calpain and Starvation

Apoptosis and autophagy play crucial roles during Bombyx mori metamorphosis and in response to various adverse conditions, including starvation. Recently, calpain, one of the major intracellular proteases, has been reported to be involved in apoptosis and autophagy in mammals. BmATG5 and BmATG6 have been identified to mediate apoptosis following autophagy induced by 20-hydroxyecdysone and starvation in B. mori. However, B. mori calpains and their functions remain unclear. In this study, phylogenetic analysis of calpains from B. mori, Drosophila melanogaster and Homo sapiens were performed and the results showed distinct close relationships of BmCalpain-A/B with DmCalpain-A/B, BmCalpain-C with DmCalpain-C, and BmCalpain-7 with HsCalpain-7. Then, the expression profiles of BmCalpains were analyzed by quantitative real-time polymerase chain reaction, and results showed that expression of BmCalpain-A/B, BmCalpain-C and BmCalpain-7 was significantly increased during B. mori metamorphosis and induced in the fat body and midgut of starved larvae, which is consistent with the expression profiles of BmAtg5, BmAtg6 and BmCaspase-1. Moreover, the apoptosis-associated cleavage of BmATG6 in Bm-12 cells was significantly enhanced when BmCalpain-A/B and BmCalpain-7 were induced by starvation, and was partially inhibited by the inhibitor of either calpain or caspase, but completely inhibited when both types of inhibitors were applied together. Our results indicated that BmCalpains, including BmCalpain-A/B, -C and -7, may be involved in autophagy and apoptosis during B. mori metamorphosis and after starvation, and may also contribute to the apoptosis-associated cleavage of BmATG6.

Topics: Animals; Apoptosis; Autophagy; Bombyx; Calpain; Caspase Inhibitors; Cell Line; Fat Body; Insect Proteins; Metamorphosis, Biological; Phylogeny; Starvation

Age-related macular degeneration (AMD) is a complex disease with multiple initiators and pathways that converge on death for retinal pigment epithelial (RPE) cells. In this study, effects of taurine on calpains, autophagy, endoplasmic reticulum (ER) stress, and apoptosis in ARPE-19 cells (a human RPE cell line) were investigated. We first confirmed that autophagy, ER stress and apoptosis in ARPE-19 cells were induced by Earle's balanced salt solution (EBSS) through starvation to induce RPE metabolic stress. Secondly, inhibition of ER stress by 4-phenyl butyric acid (4-PBA) alleviated autophagy and apoptosis, and suppression of autophagy by 3-methyl adenine (3-MA) reduced the cell apoptosis, but the ER stress was minimally affected. Thirdly, the apoptosis, ER stress and autophagy were inhibited by gene silencing of calpain-2 and overexpression of calpain-1, respectively. Finally, taurine suppressed both the changes of the important upstream regulators (calpain-1 and calpain-2) and the activation of ER stress, autophagy and apoptosis, and taurine had protective effects on the survival of ARPE-19 cells. Collectively, this data indicate that taurine inhibits starvation-triggered endoplasmic reticulum stress, autophagy, and apoptosis in ARPE-19 cells by modulating the expression of calpain-1 and calpain-2.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Calpain; Cell Line; Endoplasmic Reticulum Stress; Epithelial Cells; Gene Expression Regulation; Humans; Retinal Pigment Epithelium; Starvation; Taurine

Calpains, a superfamily of intracellular calcium-dependent cysteine proteases, are involved in the cytoskeletal remodeling and wasting of skeletal muscle. Calpains are generated as inactive proenzymes which are activated by N-terminal autolysis induced by calcium-ions.. In this study, we characterized the full-length cDNA sequences of three calpain genes, clpn1, clpn2, and clpn3 in channel catfish, and assessed the effect of nutrient restriction and subsequent re-feeding on the expression of these genes in skeletal muscle. The clpn1 cDNA sequence encodes a protein of 704 amino acids, Clpn2 of 696 amino acids, and Clpn3 of 741 amino acids. Phylogenetic analysis of deduced amino acid sequences indicate that catfish Clpn1 and Clpn2 share a sequence similarity of 61%; catfish Clpn1 and Clpn3 of 48%, and Clpn2 and Clpn3 of only 45%. The domain structure architectures of all three calpain genes in channel catfish are similar to those of other vertebrates, further supported by strong bootstrap values during phylogenetic analyses. Starvation of channel catfish (average weight, 15-20 g) for 35 days influenced the expression of clpn1 (2.3-fold decrease, P<0.05), clpn2 (1.3-fold increase, P<0.05), and clpn3 (13.0-fold decrease, P<0.05), whereas the subsequent refeeding did not change the expression of these genes as measured by quantitative real-time PCR analysis. Calpain catalytic activity in channel catfish skeletal muscle showed significant differences only during the starvation period, with a 1.2- and 1.4- fold increase (P<0.01) after 17 and 35 days of starvation, respectively.. We have assessed that fasting and refeeding may provide a suitable experimental model to provide us insight into the role of calpains during fish muscle atrophy and how they respond to changes in nutrient supply.

