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F J Doherty

Publications and source records attributed to F J Doherty.

32 records · Page 2Linked to original sources
PubMed1989

Insoluble disulfide cross-linked polypeptides accumulate in the functionally compromised lysosomes of fibroblasts treated with the cysteine protease inhibitor E-64.

Mouse fibroblasts (3T3-L1 cells) accumulate pulse-labeled long-lived polypeptides in detergent- and salt-insoluble aggregates when chased in the presence of inhibitors of lysosomal cysteine cathepsins, including E-64. Proteins found in the detergent- and salt-insoluble fraction include polypeptides which are disulfide cross-linked. E-64-induced polypeptide aggregates cofractionate with lysosomal enzyme markers on density gradients and are found in multivesicular dense bodies which by electron microscopy appear to be engaged in microautophagy. The results are discussed in relation to the possible role of polypeptide aggregation in the sequestration or trapping of cytoplasmic proteins by the lysosomal system.

Animals↗
PubMed1989

Intermediate filament-ubiquitin diseases: implications for cell sanitization.

The molecular pathology of chronic degenerative disease is not understood. Generally there must be two related, but opposing, processes: the direct deleterious effects of the pathogenic insult which can be chemical or viral and a cellular cytoprotective response to the insult. We have recently shown that there is a previously unsuspected link between the intracellular inclusions seen in some major chronic degenerative diseases: the inclusions contain ubiquitin immunoreactivity. The conditions include Parkinson's disease, motor neurone disease, Alzheimer's disease and alcoholic liver disease as well as astrocytomas and a myopathy. Protein ubiquitination is considered a signal for extra-lysosomal protein degradation although ubiquitin-protein conjugation may have several other important functions. Intermediate filaments are a component of some of the inclusions in diseased cells; we have previously reported that they are involved in protein sequestration for degradation by lysosomally mediated autophagy. Therefore, intermediate-filament-containing ubiquitinated inclusions may be hallmarks of cellular attempts to eliminate pathogenic insults by activating protein degradation mechanisms. Ubiquitinated inclusions could also be a hallmark of viral infections: they are in polio-virus-infected anterior horn neurones and Epstein-Barr-transformed lymphoblastoid cells. Some of the clinical observations can be reproduced experimentally in tissue culture cells. The implications of the combined clinical and experimental observations for cell sanitization and protein catabolism will be discussed.

Alzheimer Disease↗
PubMed1989

Ubiquitin-protein conjugates: clinical and experimental findings.

Ubiquitin has been extensively studied as a protein which is a cofactor in extralysosomal protein degradation, particularly in reticulocyte lysates. Ubiquitin is also found conjugated to nuclear histones and surface receptors in somatic cells. Until recently the occurrence of stable cellular ubiquitin-protein conjugates in physiological and pathological states had not been considered and studied. Recently we have shown that ubiquitin-protein conjugate immunoreactivity is a clinical feature in several ostensibly unrelated chronic human degenerative diseases as well as in some viral diseases. The consistent observation is the occurrence of intracellular extralysosomal inclusions containing intermediate filaments and ubiquitin conjugates as determined by immunohistochemical methods. These diseases are therefore part of a family of intermediate filament-ubiquitin diseases. The involvement of intermediate filaments with ubiquitin, a protein of known significance in protein degradation, ties in with separate evidence for a close role between intermediate filaments and protein degradation. We have previously shown that intermediate filaments may be involved in protein sequestration for degradation in the lysosomal system. Clinical immunohistochemical observations suggest that elements of the lysosomal degradation system and the ubiquitin-dependent extralysosomal system are involved in the molecular pathogenesis of some diseases. To underpin these clinical observations, we have recently shown that ubiquitin-protein conjugates accumulate in lysosome-related multivesicular bodies in cells in which lysosomal degradation is impaired. This phenomenon may result from increased ubiquitin protein-conjugate formation in cells with a compromised lysosomal system followed by chance uptake into multivesicular bodies. Alternatively, ubiquitination may normally serve as a signal for protein uptake into the lysosomal system, ubiquitinated protein-conjugates may therefore accumulate in cells with a functionally impaired lysosomal system.

