leupeptins and 3-methyladenine

The protective mechanisms of blood-brain barrier (BBB) prohibiting entry of pathogens into central nervous system (CNS) are critical for maintenance of brain homeostasis. These include various intracellular defense mechanisms that are vital to block transcytosis of neurotropic pathogens into the CNS. However, mechanistic details of coordination between these defense pathways remain unexplored. In this study, we established that BBB-driven ubiquitination acts as a major intracellular defense mechanism for clearance of Streptococcus pneumoniae, a critical neurotropic pathogen, during transit through BBB. Our findings suggest that the BBB employs differential ubiquitination with either K48- or K63-ubiquitin (Ub) chain topologies as an effective strategy to target S. pneumoniae toward diverse killing pathways. While K63-Ub decoration triggers autophagic killing, K48-Ub directs S. pneumoniae exclusively toward proteasomes. Time-lapse fluorescence imaging involving proteasomal marker LMP2 revealed that in the BBB, the majority of the ubiquitinated S. pneumoniae was cleared by proteasome. Fittingly, inhibition of proteasome and autophagy pathway led to accumulation of K48-Ub- and K63-Ub-marked S. pneumoniae, respectively, and triggered significant increases in intracellular S. pneumoniae burden. Moreover, genetic impairment of either K48- or K63-Ub chain formation demonstrated that although both chain types are key in disposal of intracellular S. pneumoniae, K48-Ub chains and subsequent proteasomal degradation have more pronounced contributions to intracellular S. pneumoniae killing in the BBB. Collectively, these observations, for the first time, illustrated a pivotal role of differential ubiquitination deployed by BBB in orchestrating a symphony of intracellular defense mechanisms for interception and degradation of S. pneumoniae, blocking its entry into the brain, which could be exploited to prevent bacterial CNS infections.

Topics: Adenine; Anti-Bacterial Agents; Autophagy; Biomarkers; Blood-Brain Barrier; Cell Line; Cell Survival; Endothelial Cells; Gene Expression Regulation, Bacterial; Gentamicins; Humans; Leupeptins; Optical Imaging; Penicillins; Proteasome Endopeptidase Complex; Streptococcus pneumoniae; Ubiquitination; Ubiquitins

N-myc downstream regulated gene 1 (NDRG1) is an intriguing metastasis suppressor protein, which plays an important role in suppressing multiple oncogenic signaling pathways. Interestingly, multiple isoforms of NDRG1 have been identified, although the molecular mechanisms involved in their generation remains elusive. Herein, we demonstrate the role of two mechanisms involving autophagic and proteasomal machinery as part of an intricate system to generate different NDRG1 isoforms. Examining multiple pancreatic cancer cell-types using immunoblotting demonstrated three major isoforms of NDRG1 at approximately 41-, 46- and 47-kDa. The top NDRG1 band at 47-kDa was shown to be processed by the proteasome, followed by autophagic metabolism of the middle NDRG1 band at 46-kDa. The role of the proteasomal and autophagic pathways in NDRG1 processing was further confirmed by co-localization analysis of confocal images using PSMD9 and LC3 as classical markers of these respective pathways. All NDRG1 isoforms were demonstrated to be, at least in part, phosphorylated forms of the protein. Inhibition of two well-characterized upstream kinases of NDRG1, namely GSK3β and SGK1, resulted in decreased levels of the top NDRG1 band. Studies demonstrated that inhibition of GSK3β decreased levels of the top 47-kDa NDRG1 band, independent of its kinase activity, and this effect was not mediated via the proteasomal pathway. In contrast, the decrease in the top NDRG1 band at 47-kDa after SGK1 inhibition, was due to suppression of its kinase activity. Overall, these studies elucidated the complex and intricate regulatory pathways involving both proteasomal and autophagic processing of the metastasis suppressor protein, NDRG1.

Topics: Adenine; Antineoplastic Agents; Autophagy; Benzoates; Bridged Bicyclo Compounds, Heterocyclic; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Epithelial Cells; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; Humans; Immediate-Early Proteins; Intracellular Signaling Peptides and Proteins; Iron Chelating Agents; Leupeptins; Macrolides; Microtubule-Associated Proteins; Pancreatic Ducts; Phosphorylation; Proteasome Endopeptidase Complex; Protein Isoforms; Protein Serine-Threonine Kinases; Proteolysis; Signal Transduction; Thiosemicarbazones

The mammalian target of rapamycin (mTOR) is a key regulator of cell growth, autophagy, translation, and survival. Dysregulation of mTOR signaling is associated with cancer, diabetes, and autism. However, a role for mTOR signaling in neuronal death is not well delineated. Here we show that global ischemia triggers a transient increase in mTOR phosphorylation at S2448, whereas decreasing p-mTOR and functional activity in selectively vulnerable hippocampal CA1 neurons. The decrease in mTOR coincides with an increase in biochemical markers of autophagy, pS317-ULK-1, pS14-Beclin-1, and LC3-II, a decrease in the cargo adaptor p62, and an increase in autophagic flux, a functional readout of autophagy. This is significant in that autophagy, a catabolic process downstream of mTORC1, promotes the formation of autophagosomes that capture and target cytoplasmic components to lysosomes. Inhibitors of the lysosomal (but not proteasomal) pathway rescued the ischemia-induced decrease in mTOR, consistent with degradation of mTOR via the autophagy/lysosomal pathway. Administration of the mTORC1 inhibitor rapamycin or acute knockdown of mTOR promotes autophagy and attenuates ischemia-induced neuronal death, indicating an inverse causal relation between mTOR, autophagy, and neuronal death. Our findings identify a novel and previously unappreciated mechanism by which mTOR self-regulates its own levels in hippocampal neurons in a clinically relevant model of ischemic stroke.

Topics: Acetylcysteine; Adenine; AMP-Activated Protein Kinases; Animals; Autophagy; Autophagy-Related Protein-1 Homolog; Beclin-1; Cells, Cultured; Hippocampus; Ischemia; Leupeptins; Lysosomes; Male; Microtubule-Associated Proteins; Neurons; Phosphorylation; Rats; RNA Interference; Sirolimus; TOR Serine-Threonine Kinases

Tau, a microtubule-binding phosphoprotein, plays a critical role in the stabilisation of microtubules and neuronal function. However, hyperphosphorylated tau is involved in the pathogenesis of Alzheimer's disease (AD) and other tauopathies. The facilitation of tau clearance is now regarded as a valid therapeutic strategy for these neurodegenerative tauopathies. Here, we provide the first demonstration that the over-expression of neuronal PAS domain protein 4 (NPAS4)-induced autophagy and effectively facilitated the clearance of endogenous total and phosphorylated tau in rat primary cortical neurons. Moreover, the activation of autophagy by serum depletion significantly decreased endogenous total and phosphorylated tau levels. Autophagy inhibitors, such as 3-methyladenine (3-MA) and chloroquine (CQ), induced tau aggregation. However, NPAS4 over-expression reversed the aggregation of tau that was induced by the inhibition of autophagy. Interestingly, proteasome inhibition by MG132, had no effect on autophagy, but did reduce tau levels, indicating that NPAS4 may also degrade tau proteins through an unknown proteasome-mediated mechanism. Furthermore, NPAS4 did not alter the activity of two major tau kinases, glycogen synthase kinase 3β (GSK3β) and cyclin-dependent kinase 5 (CDK5). Taken together, the results indicate that targeting NPAS4 could provide a therapeutic approach for the treatment of AD and other tauopathies.

