[Regulation of ubiquitin-proteasome dependent muscular proteolysis by hormones and nutrients].
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Animals↗
Biomedical subjects
D Béchet
Publications and source records attributed to D Béchet.
Leucine limitation induces autophagy and activation of lysosome-dependent proteolysis in C2C12 myotubes through a mammalian target of rapamycin-independent signaling pathway.
Loss of muscle mass usually characterizes different pathologies (sepsis, cancer, trauma) and also occurs during normal aging. One reason for muscle wasting relates to a decrease in food intake. This study addressed the role of leucine as a regulator of protein breakdown in mouse C2C12 myotubes and aimed to determine which cellular responses regulate the process. Determination of the rate of protein breakdown indicated that leucine is one key regulator of this process in myotubes because starvation for this amino acid is responsible for 30-40% of the total increase generated by total amino acid starvation. Leucine restriction rapidly accelerates the rate of protein breakdown (+11 to 15% (p < 0.001) after 1 h of starvation) in a dose-dependent manner. By using various inhibitors, evidence is provided that acceleration of protein catabolism results mainly from an induction of autophagy, activation of lysosome-dependent proteolysis, without modification of mRNA levels encoding the lysosomal cathepsins B, L, or D. Those results suggest that autophagy is an essential cellular response for increasing protein breakdown in muscle following food deprivation. Induction of autophagy precedes a decrease in global protein synthesis (-20% to -30% (p < 0.001)) that occurs after 3 h of leucine starvation. Inhibition of the mammalian target of rapamycin (mTOR) activity does not abolish the effect of leucine starvation and the level of phosphorylated ribosomal S6 protein is not affected by leucine withdrawal. These latter data provide clear evidence that the mTOR signaling pathway is not involved in the mediation of leucine effects on both protein synthesis and degradation in C2C12 myotubes.
Animals↗
Increased ATP-ubiquitin-dependent proteolysis in skeletal muscles of tumor-bearing rats.
Little information is available on proteolytic pathways responsible for muscle wasting in cancer cachexia. Experiments were carried out in young rats to demonstrate whether a small (< 0.3% body weight) tumor may activate the lysosomal, Ca(2+)-dependent, and/or ATP-ubiquitin-dependent proteolytic pathway(s) in skeletal muscle. Five days after tumor implantation, protein mass of extensor digitorum longus and tibialis anterior muscles close to a Yoshida sarcoma was significantly reduced compared to the contralateral muscles. According to in vitro measurements, protein loss totally resulted from increased proteolysis and not from depressed protein synthesis. Inhibitors of lysosomal and Ca(2+)-dependent proteases did not attenuate increased rates of proteolysis in the atrophying extensor digitorum longus. Accordingly, cathepsin B and B+L activities, and mRNA levels for cathepsin B were unchanged. By contrast, ATP depletion almost totally suppressed the increased protein breakdown. Furthermore, mRNA levels for ubiquitin, 14 kDa ubiquitin carrier protein E2, and the C8 or C9 proteasome subunits increased in the atrophying muscles. Similar adaptations occurred in the muscles from cachectic animals 12 days after tumor implantation. These data strongly suggest that the activation of the ATP-ubiquitin-dependent proteolytic pathway is mainly responsible for muscle atrophy in Yoshida sarcoma-bearing rats.
Animals↗
Effect of fasting and thyroidectomy on cysteine proteinase activities in liver and muscle.
Prolonged starvation mimics chronic negative nitrogen balance observed in many physiopathological situations. During starvation, an initial decrease in protein utilization (phase I) is followed by a long period of protein sparing (phase II) that ends with a marked rise in nitrogen excretion (phase III). Variations in protein metabolism during starvation are determined by changes in protein synthesis and degradation rates (Cherel, Y., Attaix, D. Rosolowska-Huszcz, D., Belkhou, R., Robin, J.P., Arnal, M. and Le Maho, Y. (1991) Clin. Sci. 81, 611-619), but little information is available on expression of proteolytic systems. In this study, cathepsin B, H and L activities were compared in hindlimb muscles and liver at various phases of starvation in thyroidectomized and sham-operated rats. In muscle, cathepsin activities fell from the fed state to phase II, which suggests that cathepsins may play a role in the curtailment of muscle proteolysis during protein sparing phase. This decrease of muscle cathepsin activities was reproduced by thyroidectomy alone. In contrast, liver cathepsin B and H activities fell during starvation, but were not affected by thyroidectomy alone. Liver cathepsin L decreased only during starvation in thyroidectomized animals. These observations emphasize that different mechanisms modulate cathepsin expression in skeletal muscle and liver.
Animals↗
Further characterization of a somatic cell hybrid panel: ten new assignments to the bovine genome.
Thirty-six partially characterized hamster-bovine hybrid cell lines were used for the determination of synteny groups. Sixteen additional reference loci, selected for their coverage of the bovine genome, were analysed on these hybrid cells. This increases to 25 the number of synteny groups detected. This panel was then used to make synteny assignments for 10 additional loci, eight by Southern blotting (COL1A1, COL1A2, FAS, CTSB, CTSL, CHRNG, HEXB and HTR1A) and two by polymerase chain reaction (PCR) amplification (HRH1 and ETH1112). These loci were assigned to international synteny groups U12 (HRH1), U13 (COL1A2), U17 (CHRNG), U21 (COL1A1, FAS), U29 (ETH1112), to chromosome 20 (U14 or U25) for HEXB and HTR1A, and to the same local synteny group (A), which is probably U18, for CTSB and CTSL. For three loci already mapped in humans (COL1A1, COL1A2 and CHRNG), the present results are in accordance with the predictions based on comparative mapping between the human and bovine species.
