Similarities in protein synthesis and degradation in normal and dystrophic muscle cultures

Protein synthesis and degradation were compared in cultured muscle cells obtained from normal and dystrophic chick embryos under conditions where labeled amino acid reincorporation was not a complicating factor, where fibroblast contamination was minimized, and where the animals compared were as genetically similar as possible. Under these conditions both cell types exhibited a half-time of protein turnover of 34 h. Degradation in both was inhibited 21% by leupeptin (50 μg/ml), and both showed parallel increases in degradation rates under ‘step-down’ conditions.     

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.     

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.     

Intracellular protein degradation in growing, in density-inhibited, and in serum-restricted fibroblast cultures.

Exponentially growing Balb/3T3 mouse fibroblasts contain protein populations with slow and fast turnover. These two stability classes were labelled selectively with 3H-leucine. The intracellular degradation of the proteins was then followed as the release into the medium of radioactive leucine. The degradation rate of both stability classes of protein is increased by about 55% in cultures whose growth is inhibited by high cell density. Serum-deprivation, which also halts cell growth, accelerates protein breakdown to a smaller extent, the increases for relatively stable and unstable proteins being 30% and 13%, respectively. The density-dependent increase in protein breakdown is also found in BHK21 cells but not in chick fibroblasts. Protein degradation in Balb/3T3 cells transformed by simian virus 40 is affected by serum-deprivation but not by cell density. The proteins which are relatively stable during growth were shown to become less stable in density-inhibited or serum-deprived cultures, and vice versa. Cycloheximide inhibits degradation to a variable extent. Dibutyryl adenosine-3',5'-cyclic monophosphate has no effect on the protein degradation under the conditions investigated here.     

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.     

Satellite cells of normal and dystrophic muscle

The structure, the ultrastructure and the number of myonuclei and satellite cells in Duchenne's muscular dystrophy and in control muscles were compared in order to determine the possible changes in the satellite cells population. The bioptical fragments were obtained from 16 healthy (control) and from 16 dystrophic male children from 12 to 96 months of age. The biopsies were embedded in paraffin and in Durcupan and the sections were stained with ematossilin-eosin, P.A.S. for the light microscope observation and with uranil-acetate and lead-citrate for the electron microscope study. Moreover the semithin sections were stained according to the method of Ontell (1974) that is specific for the satellite cells identification. The morphological aspects of the dystrophic muscles are the same previously reported by other authors. The quantitative analysis of the myonuclei and satellite cells in control and dystrophic muscles was carried out on five random sections of each biopsy. The whole number of nuclei (myonuclei and satellite cell nuclei) and the number of the satellite cells nuclei were evaluated and the mean values in controls and dystrophic muscles were compared with the t Student test. The obtained results show that: 1) in the control muscles the satellite cells number is nearly the same in all ages considered; 2) in the dystrophic muscles the satellite cells number is in a statistically significant way greater than in control muscles and show a moderate trend to increase with aging; 3) in the dystrophic muscles the whole number of nuclei (myonuclei and satellite cells) is greater than in control in a statistically significant way and this increase is due to the number of satellite cells.     

Sarcotubular development in dystrophic skeletal muscle cells

Summary Dilations of the sarcotubular system and misaligned myofilaments have been reported as early indicators of muscular dystrophy in skeletal muscle. Since the developing tubular component is believed instrumental in initial myofilament alignment during myogenesis, tubular development is evaluated using normal and dystrophic chick embryo skeletal muscle and cultures of normal and dystrophic embryonic pectoral muscle incubated in the presence of horse spleen ferritin. Comparisons of the findings show that periodic tubules are absent from dystrophic somitic muscle and that invaginating tubules from the sarcolemma are found in fewer, randomly located areas of dystrophic pectoral muscle cells. The results indicate that the tubular component is not involved in the bizarre vesiculations seen in mature dystrophic muscle, however, the malalignment of dystrophic myofilaments is probably the result of the poorer development of the T system in this muscle.     

Intracellular protein degradation in Neurospora crassa.

In exponentially growing cultures of Neurospora crassa, the basal rate of protein degradation increases as the constant of the rate of growth decreases, so that in slow growing cells (mu = 0.13) the rate of protein degradation is about 25% of the rate of protein accumulation. During glucose starvation and shift-down transition of growth, the rate of protein degradation is greatly enhanced, and a moderate reduction (about 30%) of the ATP level is observed. Treatment of glucose-starved cells with 2-deoxyglucose reduces the ATP content by 70% and blocks protein degradation. The addition of cycloheximide, given at the onset of glucose starvation, prevents the enhancement of protein degradation; instead cycloheximide is without effect if added when proteolysis has already started. At a supraoptimal temperature (42 degrees C) the basal rate of protein degradation is not stimulated, contrary to the behavior observed in bacteria. Guanosine nucleotides, which appear to have a regulatory role for protein degradation in bacteria, are not found in N. crassa.     

