Intracellular protein degradation in cultures of dystrophic muscle cells and fibroblasts

We have analysed protein degradation in primary cultures of normal and dystrophic chick muscle, in fibroblasts derived from normal and dystrophic chicks, and in human skin fibroblasts from normal donors and from patients with Duchenne muscular dystrophy (DMD). Our results indicate that degradative rates of both short- and long-lived proteins are unaltered in dystrophic muscle cells and in dystrophic fibroblasts. Longer times in culture and co-culturing chick fibroblasts with the chick myotubes do not expose any dystrophy-related abnormalities in protein catabolism. Furthermore, normal and dystrophic muscle cells and fibroblasts are equally able to regulate proteolysis in response to serum and insulin. We conclude that cultures of chick myotubes, chick fibroblasts, and fibroblasts derived from humans afflicted with DMD are not appropriate models for studying the enhanced protein degradation observed in dystrophy.     

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.     

Characterization of the inhibition of protein degradation by insulin in L6 cells

In muscle cells, protein degradation occurs by lysosomal and nonlysosomal mechanisms but the mechanism by which insulin inhibits protein degradation is not well understood. Using cultured L6 myotubes, the effect of insulin on muscle cell protein degradation was examined. Cells were labeled for 18 h with [3H]leucine or [3H]tyrosine and protein degradation measured by release of TCA-soluble radioactivity. Incubation with insulin for 0.5, 1, 2, or 3 h produced 0, 6, 12, and 13% inhibition, respectively, at 10(-7) M. If the cells were incubated for 3 h prior to the addition of insulin to remove short-lived proteins, the effect of insulin was enhanced, producing 26% inhibition. Very long-lived protein degradation (cells labeled for 48 h, chased for 24 h before the addition of insulin) was only inhibited 17% by insulin. This was due to serum starvation during the chase since the addition of serum to the chase medium produced a subsequent inhibition of 38% by insulin. Thus insulin had a greater effect on the degradation of longer-lived proteins. Use of inhibitors suggested that insulin requires internalization and degradation to produce inhibition of protein degradation and acts through both the proteasome and lysosomes. There appears to be no interaction with the calpains.     

Skeletal muscle protein and amino acid metabolism in hereditary mouse muscular dystrophy. Accelerated protein turnover and increased alanine and glutamine formation and release

Interactins between skeletal muscle protein and amino acid metabolism were investigated using C57BL and 129ReJ mice with hereditary muscular dystrophy. On incubation, hind limb muscle preparations from dystrophic mice released large quantities of amino acids, particularly alanine and glutamine which were increased 70% and 40% compared to muscles from carrier or control mice. The increased alanine release did not result from altered alanine oxidation to CO2 or reincorporation into protein. Alanine and glutamine formation from added amino acids were equal with dystrophic and control muscles. Incorporation in vitro of leucine, alanine, and glutamate into proteins of dystrophic muscle was 3- to 7-fold greater than control muscle, and the incorporation in vivo of [3H]- or [14C]arginine into muscle proteins was greater in extent and earlier in time with dystrophic as compared to control muscle. Proteins were also labeled in vivo using [guanido-14C]arginine. On incubation of these muscles in vitro, a 100% greater loss of label from protein was observed with dystrophic as compared to control preparations, and the appearance of label in the media was correspondingly increased. Sodium dodecyl sulfate-gel electrophoresis of dystrophic skeletal muscle showed numerous protein bands to be reduced in density, but autoradiographic studies demonstrated that these same bands were more highly labeled in vitro by [35S]methionine in dystrophic than in control muscle. Although insulin stimulation of glucose uptake was markedly blunted in dystrophic muscle, insulin inhibited alanine and glutamine release equally from both control and dystrophic muscle. These data indicate that alanine and glutamine formation and release are increased in hereditary mouse muscular dystrophy. An accelerated degradation and an increased resynthesis of many muscle proteins were also observed in dystrophic compared to control animals. This increased proteolysis may account for the increased alanine and glutamine formation in dystrophic muscle.     

Degradation of proteins in steady-state cultures of Escherichia coli.

