المؤلفون: Trond Berg, Per Ottar Seglen, Per E. Stromhaug, Monica Fengsrud

المصدر: Biochemical Journal. 335:217-224

مصطلحات موضوعية: Male, Autophagosome, Endosome, Liver cytology, Immunoblotting, Centrifugation, Mitochondria, Liver, Endosomes, Biology, Endoplasmic Reticulum, Vinblastine, Biochemistry, Cathepsin B, Osmotic Pressure, Phagosomes, Autophagy, Animals, Rats, Wistar, Molecular Biology, Differential centrifugation, Endoplasmic reticulum, Proteins, Cell Biology, Enzymes, Rats, Cell biology, Cytoskeletal Proteins, Liver, Cytoplasm, Lysosomes, Percoll, Research Article

الوصف: To investigate the properties and intracellular origin of autophagosomes, a procedure for the purification and isolation of these organelles from rat liver has been developed. Isolated hepatocytes were incubated with vinblastine to induce autophagosome accumulation; the cells were then homogenized and treated with the cathepsin C substrate glycyl-l-phenylalanine 2-naphthylamide to cause osmotic disruption of the lysosomes. Nuclei were removed by differential centrifugation, and the postnuclear supernatant was fractionated on a discontinuous Nycodenz density gradient. The autophagosomes, recognized by their content of autophagocytosed lactate dehydrogenase (LDH), could be recovered in an intermediate-density fraction, free from cytosol and mitochondria. Finally, the autophagosomes were separated from the endoplasmic reticulum and other membranous elements by centrifugation in a Percoll colloidal density gradient, followed by flotation in iodixanol to remove the Percoll particles. The final autophagosome preparation represented a 24-fold purification of autophagocytosed LDH relative to intact cells, with a 12% recovery. The purified autophagosomes contained sequestered cytoplasm with a normal ultrastructure, including mitochondria, peroxisomes and endoplasmic reticulum in the same proportions as in intact cells. However, immunoblotting indicated a relative absence of cytoskeletal elements (tubulin, actin and cytokeratin), which may evade autophagic sequestration. The autophagosomes showed no enrichment in protein markers typical of lysosomes (acid phosphatase, cathepsin B, lysosomal glycoprotein of 120 kDa), endosomes (early-endosome-associated protein 1, cation-independent mannose 6-phosphate receptor, asialoglycoprotein receptor) or endoplasmic reticulum (esterase, glucose-regulated protein of 78 kDa, protein disulphide isomerase), suggesting that the sequestering membranes are not derived directly from any of these organelles, but rather represent unique organelles (phagophores).

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::1acd7edf3918c74661f1fc96fe9b5fa7Test
https://doi.org/10.1042/bj3350217Test

المؤلفون: Paul B. Gordon, Per Ottar Seglen, Ingunn Holen

المصدر: European Journal of Biochemistry. 215:113-122

مصطلحات موضوعية: Male, Phosphatase, Biochemistry, chemistry.chemical_compound, Raffinose, Ethers, Cyclic, Okadaic Acid, Autophagy, Phosphoprotein Phosphatases, Animals, Protein phosphorylation, Rats, Wistar, Protein kinase A, Oxazoles, Cytoskeleton, Protein kinase C, biology, Kinase, Acid phosphatase, Proteins, Okadaic acid, Rats, Calphostin C, Liver, chemistry, Vacuoles, biology.protein, Marine Toxins, Energy Metabolism, Lysosomes, Protein Kinases