Cell Compartmentation↗
PubMed1988

A filamentous inclusion body within anterior horn neurones in motor neurone disease defined by immunocytochemical localisation of ubiquitin.

Using an immunocytochemical method to localise antibodies to ubiquitin, filamentous inclusion bodies were seen in spinal anterior horn neurones in cases of motor neurone disease (MND) but not in any control cases. These inclusion bodies appeared to be closely associated with classical Bunina bodies and immuno-electron microscopy suggested that they were based on arrays of straight 10-15 nm filaments together with some granular material. These observations link the protein ubiquitin with a chronic neurodegenerative disease and extend previous observations of a close association between filamentous inclusion bodies and ubiquitin. Ubiquitin-filament inclusions should be regarded as a new hallmark in the histological diagnosis of MND.

Adult↗
PubMed1987

A putative protein-sequestration site involving intermediate filaments for protein degradation by autophagy. Studies with microinjected purified glycolytic enzymes in 3T3-L1 cells.

Several glycolytic enzymes (lactate dehydrogenase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase) were radiolabelled by [125I]iodination, conjugation with 125I-labelled Bolton & Hunter reagent and reductive [3H]methylation, and their degradative rates after microinjection into 3T3-L1 cells compared with that of the extracellular protein bovine serum albumin. Although the albumin remains largely cytosolic in recipient cells, the glycolytic enzymes rapidly (less than 30 min) become insoluble, as measured by detergent and salt extractions. The microinjected glycolytic enzymes appear to form disulphide-linked aggregates, are found in a cell fraction rich in vimentin-containing intermediate filaments and histones (nuclear-intermediate-filament fraction), and are degraded slowly by a lysosomal mechanism, as judged by the effects of inhibitors (NH4Cl, leupeptin, 3-methyladenine). 125I-labelled bovine serum albumin appears to be degraded rapidly and non-lysosomally. Prolonged treatment (96 h) of cultured cells with leupeptin results in the accumulation of pulse-labelled ([35S]methionine for 24 h) endogenous cell proteins in the detergent-and salt-non-extractable residue, but NH4Cl and 3-methyladenine do not have this effect. The findings are in terms of the interpretation of experiments involving microinjection of proteins to study intracellular protein protein degradation by autophagy.

Autophagy↗
PubMed1986

Intracellular protein catabolism: evidence for sequestration of proteins into an intermediate-filament fraction before lysosomal degradation.

Iodinated [125I] glycolytic enzymes (lactate dehydrogenase, pyruvate kinase and glyceraldehyde phosphate dehydrogenase) and bovine serum albumin have been erythrocyte microinjected or scrape loaded into confluent 3T3-L1 cells. The glycolytic enzymes are rapidly (within 30 min) sequestered into a form which is inextractable with sequential treatments with digitonin, Triton X-100 and potassium iodide. Bovine serum albumin is readily extractable by digitonin. Glycolytic enzymes are degraded relatively slowly (t1/2's 124-330 h) by a lysosomal mechanism inhibitable by NH4Cl (50-91%), 3-methyl adenine (62-70%) and leupeptin (47-80%). Bovine serum albumin is degraded relatively rapidly (t1/2 17-20 h) by a non-lysosomal mechanism. Nycodenz density gradient fractionation of homogenates of 3T3-L1 cells after microinjection shows that [125I]-lactate dehydrogenase sediments in a dense fraction containing nuclei (DNA) and lacking cytosolic, lysosomal, plasma membrane or mitochondrial marker enzymes. The [125I]-enzyme is not released from this fraction by the sequential detergent/salt fractionation described above. Analysis of the detergent/salt inextractable residue by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and 2-mercaptoethanol shows nuclear histones and vimentin. [125I]-lactate dehydrogenase does not enter the polyacrylamide gel in the absence of 2-mercaptoethanol. Importantly endogenous proteins are found in the residue in the presence of leupeptin. The data suggest that sequestration of proteins into a nuclear-intermediate filament fraction may precede lysosomal degradation. Iodinated [125]-Sendai virus HN and F membrane glycoproteins in reconstituted Sendai virus envelope (RSVE) have been fused with growing HTC cells. HN and F are rapidly internalised (within 5 h) to assume a perinuclear distribution before lysosomal degradation. HN and F proteins are either extractable predominantly with triton X-100 or are inextractable by the treatments described above. Nycodenz fractionation of HTC cell homogenates after RSVE-cell fusion shows that HN and F sediment predominantly as a dense peak, enriched in DNA and free from the markers indicated above, and a less dense peak enriched in lysosomal marker enzymes. HN and F proteins translocate from the dense to the less dense peak and are degraded lysosomally (t1/2 av. 70 h). Leupeptin slows the translocation. The detergent/salt inextractable residue is enriched in nuclear histones and vimentin. The data suggest that Sendai viral membrane glycoproteins are sequestered into a nuclear-intermediate filament fraction before lysosomal degradation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Degradation of erythrocyte-microinjected and scrape-loaded homologous cytosolic proteins by 3T3-L1 cells.