The degradation of proteins in Escherichia coli was investigated in cells grown under steady-state conditions in a glucose-limited chemostat. During the first 24 h, approximately 25% of pulse-labeled proteins were degraded and after 72 h up to 58% of the proteins were broken down. To examine the stability of subcellular components steady-state cultures were labeled with an initial pulse of [14C]leucine, 24 h were allowed for turnover of these proteins, and the cells were then labeled with a short pulse of [3H]leucine. By this double-label protocol, the labile proteins were preferentially labeled with [H]leucine and had high 3H/14C ratios, while the more stable proteins had lower 3//14C ratios. The 3/-labeled proteins were degraded approximately five times as rapidly as the 14C-labeled proteins in exponentially growing cells. The relative stability of subcellular fractions was determined by comparing their 3H/14C ratios to the ratio of the cells at harvest. The soluble fraction contained the most labile proteins, while the ribosomal and membrane fractions were at least as stable as the average cell protein.     

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.     

Expression of an X-linked muscular dystrophy in a female due to translocation involving Xp21 and non-random inactivation of the normal X chromosome

Summary A young female was diagnosed as having X-linked muscular dystrophy of the Duchenne type. Chromosome studies, including trypsin-Giemsa banding, Quinacrine fluorescence, and nucleolus organizer region (NOR) silver staining revealed an X-autosome reciprocal translocation t(X;21) (p21;p12). Utilizing both [³H] thymidine autoradiography and the BrdU-Hoechst 33258-Giemsa technique, lymphocytes and fibroblasts were found to show a preferential inactivation of the normal X suggesting the presence of a single mutant gene on the translocated X. This patient is one of seven reported cases of an X-linked muscular dystrophy associated with an X-autosome translocation. In all seven cases the exchange point in the X chromosome is in band p21 at or near the site of the Duchenne gene.     

Heat stress-induced alterations in the synthesis and secretion of proteins and prostaglandins by cultured bovine conceptuses and uterine endometrium

Effect of in vitro heat stress on protein and prostaglandin synthesis and secretion by bovine conceptuses and endometrium was examined. Conceptuses (n = 11) and endometrium (n = 10) obtained on Day 17 of pregnancy were cultured at thermoneutral (39 degrees C, 24 h) or heat stress (39 degrees C, 6 h; 43 degrees C, 18 h) temperatures in medium supplemented with L-[4,5-3H]leucine (100 microCi) and arachidonic acid (10 micrograms/ml). Radiolabeled protein secreted into culture medium increased with time in both groups. Heat stress reduced (p less than 0.001) incorporation of [3H]leucine into intracellular and secreted proteins by conceptuses but did not alter incorporation of [3H]leucine by endometrium. In particular, heat stress reduced by 72% the secretion of bovine trophoblast protein-1, the conceptus polypeptide believed to cause extension of luteal lifespan. Two-dimensional, sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that heat stress altered the array of proteins in endometrial and conceptus tissues, as evidenced by the induction of "heat-shock proteins." Endometrial secretion of prostaglandin F (p less than 0.001) and conceptus secretion of prostaglandin E2 (p less than 0.05) increased in response to heat stress. Sensitivity of bovine conceptuses and endometrium to heat stress in vitro suggests that infertility associated with maternal heat stress may be caused, partially by alterations in signals required for maintenance of the corpus luteum during early pregnancy.     

Intracellular protein degradation in serum-deprived human fibroblasts.

IMR90 human fibroblasts were labelled by incubation of cells for 48 h in medium containing 10% serum and [3H]leucine. The labelled protein was degraded at a rate of 1%/h during a subsequent incubation in medium with 10% serum. Incubation in medium without serum caused a transient enhancement of the degradation of endogenous protein, which was also found in cells labelled in medium without serum. The degradation of micro-injected haemoglobin was enhanced by serum deprivation in a non-transient manner. These results suggest that enhanced degradation in serum-free medium occurs only for a subpopulation of cell proteins and that it appears transient because the major part of the pool of susceptible endogenous proteins is being degraded during the first 20-30 h in serum-free unlabelled medium. Protein turnover in various cell compartments was measured by a double-labelling technique. Most of the enhanced degradation in serum-deprived cultures (73-83%) was due to breakdown of cytosolic proteins. The enhanced degradation of cytosolic proteins seemed to affect several proteins irrespective of their molecular mass or metabolic stability.     