Topics: Analysis of Variance; Animal Nutritional Physiological Phenomena; Animals; Base Sequence; Body Weight; Calpain; Cluster Analysis; DNA Primers; DNA, Complementary; Gene Expression Regulation; Ictaluridae; Molecular Sequence Data; Multigene Family; Muscle, Skeletal; Phylogeny; Real-Time Polymerase Chain Reaction; Sequence Analysis, DNA; Sequence Homology; Spectrophotometry; Starvation

Here, we show that FoxO3A transcription factor is upregulated upon calpain small-1 (CAPNS1) depletion both in mouse embryonic fibroblasts (MEFs) and in the human mammary carcinoma cell line MCF-7. On starvation, CAPNS1 depletion is associated with a higher rate of FoxO3A dephosphorylation and translocation to the nucleus and to a sharper increase in the levels of p27Kip1 and Bim, the products of two FoxO target genes. Notably, FoxO3A depletion in CAPNS1-/- MEFs reduces both the induction of Bim and apoptosis. Both okadaic acid treatment and silencing of the protein phosphatase 2A (PP2A) catalytic subunit can partially reduce starvation-induced FoxO3A activation and apoptosis in CAPNS1-/- fibroblasts. PP2A associates more tightly with Akt in CAPNS1 knockout cells, indicating that PP2A is involved in calpain-mediated FoxO regulation. Finally, we show that PP2A regulatory subunits B56 alpha and gamma are in vitro substrates of calpain, and calpain regulates B56 alpha stability in vivo, suggesting a direct role of calpain in the regulation of PP2A function. In conclusion, for the first time we report that CAPNS1 interferes with PP2A-Akt interaction consequently affecting FoxO3A-dependent cell death. Calpain inhibition might therefore be exploited as a tool to induce apoptosis in tumors sensitive to FoxO activation.

Topics: Animals; Apoptosis; Calpain; Cell Nucleus; Cells, Cultured; Forkhead Box Protein O3; Forkhead Transcription Factors; Gene Knockdown Techniques; Humans; Mice; Oncogene Protein v-akt; Phosphorylation; Protein Phosphatase 2; Protein Transport; Signal Transduction; Starvation

Fast, efficiently growing animals have increased protein synthesis and/or reduced protein degradation relative to slow, inefficiently growing animals. Consequently, minimizing the energetic cost of protein turnover is a strategic goal for enhancing animal growth. Characterization of gene expression profiles associated with protein turnover would allow us to identify genes that could potentially be used as molecular biomarkers to select for germplasm with improved protein accretion.. We evaluated changes in hepatic global gene expression in response to 3-week starvation in rainbow trout (Oncorhynchus mykiss). Microarray analysis revealed a coordinated, down-regulated expression of protein biosynthesis genes in starved fish. In addition, the expression of genes involved in lipid metabolism/transport, aerobic respiration, blood functions and immune response were decreased in response to starvation. However, the microarray approach did not show a significant increase of gene expression in protein catabolic pathways. Further studies, using real-time PCR and enzyme activity assays, were performed to investigate the expression of genes involved in the major proteolytic pathways including calpains, the multi-catalytic proteasome and cathepsins. Starvation reduced mRNA expression of the calpain inhibitor, calpastatin long isoform (CAST-L), with a subsequent increase in the calpain catalytic activity. In addition, starvation caused a slight but significant increase in 20S proteasome activity without affecting mRNA levels of the proteasome genes. Neither the mRNA levels nor the activities of cathepsin D and L were affected by starvation.. These results suggest a significant role of calpain and 20S proteasome pathways in protein mobilization as a source of energy during fasting and a potential association of the CAST-L gene with fish protein accretion.

Topics: Animals; Calcium-Binding Proteins; Calpain; Computer Systems; Energy Metabolism; Fish Diseases; Gene Expression Profiling; Gene Expression Regulation; Oligonucleotide Array Sequence Analysis; Oncorhynchus mykiss; Peptide Hydrolases; Polymerase Chain Reaction; Proteasome Endopeptidase Complex; Protein Biosynthesis; Proteins; Starvation

While the apoptotic and necrotic cell death pathways have been well studied, there lacks a comprehensive understanding of the molecular events involving autophagic cell death. We examined the potential roles of the apoptosis-linked caspase-3 and the necrosis/apoptosis-linked calpain-1 after autophagy induction under prolonged amino acid (AA) starvation conditions in PC-12 cells. Autophagy induction was observed as early as three hours following amino acid withdrawal. Cell death, measured by lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays occurred within 24 h following starvation and was accompanied by an upregulation in caspase-3 activity but not calpain-1. The cell death that occurred following AA starvation was significantly alleviated by treatment with the autophagy inhibitor 3-methyl adenine but not with the broad spectrum caspase inhibitors. Thus, this study demonstrates that 3-methyladenine-sensitive autophagic cell death due to AA starvation in PC-12 cells is mechanistically and biochemically similar to, yet distinct from, classic caspase dependent apoptosis.

