Biomedical subjects
Publications and source records attributed to J M Fagan.
We have purified the multicatalytic proteinase from the nucleus and cytoplasm of chicken red blood cells. Both enzymes have an Mr of 700 kDa and can be separated into 8-10 bands (22-32 kDa) on 1D-SDS-PAGE.
Previously, we isolated an ATP-dependent proteolytic pathway in muscle, liver, and reticulocytes that requires ubiquitin and the enzymes which conjugate ubiquitin to proteins. We report here that skeletal muscle contains another soluble alkaline energy-dependent (but ubiquitin-independent) proteolytic activity. The cleavage of non-ubiquitinated protein substrates by the partially purified protease requires ATP hydrolysis since ATP in the absence of Mg2+, nonhydrolyzable ATP analogs, and pyrophosphate all fail to stimulate proteolysis. Proteolytic activity is also stimulated by UTP, CTP, and GTP, although not as effectively as by ATP (Km(ATP) = 0.027 mM). The enzyme is inactivated by the serine protease inhibitors diisopropyl fluorophosphate and 3,4-dichloroisocoumarin, but not by specific inhibitors of aspartic, thiol, or metalloproteases. It is maximally active at pH 8 and has a molecular weight of approximately 600,000. This new activity differs from the 720-kDa multicatalytic proteinase, but resembles the soluble ATP-dependent proteolytic system that we previously isolated from murine erythroleukemia cells.
Reticulocytes contain a soluble nonlysosomal proteolytic pathway that requires ATP and ubiquitin. Polyamines at physiological concentrations were found to inhibit rapidly the ATP-dependent proteolytic system in reticulocyte lysates; spermidine and putrescine inhibited this process by 26-72% and spermine by 71-96%. Spermine had little effect on the ATP-independent breakdown of oxidant-treated hemoglobin. By fractionating the ATP-dependent system, we show that polyamines inhibit the ATP-dependent degradation of ubiquitin-protein conjugates.
Isolated soleus muscle deprived of oxygen produces more lactate and alanine than oxygen-supplied muscle. Oxygenated muscle synthesized glutamine, while anoxic muscle used this amino acid. Oxygen deprivation decreased adenine nucleotides leading to the efflux of nucleosides. Protein synthesis and degradation responded differently to anoxia. Synthesis almost completely ceased, while proteolysis increased. Therefore, protein degradation in soleus muscle is enhanced when energy supplies and oxygen tension are low.
Dorsiflexion of one unloaded hind limb caused hypertrophy of the soleus relative to weight-bearing controls and faster growth of the plantaris and gastrocnemius muscles relative to the contralateral freely moving muscles. Unloading of the soleus muscle diminished primarily myofibrillar proteins whereas stretching increased all proteins. Stretching the soleus increased RNA, accelerated, especially, in vitro synthesis of sarcoplasmic proteins, and diminished in vitro proteolysis. Both in vivo and in vitro results showed slower synthesis and faster degradation in the freely moving than in the weight-bearing soleus muscle, faster synthesis and slower degradation in the stretched than in the freely moving soleus muscle, and faster degradation in the stretched than in the weight-bearing soleus muscle. Hence, stretching of the soleus muscle prevented changes in mass and protein metabolism produced by unloading. Shortening of the extensor digitorum longus muscle produced less muscle growth, slowed in vitro protein synthesis, and lowered RNA relative to the contralateral, freely moving muscle.
During sepsis or after injection of endotoxin into rats, there is a large increase in muscle protein breakdown and prostaglandin E2 (PEG2) production. Prior studies showed that partially purified interleukin 1 (IL-1) from human monocytes can stimulate these processes when added to isolated rat muscles. The availability of pure recombinant IL-1 and other monokines has allowed us to investigate the identity of the active agent in this process. Incubation of muscles with recombinant human or murine IL-1 alpha or IL-1 beta or with IL-1 plus a phorbol ester did not stimulate muscle proteolysis or PGE2 production. Homogeneous natural porcine IL-1 ("catabolin") and mouse or human IL-1 beta were also not effective in vitro. In addition, a variety of other human cytokines, including tumor necrosis factor ("cachectin"), epidermal thymocyte-activating factor, eosinophil cytotoxicity-enhancing factor, interferon-alpha, beta, and gamma, platelet-derived growth factor, and transforming growth factor (TGF) beta, which are all released by activated macrophages, TGF-alpha, or mixtures of these polypeptides, also failed to activate proteolysis or PGE2 production. By contrast, a large increase in net protein breakdown could be induced in the rat soleus by polypeptides released from porcine monocytes or by the serum from febrile cattle which had been injected with Pasteurella haemolytica or bovine rhinotracheitis virus. Therefore, a still-unidentified product of activated monocytes appears to be responsible for the negative nitrogen balance that accompanies infectious illness.
