sirolimus and Protein-Energy-Malnutrition

Peroxisomes are metabolic organelles necessary for anabolic and catabolic lipid reactions whose numbers are highly dynamic based on the metabolic need of the cells. One mechanism to regulate peroxisome numbers is through an autophagic process called pexophagy. In mammalian cells, ubiquitination of peroxisomal membrane proteins signals pexophagy; however, the E3 ligase responsible for mediating ubiquitination is not known. Here, we report that the peroxisomal E3 ubiquitin ligase peroxin 2 (PEX2) is the causative agent for mammalian pexophagy. Expression of PEX2 leads to gross ubiquitination of peroxisomes and degradation of peroxisomes in an NBR1-dependent autophagic process. We identify PEX5 and PMP70 as substrates of PEX2 that are ubiquitinated during amino acid starvation. We also find that PEX2 expression is up-regulated during both amino acid starvation and rapamycin treatment, suggesting that the mTORC1 pathway regulates pexophagy by regulating PEX2 expression levels. Finally, we validate our findings in vivo using an animal model.

Topics: Amino Acids; Animals; ATP-Binding Cassette Transporters; Autophagy; Disease Models, Animal; HEK293 Cells; HeLa Cells; Humans; Intracellular Signaling Peptides and Proteins; Male; Mechanistic Target of Rapamycin Complex 1; Membrane Proteins; Mice, Inbred C57BL; Multiprotein Complexes; Peroxisomal Biogenesis Factor 2; Peroxisome-Targeting Signal 1 Receptor; Peroxisomes; Protein-Energy Malnutrition; Proteins; Proteolysis; Rats; Receptors, Cytoplasmic and Nuclear; RNA Interference; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Transfection; Ubiquitination

Protein malnutrition occurs when there is insufficient protein to meet metabolic demands. Previous works have indicated that cycles of protein fasting/refeeding enhance the incidence of early lesions during chemical carcinogenesis in rat liver. The general objective of this work was to study the effect of aminoacids (Aa) deprivation on the proliferation and survival of hepatocytes, to understand its possible involvement in the generation of pre-neoplastic stages in the liver. Lack of Aa in the culture medium of an immortalized mice hepatocyte cell line induced loss in cell viability, correlating with apoptosis. However, a subpopulation of cells was able to survive, which showed a more proliferative phenotype and resistance to apoptotic stimuli. Escaping to Aa deprivation-induced death is coincident with an activated mTOR signaling and higher levels of phospho-AKT and phospho-ERKs, which correlated with increased activation of EGFR/SRC pathway and overexpression of EGFR ligands, such as TGF-α and HB-EGF. Lack of Aa induced a rapid increase in reactive oxygen species (ROS) production. However, cells that survived showed an enhancement in the levels of reduced glutathione and a higher expression of γ-GCS, the regulatory enzyme of glutathione synthesis, which can be interpreted as an adaptation of the cells to counteract the oxidative stress. In conclusion, results presented in this paper indicate that it is possible to isolate a subpopulation of hepatocytes that are able to grow in the absence of Aa, showing higher capacity to proliferate and survive, reminiscent of a preneoplastic phenotype.

Topics: Amino Acids; Animals; Caspase 3; Cell Line, Transformed; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Culture Media; Enzyme Activation; Enzyme Assays; Hepatocytes; Liver; Mice; Oxidation-Reduction; Oxidative Stress; Phosphoproteins; Phosphorylation; Precancerous Conditions; Protein-Energy Malnutrition; Signal Transduction; Sirolimus