Sylvie Mordier

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.

Recent Advances in the Understanding of Amino Acid Regulation of Gene Expression

In mammals, the impact of nutrients on gene expression has become an important area of research. Because amino acids have multiple and important functions, their homeostasis has to be finely maintained. However, amino acidemia can be affected by certain nutritional conditions or various forms of stress. Consequently, mammals must adjust several of the physiological functions involved in the adaptation to amino acid availability by regulating expression of numerous genes. It has been shown that amino acids alone can modify the expression of target genes. However, understanding of amino acid-dependent control of gene expression has just started to emerge. This review focuses on recent advances in the understanding of mechanisms involved in the amino acid control of gene expression.

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.

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.

Short sequence-paperNucleotide 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.

Phospho-proteomic approach to identify new targets of leucine deprivation in muscle cells

The aim of this study was to optimize a protocol that allows identifying changes at the phosphorylation level of specific proteins in response to cell stimulation by leucine starvation. To make possible the identification of differentially phosphorylated proteins by the combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), we prepared fraction enriched in phosphoproteins. For that purpose, we adapted the immobilized metal affinity chromatography (IMAC) technique to make it compatible with 2D-PAGE. On the whole, this procedure allowed identifying regulated targets of leucine deprivation: molecular chaperones glucose-regulated protein 58 kDa (GRP58) and BiP (GRP78), RNA helicase DEAD box polypeptide 3, and eukaryotic translation initiation factor 4B (eIF4B).

Amino-acid limitation induces the GCN2 signaling pathway in myoblasts but not in myotubes

There is a growing body of evidence that suggests that amino acids play an important role in controlling gene expression, but the cell specificity of the amino-acid-mediated regulation of gene expression in mammals remains unknown. Using a model of muscle cells (C2C12) at two stages of differentiation, i.e. myoblasts and myotubes, we employed transcriptional profiling to show that amino-acid deficiency does not regulate the same set of gene in differentiated and non-differentiated cells. Furthermore, in myotubes, the GCN2 pathway is not activated by amino-acid starvation due to an amino-acid supply from intracellular proteolysis associated with a low GCN2 expression.