الوصف: Autophagy, measured as the sequestration of electroinjected [3H]raffinose or endogenous lactate dehydrogenase, was inhibited in isolated rat hepatocytes by the protein phosphatase inhibitors okadaic acid, calyculin A and microcystin-LR. Okadaic acid, the most potent inhibitor, suppressed autophagy almost completely at 15 nM, suggesting inhibition of a protein phosphatase of type 2A. Okadaic acid had no effect on ATP levels, protein synthesis or cellular viability at this concentration, but caused a disruption of the hepatocytic cytoskeleton and a consequent reduction in organelle sedimentability, potentially interfering with the autophagy assay unless the necessary precautions are taken. Lysosomal (propylamine-sensitive) degradation of endogenous protein was inhibited by okadaic acid, whereas non-lysosomal (propylamine-resistant) degradation was unaffected. The autophagy-inhibitory effect of okadaic acid was not affected by inhibitors of cAMP-dependent protein kinase or protein kinase C (H-7, H-89, calphostin C) but eliminated by the non-specific inhibitor K-252a and its analogues (KT-5720, KT-5823, KT-5926) and by KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II. Protein phosphorylation by this kinase would thus seem to play a role in regulation of the autophagic-lysosomal degradation pathway.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::dc1d1d94c8521bff087f818b390221d3Test
https://doi.org/10.1111/j.1432-1033.1993.tb18013.xTest

المؤلفون: Daniel J. Klionsky, Ana Maria Cuervo, Per Ottar Seglen

المصدر: Autophagy. 3(3)

مصطلحات موضوعية: Microscopy, Extramural, Autophagy database, Autophagy, Cytological Techniques, Proteins, Cell Biology, Computational biology, Protein degradation, Biology, Biochemistry, Yeast, Cell biology, Proteins metabolism, Yeasts, Animals, Humans, Biological Assay, Microautophagy, Molecular Biology, Metabolic Networks and Pathways

الوصف: The increasing interest in autophagy in a wide range of organisms, accompanied by an ever-growing influx of researchers into this field, necessitates a good understanding of the methodologies available to monitor this process. In this review we discuss current approaches that can be used to follow the overall process of autophagy, as well as individual steps, from yeast to human. The majority of the review considers methods that apply to macroautophagy; however, we also consider alternative types of degradation including chaperone-mediated autophagy and microautophagy. This information is meant to provide a resource for newcomers as well as a stimulus for experienced researchers who may be prompted to develop additional assays to examine autophagy-related pathways.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::0a764ae3813665dfad26fc54b1fe4638Test
https://pubmed.ncbi.nlm.nih.gov/17224625Test

المؤلفون: Ingunn Holen, Per E. Stromhaug, Paul B. Gordon, Per Ottar Seglen

المصدر: European journal of biochemistry. 236(1)

مصطلحات موضوعية: Male, Myosin light-chain kinase, medicine.drug_class, Cell Survival, Biology, Protein degradation, Biochemistry, Adenylyl cyclase, chemistry.chemical_compound, medicine, Autophagy, Cyclic AMP, Animals, Enzyme Inhibitors, Rats, Wistar, Protein kinase A, Protein Kinase Inhibitors, Dose-Response Relationship, Drug, Kinase, Activator (genetics), Colforsin, Proteins, Protein kinase inhibitor, Glucagon, Cell biology, Rats, chemistry, Bucladesine, Liver