Topics: Adenine; Animals; Autophagy; Basic Helix-Loop-Helix Transcription Factors; Cells, Cultured; Cerebral Cortex; Chloroquine; Cyclin-Dependent Kinase 5; Glycogen Synthase Kinase 3; Leupeptins; Neurons; Proteolysis; Rats; Rats, Sprague-Dawley; tau Proteins

The aim of the present study was to investigate whether endoplasmic reticulum (ER) stress is involved in MG‑132‑induced autophagy, and to determine the effects of the inhibition of autophagy and ER stress on cell viability following MG‑132 treatment. The proteasome inhibitor, MG‑132, was used to induce autophagy in MCF‑7 cells, and 3‑methyladenine (3‑MA) and salubrinal were used to inhibit autophagy and ER stress, respectively. An MTT assay was used to analyze cell viability. Apoptosis and the cell cycle were analyzed using flow cytometry. The expression levels of apoptosis‑ and ER stress‑associated genes were investigated using western blot and reverse transcription‑quantitative polymerase chain reaction analyses. MG‑132 inhibited cell proliferation, and induced apoptosis and cell cycle arrest at the G2 phase of the cell cycle. Notably, MG‑132 increased the autophagy‑associated conversion of microtubule‑associated protein 1 light chain 3 (LC3)‑I to LC3‑II, which was partially attenuated by the ER stress inhibitor, salubrinal. In addition, MG‑132 inhibited the protein expression of the anti‑apoptotic protein, B‑cell lymphoma (Bcl)‑2, whereas the expression levels of Bcl‑2‑associated X protein and caspase‑3 were upregulated. These effects were enhanced by co‑treatment with either 3‑MA or salubrinal. Furthermore, the mRNA and protein levels of the ER stress‑associated genes, glucose‑regulated protein 78, growth arrest and DNA damage induced gene‑153, and caspase‑12, were upregulated by MG132, and these levels were significantly inhibited by co‑treatment of the cells with salubrinal. Taken together, the results of the present study indicated that the induction of autophagy by the proteasome inhibitor was associated with ER stress in the MCF‑7 cells, and that the inhibition of autophagy or ER stress enhanced MG‑132‑induced apoptosis. These findings suggest the potential application of inhibitors of ER stress and autophagy, in combination with proteasomal inhibitors, for the development of combinatorial targeted cancer therapy.

Topics: Adenine; Apoptosis; Autophagy; Cinnamates; Drug Synergism; Endoplasmic Reticulum Stress; Humans; Leupeptins; MCF-7 Cells; Proteasome Inhibitors; Thiourea

The activation of autophagy has been demonstrated to exert protective roles during hypoxia-reoxygenation (H/R)-induced brain injuries. This study aimed to investigate whether and how preconditioning with a proteasome inhibitor (MG-132), a proteasome promoter (Adriamycin, ADM), an autophagy inhibitor (3-methyladenine, 3-MA) and an autophagy promoter (Rapamycin, Rap) affected endoplasmic reticulum stress (ERS), the ubiquitin-proteasome system (UPS), autophagy, inflammation and apoptosis. Ubiquitin protein and 26S proteasome activity levels were decreased by MG-132 pretreatment but increased by ADM pretreatment at 2h, 4h and 6h following H/R treatment. MG-132 pretreatment led to the increased expression of autophagy-related genes, ER stress-associated genes and IκB but decreased the expression levels of NF-κB and caspase-3. ADM pretreatment led to the decreased expression of autophagy-related genes, ERS-associated genes and IκB but increased the expression of NF-κB and caspase-3. Pretreatment with 3-MA reduced the expression of autophagy-related genes, autophagy and UPS co-related genes, as well as apoptosis-related although the latter was increased by Rap pretreatment at 2h, 4h and 6h following H/R treatment. In vivo, pretreatment of rats with ADM, MG-132, 3-MA or Rap followed by ischemia-reperfusion (I/R) treatment resulted in similar changes. Proteasome inhibition preconditioning strengthened autophagy and ER stress but decreased apoptosis and inflammation. Autophagy promotion preconditioning exhibited similar changes. The combination of a proteasome inhibitor and an autophagy promoter might represent a new possible therapy to treat H/R or I/R injury-related diseases.

Topics: Adenine; Animals; Apoptosis; Autophagy; Cell Hypoxia; Cell Line; Cell Survival; Doxorubicin; Endoplasmic Reticulum Stress; Histone Deacetylase 6; Histone Deacetylases; Leupeptins; Lung; Male; NF-kappa B; Oxygen; Proteasome Endopeptidase Complex; Rats; Reperfusion Injury; Sirolimus; Ubiquitin

The aim of the present study was to examine the effects of proteasome inhibitor (PI)‑induced autophagy on PC12 cells overexpressing A53T mutant α‑synuclein (α‑syn) by detecting alterations in the levels of microtubule‑associated protein 1A/1B light chain (LC3)+ autophagosomes and the lysotracker‑positive autolysosomes using immunofluorescence, the expression of LC3‑II using western blot analysis and the morphology of PC12 cells using transmission electron microscopy. It was found that the addition of MG132 (500 nmol/l) significantly increased the number of autophagosomes and autolysosomes and upregulated the expression of LC3‑II. The autophagy inhibitor 3‑methyladenine (3‑MA) completely inhibited the autophagy induced by MG132 (500 nmol/l). The autophagy enhancer trehalose significantly increased the number of autophagosomes and autolysosomes and improved the protein level of LC3‑II induced by MG132. To examine the effect of PI‑induced autophagy on the degradation of A53T mutant α‑syn, the expression of α‑syn was detected by western blot analysis. It was revealed that MG132 increased the expression of A53T α‑syn and trehalose counteracted the increase of A53T α‑syn induced by MG132. Combined inhibition of 3‑MA and PI significantly increased the accumulation of A53T α‑syn as compared with treatment using either single agent. In addition, combination of MG132 (500 nmol/l) with trehalose (50 mmol/l) or 3‑MA (2 mmol/l) markedly decreased the cell viability as compared with treatment using either single agent individually as demonstrated using a 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay. These results suggest that the PI, MG132, could induce autophagy in PC12 cells overexpressing A53T mutant α‑syn and this autophagy could be completely inhibited by 3‑MA, indicating that PI‑induced autophagy is mediated by the upregulation of the macroautophagy class III PI3K pathway. PI‑induced autophagy may act as a compensatory degradation system for degradation of A53T α‑syn when the ubiquitin‑proteasome system is impaired. Autophagy activation may directly contribute to the survival of PC12 cells treated with proteasome inhibitors. The present study may assist in illuminating the association between PI and autophagy in the pathogenesis of Parkinson's disease.

Topics: Adenine; alpha-Synuclein; Animals; Autophagy; Cell Death; Gene Expression; Leupeptins; Mutation; PC12 Cells; Proteasome Inhibitors; Rats; TOR Serine-Threonine Kinases

Several pathologies are associated with elevated levels of serum ferritin, for which growth inhibitory properties have been reported; however, the underlying mechanisms are still poorly defined. Previously we have described cytotoxic properties of isoferritins released from primary hepatocytes in vitro, which induce apoptosis in an iron and oxidative stress-dependent mode. Here we show that this ferritin species stimulates endosome clustering and giant endosome formation in primary hepatocytes accompanied by enhanced lysosomal membrane permeability (LMP). In parallel, protein modification by lipid peroxidation-derived 4-hydroxynonenal (HNE) is strongly promoted by ferritin, the HNE-modified proteins (HNE-P) showing remarkable aggregation. Emphasizing the prooxidant context, GSH is rapidly depleted and the GSH/GSSG ratio is substantially declining in ferritin-treated cells. Furthermore, ferritin triggers a transient upregulation of macroautophagy which is abolished by iron chelation and apparently supports HNE-P clearance. Macroautophagy inhibition by 3-methyladenine strongly amplifies ferritin cytotoxicity in a time- and concentration-dependent mode, suggesting an important role of macroautophagy on cellular responses to ferritin endocytosis. Moreover, pointing at an involvement of lysosomal proteolysis, ferritin cytotoxicity and lysosome fragility are aggravated by the protease inhibitor leupeptin. In contrast, EGF which suppresses ferritin-induced cell death attenuates ferritin-mediated LMP. In conclusion, we propose that HNE-P accumulation, lysosome dysfunction, and macroautophagy stimulated by ferritin endocytosis provoke lysosomal "metastability" in primary hepatocytes which permits cell survival as long as in- and extrinsic determinants (e.g., antioxidant availability, damage repair, EGF signaling) keep the degree of lysosomal destabilization below cell death-inducing thresholds.