Accelerated degradation of glycogen phosphorylase in denervated and dystrophic mouse skeletal muscle

Pyridoxal phosphate, the cofactor of glycogen phosphorylase, fulfils the criteria needed of a turnover label for this enzyme. The decay of protein-bound label following administration of [³H]pyridoxine is a good index of the rate of degradation of the enzyme in vivo. This method has been applied to the study of catabolism of the enzyme in normal, denervated and dystrophic mouse skeletal muscle. In both of the pathological conditions the enzyme is degraded more rapidly than normal.     

Effect of leupeptin of protein turnover in normal and dystrophic chicken skeletal muscle cells in culture

The effect of leupeptin on turnover of the soluble protein fraction, the myofibrillar protein fraction, and myosin heavy chain was evaluated in muscle cell cultures. Cultures were prepared from the breast muscle of 12-day normal (white leghorn) and dystrophic (line 307) chick embryos. After 7 days in culture, cells were labeled for 16 hr with 1 μCi/ml of [³H]Leu and then “chased” for an additional 24 hr in culture medium containing no [³H]Leu and 0–75 μg/ml leupeptin. Cultures were analyzed for radioactivity in the soluble protein fraction, the myofibrillar protein fraction, and myosin heavy chain. Leupeptin (75 μg/ml) virtually eliminated loss of radioactivity from the soluble protein fraction, but only minimally affected loss of radioactivity from the myofibrillar fraction or myosin heavy chain. Normal and dystrophic muscle cells responded identically to leipeptin treatment. Thus, the muscle proteases that are specifically inhibited by leupeptin seem to have no major role in initiating myofibrillar protein turnover.     

Intracellular degradation by liver endothelial cells

Investigations were carried out on the intracellular fate of formaldehyde treated bovine serum albumin (F-BSA), in liver non-parenchymal cells. This paper reports the observations and results obtained by us. The first part of our work involved the injecting of the compound into either a) normal rats, b) rats injected with Triton WR 1339 or c) rats treated with mannan. Fractions obtained after differential and isopycnic centrifugation in sucrose gradients, were analysed by SDS-gel electrophoresis and fluorography. The degradation takes place in a two step process. The molecule is first split into radiolabeled compounds that are still acid precipitable. This is followed by the appearance of acid soluble radioactive molecules. In a sucrose gradient the first kind of degradation products exhibit a distribution totally different from that of acid soluble degradation compounds. In the second part of our experiments, fairly pure fractions of the organelles, known to be involved in the endocytic pathway i.e. endosomes, transfer lysosomes and accumulation lysosomes (marked by the presence of either Triton WR 1339 or mannan) were isolated and incubated with [¹²⁵I]-F-BSA. These experiments revealed that endosomes, isolated by us, are incapable of degradation. Accumulation lysosomes arising exclusively from liver non-parenchymal cells (in which mannan had accumulated) though rich in certain hydrolases eg. arylsulfatase did not have an efficient proteolytic machinery. Our results, both fromin vivo andin vitro studies, suggest that the first degradation step occurs in one type of structure (probably not endosomes), a sort of hybrid endosome-lysosome (as they are not affected by glycyl-l-phenyl-2-napthylamide [1]) and the second step in a different type of lysosomes, what we have designated transfer lysosomes.     

Intracellular proteolysis: Signals of selective protein degradation

Selective proteolysis is one of the mechanisms for the maintenance of cell homeostasis via rapid degradation of defective polypeptides and certain short-lived regulatory proteins. In prokaryotic cells, high-molecular-mass oligomeric ATP-dependent proteases are responsible for selective protein degradation. In eukaryotes, most polypeptides are attacked by the multicatalytic 26S proteasome, and the degradation of the majority of substrates involves their preliminary modification with the protein ubiquitin. The proteins undergoing the selective proteolysis often contain specific degradation signals necessary for their recognition by the corresponding proteases. This article is dedicated to the 25th Anniversary of the journal Bioorganicheskaya Khimiya     

Accelerated protein turnover in the skeletal muscle of dystrophic mice

The excretion of 3-methylhistidine increased in the urine of dystrophic mice C57BL/6J. The content of 3-methylhistidine residue decreased in the muscle proteins of dystrophic mice, but not in other organs. Methylated proteins in the skeletal muscle, actin and myosin, were partially purified from the dystrophic and control muscles. The amount of 3-methylhistidine residue in unit weight of the actin and myosin preparations was normal in dystrophic muscle. These three facts indicate that the turnover rates of actin and myosin are increased in the muscle of the dystrophic mice.     

Intracellular proteolysis: signals of selective protein degradation

Selective proteolysis is one of the mechanisms for the maintenance of cell homeostasis via rapid degradation of defective polypeptides and certain short-lived regulatory proteins. In prokaryotic cells, high-molecular-mass oligomeric ATP-dependent proteases are responsible for selective protein degradation. In eukaryotes, most polypeptides are attacked by the multicatalytic 26S proteasome, and the degradation of the majority of substrates involves their preliminary modification with the protein ubiquitin. The proteins undergoing the selective proteolysis often contain specific degradation signals necessary for their recognition by the corresponding proteases.     

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.