ABSTRACT
Glucocorticoids, such as dexamethasone, enhance protein breakdown via ubiquitin-proteasome system. However, the role of autophagy in organelle and protein turnover in the glucocorticoid-dependent atrophy program remains unknown. Here, we show that dexamethasone stimulates an early activation of autophagy in L6 myotubes depending on protein kinase, AMPK, and glucocorticoid receptor activity. Dexamethasone increases expression of several autophagy genes, including ATG5, LC3, BECN1, and SQSTM1 and triggers AMPK-dependent mitochondrial fragmentation associated with increased DNM1L protein levels. This process is required for mitophagy induced by dexamethasone. Inhibition of mitochondrial fragmentation by Mdivi-1 results in disrupted dexamethasone-induced autophagy/mitophagy. Furthermore, Mdivi-1 increases the expression of genes associated with the atrophy program, suggesting that mitophagy may serve as part of the quality control process in dexamethasone-treated L6 myotubes. Collectively, these data suggest a novel role for dexamethasone-induced autophagy/mitophagy in the regulation of the muscle atrophy program.
Subject(s)
Autophagy/drug effects , Dexamethasone/toxicity , Glucocorticoids/toxicity , Mitochondria, Muscle/drug effects , Muscle Fibers, Skeletal/drug effects , Muscular Atrophy/chemically induced , AMP-Activated Protein Kinases/metabolism , Animals , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Autophagy-Related Protein 5 , Beclin-1 , Cell Line , Dose-Response Relationship, Drug , Dynamins/genetics , Dynamins/metabolism , Heat-Shock Proteins/deficiency , Heat-Shock Proteins/metabolism , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Mitochondria, Muscle/metabolism , Mitochondria, Muscle/pathology , Mitophagy/drug effects , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/pathology , Muscular Atrophy/genetics , Muscular Atrophy/metabolism , Muscular Atrophy/pathology , Proteins/genetics , Proteins/metabolism , Quinazolinones/pharmacology , RNA Interference , Rats , Receptors, Glucocorticoid/agonists , Receptors, Glucocorticoid/metabolism , Sequestosome-1 Protein , Signal Transduction/drug effects , Time Factors , TransfectionABSTRACT
Maize heat shock protein HSP101 accumulates during embryo maturation and desiccation and persists at high levels during the first 24 h following kernel imbibition in the absence of heat stress. This protein has a known function in disaggregation of high molecular weight complexes and has been proposed to be a translational regulator of specific mRNAs. Here, a global proteomic approach was used to identify changes in the maize proteome due to the absence of HSP101 in embryos from mature-dry or 24 h-imbibed kernels. A total of 26 protein spots from the mature dry embryo exhibited statistically significant expression changes in the L10 inbred hsp101 mutant (hsp101-m5::Mu1/hsp101-m5::Mu1) line as compared to the corresponding wild type (Hsp101/Hsp101). Additional six spots reproducibly showed qualitative changes between the mutant and wild-type mature and germinating embryos. Several chaperones, translation-related proteins, actin, and enzymes participating in cytokinin metabolism were identified in these spots by tandem mass-spectrometry (MS). The proteomic changes partially explain the altered root growth and architecture observed in young hsp101 mutant seedlings. In addition, specific protein de novo synthesis was altered in the 24 h-imbibed mutant embryos indicating that maize HSP101 functions as both chaperone and translational regulator during germination. Supporting this, HSP101 was found as part of Cap-binding and translation initiation complexes during early kernel imbibition. Overall, these findings expose the relevance of maize HSP101 for protein synthesis and balance mechanisms during germination.