Topics: Adenine; Aldehydes; Animals; Apoptosis; Autophagy; Culture Media, Conditioned; Endocytosis; Epidermal Growth Factor; Female; Ferritins; Glutathione; Glutathione Disulfide; Hepatocytes; Intracellular Membranes; Iron Chelating Agents; Leupeptins; Liver; Lysosomes; Molecular Imaging; Permeability; Primary Cell Culture; Protease Inhibitors; Protein Aggregates; Rats; Rats, Inbred F344

High insulin/IGF is a biologic link between diabetes and cancers, but the underlying molecular mechanism remains unclear. Here we report a previously unrecognized tumour-promoting mechanism for stress protein TRB3, which mediates a reciprocal antagonism between autophagic and proteasomal degradation systems and connects insulin/IGF to malignant promotion. We find that several human cancers express higher TRB3 and phosphorylated insulin receptor substrate 1, which correlates negatively with patient's prognosis. TRB3 depletion protects against tumour-promoting actions of insulin/IGF and attenuates tumour initiation, growth and metastasis in mice. TRB3 interacts with autophagic receptor p62 and hinders p62 binding to LC3 and ubiquitinated substrates, which causes p62 deposition and suppresses autophagic/proteasomal degradation. Several tumour-promoting factors accumulate in cancer cells to support tumour metabolism, proliferation, invasion and metastasis. Interrupting TRB3/p62 interaction produces potent antitumour efficacies against tumour growth and metastasis. Our study opens possibility of targeting this interaction as a potential novel strategy against cancers with diabetes.

Topics: Adaptor Proteins, Signal Transducing; Adenine; Animals; Autophagy; Cell Cycle Proteins; Cell Movement; Gene Expression Regulation, Neoplastic; Humans; Insulin; Insulin Receptor Substrate Proteins; Insulin-Like Growth Factor I; Leupeptins; Male; Mice; Mice, Inbred C57BL; Mice, Nude; Neoplasm Metastasis; Neoplasms, Experimental; Proteasome Endopeptidase Complex; Protein Serine-Threonine Kinases; Repressor Proteins; Sequestosome-1 Protein; Tissue Array Analysis; Ubiquitin

Cells rely on complementary proteolytic pathways including the ubiquitin-proteasome system and autophagy to maintain proper protein degradation. There is known to be considerable interplay between them, whereby the loss of one clearance system results in compensatory changes in other proteolytic pathways of the cell. Disturbances in proteolysis are known to occur in Alzheimer's disease, and potentially contribute to neurophysiological and neurodegenerative processes. Currently, few data are available on how the presence of wild type and mutant amyloid precursor protein (APPwt and APPmut) potentially alters the reciprocal interplay between the different intracellular proteolytic pathways. This study used human SH-SY5Y neuronal cell lines, and SH-SY5Y transfected with either APPwt or APPmut (valine-to-glycine substitution at position 717), in order to explore if the presence of APPwt or APPmut altered the downstream effects of pharmacological proteasome or autophagy inhibition. The occurrence of APPwt or APPmut was observed to disturb proteasome or autophagy activities upon treatment with proteasome inhibitors or authophagy inhibitors. Interestingly, APPwt and APPmut expression was observed to significantly and robustly enhance the induction in cathepsin B following the administration of an established proteasome inhibitor. The presence of APPwt and APPmut also significantly reduced the elevation in ubiquitinated proteins following proteasome inhibitor treatments. Our data strongly suggest that APP is able to affect the downstream effects of protease inhibition in neural cells including enhancement of cathepsin B activity, with these changes in cathepsin B significantly and inversely related to the levels of ubiquitinated protein.

Topics: Adenine; Amino Acid Substitution; Amyloid beta-Protein Precursor; Autophagy; Blotting, Western; Cathepsin B; Cell Line, Tumor; Cell Survival; Cysteine Proteinase Inhibitors; Humans; Immunohistochemistry; Leupeptins; Mutant Proteins; Mutation; Proteasome Endopeptidase Complex; Proteolysis

We have shown that extracellular adenosine diphosphate (ADP) affects lipoprotein secretion from liver cells by stimulating cellular autophagic degradation. In this study, we investigated the effect of ADP and cellular autophagy on hepatic lipase (HL) release from human liver cells.. Depletion of media serum stimulates an autophagic response in liver cells, which parallels an 8-fold increase in the release of ADP into the media and a complete inhibition of HL release. Treatment of cells with exogenous ADP stimulates cellular autophagy and also blocks HL release. Treatment with the autophagic stimulant and proteasomal inhibitor, ALLN (25 µM), reduces cellular HL levels and blocks HL release at 4h. In contrast, treatment with the autophagy inhibitor, 3-methyladenine (3-MA) (5 mM), increases cellular HL levels and stimulates HL release. ADP acts through the G-protein coupled receptor, P2Y13, to stimulate autophagy. siRNA-targeted reduction in P2Y13 protein expression stimulates the release of HL by 5 to 8-fold, while overexpression of P2Y13 blocks HL release. HL release from liver cells is therefore inhibited by a purinergic induction of autophagy. To evaluate the effect of extracellular ADP on the processing of HL, we expressed a V5-epitope tag-labeled HL (HL-V5) and then measured secretion, uptake and degradation. Two isoforms of HL-V5, at 62 and 68 kDa, are released from HepG2 cells, but only the 62 kDa protein undergoes reuptake / internalization. The 62 kDa HL-V5 isoform progressively accumulates in the cell over 24h, with no detectible modification or degradation. Treatment of liver cells with ADP has no effect on HL-V5 internalization or degradation at 30 min and 4h.. These studies show that extracellular nucleotides act to prevent HL accumulation in the media by stimulating cellular autophagic degradation and blocking HL release.

Topics: Adenine; Adenosine Diphosphate; Autophagy; Endocytosis; Hep G2 Cells; Hepatocytes; Humans; Leupeptins; Lipase; Microtubule-Associated Proteins; Protein Isoforms; Receptors, Purinergic P2; RNA Interference; RNA, Small Interfering; Time Factors

Spinal and bulbar muscular atrophy (SBMA) is an X-linked motoneuron disease caused by an abnormal expansion of a tandem CAG repeat in exon 1 of the androgen receptor (AR) gene that results in an abnormally long polyglutamine tract (polyQ) in the AR protein. As a result, the mutant AR (ARpolyQ) misfolds, forming cytoplasmic and nuclear aggregates in the affected neurons. Neurotoxicity only appears to be associated with the formation of nuclear aggregates. Thus, improved ARpolyQ cytoplasmic clearance, which indirectly decreases ARpolyQ nuclear accumulation, has beneficial effects on affected motoneurons. In addition, increased ARpolyQ clearance contributes to maintenance of motoneuron proteostasis and viability, preventing the blockage of the proteasome and autophagy pathways that might play a role in the neuropathy in SBMA. The expression of heat shock protein B8 (HspB8), a member of the small heat shock protein family, is highly induced in surviving motoneurons of patients affected by motoneuron diseases, where it seems to participate in the stress response aimed at cell protection. We report here that HspB8 facilitates the autophagic removal of misfolded aggregating species of ARpolyQ. In addition, though HspB8 does not influence p62 and LC3 (two key autophagic molecules) expression, it does prevent p62 bodies formation, and restores the normal autophagic flux in these cells. Interestingly, trehalose, a well-known autophagy stimulator, induces HspB8 expression, suggesting that HspB8 might act as one of the molecular mediators of the proautophagic activity of trehalose. Collectively, these data support the hypothesis that treatments aimed at restoring a normal autophagic flux that result in the more efficient clearance of mutant ARpolyQ might produce beneficial effects in SBMA patients.