A method for the detection of carriers of duchenne muscular dystrophy—A preliminary report

This report of a method to detect carriers of Duchenne muscular dystrophy describes a technique of tissue autoradiography that identifies increased uptake of [³H]leucine into some individual muscle fibers in muscle biopsies. Three obligate and seven of 11 sisters of affected boys are identified as carriers in this series. This method of identification may be of value in identifying those carriers undetected by other means, but remains to be further evaluated.     

Chromatin proteins as a possible target for antitumour agents: alterations of chromatin proteins in dibromodulcitol treated Yoshida tumors

The effect of dibromodulcitol (DBD) on Yoshida sarcoma chromatin components has been investigated. Measurements on the radioactivity of nuclear components after in vivo treatment with [3H]DBD for 1 h indicated preferential drug binding to the high molecular weight component of the nuclear residual acidic protein (non-histones) and also to Histone 1 (H1) (very lysine rich, F1). Two-hour DBD treatment resulted in partial degradation and reduced [3H]leucine incorporation into the same fractions of chromatin. However, 6 h after DBD treatment, the synthesis of the degraded chromatin proteins began and by 24 h was completed. During the same treatment period the composition of chromatin showed a remarkable alteration; 2 h after DBD treatment the amount of the nuclear residual acidic proteins relative to DNA decreased by approx. 50%, but returned to control value 24 h after drug treatment. This in conjunction with the data on [3H]leucine incorporation suggests that certain chromatin proteins are degraded and subsequently newly synthesised after DND treatment resulting in an exchange of chromatin components. The formation of a nucleohistone complex between H1 and DNA was inhibited by pretreatment of H1 and DBD, dianhydrodulcitol (DAD) and bischloroethylnitrosourea (BCNU).     

Kinetics of protein degradation in diploid and trisomic human fibroblasts

The degradation rate of long-lived and short-lived proteins was determined in diploid fibroblasts and fibroblasts with trisomy 7 derived from human embryos. Two fractions of proteins were detected in the exponentially growing diploid fibroblasts with half-lives (T 1/2) 37 and 19 hours. The rate of protein degradation increases in diploid fibroblasts as they approach confluence and protein fractions with T 1/2 30, 18 and 12 hours appear. The rate of protein degradation in trisomic fibroblasts does not change for the long-lived and short-lived proteins and is the same in both exponential (T 1/2 31 and 14 hours) and stationary phase (T 1/2 33 and 17 hours). The relative amount of the short-lived proteins in trisomic fibroblasts in the stationary phase decreased as compared with the one in diploid fibroblasts. It is apparent that a mechanism of regulation of protein catabolism in trisomic fibroblasts is impaired.     

Secretion-coupled protein degradation: studies on mammary casein

Mammary explants from midpregnant rabbits were cultured for 18 h at 37 degrees C with insulin, prolactin and cortisol. Subsequently, explants were labelled for 2 h with inorganic [32P]phosphate, L-[5-3H]proline or L-[4,5-3H]leucine, washed and chased for up to 3 h. The radiolabelling profile of [32P]casein or [3H]casein during the chase period, obtained by isoelectric focussing or immunoprecipitation indicates extensive destruction of neosynthesized casein. The extent of casein destruction in mammary explants in culture (measured after radiolabelling with L-[5-3H]proline), is inversely related to casein secretion. Least casein degradation is observed in explants after 48 h in culture when casein secretion is maximal (observed histochemically). Subsequently, when the extracellular alveolar lumen is filled with secretion products (72 h), rapid intracellular casein destruction is again observed. When the chase was carried out in the presence of drugs which inhibit degradation and/or secretion, the results indicate that secretion-coupled casein degradation is dependent on an intact functional microfilamentous-microtubular network, casein is not degraded by an autophagosome requiring process, degradation is inhibited by leupeptin, amino-acid analogue containing casein does not undergo secretion-coupled degradation and inhibition of N-glycosylation of intracellular vesicular membrane proteins prevents secretion-coupled degradation. Secretion-coupled protein destruction is discussed in relation to the post-translational regulation of the net production of secretory proteins in eukaryotic cells.     