الوصف: To assess the role of cAMP in the regulation of autophagy, we examined the effects of cAMP analogues and cAMP-elevating agents on freshly isolated rat hepatocytes, using electroinjected [3H]raffinose as an autophagy probe. Glucagon was found to stimulate, inhibit or have no effect on autophagy, depending on the inclusion of metabolites like pyruvate (which caused ATP depletion and autophagy suppression) and amino acids (a complete mixture that antagonized pyruvate) in the incubation medium. Inhibition was also observed with theophylline, a cAMP-elevating inhibitor of cyclic nucleotide phosphodiesterases, and with the adenylyl cyclase activator deacetylforskolin. At low concentrations of deacetylforskolin, the inhibition could be abolished by amino acids. N6,2'-O-Dibutyryladenosine 3',5'-monophosphate (Bt2-cAMP) strongly inhibited both autophagic sequestration of [3H]raffinose and overall autophagic protein degradation; again, amino acids abolished the autophagy-inhibitory effect of low Bt2-cAMP concentrations. Several other cAMP analogues (8-thiomethyl-cAMP, N6-benzoyl-cAMP, (S)-5,6-dichloro-1-D-ribofuranosylbenzimidazole 3',5'-[thio]monophosphate, (S)-8-bromoadenosine 3',5'-[thio]monophosphate) inhibited autophagy as well. The effect of Bt2-cAMP was rapid, dose-dependent, reversible and did not require concomitant protein synthesis. Neither Bt2-cAMP nor deacetylforskolin reduced intracellular ATP levels or cell viability, ruling out inhibition of autophagy by non-specific cytotoxicity. The autophagy-inhibitory effect of Bt2-cAMP could be substantially antagonized (40-50%) by KT-5720, a specific inhibitor of the cAMP-dependent protein kinase A, and by the nonspecific protein kinase inhibitor K-252a. Somewhat surprisingly, KN-62 and KT-5926, allegedly specific inhibitors of Ca2+/calmodulin-dependent protein kinase II and myosin light chain kinase, respectively, were also Bt2-cAMP-antagonistic. These results suggest that cAMP regulates the early, sequestrational step of hepatocytic autophagy by a highly conditional, dual mechanism, inhibition being predominant under most conditions in freshly isolated hepatocytes, whereas stimulation reportedly predominates in vivo. The effect of cAMP is probably mediated by protein kinase A, but other protein kinases would appear to participate in the regulation of autophagic sequestration as well.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::9453c0ced96092ba476ea7e5dba979fdTest
https://pubmed.ncbi.nlm.nih.gov/8617261Test

المؤلفون: P. Bohley, Per Ottar Seglen

المصدر: Experientia. 48(2)

مصطلحات موضوعية: Vacuole, Biology, Protein degradation, Endoplasmic Reticulum, Models, Biological, Cellular and Molecular Neuroscience, Cyclic nucleotide, chemistry.chemical_compound, Lysosome, Endopeptidases, medicine, Animals, Protein phosphorylation, Molecular Biology, Pharmacology, Autophagy, Phosphodiesterase, Proteins, Cell Biology, Okadaic acid, Endocytosis, Cell biology, medicine.anatomical_structure, chemistry, Biochemistry, Vacuoles, Molecular Medicine, Lysosomes

الوصف: Autophagic degradation of cytoplasm (including protein, RNA etc.) is a non-selective bulk process, as indicated by ultrastructural evidence and by the similarity in autophagic sequestration rates of various cytosolic enzymes with different half-lives. The initial autophagic sequestration step, performed by a poorly-characterized organelle called a phagophore, is subject to feedback inhibition by purines and amino acids, the effect of the latter being potentiated by insulin and antagonized by glucagon. Epinephrine and other adrenergic agonists inhibit autophagic sequestration through a prazosin-sensitive alpha 1-adrenergic mechanism. The sequestration is also inhibited by cAMP and by protein phosphorylation as indicated by the effects of cyclic nucleotide analogues, phosphodiesterase inhibitors and okadaic acid. Asparagine specifically inhibits autophagic-lysosomal fusion without having any significant effects on autophagic sequestration, on intralysosomal degradation or on the endocytic pathway. Autophaged material that accumulates in prelysosomal vacuoles in the presence of asparagine is accessible to endocytosed enzymes, revealing the existence of an amphifunctional organelle, the amphisome. Evidence from several cell types suggests that endocytosis may be coupled to autophagy to a variable extent, and that the amphisome may play a central role as a collecting station for material destined for lysosomal degradation. Protein degradation can also take place in a 'salvage compartment' closely associated with the endoplasmic reticulum (ER). In this compartment unassembled protein chains are degraded by uncharacterized proteinases, while resident proteins return to the ER and assembled secretory and membrane proteins proceed through the Golgi apparatus. In the trans-Golgi network some proteins are proteolytically processed by Ca(2+)-dependent proteinases; furthermore, this compartment sorts proteins to lysosomes, various membrane domains, endosomes or secretory vesicles/granules. Processing of both endogenous and exogenous proteins can occur in endosomes, which may play a particularly important role in antigen processing and presentation. Proteins in endosomes or secretory compartments can either be exocytosed, or channeled to lysosomes for degradation. The switch mechanisms which decide between these options are subject to bioregulation by external agents (hormones and growth factors), and may play an important role in the control of protein uptake and secretion.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::b99692fb917e92f248fc64f350aaea75Test
https://pubmed.ncbi.nlm.nih.gov/1740188Test