Topics: Adaptor Proteins, Signal Transducing; Adenine; Animals; Autophagy; Cell Line, Transformed; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Gene Expression Regulation; Green Fluorescent Proteins; Heat-Shock Proteins; HSP20 Heat-Shock Proteins; Humans; Leupeptins; Mice; Molecular Chaperones; Motor Neurons; Muscle Proteins; Mutation; Receptors, Androgen; RNA, Small Interfering; Sequestosome-1 Protein; Signal Transduction; Testosterone; Trehalose

NTE-related esterase (NRE) is a novel endoplasmic reticulum-anchored lysophospholipase with high homology to neuropathy target esterase (NTE). However, little is known about the regulation of NRE protein. In the current study, we investigated the degradation pathways of mouse NRE (mNRE) in mammalian cells. Based on experiments with inhibitors and inducer of protein degradation pathways, we provide here the first evidence that mNRE is degraded by macroautophagy as well as by the proteasome. Moreover, the contribution of protein domains to the degradation of mNRE was investigated, which showed that the transmembrane and regulatory domain played a role in the degradation of mNRE by macroautophagy and the proteasome respectively. In contrast the C-terminal catalytic domain was not involved in both degradation pathways of mNRE. These findings showed for the first time that the degradation pathways in controlling mNRE quantity and may provide further insight into structure and regulation of mNRE.

Topics: Adenine; Animals; Autophagy; Carboxylic Ester Hydrolases; Cycloheximide; Green Fluorescent Proteins; HEK293 Cells; Humans; Leupeptins; Mice; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Biosynthesis; Protein Structure, Tertiary; Proteolysis; Recombinant Fusion Proteins

The ubiquitin-proteasome system and autophagy-lysosome system are 2 major protein degradation pathways in eukaryotic cells, which are tightly linked to cancer. Proteasome inhibitors have been approved in clinical use against hematologic malignancies, but their application in solid tumors is uncertain. Moreover, the role of autophagy after proteasome inhibition is controversial.. Two proteasome inhibitors, 2 autophagy inhibitors, and 3 hepatocellular carcinoma (HCC) cell lines were investigated in the current study. In vitro, cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell apoptosis was evaluated by flow cytometry analysis of annexin-V/propidium iodide staining, and autophagy was evaluated by green fluorescent protein-light chain 3 (GFP-LC3) redistribution and LC3 Western blot analysis. In vivo, Ki-67 staining was used to detect cell proliferation, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was used to detect apoptosis, and electron microscopy and p62 immunohistochemical staining were used to detect autophagy.. Proteasome inhibitors suppressed proliferation, induced apoptosis, and activated autophagy in HCC cell lines in vitro, and autophagy exerted a protective role after proteasome inhibition. In vivo, anticancer effects of bortezomib on the MHCC-97H orthotopic model (human HCC cells) were different from the effects observed on the Huh-7 subcutaneous model (human HCC cells). The autophagy inhibitor chloroquine interacted synergistically with bortezomib to suppress proliferation and induce apoptosis in both tumor models.. The current results indicated that simultaneous targeting of the proteasome and autophagy pathways may represent a promising method for HCC treatment.

Topics: Adenine; Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Boronic Acids; Bortezomib; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Chloroquine; Cysteine Proteinase Inhibitors; Humans; Leupeptins; Membrane Proteins; Microtubule-Associated Proteins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyrazines

Congenital muscular dystrophy caused by laminin α2 chain deficiency (also known as MDC1A) is a severe and incapacitating disease, characterized by massive muscle wasting. The ubiquitin-proteasome system plays a major role in muscle wasting and we recently demonstrated that increased proteasomal activity is a feature of MDC1A. The autophagy-lysosome pathway is the other major system involved in degradation of proteins and organelles within the muscle cell. However, it remains to be determined if the autophagy-lysosome pathway is dysregulated in muscular dystrophies, including MDC1A. Using the dy(3K)/dy(3K) mouse model of laminin α2 chain deficiency and MDC1A patient muscle, we show here that expression of autophagy-related genes is upregulated in laminin α2 chain-deficient muscle. Moreover, we found that autophagy inhibition significantly improves the dystrophic dy(3K)/dy(3K) phenotype. In particular, we show that systemic injection of 3-methyladenine (3-MA) reduces muscle fibrosis, atrophy, apoptosis and increases muscle regeneration and muscle mass. Importantly, lifespan and locomotive behavior were also greatly improved. These findings indicate that enhanced autophagic activity is pathogenic and that autophagy inhibition holds a promising therapeutic potential in the treatment of MDC1A.

Topics: Adenine; Animals; Apoptosis; Autophagy; Behavior, Animal; Disease Models, Animal; Drug Therapy, Combination; Fibrosis; Gene Expression Regulation; Injections; Laminin; Leupeptins; Mice; Motor Activity; Muscles; Muscular Atrophy; Muscular Dystrophies; Peripheral Nervous System Diseases; Phenotype; Phosphorylation; Proto-Oncogene Proteins c-akt; Regeneration; Survival Analysis

TAR DNA-binding protein-43 (TDP-43) is a nuclear protein functioning in the regulation of transcription and mRNA splicing. TDP-43 is accumulated in ubiquitinated inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS) diseased brains. However, the pathways involved in the clearance of TDP-43 and its pathogenic form (TDP-25), a truncated form of TDP-43, are still not elucidated. In this study, we demonstrated that the protein levels of TDP-43 and TDP-25 were increased in cells treated with a proteasome inhibitor, MG132, or an autophagy inhibitor, 3-MA, whereas, they were decreased in cells treated with an enhancer of autophagy, trehalose. Furthermore, more protein level changes of TDP-25 than TDP-43 were observed in cells treated with above inhibitors or enhancer. Thus, our data suggest that TDP-43 and TDP-25 are degraded by both proteasome and autophagy with TDP-25 being more regulated.