Intracellular degradation of hemoglobin transferred into fibroblasts by fusion with red blood cells

Hamster fibroblast protein and rabbit hemoglobin were labelled by incubation of fibroblasts (BHK21) or reticulocytes with [³H]leucine. Alternatively, human or rabbit hemoglobin was labelled by carbamoylation of erythrocytes with K¹⁴CNO. The labelled hemoglobins were introduced into fibroblasts by virus-mediated fusion between the blood cells and fibroblasts. The hemoglobins became uniformly distributed throughout the cytoplasm. Degradation was assessed from release of acid-soluble radioactivity into the medium. Radioactivity from [¹⁴C]-carbamoylhemoglobin was released as carbamoylvaline and homocitrulline, and these compounds were not metabolized or reincorporated by the cells. Intermediate degradation products could not be detected. The degradation of hemoglobin followed first-order kinetics. The half-life of both carbamoylated and native rabbit hemoglobin in hamster fibroblasts was 28 h, and the half-life of carbamoylated human hemoglobin was about 150 h in fibroblasts from hamster (BHK21), mouse (Balb/3T3), and man (MRC 5), corresponding to that of the more stable endogenous proteins. Phenylhydrazine increased the intracellular degradation of carbamoylated human hemoglobin about 13 times, whereas the degradation of endogenous proteins was little affected. Hemoglobin was degraded in homogenates at 31% h^?1 at pH 5 and 0.3% h^?1 at pH 7.4. Phenylhydrazine increased these rates to 45% h^?1 and 9.7% h^?1, respectively. Growing hamster fibroblasts, which are brought into quiescence by serum deprivation or by high culture density, increase the degradation of endogenous protein and of hemoglobin in parallel.     

Mechanisms of intracellular protein catabolism. Intracellular fate of microinjected polypeptides translated in vitro.

Erythrocyte-mediated microinjection was used to introduce [35S]polypeptides translated in vitro into 3T3-L1 cells. Such [35S]polypeptides are not degraded after loading into erythrocytes and are stable for the first 2 h after microinjection into growing 3T3-L1 cells. Similarly, little or no degradation of microinjected [35S]polypeptides is observed in either growing or confluent 3T3-L1 cells over a 70 h period. Microinjection of reticulocyte lysate alone does not affect the rate of degradation of long-lived endogenous protein. Reductively [3H]methylated lysate haemoglobin is degraded after microinjection by a cytosolic mechanism. Microinjected 125I-labelled bovine serum albumin is rapidly degraded by a cytosolic mechanism at the same rate in the absence or presence of reticulocyte lysate. The data do not support the notion that the observed lack of degradation of microinjected [35S]polypeptides translated in vitro is due to the presence of proteolytic inhibitors in reticulocyte lysates which can inhibit the degradation of microinjected or cellular proteins.     

Improved Muscle Function in Duchenne Muscular Dystrophy through L-Arginine and Metformin: An Investigator-Initiated,Open-Label,Single-Center,Proof-Of-Concept-Study

Altered neuronal nitric oxide synthase function in Duchenne muscular dystrophy leads to impaired mitochondrial function which is thought to be one cause of muscle damage in this disease. The study tested if increased intramuscular nitric oxide concentration can improve mitochondrial energy metabolism in Duchenne muscular dystrophy using a novel therapeutic approach through the combination of L-arginine with metformin. Five ambulatory, genetically confirmed Duchenne muscular dystrophy patients aged between 7–10 years were treated with L-arginine (3 x 2.5 g/d) and metformin (2 x 250 mg/d) for 16 weeks. Treatment effects were assessed using mitochondrial protein expression analysis in muscular biopsies, indirect calorimetry, Dual-Energy X-Ray Absorptiometry, quantitative thigh muscle MRI, and clinical scores of muscle performance. There were no serious side effects and no patient dropped out. Muscle biopsy results showed pre-treatment a significantly reduced mitochondrial protein expression and increased oxidative stress in Duchenne muscular dystrophy patients compared to controls. Post-treatment a significant elevation of proteins of the mitochondrial electron transport chain was observed as well as a reduction in oxidative stress. Treatment also decreased resting energy expenditure rates and energy substrate use shifted from carbohydrates to fatty acids. These changes were associated with improved clinical scores. In conclusion pharmacological stimulation of the nitric oxide pathway leads to improved mitochondria function and clinically a slowing of disease progression in Duchenne muscular dystrophy. This study shall lead to further development of this novel therapeutic approach into a real alternative for Duchenne muscular dystrophy patients.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02516085","term_id":"NCT02516085"}}NCT02516085     

