATP/ubiquitin-dependent proteolysis in order to establish the presence of this degradation mechanism in A. franciscana embryos, and to describe some characteristics that may regulate its function during anoxia-induced quiescence. For lysates experimentally depleted of adenylates, supplementation with ATP and ubiquitin stimulated protein degradation rates by 92 ± 17% (mean ± SE) compared to control rates. The stimulation by ATP was maximal at concentrations ≥11 μmol · l⁻¹. In the presence of ATP and ubiquitin, ubiquitin-conjugated proteins were produced by lysates during the course of the 4-h assays, as detected by Western blotting. Acute acidification of lysates to values approximating the intracellular pH observed under anoxia completely inhibited ATP/ubiquitin-dependent proteolysis. Depressed degradation was also observed under conditions where net ATP hydrolysis occurred. These results suggest that ATP/ubiquitin-dependent proteolysis is markedly inhibited under cellular conditions promoted by anoxia. Inhibition of proteolysis during quiescence may be one critical factor that increases macromolecular stability, which may ultimately govern the duration of embryo survival under anoxia. Accepted: 2 November 1999     

Effects of weak bases on the degradation of endogenous and exogenous proteins by rat yolk sacs.

In rat yolk sacs incubated in vitro, the rates of degradation of endogenous [3H]leucine-labelled proteins and of pinocytically ingested 125I-labelled bovine serum albumin were both decreased in the presence of either ammonium, methylammonium or ethylammonium ions (0-20 mM) or much lower concentrations of chloroquine (0-500 microM). These effects were also accompanied by an inhibition of pinocytosis, as measured by the rate of uptake of 125I-labelled polyvinylpyrrolidone, and by a fall in the [ATP]/[ADP] ratio within the tissue. Re-incubation in inhibitor-free medium of yolk sacs previously exposed to a weak base restored pinocytic and proteolytic capacities, except for tissues exposed to chloroquine at concentrations above 0.1 mM (these appeared to be cytotoxic); an attendent rise in [ATP]/[ADP] ratios to near normal values was also observed. Weak bases, at concentrations that fully arrested the breakdown of 125I-labelled albumin, failed to inhibit by more than 45% the degradation of [3H]leucine-labelled endogenous proteins. Since 125I-labelled bovine serum albumin has been shown to be degraded entirely intralysosomally by yolk sacs, this suggests either that the hydrolysis of endogenous proteins is shared between lysosomes and some other site or that, unlike 125I-labelled albumin, some endogenous proteins can be degraded within lysosomes at abnormally high pH.     

Inhibition of ubiquitin-dependent proteolysis by non-ubiquitinatable proteins

The effect in reticulocyte lysates of proteins with blocked amino groups on the ATP-dependent degradation of casein and serum albumin was studied in order to assess the extent to which proteins with blocked and with free amino groups share common paths of proteolytic degradation. Completely acetylated or succinylated casein and acetylated or succinylated serum albumin (reduced and carboxymethylated), in addition to other amino-modified proteins, inhibited the ATP-dependent proteolysis of both casein and reduced carboxymethylated serum albumin. Inhibition of serum albumin degradation by acetylated serum albumin was competitive, whereas inhibition of casein degradation by acetylated casein was largely competitive with evidence of mixed kinetics. The different amino-blocked proteins studied, which were largely unfolded under assay conditions, were similarly effective as inhibitors on a weight basis, with Ki values in the range 0.2-0.6 mg/ml; there was no correlation between the ability of the blocked proteins to serve as proteolysis substrates and their effectiveness as inhibitors. Studies of the effects of acetylated proteins on the conjugation of ubiquitin to serum albumin and casein demonstrated that the acetylated proteins blocked formation of ubiquitin-albumin conjugates and of selected casein conjugates; the steady state concentration of selected conjugates of endogenous lysate proteins was increased in the presence of amino-blocked proteins. The results suggest that proteins with blocked amino groups, which cannot serve as substrates for ubiquitin conjugation, can compete for binding to those ubiquitin conjugation factors that recognize and ubiquitinate potential substrates of the ubiquitin pathway. The similar inhibitory properties of the different blocked proteins in turn suggest that a common factor in binding to these conjugation factors may be recognition of the polypeptide backbone.     