Topics: Adenine; Autophagy; Cell Line; DNA-Binding Proteins; Humans; Leupeptins; Pepstatins; Peptide Fragments; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Trehalose; Ubiquitin

The sphingolipids ceramide, sphingosine, and sphingosine 1-phosphate (S1P) regulate cell signaling, proliferation, apoptosis, and autophagy. Sphingosine kinase-1 and -2 (SK1 and SK2) phosphorylate sphingosine to form S1P, shifting the balanced activity of these lipids toward cell proliferation. We have previously reported that pharmacological inhibition of SK activity delays tumor growth in vivo. The present studies demonstrate that the SK2-selective inhibitor 3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide (ABC294640) induces nonapoptotic cell death that is preceded by microtubule-associated protein light chain 3 cleavage, morphological changes in lysosomes, formation of autophagosomes, and increases in acidic vesicles in A-498 kidney carcinoma cells. ABC294640 caused similar autophagic responses in PC-3 prostate and MDA-MB-231 breast adenocarcinoma cells. Simultaneous exposure of A-498 cells to ABC294640 and 3-methyladenine, an inhibitor of autophagy, switched the mechanism of toxicity to apoptosis, but decreased the potency of the SK2 inhibitor, indicating that autophagy is a major mechanism for tumor cell killing by this compound. Induction of the unfolded protein response by the proteasome inhibitor N-(benzyloxycarbonyl)leucinylleucinylleucinal Z-Leu-Leu-Leu-al (MG-132) or the heat shock protein 90 inhibitor geldanamycin synergistically increased the cytotoxicity of ABC294640 in vitro. In severe combined immunodeficient mice bearing A-498 xenografts, daily administration of ABC294640 delayed tumor growth and elevated autophagy markers, but did not increase terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive cells in the tumors. These data suggest that ABC294640 promotes tumor cell autophagy, which ultimately results in nonapoptotic cell death and a delay of tumor growth in vivo. Consequently, ABC294640 may effectively complement anticancer drugs that induce tumor cell apoptosis.

Topics: Adamantane; Adenine; Animals; Apoptosis; Autophagy; Blotting, Western; Cell Cycle; Electrophoresis, Polyacrylamide Gel; Humans; Leupeptins; Mice; Mice, SCID; Microscopy, Confocal; Microtubule-Associated Proteins; Mitochondrial Membranes; Neoplasm Transplantation; Neoplasms, Experimental; Phosphotransferases (Alcohol Group Acceptor); Polymerase Chain Reaction; Pyridines; RNA, Small Interfering; Tumor Cells, Cultured; Unfolded Protein Response

The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Blockade of UPS by proteasome inhibitors has been shown to activate autophagy. Recent evidence also suggests that proteasome inhibitors may inhibit cancer growth. In this study, the effect of a proteasome inhibitor MG-132 on the proliferation and autophagy of cultured colon cancer cells (HT-29) was elucidated. Results showed that MG-132 inhibited HT-29 cell proliferation and induced G(2)/M cell cycle arrest which was associated with the formation of LC3(+) autophagic vacuoles and the accumulation of acidic vesicular organelles. MG-132 also increased the protein expression of LC3-I and -II in a time-dependent manner. In this connection, 3-methyladenine, a Class III phosphoinositide 3-kinase inhibitor, significantly abolished the formation of LC3(+) autophagic vacuoles and the expression of LC3-II but not LC3-I induced by MG-132. Taken together, this study demonstrates that inhibition of proteasome in colon cancer cells lowers cell proliferation and activates autophagy. This discovery may shed a new light on the novel function of proteasome in the regulation of autophagy and proliferation in colon cancer cells.

Topics: Adenine; Autophagy; Cell Division; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cysteine Proteinase Inhibitors; Cytoplasmic Vesicles; G2 Phase; Humans; Leupeptins; Microtubule-Associated Proteins; Proteasome Inhibitors; Vacuoles

Autophagy is simultaneously a mode of programmed cell death and an important physiological process for cell survival, but its pathophysiological significance in cardiac myocytes remains largely unknown. We induced autophagy in isolated adult rat ventricular cardiomyocytes (ARVCs) by incubating them in glucose-free, mannitol-supplemented medium for up to 4 days. Ultrastructurally, intracellular vacuoles containing degenerated subcellular organelles (e.g., mitochondria) were markedly apparent in the glucose-starved cells. Microtubule-associated protein-1 light chain 3 was significantly upregulated among the glucose-starved ARVCs than among the controls. After 4 days, glucose-starved ARVCs showed a significantly worse survival rate (19+/-5.2%) than the controls (55+/-8.3%, P<0.005). Most dead ARVCs in both groups showed features of necrosis, and the rate of apoptosis did not differ between the groups. Two inhibitors of autophagy, 3-methyladenine (3-MA) and leupeptin, significantly and dose-dependently reduced the viability of both control and glucose-starved ARVCs and caused specific morphological alterations; 3-MA reduced autophagic findings, whereas leupeptin greatly increased the numbers and the sizes of vacuoles that contained incompletely digested organelles. The knockdown of the autophagy-related genes with small interfering RNA also reduced the glucose-starved ARVCs viability, but rapamycin, an autophagy enhancer, improved it. Reductions in the ATP content of ARVCs caused by glucose depletion were exacerbated by the inhibitors while attenuated by rapamycin, suggesting that autophagy inhibition might accelerate energy depletion, leading to necrosis. Taken together, our findings suggest that autophagy in cardiomyocytes reflects a prosurvival, compensatory response to stress and that autophagic cardiomyocyte death represents an unsuccessful outcome due to necrosis.

Topics: Adenine; Adenosine Triphosphate; Animals; Autophagy; Cell Shape; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Glucose; Leupeptins; Male; Microtubule-Associated Proteins; Myocytes, Cardiac; Necrosis; Rats; Rats, Sprague-Dawley; RNA Interference; RNA, Small Interfering; Sirolimus; Time Factors; Vacuoles

Mammalian hepatic cytochromes P450 (P450s) are endoplasmic reticulum (ER)-anchored hemoproteins engaged in the metabolism of numerous xeno- and endobiotics. P450s exhibit widely ranging half-lives, utilizing both autophagic-lysosomal (ALD) and ubiquitin-dependent 26S proteasomal (UPD) degradation pathways. Although suicidally inactivated hepatic CYPs 3A and "native" CYP3A4 in Saccharomyces cerevisiae are degraded via UPD, the turnover of native hepatic CYPs 3A in their physiological milieu has not been elucidated. Herein, we characterize the degradation of native, dexamethasone-inducible CYPs 3A in cultured primary rat hepatocytes, using proteasomal (MG-132 and MG-262) and ALD [NH4Cl and 3-methyladenine (3-MA)] inhibitors to examine their specific degradation route. Pulse-chase with immunoprecipitation analyses revealed a basal 52% 35S-CYP3A loss over 6 h, which was stabilized by both proteasomal inhibitors. By contrast, no corresponding CYP3A stabilization was detected with either ALD inhibitor NH4Cl or 3-MA. Furthermore, MG-262-induced CYP3A stabilization was associated with its polyubiquitylation, thereby verifying that native CYPs 3A were also degraded via UPD. To identify the specific participants in this process, cellular proteins were cross-linked in situ with paraformaldehyde (PFA) in cultured hepatocytes. Immunoblotting analyses of CYP3A immunoprecipitates after PFA-cross-linking revealed the presence of p97, a cytosolic AAA ATPase instrumental in the extraction and delivery of ubiquitylated ER proteins for proteasomal degradation. Such native CYP3A-p97 interactions were greatly magnified after CYP3A suicidal inactivation (which accelerates UPD), and/or proteasomal inhibition, and were confirmed by proteomic and confocal immunofluorescence microscopic analyses. These findings clearly reveal that native CYPs 3A undergo UPD and implicate a role for p97 in this process.