Differential degradation of intracellular proteins in human skin fibroblasts of mitotic and mitomycin-C (MMC)-induced postmitotic differentiation states in vitro

Rates of degradation of short- and long-lived proteins were analysed in homogeneous fibroblast cultures of mitotic or mitomycin C (MMC)-induced postmitotic states. When the highly mitotic MFII type cells--the major cell type of so called "early passage" or "young" fibroblasts--differentiate into MFIII type cells, the last mitotic fibroblast type, and further into MMC-induced postmitotic fibroblasts, the degradation of short-lived proteins increases by a factor of 1.4, resulting in significantly reduced half-lives of these proteins in the postmitotic fibroblasts. From the highly mitotic MFII to the final postmitotic PMFVI-type cells via the intermediates MFIII, PMFIV and PMFV, the half lives (t1/2) of short-lived proteins decrease by a total of 122 min in average, from 362 to 240 min. Degradation of long-lived proteins did not change significantly from cell type MFII to PMFVI. As analysed by two-dimensional (2D)-gel electrophoresis the half-lives of the mitotic and postmitotic cell-type-specific proteins except one, protein PIVa (33 kDa; Pi 5.0), range between 33.2 h and 62.9 h. Protein PIVa, the first protein specific for postmitotic cells, is initially expressed 18 h after the induction of the postmitotic state by mitomycin C (MMC) and has a half-life of approximately 66 min. This may indicate that protein PIVa could function as one possible regulatory factor controlling the postmitotic differentiation state.     

Paradoxical stimulation by amino acids of the degradation of [35S]methionine-labelled, short-lived protein in isolated rat hepatocytes

A complete amino acid mixture inhibited the degradation of long-lived and [14C]valine-labelled short-lived protein in isolated rat hepatocytes, but paradoxically stimulated the degradation of [35S]methionine-labelled short-lived protein. The stimulation persisted in the presence of autophagiclysosomal pathway inhibitors like 3-methyladenine and propylamine, indicating the existence of an hitherto unrecognized non-lysosomal degradation mechanism with selectivity towards methionine-rich proteins or peptide regions.     

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.     

Inhibition of protein degradation in isolated rat hepatocytes

1. Isolated parenchymal cells were prepared by collagenase perfusion of livers from fed rats that had been previously injected with [³H]leucine to label liver proteins. When these cells were incubated in a salts medium containing glucose, gelatin and EDTA, cellular integrity was maintained over a period of 6h. 2. Cells incubated in the presence of 2mm-leucine to minimize radioactive isotope reincorporation released [³H]leucine into the medium at a rate accounting for the degradation of 4.5% of the labelled cell protein per h. 3. Degradation of [³H]protein in these cells was inhibited by insulin and by certain amino acids, of which tryptophan and phenylalanine were the most effective. 4. Protein degradation was decreased by several proteinase inhibitors, particularly those that are known to inhibit lysosomal cathepsin B, and by inhibitors of cell-energy production. 5. Ammonia inhibited degradation, but only at concentrations above 1.8mm. Aliphatic analogues of ammonia were effective at lower concentrations than was ammonia. 6. High concentrations of ammonia inhibited degradation by 50%. The extent of this inhibition could not be increased further by the addition of the cathepsin B inhibitor leupeptin, which by itself inhibited degradation by approx. 30%. 7. The sensitivity of proteolysis in isolated hepatocytes to these various inhibitory agents is discussed in relation to their possible modes of action.