Topics: Amino Acids; Animals; Autophagy; Calpain; Caspase 3; Caspase Inhibitors; Cathepsins; Cell Nucleus; L-Lactate Dehydrogenase; Rats; Spectrin; Starvation; Time; Tumor Cells, Cultured

Calpains are calcium regulated proteases involved in cellular functions that include muscle proteolysis both ante- and postmortem. Here, we describe the molecular characterization of the rainbow trout catalytic subunits of the mu- and m-calpains, respectively. The cDNA sequence for Capn1 encodes a protein of 704 amino acids with a calculated molecular mass of 79.9 kDa. The amino acid sequence shows 66% and 86% identity with the mouse and zebrafish Capn1, respectively. The Capn2 cDNA codes for a protein consisting of 701 amino acid residues with a calculated molecular mass of 78.2 kDa. The protein shows 65% amino acid sequence identity with the mouse and chicken Capn2. The two isozymes of rainbow trout have the characteristic domains: I (propeptide), II (cysteine catalytic site), III (electrostatic switch), and IV (contains five EF-hands). Because starvation induces muscle wasting, the hypothesis of this study was that starvation could affect regulation of the calpain system in muscle. Starvation of rainbow trout fingerlings (15-20 g) for 35 days stimulated the expression of Capn1 (2.2-fold increase, P < 0.01), Capn2 (6.0-fold increase, P < 0.01), and calpastatins (1.6-fold increase, P < 0.05) as measured by quantitative real-time RT-PCR. The mRNA changes led to a 1.23-fold increase in the calpain catalytic activity. The results suggest a potential role of calpains in protein mobilization as a source of energy under fasting condition.

Topics: Amino Acid Sequence; Animals; Calpain; Cloning, Molecular; DNA, Complementary; Fish Proteins; Molecular Sequence Data; Muscular Atrophy; Oncorhynchus mykiss; RNA, Messenger; Sequence Homology, Amino Acid; Starvation; Tissue Distribution

The calpain system is a family of calcium activated proteases that degrade myofibrillar protein. Male broiler chickens (Ross) were provided a standard starter diet top-dressed with Oasis((R)) nutritional supplement (fed; Novus International, St. Louis, MO, USA), or they were not provided any feed (starved) for the first 3 days posthatch. Subsequently, the standard starter diet was provided to all chickens between 3 and 7 days posthatch. RNA was extracted from the Pectoralis thoracicus, and skeletal muscle-specific n-calpain-1 (p94) calpain, mu-calpain, and m-calpain expression was evaluated using quantitative Northern analysis. Early posthatch starvation did not (P>0.05) affect calpain mRNA levels on each day examined. Similarly, there were no (P>0.05) changes in mu-calpain or m-calpain mRNA levels between 0 and 7 days posthatch in fed birds. However, p94 calpain mRNA levels were significantly (P<0.05) lower at 7 days posthatch compared to 0 or 2 days posthatch. Therefore, in the early posthatch chicken, it appears that the calpain system may not be affected by the presence of oral nutrition, and that there is an age-related downregulation of p94 calpain mRNA expression.

Topics: Age Factors; Animals; Animals, Newborn; Blotting, Northern; Calpain; Chickens; Down-Regulation; Food Deprivation; Male; Muscle, Skeletal; RNA, Messenger; Starvation

Changes in the activity of proteases (cathepsin D and calpains) caused by 48-h food withdrawal were studied in the brain, liver, kidney, spleen, and heart of 3-, 12-, and 24-month-old Fischer rats. Cathepsin D activity was similar in brain, liver, and heart of control animals; in kidney it was 5-fold higher and in spleen about 10-fold higher. With age, activity increased in all organs tested except spleen. Brief starvation caused no change of cathepsin D activity in brain, but caused an increase in liver and a decrease in spleen. Neutral proteolytic activity in control was highest in the pons-medulla-cerebellum fraction of brain, and activity in liver and heart was below that in brain. Activity increased with age in brain and decreased in other organs. Brief starvation in young animals caused an increase in activity in brain, and a decrease in liver and spleen. Isolated calpain II activity was high in control brain. It increased with age in the cerebrum. Brief starvation resulted in a decrease in the brain. The results indicate that the protease content of the brain is altered with age and in malnutrition, with changes not being the same for all proteases, and changes in brain being different from those in other organs.

Topics: Aging; Animals; Brain; Calpain; Cathepsin D; Endopeptidases; Organ Specificity; Proteins; Rats; Rats, Inbred F344; Starvation; Time Factors; Tissue Distribution