Circular pancreatic trypsin inhibitor. A novel substrate for studies on intracellular proteolysis

Several recent studies indicate that substrates for ubiquitin-dependent proteolysis must possess unblocked alpha-amino termini. To examine further the importance of free amino groups for proteolytic susceptibility we selected pancreatic trypsin inhibitor (PTI) as a test substrate. PTI can be circularized to form cPTI, a molecule that lacks alpha-amino groups in the absence of an endoproteolytic cleavage. We compared the breakdown of radioiodinated PTI and cPTI in rabbit reticulocyte lysate and found that cPTI was not stabilized relative to PTI. In addition, proteolysis of PTI or cPTI was not inhibited upon conversion of their lysine residues to homoarginine. However, neither degradation of PTI nor cPTI required ATP, and ubiquitin conjugation to either molecule was minor relative to known substrates of the ubiquitin pathway. Thus, PTI and cPTI are cleaved by an ATP-independent endoprotease(s) that does not require the substrate to be ubiquitinated. Such an activity was identified in low salt fractions obtained upon DEAE chromatography of reticulocyte lysate. The ubiquitin/ATP-dependent protease and another large multisubunit protease, both of which elute from DEAE-Fractogel at higher salt concentrations, do not degrade PTI or cPTI. Although monomeric PTI was rapidly degraded in reticulocyte lysate, cross-linked PTI molecules were stable both in reticulocyte lysate and following introduction into cultured cells using red blood cell-mediated microinjection. Thus, increased rates of turnover do not necessarily correlate with greater molecular mass of the substrate.     

Proteins damaged by oxygen radicals are rapidly degraded in extracts of red blood cells

We have suggested that red blood cell proteolytic systems can degrade oxidatively damaged proteins, and that both damage and degradation are independent of lipid peroxidation (Davies, K. J. A., and Goldberg, A. L. (1987) J. Biol. Chem. 262, 8220-8226. These ideas have now been tested in cell-free extracts of rabbit erythrocytes and reticulocytes. Exposure to oxygen radicals or H2O2 increases the degradation of endogenous proteins in cell-free extracts, as in intact cells. Various radical-generating systems (acetaldehyde or xanthine + xanthine oxidase, ascorbic acid + iron, H2O2 + iron) and H2O2 alone enhanced the rates of proteolysis severalfold. Since these extracts were free of membrane lipids, protein damage and degradation must be independent of lipid peroxidation. An antioxidant buffer consisting of HEPES, glycerol, and dithiothreitol inhibited the increased proteolysis by 60-100%. Mannitol caused a 50-80% reduction in proteolysis suggesting that the hydroxyl radical (.OH), or a species with similar reactivity, may be the initiator of protein damage. When casein or bovine serum albumin were exposed to .OH (generated by H2O2 + Fe2+, or COCo radiation) these proteins were degraded up to 50 times faster than untreated proteins during subsequent incubations with red cell extracts. Mannitol inhibited this increase in proteolysis only if present during .OH exposure; mannitol did not affect the degradative system. Although ATP increased the degradation of untreated proteins 4- to 6-fold in reticulocyte extracts, it had little or no effect on the degradation of proteins exposed to .OH. ATP also did not stimulate hydrolysis of .OH-treated proteins in erythrocyte extracts. Leupeptin did not affect the degradative processes in either extract; thus lysosomal or Ca2+-activated thiol proteases were not involved. We propose that red cells contain a soluble, ATP-independent proteolytic pathway which may protect against the accumulation of proteins damaged by .OH or other active oxygen species.     

An ATP-dependent system specific for degradation of long-lived proteins in permeabilized cells

We have characterized a digitonin-permeabilized cell system for the ATP-dependent degradation of endogenous long-lived proteins. Proteolysis requires Mg2+ and ATP hydrolysis. Other nucleotide triphosphates (CTP, UTP) can partially replace the ATP requirement. The enhanced rate of degradation of long-lived proteins in response to serum starvation is maintained in the permeabilized cell system and can be partially inhibited by lysosomal inhibitors. The maintenance of intracellular architecture and ease of manipulation of soluble components make the permeabilized cell system ideal for studying the proteolysis of both endogenous and exogenous substrates.