Topics: Adenine; Adenosine Triphosphatases; Ammonium Chloride; Animals; Autophagy; Boronic Acids; Cell Cycle Proteins; Cross-Linking Reagents; Cytochrome P-450 CYP3A; Cytochrome P-450 CYP3A Inhibitors; Dexamethasone; Dicarbethoxydihydrocollidine; Formaldehyde; Hepatocytes; Leupeptins; Lysosomes; Male; Nuclear Proteins; Polymers; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Saccharomyces cerevisiae Proteins; Troleandomycin; Ubiquitin; Valosin Containing Protein

EGF suppresses proteolysis via class 1 phosphatidylinositol 3-kinase (PI3K) in renal tubular cells. EGF also increases the abundance of glycolytic enzymes (e.g., glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) and transcription factors (e.g., pax2) that are degraded by the lysosomal pathway of chaperone-mediated autophagy. To determine if EGF regulates chaperone-mediated autophagy through PI3K signaling, this study examined the effect of inhibiting PI3K and its downstream mediators Akt and the mammalian target of rapamycin (mTOR). Inhibition of PI3K with LY294002 prevented EGF-induced increases in GAPDH and pax2 abundance in NRK-52E renal tubular cells. Similar results were seen with an adenovirus encoding a dominant negative Akt (DN Akt). Expression of a constitutively active Akt increased GAPDH and pax2 abundance. An mTOR inhibitor, rapamycin, did not prevent EGF-induced increases in these proteins. Neither DN Akt nor rapamycin alone had an effect on total cell protein degradation, but both partially reversed EGF-induced suppression of proteolysis. DN Akt no longer affected proteolysis after treatment with a lysosomal inhibitor, methylamine. In contrast, methylamine or the inhibitor of macroautophagy, 3-methyladenine, did not prevent rapamycin from partially reversing the effect of EGF on proteolysis. Notably, rapamycin did not increase autophagasomes detected by monodansylcadaverine staining. Blocking the proteasomal pathway with either MG132 or lactacystin prevented rapamycin from partially reversing the effect of EGF on proteolysis. It is concluded that EGF regulates pax2 and GAPDH abundance and proteolysis through a PI3K/Akt-sensitive pathway that does not involve mTOR. Rapamycin has a novel effect of regulating proteasomal proteolysis in cells that are stimulated with EGF.

Topics: Acetylcysteine; Adenine; Animals; Autophagy; Cell Line; Chromones; Epidermal Growth Factor; Glyceraldehyde-3-Phosphate Dehydrogenases; Kidney Tubules; Leupeptins; Lysosomes; Methylamines; Morpholines; PAX2 Transcription Factor; Peptide Hydrolases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proteasome Endopeptidase Complex; Protein Kinases; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Imperfect autophagic degradation of oxidatively damaged macromolecules and organelles (so-called biological "garbage") is considered an important contributor to ageing and consequent death of postmitotic (non-dividing) cells, such as neurons and cardiac myocytes. In contrast, proliferating cells apparently escape senescence by a continuous dilution and repair of damaged structures during division. Postmitotic ageing can be mimicked and studied in cultures of potentially dividing cells if their mitotic activity is inhibited. To test the "garbage accumulation" theory of ageing, we compared survival of density-dependent growth-arrested (confluent) and proliferating human fibroblasts and astrocytes following inhibition of autophagic sequestration with 3-methyladenine (3MA). Exposure of confluent fibroblast cultures to 3MA for two weeks resulted in a significantly increased proportion of dying cells compared to both untreated confluent cultures and dividing cells with 3MA-inhibited autophagy. Similar results were obtained when autophagic degradation was suppressed by the protease inhibitor leupeptin. In 3MA- or leupeptin-exposed cultures, dying cells were overloaded with undegraded autofluorescent material. The results support a key role of biological lysosomal "garbage" accumulation in the triggering of ageing and death of postmitotic cells, as well as the anti-ageing role of cell division.

Topics: Adenine; Aging; Astrocytes; Autophagy; Cell Line; Cell Survival; Cellular Senescence; Cysteine Proteinase Inhibitors; Fibroblasts; Humans; Leupeptins; Mitosis; Phosphoinositide-3 Kinase Inhibitors

Endostatin, a proteolytic fragment of collagen XVIII, is a potent inhibitor of angiogenesis and suppresses neovascularization and tumor growth. However, the inhibitory mechanism of endostatin in human endothelial cells has not been characterized yet. Electron microscopic analysis revealed that endostatin induced formation of numerous autophagic vacuoles in endothelial in 6 to 24 h after treatment. Moreover, there was only a 2- to 3-fold increase in intracellular reactive oxygen species after endostatin treatment. Endostatin-induced cell death was not prevented by antioxidants (vitamin C, vitamin E, or propyl gallate) or caspase inhibitors, suggesting that the increase of oxidative stress or the activation of caspases may not be the crucial factors in the anti-angiogenic mechanism of endostatin. However, the cytotoxicity of endostatin was significantly reduced by 3-methyladenine (a specific inhibitor of autophagy) and serine and cysteine lysosomal protease inhibitors (leupeptin and aprotinin). Taken together, these results suggest that in human endothelial cells: (1) endostatin predominantly causes autophagic, rather than apoptotic, cell death, (2) endostatin-induced autophagic cell death occurs in the absence of caspase activation and through an oxidative-independent pathway, and (3) endostatin-induced "autophagic cell death" or "type 2 physiological cell death" is regulated by serine and cysteine lysosomal proteases.

Topics: Acridine Orange; Adenine; Animals; Antineoplastic Agents; Apoptosis; Aprotinin; Blotting, Western; Caspases; Cell Death; Cells, Cultured; CHO Cells; Cloning, Molecular; Cricetinae; Cysteine; Cysteine Endopeptidases; Dose-Response Relationship, Drug; Endostatins; Endothelial Cells; Endothelium, Vascular; Enzyme Activation; Flow Cytometry; Glutathione Transferase; Humans; Immunohistochemistry; Leupeptins; Lysosomes; Microscopy, Electron; Microscopy, Phase-Contrast; Oxidative Stress; Reactive Oxygen Species; Recombinant Proteins; Serine; Time Factors

Previously we showed that the redox active Cu(2+) was much more effective than Cd(2+) at inducing reactive oxygen species ("ROS") formation in hepatocytes and furthermore "ROS" scavengers prevented Cu(2+)-induced hepatocyte cytotoxicity (Pourahmad and O'Brien, 2000). In the following it is shown that hepatocyte cytotoxicity induced by Cu(2+), but not Cd(2+), was preceded by lysosomal membrane damage as demonstrated by acridine orange release. Cytotoxicity, "ROS" formation, and lipid peroxidation were also readily prevented by methylamine or chloroquine (lysosomotropic agents) or 3-methyladenine (an inhibitor of autophagy). Hepatocyte lysosomal proteolysis was also activated by Cu(2+), but not Cd(2+), as tyrosine was released from the hepatocytes and was prevented by leupeptin and pepstatin (lysosomal protease inhibitors). Cu(2+)-induced cytotoxicity was also prevented by leupeptin and pepstatin. A marked increase in Cu(2+)-induced hepatocyte toxicity also occurred if the lysosomal toxins gentamicin or aurothioglucose were added at the same time as the Cu(2+). Furthermore, destabilizing lysosomal membranes beforehand by preincubating the hepatocytes with gentamicin or aurothioglucose prevented Cu(2+)-induced hepatocyte cytotoxicity. It is proposed that Cu(2+)-induced cytotoxicity involves lysosomal damage that causes the release of cytotoxic digestive enzymes as a result of lysosomal membrane damage by "ROS" generated by lysosomal Cu(2+) redox cycling.

Topics: Acridine Orange; Adenine; Animals; Aurothioglucose; Cadmium; Cell Death; Chloroquine; Copper; Endopeptidases; Enzyme Activation; Gentamicins; Leupeptins; Lipid Peroxidation; Liver; Lysosomes; Male; Methylamines; Monensin; Oxidation-Reduction; Pepstatins; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species

Dihydrotestosterone (DHT) decreases rat liver alcohol dehydrogenase (ADH) due principally to an increased rate of degradation of the enzyme. The pathway of degradation of ADH was investigated. Exposure of hepatocytes in culture to lactacystin or to MG132, which are inhibitors of the ubiquitin-proteasome pathway of protein degradation, resulted in higher ADH. Furthermore, both lactacystin and MG132 prevented the decrease in ADH caused by DHT. By contrast, the lysosomal proteolytic inhibitors 3-methyladenine and leupeptin as well as inhibitors of the calcium-activated neutral protease calpain system had no effect on ADH in the absence or presence of DHT. ADH isolated by immunoprecipitation from hepatocytes exposed to DHT reacted specifically with anti-ubiquitin antibody. Ubiquitinated ADH was also demonstrated in hepatocytes exposed to MG132. The combination of DHT and MG132 resulted in more ubiquitinated ADH than exposure to either compound alone. These results suggest that the ubiquitin-proteasome pathway plays a role in the degradation of ADH and in the enhanced degradation of this enzyme by DHT.