Although protein breakdown in most cells seems to require metabolic energy, it has only been possible to establish a soluble ATP-dependent proteolytic system in extracts of reticulocytes and erythroleukemia cells. We have now succeeded in demonstrating in soluble extracts and more purified preparations from rabbit skeletal muscle a 12-fold stimulation by ATP of breakdown of endogenous proteins and a 6-fold stimulation of 125I-lysozyme degradation. However, it has still not been possible to demonstrate such large effects of ATP in similar preparations from liver. Nevertheless, after fractionation by DEAE-chromatography and gel filtration, we found that extracts from liver as well as muscle contain both the enzymes which conjugate ubiquitin to 125I-lysozyme and an enzyme which specifically degrades the ubiquitin-protein conjugates. When this proteolytic activity was recombined with the conjugating enzymes, ATP + ubiquitin-dependent degradation of many proteins was observed. This proteinase is unusually large, approx. 1500 kDa, requires ATP hydrolysis for activity and resembles the ubiquitin-protein-conjugate degrading activity isolated from reticulocytes. Thus the ATP + ubiquitin-dependent pathway is likely to be present in all mammalian cells, although certain tissues may contain inhibitory factors.
Reticulocytes contain a nonlysosomal proteolytic pathway that requires ATP and ubiquitin. By DEAE chromatography and gel filtration, we were able to fractionate the ATP-dependent system into a 30-300-kDa fraction that catalyzes the ATP-dependent conjugation of ubiquitin to substrates ("Conjugation Fraction") and a high mass fraction (greater than 450 kDa) necessary for hydrolysis of the conjugated proteins. The latter contains two distinct proteases. One protease is unusually large, approximately 1500 kDa, and degrades proteins only when ATP and the conjugating fractions are added. This activity precipitates at 0-38% (NH4)2SO4 saturation and is essential for ATP-dependent proteolysis. Like crude extracts, it is labile in the absence of nucleotides and is inhibited by heparin, poly(Glu-Ala-Tyr), 3,4-dichloroisocoumarin, hemin, decavanadate, N-ethylmaleimide, and various peptide chloromethyl ketones. It lacks amino-peptidase and insulin-degrading activities and does not require tRNA for activity. The ubiquitin-conjugate degrading enzyme, which we suggest be named UCDEN, is inactive against substrates that cannot undergo ubiquitin conjugation. The smaller protease (670 kDa), which precipitates at 40-80% (NH4)2SO4 saturation, does not require ATP or ubiquitin and is therefore not required for ATP-dependent proteolysis. It is stimulated by N-ethylmaleimide and 3,4-dichloroisocoumarin and is stable at 37 degrees C. It hydrolyzes fluorometric tetrapeptides and proteins, including proteins which cannot be conjugated to ubiquitin. Thus, reticulocytes contain two large cytosolic proteases: one is essential for the degradation of ubiquitin conjugates, while the function of the other is uncertain.
Protein synthesis and degradation, and redox state were measured in soleus and extensor digitorum longus muscles of rats up to 12 days after injection of streptozotocin. Muscle growth was slower in these animals apparently due to slower protein synthesis throughout the duration of diabetes. Up to day 4 after injection of streptozotocin or withdrawal of insulin from treated, diabetic animals, the muscle ratio of lactate/pyruvate, an indicator of the cytoplasmic NAD+ redox couple, was lower and protein degradation was faster than in control muscles. Thereafter, the ratio of lactate/pyruvate was greater and protein degradation was slower than in size- or age-matched control muscles. Insulin treatment in vitro or in vivo increased lactate/pyruvate and decreased proteolysis. Therefore, in muscles of streptozotocin-diabetic rats, the initial increase and later fall in proteolysis, and the inhibition of proteolysis by insulin, may correlate with opposite changes in NADH/NAD+.
The decrease in plasma lactate during dichloroacetate (DCA) treatment is attributed to stimulation of lactate oxidation. To determine whether DCA also inhibits lactate production, we measured glucose metabolism in muscles of fed and fasted rats incubated with DCA and insulin. DCA increased glucose-6-phosphate, an allosteric modifier of glycogen synthase, approximately 50% and increased muscle glycogen synthesis and glycogen content greater than 25%. Lactate release fell; inhibition of glycolysis accounted for greater than 80% of the decrease. This was associated with a decrease in intracellular AMP, but no change in citrate or ATP. When lactate oxidation was increased by raising extracellular lactate, glycolysis decreased (r = - 0.91), suggesting that lactate oxidation regulates glycolysis. When muscle lactate production was greatly stimulated by thermal injury, DCA increased glycogen synthesis, normalized glycogen content, and inhibited glycolysis, thereby reducing lactate release. The major effect of DCA on lactate metabolism in muscle is to inhibit glycolysis.