المؤلفون: Per Ottar Seglen, Per E. Schwarze

المصدر: Experimental Cell Research. 157:15-28

مصطلحات موضوعية: Male, Sucrose, Cell Survival, Liver cytology, Cell, In Vitro Techniques, Biology, Protein degradation, Phagocytosis, Lysosome, Autophagy, medicine, Animals, Hepatectomy, Diethylnitrosamine, Amino Acids, Carcinogen, Adenosine Triphosphatases, Histocytochemistry, Adenine, Proteins, Rats, Inbred Strains, gamma-Glutamyltransferase, Cell Biology, 2-Acetylaminofluorene, Molecular biology, In vitro, Rats, Kinetics, Cell Transformation, Neoplastic, medicine.anatomical_structure, Liver, Biochemistry, Hepatocyte, Carcinogens, Lysosomes

الوصف: Sequential carcinogen treatment (diethylnitrosamine/partial hepatectomy followed by 2-acetylaminofluorene (2-AAF] induced multiple hepatocarcinomas in rats with 100% certainty within a year. Enzyme-altered lesions, i.e. gamma-glutamyltranspeptidase (GGT)-positive and/or ATPase-negative cell foci, were numerous already at 8 weeks, and suspensions of purified hepatocytes isolated (by collagenase perfusion) at this time contained 30-40% GGT-positive cells. These hepatocyte suspensions were markedly deficient with respect to autophagic protein degradation (in comparison with cell suspensions from normal rats), and the cells lost less protein and survived much better than normal hepatocytes in culture under conditions of amino acid deprivation (which activates the autophagic mechanism). The anabolic advantage of reduced autophagy may possibly contribute to the selective outgrowth of preneoplastic cells during the earliest stage of liver carcinogenesis. Inclusion of the autophagy inhibitor 3-methyladenine in the culture medium elevated the survival of normal hepatocytes up to the level seen with hepatocytes from carcinogen-treated animals, suggesting that protection of normal cells by autophagy suppression may be a potentially interesting therapeutic principle.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::f2caacc602ec25d6e976ab27de2c5c57Test
https://doi.org/10.1016/0014-4827Test(85)90148-x

المؤلفون: Bjørn Grinde, Per Ottar Seglen

المصدر: Biochimica et Biophysica Acta (BBA) - General Subjects. 676:43-50

مصطلحات موضوعية: Male, Stereochemistry, Glutamine, Phenylalanine, Biophysics, Biology, Protein degradation, Biochemistry, Structure-Activity Relationship, Leucine, Autophagy, Animals, Histidine, Asparagine, Amino Acids, Molecular Biology, chemistry.chemical_classification, Tryptophan, Proteins, Photo-reactive amino acid analog, Rats, Amino acid, Liver, chemistry

الوصف: Analogues and derivatives of six of the amino acids which most effectively inhibit protein degradation in isolated rat hepatocytes (leucine, asparagine, glutamine, histidine, phenylalanine and tryptophan) were investigated to see if they could antagonize or mimic the effect of the parent compound. No antagonists were found. Amino alcohols and amino acid amides tended to inhibit protein degradation strongly, apparently by a direct lysosomotropic effect as indicated by their ability to cause lysosomal vacuolation. Amino acid alkyl esters and dipeptides inhibited degradation to approximately the same extent as did their parent amino acids, possibly by being converted to free amino acids intracellularly. Of several leucine analogues tested, four (L-norleucine, L-norvaline, D-norleucine and L-allo-isoleucine) were found to be as effective as leucine in inhibiting protein degradation. None of the analogues had any effect on protein synthesis. Since leucine appears to play a unique role as a regulator of bulk autophagy in hepatocytes, the availability of active leucine agonists may help tj elucidate the biochemical mechanism for control of this important process.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::4ad0b925eef389ee278b49cac7dc7ad0Test
https://doi.org/10.1016/0304-4165Test(81)90007-6