Topics: Acetylcysteine; Adenine; Alcohol Dehydrogenase; Animals; Calpain; Cells, Cultured; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dihydrotestosterone; Electrophoresis, Polyacrylamide Gel; Hepatocytes; Leupeptins; Liver; Lysosomes; Male; Multienzyme Complexes; Precipitin Tests; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Ubiquitins

Simultaneous investigation of protein degradation and autophagy of isolated exocrine pancreatic cells is carried out here for the first time in a systematic way by a complex biochemical, morphological and morphometrical approach. Protein degradation proceeds with a decreasing rate of 4-1.5 per cent per h over a 4-h period indicating a comparatively low degradation capacity. Cells in freshly isolated acini do not contain autophagic vacuoles but the latter appear within an hour in vitro and their quantity remains close to a steady state during the subsequent 3 h. Both traditional inhibitors of the autophagic-lysosomal pathway, e.g. vinblastine, leupeptin, and lysosomotropic amines together with the recently introduced 3-methyladenine, inhibit degradation to a similar maximal extent, offering the possibility of the estimation of the ratio of lysosomal/non-lysosomal degradation. In pancreatic acinar cells autophagic sequestration is unaffected and protein degradation is inhibited inside secondary lysosomes by leupeptin and lysosomotropic amines, while 3-methyladenine prevents the formation of autophagosomes. Vinblastine seems to act by inhibiting the fusion of autophagosomes with lysosomes and there is no evidence for the stimulation of autophagic sequestration by vinblastine in the present system. The effect of inhibitors of protein breakdown on protein synthesis is variable and does not correlate with their influence on degradation. Amino acids strongly stimulate protein synthesis, but in contrast to what is found in liver cells, they do not seem to affect protein degradation or autophagy significantly, thus indicating major regulatory differences of these processes between pancreatic acinar cells and hepatocytes.

Topics: Adenine; Amines; Amino Acids; Animals; Autophagy; Cells, Cultured; Enzyme Inhibitors; Kinetics; Leupeptins; Lysosomes; Male; Microscopy, Electron; Pancreas; Protein Biosynthesis; Proteins; Rats; Rats, Long-Evans; Vinblastine

The major histocompatibility complex class II-associated invariant chain is believed to direct newly synthesized class II to endocytic compartments. Invariant chain synthesized at high levels in transiently transfected cells induces formation of large vesicular structures. We have examined the effect of stable expression of invariant chain in human fibroblasts by light and electron microscopy. Invariant chain expression dramatically modified endocytic compartments and induced the formation of greatly enlarged structures. These modifications were not lethal. Ultrastructurally, at least three morphologically distinct enlarged compartments could be discerned in the cells. These three compartments may represent early and late endosomes and lysosomes. Internalization of anti-invariant chain antibodies shows that invariant chain may reach the large endosomes via rapid internalization from the plasma membrane. Internalized protein remained in the enlarged vesicles for 4-6 h, indicating an invariant chain-induced delay in the pathway to lysosomes. Although the large invariant chain-induced vesicles have not yet been seen in professional antigen-presenting cells, the invariant chain-induced effects may play a role in regulating the endocytic pathway, creating a special environment for MHC class II to bind antigen.

Topics: Adenine; Antigens, CD; Brefeldin A; Cell Line; Chloroquine; Cyclopentanes; Endocytosis; Endosomes; Fibroblasts; HLA-DR alpha-Chains; HLA-DR Antigens; Humans; Immunohistochemistry; Leupeptins; Lysosomal Membrane Proteins; Lysosomes; Macromolecular Substances; Membrane Glycoproteins; Receptor, IGF Type 2; Recombinant Proteins; Transfection

Previous studies have shown that in fibroblasts from patients with the Zellweger syndrome (ZS) aberrant membrane structures are present which contain peroxisomal membrane proteins (Santos, M. J. et al., Science 239, 1536-1538 (1988)). In order to characterize these structures we have performed double labeling immunoelectron microscopy experiments using antisera directed against the 69 kDa peroxisomal integral membrane protein (PMP) and lysosomal hydrolases. The results indicate that at least 80% of the structures earlier referred to as 'peroxisomal ghosts' contain lysosomal hydrolases. In addition, we have studied the effect of culture of ZS fibroblasts in the presence of 3-methyladenine, an inhibitor of autophagy, on the intracellular distribution of the 69 kDa PMP. Immunofluorescence experiments showed that in the presence of 3-methyladenine there is an increase in fluorescent spots and a change in the distribution of the spots from mainly perinuclear to randomly distributed throughout the cytoplasm. Double labeling immunoelectron microscopy revealed that after culture in the presence of 3-methyladenine the 69 kDa PMP also accumulates mainly in compartments containing lysosomal hydrolases. In one ZS cell line we found that after culture in the presence of 3-methyladenine there was also an accumulation of structures which were as small as normal microperoxisomes. We conclude that in ZS fibroblasts the 69 kDa PMP is mainly present in lysosomal compartments, presumably degradative autophagic vacuoles. Furthermore, in ZS fibroblasts peroxisomes of apparently normal morphology may be synthesized, but they are degraded by autophagic proteolysis.

Topics: Adenine; Autophagy; Cell Line; Fibroblasts; Fluorescent Antibody Technique; Humans; Hydrolases; Leupeptins; Lysosomes; Membrane Proteins; Microbodies; Zellweger Syndrome

1. The regulation of protein breakdown as well as the generation of intermediates in the pathway from intact protein to amino acids was investigated by using 3H-labelled N-ethylmaleimide-modified aldolase (NEM-aldolase) as an indicator protein after its microinjection into HeLa cells. 2. NEM-aldolase degradation to acid-soluble products proceeded at a slower rate than that of endogenously labelled total cell protein, and was inhibited to a greater extent by 3-methyladenine, leupeptin and NH4Cl. The combination of leupeptin plus NH4Cl was particularly effective, decreasing the NEM-aldolase breakdown rate by 90%. 3. Measurements of the loss of radioactivity from the aldolase band located from fluorograms after SDS/polyacrylamide-gel electrophoresis showed that NEM-aldolase breakdown was much more rapid when measured by this method. The effects of insulin, 3-methyladenine, leupeptin and NH4Cl on this breakdown were also substantial. 4. Substantial amounts of peptide intermediates in the breakdown pathway of NEM-aldolase accumulated in cells. The production of small intermediates (less than 30 kDa) accounted for approx. 40% of the NEM-aldolase degraded in control cultures. Addition of NH4Cl increased the proportion of these intermediates. Large intermediates, between 31 and 38 kDa, were particularly evident in the presence of the cysteine proteinase inhibitor leupeptin, but almost no small intermediates were detected. 5. The results are best explained by the degradation of NEM-aldolase being predominantly a lysosomal process, with cysteine proteinases involved in early proteolytic steps and other proteinases that have acid pH optima required for the complete catabolism of small intermediates.