It is generally accepted that ATP is required for intracellular protein breakdown. Reticulocytes contain a soluble ATP-dependent pathway for the degradation of highly abnormal proteins and for the elimination of certain proteins during cell maturation. Reticulocytes and erythrocytes also selectively degrade proteins damaged by oxidation. When these cells were exposed to oxidants, such as phenylhydrazine or nitrite, they showed a large increase in protein breakdown. This oxidant-induced proteolysis was not inhibited in cells depleted of ATP. However, ATP depletion did prevent the degradation of pre-existent cell proteins. In reticulocyte extracts, phenylhydrazine-treated hemoglobin is also degraded rapidly by an ATP-independent process, unlike endogenous proteins and many exogenous polypeptides. This lack of an ATP requirement means that the degradation of oxidant-damaged proteins does not require ligation to ubiquitin (even though phenylhydrazine treatment does make hemoglobin a very good substrate for ubiquitin conjugation). In many respects, the pathway for breakdown of oxidant-treated hemoglobin differs from the ATP-dependent process. The latter has a much higher activation energy than the degradation of oxidized proteins. The ATP-dependent process is inhibited by hemin, 3,4-dichloroisocoumarin, diisopropylfluorophosphate and N-ethylmaleimide. The ATP-independent pathway is less sensitive to N-ethylmaleimide, hemin, and 3,4-dichloroisocoumarin and is not affected by diisopropylfluorophosphate. In addition, only the ATP-dependent proteolytic process is inactivated by dilution or incubation at 37 degrees C in the absence of nucleotides. Reticulocytes thus contain multiple soluble systems for degrading proteins and can rapidly hydrolyze certain types of abnormal proteins by either an ATP-independent or ATP-dependent process. Erythrocytes lack the ATP-dependent process present in reticulocytes; however, erythrocytes retain the capacity to degrade oxidant-damaged hemoglobin. These two processes probably are active in the elimination of different types of abnormal proteins.
Inhibitors of protein or RNA synthesis prevented prostaglandin (PG) production in isolated skeletal muscles, brain, and spleen. Incubation of rat muscles with cycloheximide prevented the stimulation of PGE2 production induced in vitro by the Ca2+ ionophore A23187 and in vivo by injection of endotoxin. Cycloheximide also inhibited the stimulation by arachidonic acid of PGE2, PGF2 alpha, and prostacyclin. These observations suggest that the block in prostanoid production results from a loss of PG synthase activity (EC 1.14.99.1). These effects were detectable within 10 min after exposure of the muscle to cycloheximide. The degree of inhibition of PG production correlated with the degree of inhibition of protein synthesis. Other inhibitors of protein synthesis, puromycin and emetine, also prevented conversion of arachidonate into PGE2 in these tissues, but they did not inhibit purified PG synthase. Exposure of muscles to actinomycin D for 20 min also reduced PGE2 production from arachidonate by 90%. Thus, both the PG synthase and its mRNA appear to be inactivated rapidly (t1/2 less than 10 min) in muscle and other mammalian tissues. The block in PG production induced by inhibitors of protein and RNA synthesis may account for their antipyrogenic actions and certain of their other physiological effects.
Redox state and protein degradation were measured in isolated muscles of fasted (up to 10 d) and refed (up to 4 d) 7- to 14-wk-old rats. Protein degradation in the extensor digitorum longus muscle, but not in the soleus muscle, was greater in the fasted rats than in weight-matched muscle from fed rats. The NAD couple was more oxidized in incubated and fresh extensor digitorum longus muscles and in some incubated soleus muscles of fasted rats than in weight-matched muscle from fed rats. In the extensor digitorum longus muscle of refed or prolonged fasted rats, protein degradation was slower and the NAD couple was more reduced than in the fed state. Therefore, oxidation of the NAD couple was associated with increased muscle breakdown during fasting, whereas reduction of the NAD couple was associated with muscle conservation and deposition.
The relationship between the NAD redox state and protein degradation during growth was studied in isolated soleus and extensor digitorum longus muscles of 4- to 14-week-old rats. As muscle size increased with age, protein breakdown slowed and the muscles became progressively more reduced as shown by higher ratios of lactate/pyruvate in incubated and fresh-frozen muscle. Correlations were strong between redox state of protein degradation, and muscle mass, and between redox state and protein degradation. This relationship may be important in the slowing of muscle breakdown that occurs with age.