Homologous cytosol was introduced into 3T3-L1 cells by two different methods. Erythrocytes loaded with radiolabelled cytosolic proteins extracted from 3T3-L1 cells were fused with the aid of Sendai virus to 3T3-L1 cells, which were then seeded to confluent and non-confluent cultures. Cytosolic proteins were also introduced into cells by the technique of scrape-loading. In confluent cells, injected cytosolic proteins were recovered largely (54-93%) in a sedimentable (6 X 10(6) gav.-min) fraction from recipient cells irrespective of the method of introduction or of radiolabelling of the injected proteins [( 125I]iodination, reductive methylation with NaB3H4 and backbone labelling with L-[4,5-3H]leucine). The degradation of microinjected cytosolic proteins was in all cases inhibited by the lysosomotropic agent NH4Cl to a greater extent (32-75%) than that observed for endogenous cytosolic (less than or equal to 19%) proteins (labelled with L-[4,5-3H]leucine). In growing cells both endogenous total cell proteins and microinjected proteins were degraded at a slower rate than in confluent cell monolayers. The inhibition by NH4Cl of the degradation of both the endogenous and microinjected proteins is decreased compared with the inhibition observed in confluent monolayers. The results are discussed in terms of the cytoplasmic capacity to segregate microinjected homologous proteins before protein degradation can take place.

Ammonium Chloride↗
PubMed1980

The intracellular location of a Ca2+-stimulated phospholipase A1 in rat brain.

Rat brain contains a phospholipase A1 which hydrolyzes phosphatidylethanolamine with a pH optimum of 9.5. The enzyme is stimulated by CaCl2 and inhibited by sodium taurocholate and Triton X-100. The subcellular distibution of the enzyme is similar to that of lactate dehydrogenase indicating the location of the phospholipase in the soluble cytoplasm in vitro.

Animals↗
PubMedDate not supplied

The possible biological and reproductive functions of ubiquitin.

The protein ubiquitin (Ub) appears to be present in all eukaryotic cells. Its widespread presence and extremely conserved structure indicate that it may play a vital role in cell metabolism. The roles of Ub are mediated by its covalent attachment to target proteins, a process known as ubiquitylation, a form of protein modification which may lead to degradation of the modified protein. A number of proteins with similar structure to Ub but varying in function have been isolated. Recently, there has been much interest in the role of Ub and its related proteins in reproductive processes. Ub and Ub-related proteins may be involved in gametogenesis, modulation of steroid receptor concentrations, placental development and endometrial modification at the beginning of pregnancy. These wide-ranging effects have led to extensive research which will be reviewed in this article.

Animals↗