Topics: Adenine; Ammonium Chloride; Ethylmaleimide; Fructose-Bisphosphate Aldolase; HeLa Cells; Humans; Insulin; Leupeptins; Microinjections

1. Degradation rate constants for individual biotin-labelled proteins were measured in Swiss 3T3-L1 adipocytes that had been incubated with inhibitors of autophagy or of lysosomal proteolysis. 2. Inhibitory effects produced by 10 mM-3-methyladenine and a combination of 5 mM-NH4Cl and leupeptin (50 micrograms/ml) were approximately equal. The inclusion of NH4Cl did not significantly enhance the responses to 3-methyladenine, suggesting that autophagy was already maximally inhibited. 3. The extent of inhibition by 3-methyladenine or by the NH4Cl/leupeptin mixture was similar for the cytosolic enzyme acetyl-CoA carboxylase and for the three mitochondrial carboxylases. This inhibition averaged 50%. The breakdown rate of a more-stable 38 kDa biotin-containing mitochondrial protein was more responsive to the inhibitory agents. These results are best explained by mitochondrial proteolysis occurring via a combination of the degradation of whole mitochondria within autophagic vacuoles, supplemented by the selective intramitochondrial breakdown of more labile proteins. 4. A number of intermediate products in the degradation of biotin-containing proteins were detected. Differences in the patterns of radioactivity between these peptides after incubation of cells in the presence of inhibitors of the breakdown process provided evidence that some peptides were produced before autophagy, others as a result of intralysosomal inhibition, while at least one was associated with intramitochondrial proteolysis.

Topics: Adenine; Biotin; Cell Line; Electrophoresis, Polyacrylamide Gel; Insulin; Leucine; Leupeptins; Proteins

Mammary explants from midpregnant rabbits were cultured for 18 h at 37 degrees C with insulin, prolactin and cortisol. Subsequently, explants were labelled for 2 h with inorganic [32P]phosphate, L-[5-3H]proline or L-[4,5-3H]leucine, washed and chased for up to 3 h. The radiolabelling profile of [32P]casein or [3H]casein during the chase period, obtained by isoelectric focussing or immunoprecipitation indicates extensive destruction of neosynthesized casein. The extent of casein destruction in mammary explants in culture (measured after radiolabelling with L-[5-3H]proline), is inversely related to casein secretion. Least casein degradation is observed in explants after 48 h in culture when casein secretion is maximal (observed histochemically). Subsequently, when the extracellular alveolar lumen is filled with secretion products (72 h), rapid intracellular casein destruction is again observed. When the chase was carried out in the presence of drugs which inhibit degradation and/or secretion, the results indicate that secretion-coupled casein degradation is dependent on an intact functional microfilamentous-microtubular network, casein is not degraded by an autophagosome requiring process, degradation is inhibited by leupeptin, amino-acid analogue containing casein does not undergo secretion-coupled degradation and inhibition of N-glycosylation of intracellular vesicular membrane proteins prevents secretion-coupled degradation. Secretion-coupled protein destruction is discussed in relation to the post-translational regulation of the net production of secretory proteins in eukaryotic cells.

Topics: Adenine; Amino Acids; Animals; Biological Transport; Caseins; Colchicine; Cytochalasin B; Female; Leupeptins; Mammary Glands, Animal; Organ Culture Techniques; Rabbits; Tunicamycin

Dantrolene, an agent that inhibits Ca2+ mobilization, improved protein balance in skeletal muscle, as thyroid status was increased, by altering rates of protein synthesis and degradation. Thyroxine (T4) caused increases in protein degradation that were blocked by leupeptin, a proteinase inhibitor previously shown to inhibit Ca2+-dependent non-lysosomal proteolysis in these muscles. In addition, T4 abolished sensitivity to the lysosomotropic agent methylamine and the autophagy inhibitor 3-methyladenine, suggesting that T4 inhibits autophagic/lysosomal proteolysis.

Topics: Adenine; Animals; Calcium; Dantrolene; Female; Leupeptins; Methylamines; Muscle Proteins; Muscles; Potassium; Rats; Rats, Inbred Strains; Thyroxine

Addition of epidermal growth factor (EGF) to cultures of A431 human epidermoid carcinoma cells produces an increase in the rate of intracellular protein breakdown that cannot be accounted for by increased proteolysis in lysates from EGF-treated cells. In support of this observation, inhibition of protein synthesis with cycloheximide does not reduce the EGF response in cell monolayers. On the other hand, inhibitors of lysosomal proteolytic function such as leupeptin, vinblastine and especially the weak base, ammonia, are able to block the ability of EGF to increase protein breakdown. Additional results suggest that the EGF effect is mediated via a stimulation of autophagy. First, the autophagocytosis inhibitor, 3-methyladenine, reduces the EGF response, and second, the ability of insulin to inhibit protein breakdown by preventing the formation of autophagic vacuoles is overcome by EGF. Moreover, the actions of inhibitors and competing hormones are similar to those reported for glucagon, a hormone known to increase autophagy. The EGF response on protein breakdown persists for at least 6 h after thorough washing of the A431 monolayers. This result contrasts with the rapid reversal of EGF effects in other cell lines. Examination of the fate of bound EGF in cells washed and incubated for 2 h at 37 degrees C shows that some 500-fold more EGF per mg protein is retained on the surface of A431 cells compared to AG2804-transformed fibroblasts, a difference which probably explains the unusual persistence of the EGF effect on protein breakdown.

Topics: Adenine; Ammonium Chloride; Autolysis; Carcinoma, Squamous Cell; Cell Line; Cycloheximide; Epidermal Growth Factor; Fibroblasts; Humans; Hydrogen-Ion Concentration; Insulin; Leupeptins; Lung; Proteins; Time Factors; Vinblastine

Lysosomes are presumed to be involved in protein degradation in heart, but their exact role is poorly understood. Several interventions that are known to alter cardiac proteolysis (e.g., insulin) also produce lysosomal changes that might account for the observed changes in protein degradation; but many other interventions appear not to do so. Agents that interfere with lysosomal function (e.g., sucrose, chloroquine, methyladenine, leupeptin) cause a 25% reduction in the rate of degradation of total protein in fetal mouse hearts in organ culture; however, in the same hearts the rate of degradation of myosin and other myofibrillar proteins remains unchanged. Thus, it appears that lysosomes are involved in cardiac proteolysis, but may not play a rate-limiting or regulatory role in many circumstances. The regulation of proteolysis by insulin appears to involve non-lysosomal pathways in addition to any lysosomal alterations it may cause. Furthermore, the initial cleavage of myofibrillar proteins appears no to be dependent on normal lysosomal function.

Topics: Adenine; Animals; Chloroquine; Fetus; Insulin; Kinetics; Leupeptins; Lysosomes; Mice; Myocardium; Myofibrils; Proteins

Lymphocytes from normal human subjects and from patients with chronic lymphocytic leukemia were found to degrade their endogenous protein at similar rates (2.5-3.0%/h) when incubated in an amino acid-free buffer. Protein degradation was inhibited 20-35% by inhibitors of autophagic sequestration (amino acids, 3-methyladenine) and by inhibitors of intra-lysosomal proteolysis (leupeptin, propylamine), the extent of inhibition being similar in normal and leukemic lymphocytes. The inhibitor effects, together with the electronmicroscopic demonstration of autophagosomes in the lymphocyte cytoplasm, is taken as evidence for the existence of an autophagic-lysosomal pathway in human lymphocytes, potentially responsible for as much as one-third of their overall protein degradation.

Topics: Adenine; Amino Acids; Autophagy; Humans; Leukemia, Lymphoid; Leupeptins; Lymphocytes; Lysosomes; Neoplasm Proteins; Phagocytosis; Propylamines; Proteins