Animals↗
Nucleotide sequence of bovine preprocathepsin B. A study of polymorphism in the protein coding region.
A cDNA encoding bovine procathepsin B was isolated. The deduced amino acid sequence revealed that a stop (TAG) codon, instead of a Trp-257 codon (TGG), generates in bovine a cathepsin B precursor four amino acids shorter than in other species. Because micro-heterogeneities were previously reported in the cathepsin B primary structure, sequence polymorphism in the protein coding region was then investigated by PCR sequencing of genomic fragments and RNase protection assays. Experiments performed with 12-15 animals of three breeds did not reveal any difference with our cDNA sequence. We conclude that sequence polymorphism in bovine cathepsin B is a rare event, and can only result from the expression of different alleles of a unique gene.
Animals↗
Systematic degradation of cellular proteins: an introduction to the workshop.
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Amino Acid Sequence↗
Multiple leader sequences for mouse cathepsin B mRNA?
We previously described the gene structure of murine cathepsin B. Our results suggested that the 5'-untranslated region (leader) is interrupted by a large intron. The second exon (exon-2) contains the translation initiation site. To characterize the leader region, a rapid amplification of cDNA ends (RACE) procedure was developed. The PCR products were directly cloned and sequenced. Nucleotide sequence analyses revealed three different 5'-cDNA ends, suggesting the existence of three different leader regions. In addition to the leader (LA) previously characterized, we now describe two other 5'-untranslated regions, LB and LC. Leader LB is located 2.3 kb upstream exon-2, and leader LC corresponds to the 3'-end of the first intron and is thus contiguous to exon-2. Our results suggest for murine cathepsin B gene the presence of multiple promoters, and possibly the expression of multiple mRNAs differing in their leader region.
3T3 Cells↗
Muscle differentiation in the bovine fetus: a histological and histochemical approach.
The chronology of muscle fiber differentiation was analysed in 37 fetal calves of 69 to 266 days of age. Semitendinosus muscle weight was measured throughout the experimental period and biochemical, histological and histochemical investigations were made to determine respectively the protein and DNA content of the muscle, the size and the number of the fibers and their ATPase and SDH activity. The relative growth of all the quantitative characteristics (muscle weight, protein and DNA content) was much greater in the early stages of gestation than in the new-born animal. In the younger fetuses DNA relative growth was faster than protein relative growth, whereas at the end of gestation the reverse progression was observed. Before 90 days, the muscle tissue was composed of myotube-like cells without any clear organization. The organization of muscle tissue into clear bundles occurred around 120 days of age, and about 30 days later the large myotubes transformed into myofibers. The myotubes reacted positively for acid-ATPase activity, whereas the large population of smaller cells which developed in parallel did not. The number of muscle cells increased up to 240 days of age, as did the percentage of fibers positive for acid-ATPase activity. Finally, oxidative differentiation occurred around 260 days of age, with the appearance of a population of cells characterized by increased SDH activity. A comparison of these results with previous findings suggests that the muscular tissue differentiates through similar stages in various species, but over different lengths of time. The percentage of mature weight might provide a better inter-species time scale than chronological age.
Adenosine Triphosphatases↗
Gene structure of mouse cathepsin B.
The structure of a genomic DNA fragment encoding mouse cathepsin B was characterized. The genomic insert spans 15 kbp and contains 9 exons encoding the 339 amino acid residues of mouse preprocathepsin B. Intron break-points are not found at the junctions of the pre-peptide, pro-peptide and mature enzyme. Like other cysteine proteinase genes, the region around the cysteinyl active site is split by an intron, but in contrast with cathepsins L and H the intron break-point is located immediately after the active site.
Amino Acid Sequence↗
Purification and amino acid sequence of chicken liver cathepsin L.
Cathepsin L was purified from chicken liver lysosomes by a two-step procedure. Cathepsin L exhibited a single band of Mr 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, presented a high affinity for the substrate Z-Phe-Arg-NMec, was very unstable at neutral pH, and was inhibited by Z-Phe-Phe-CHN2. The complete amino acid sequence of cathepsin L has been determined and consists of 215 residues. The sequence was deduced from analysis of peptides generated by enzymatic digestions and by chemical cleavage at methionyl bonds. Comparison of the amino acid sequence of cathepsin L with those of rat liver cathepsins B and H and papain demonstrates a striking homology among their primary structures.
Amino Acid Sequence↗
Species variations amongst proteinases in liver lysosomes.
Cathepsin B, H, L and D activities in liver lysosomes were compared between species. Although cathepsin B and D were detected in bovine, pig, chicken and rat liver, striking species differences were evident for cathepsin H and L. Cathepsin L activity was particularly high in chicken lysosomal extracts, but could not be detected in bovine and pig extracts. Whereas there was no significant cathepsin H activity in bovine extracts, rat liver lysosomal extracts contained large amounts of cathepsin H activity.
Animals↗
Control of gene expression by steroid hormones.
The mechanism of action of steroid hormones involves their interaction with tissue-specific binding sites, and results in a precise modulation of gene expression. Both high-affinity receptors and secondary binding sites exist for steroid hormones in target tissues. Only steroid-receptor complexes were, in several cases, clearly shown to directly regulate transcription by interacting with DNA region(s) close to steroid-controlled genes. However other indications suggest that steroid hormones could also modulate transcription by altering chromatin conformation. These modifications encompass post-traductional modifications of histones and non-histone proteins, as well as changes in the pattern of histone variants. Beside transcription, there are also evidences that steroid hormones can modulate gene expression by regulating some RNA processing events. Whether high-affinity receptors or secondary binding sites directly regulate these events is not known. These observations however suggest that several levels of control might exist for steroid hormones to precisely regulate gene expression.
Animals↗