To determine if vanadate has insulin-like actions in skeletal muscle, we measured its effects on glucose and protein metabolism in epitrochlearis muscles of rats. Compared with insulin, vanadate increased glucose uptake, glycogen synthesis and glycolysis to a lesser degree, but caused a greater stimulation of lactate and glucose oxidation. Unlike insulin, vanadate did not change either protein synthesis or degradation. These different metabolic responses could be related to the different pattern of insulin-receptor phosphorylation caused by insulin and vanadate.
A soluble ATP-dependent system for protein degradation has been demonstrated in reticulocyte lysates, but not in extracts of nucleated cells. We report that extracts of undifferentiated murine erythroleukemia (MEL) cells contain a labile ATP-stimulated proteolytic system. The addition of ATP to MEL cell extracts at alkaline pH enhances degradation of endogenous cell proteins and various radiolabeled exogenous polypeptides from 2-15-fold. Nonhydrolyzable ATP analogs had no effect. In reticulocytes, one role of ATP in proteolysis is for ubiquitin conjugation to protein substrates. MEL cells also contain ubiquitin and extracts can conjugate 125I-ubiquitin to cell proteins; however, this process in MEL cells seems unrelated to protein breakdown. After removal of ubiquitin from these extracts by DEAE- or gel chromatography, the stimulation of proteolysis by ATP was maintained and readdition of purified ubiquitin had no further effect. In addition, these extracts degraded in an ATP-dependent fashion casein whose amino groups were blocked and could not be conjugated to ubiquitin. After gel filtration or DEAE-chromatography of the MEL cell extracts (unlike those from reticulocytes), we isolated a high molecular weight (600,000) ATP-dependent proteolytic activity, which exhibits many of the properties of energy-dependent proteolysis seen in crude cell extracts. For example, both the protease and crude extracts are inhibited by hemin and N-ethylmaleimide and both hydrolyze casein, globin, and lysozyme rapidly and denatured albumin relatively slowly. The protease, like the crude extracts, is also stimulated by UTP, CTP, and GTP, although not as effectively as ATP. Also, nonhydrolyzable ATP analogs and pyrophosphate do not stimulate the protease. Thus, some mammalian cells contain a cytosolic proteolytic pathway that appears independent of ubiquitin and involves and ATP-dependent protease, probably similar to that found in Escherichia coli or mitochondria.
It has been suggested that the cytoplasmic reduction-oxidation state correlates with, and may regulate, rates of protein breakdown in skeletal muscle. To test whether an increased lactate/pyruvate ratio is in fact generally associated with low proteolytic rates, this ratio was measured in rat extensor digitorum longus muscles incubated under conditions that rates of protein breakdown. Treatment with the calcium ionophore A23187 caused similar large increases in the lactate/pyruvate ratio at 2 microM, where proteolysis did not change, and at 20 microM, where proteolysis was greatly accelerated. Omission of Ca2+ from the medium slowed proteolysis, but decreased the lactate/pyruvate ratio. In muscles incubated at 40 degrees C, rates of proteolysis were faster, but the lactate/pyruvate ratios were higher than 37 degrees C. Thus alterations in the redox status do not necessarily correlate with, and can occur independently of, changes in proteolysis. Furthermore, insulin and inhibitors of lysosomal proteinases decreased proteolysis but, in contrast with previous reports, failed to alter the lactate/pyruvate ratio. In addition, protein breakdown decreased in muscles maintained under tension, although redox state did not change. Thus protein degradation can fall without a concomitant change in the reduction-oxidation state.
One leg of tail-casted suspended rats was immobilized in a plantar-flexed position to test whether chronic shortening of posterior leg muscles affected the metabolic response to unloading. The immobilized plantaris and gastrocnemius muscles of these animals showed approximately 20% loss of muscle mass in contrast to simply a slower growth rate with unloading. Loss of mass of the soleus muscle during suspension was not accentuated by chronic shortening. Although protein degradation in the isolated soleus muscle of the plantar-flexed limb was slightly faster than in the contralateral free limb, this difference was offset by faster synthesis of the myofibrillar protein fraction of the chronically shortened muscle. Total adenine nucleotides were 17% lower (P less than 0.005) in the chronically shortened soleus muscle following incubation. Glutamate, glutamine, and alanine metabolism showed little response to chronic shortening. These results suggest that, in the soleus muscle, chronic shortening did not alter significantly the metabolic responses to unloading and reduced activity.