المؤلفون: Helge Tolleshaug, Paul B. Gordon, Henrik Hoyvik, Per Ottar Seglen

المصدر: Biochemical Society Transactions. 13:1007-1010

مصطلحات موضوعية: Sucrose, Chemistry, Adenine, Autophagy, Proteins, Mitochondria, Liver, Protein degradation, Cell Fractionation, Biochemistry, Cell Compartmentation, Rats, Cell biology, Liver, Phagocytosis, Animals, Autolysis, Lysosomes

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::392aa28c27175787e5455cf8cba2dcccTest
https://doi.org/10.1042/bst0131007Test

المؤلفون: Paul B. Gordon, Per Ottar Seglen

المصدر: Archives of Biochemistry and Biophysics. 217:282-294

مصطلحات موضوعية: Male, Adenosine, Biophysics, Endogeny, Puromycin Aminonucleoside, Biology, Protein degradation, Biochemistry, chemistry.chemical_compound, Adenosine Triphosphate, medicine, Protein biosynthesis, Animals, Purine metabolism, Molecular Biology, chemistry.chemical_classification, Dose-Response Relationship, Drug, Mercaptopurine, Adenine, Autophagy, Proteins, Rats, Inbred Strains, Riboside, Rats, Amino acid, Liver, chemistry, Purines, Lysosomes, medicine.drug

الوصف: About 100 different purine derivatives and analogs were tested for their effect on protein synthesis and protein degradation in isolated rat hepatocytes. These included 6-aminopurines (adenine and adenosine analogs), 6-mercaptopurines, chloropurines, oxypurines, cytokinins, methylxanthines, methylindoles, benzimidazoles, and benzodiazepines. Most of the compounds were either inactive or inhibited protein synthesis as much as or more than they inhibited protein degradation. However, three methylated 6-aminopurines (3-methyladenine, 6-dimethylaminopurine riboside, and puromycin aminonucleoside) and four 6-mercaptopurines (6-methylmercaptopurine, 6-methylmercaptopurine riboside, 6-mercaptopurine riboside, and 2′,3′,5t - triacetyl-6-mercaptopurine riboside) had a markedly stronger effect on protein degradation than on synthesis, and might therefore be potentially useful as selective degradation inhibitors. None of the seven above-mentioned purines had any significant effect on the degradation of the exogenous protein, asialofetuin, and would therefore seem to selectively inhibit endogenous protein degradation. Since the degradation was not further affected by purines in the presence of amino acids or lysosomotropic amines, it is suggested that the purines exert their effect specifically upon the autophagic/lysosomal pathway. All the mercaptopurines significantly depressed cellular ATP levels, whereas the methylated aminopurines did not. For this reason, the latter are probably more useful as degradation inhibitors. 3-Methyladenine had no effect on protein synthesis at a concentration (5 m m ) which inhibited protein degradation by more than 60%, and may therefore be regarded as a highly specific inhibitor of autophagy.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::cbb3d1d32cf246940c91647e9d840220Test
https://doi.org/10.1016/0003-9861Test(82)90504-5

المؤلفون: Amy C. Munthe-Kaas, Mary Ann S. Dybedal, Per Ottar Seglen

المصدر: Experimental Cell Research. 155:121-128

مصطلحات موضوعية: Leupeptins, Proteolysis, Chronic lymphocytic leukemia, Lymphocyte, Endogeny, Protein degradation, Biology, chemistry.chemical_compound, Phagocytosis, Autophagy, medicine, Humans, Lymphocytes, Amino Acids, chemistry.chemical_classification, Propylamines, medicine.diagnostic_test, Adenine, Leupeptin, Proteins, Cell Biology, medicine.disease, Leukemia, Lymphoid, Neoplasm Proteins, Amino acid, medicine.anatomical_structure, chemistry, Biochemistry, Cytoplasm, Lysosomes

الوصف: 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.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::dc496afd1eb90b5bb6fc506dc9d3968eTest
https://doi.org/10.1016/0014-4827Test(84)90773-0