Ubiquitin and its role in neurodegeneration.
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Amino Acid Sequence↗
Biomedical subjects
M Landon
Publications and source records attributed to M Landon.
Prion protein fragments spanning helix 1 and both strands of beta sheet (residues 125-170) show evidence for predominantly helical propensity by CD and NMR.
BACKGROUND: Transmissible spongiform encephalopathies are a group of neurodegenerative disorders of man and animals that are believed to be caused by an alpha-helical to beta-sheet conformational change in the prion protein, PrP. Recently determined NMR structures of recombinant PrP (residues 121-231 and 90-231) have identified a short two-stranded anti-parallel beta sheet in the normal cellular form of the protein (PrPC). This beta sheet has been suggested to be involved in seeding the conformational transition to the disease-associated form (PrPSc) via a partially unfolded intermediate state. RESULTS: We describe CD and NMR studies of three peptides (125-170, 142-170 and 156-170) that span the beta-sheet and helix 1 region of PrP, forming a large part of the putative PrPSc-PrPC binding site that has been proposed to be important for self-seeding replication of PrPSc. The data suggest that all three peptides in water have predominantly helical propensities, which are enhanced in aqueous methanol (as judged by deviations from random-coil Halpha chemical shifts and 3JHalpha-NH values). Although the helical propensity is most marked in the region corresponding to helix 1 (144-154), it is also apparent for residues spanning the two beta-strand sequences. CONCLUSIONS: We have attempted to model the conformational properties of a partially unfolded state of PrP using peptide fragments spanning the region 125-170. We find no evidence in the sequence for any intrinsic conformational preference for the formation of extended beta-like structure that might be involved in promoting the PrPC-PrPSc conformational transition.
Animals↗
Ubiquitin superfolds: intrinsic and attachable regulators of cellular activities?
Ubiquitinylation, the post-translational covalent conjugation of ubiquitin to other proteins, mediates diverse cellular processes in addition to the proteasome-catalysed degradation signalled by multiple ubiquitinylation. Ubiquitin superfolds have also been found in other proteins. The amino acid sequences of these superfolds are unrelated to ubiquitin, but they have an almost identical three-dimensional shape to that of ubiquitin. Additionally, a number of 'ubiquitin-like' proteins, some of which can be conjugated to other proteins, may also contain the ubiquitin superfold. Intrinsic and attachable ubiquitin superfolds can act as powerful ligands and probably have important roles in protein-protein interactions in the cell.
Protein Folding↗
Application of formalin fixation to the purification of amyloid proteins.
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Aged↗
Capillary electrophoresis assay for ubiquitin carboxyl-terminal hydrolases with chemically synthesized ubiquitin-valine as substrate.
Ubiquitin is expressed in eukaryotic cells as precursors, fused via its carboxyl terminus either to other ubiquitin sequences in linear polyubiquitin arrays or to specific ribosomal proteins. In some of the polyubiquitin fusions a single amino acid (e.g., valine in humans) is attached to the carboxyl terminus. These gene products are rapidly (probably cotranslationally) cleaved by ubiquitin carboxyl-terminal hydrolase (UCH) enzymes; therefore, although ubiquitin precursors are suitable substrates for assays of UCH activity, they are difficult to isolate from nucleated cells. While the recombinant approach allows the production of ubiquitin precursors in prokaryotic cells (which do not contain the ubiquitin system), proteins produced in this manner require purification and may also be susceptible to modification by bacterial enzymes, e.g., adventitious proteolysis. As an alternative we have chemically synthesized human ubiquitin-valine. In the assay described here the cleavage of ubiquitin-valine to ubiquitin (77 and 76 residue proteins, respectively) by a purified recombinant Drosophila UCH was monitored by capillary electrophoresis. Mass spectrometry verified the precise cleavage of ubiquitin-valine, confirming that this synthetic protein is a UCH substrate. Synthetic ubiquitin-valine may serve as a generic substrate for UCHs allowing the purification and identification of new members of this enzyme family.
Animals↗
Cancer incidence and mortality around the Pan Britannica Industries pesticide factory, Waltham Abbey.
OBJECTIVES: To examine the incidence and mortality of cancer near the Pan Britannica Industries factory, Waltham Abbey, after reports of a possible cluster of all cancers and brain cancer in the vicinity. METHOD: Small area study of cancer incidence 1977-89, and mortality 1981-92, within a 7.5 km radius of the factory site. Postcoded cancer registrations and deaths in the study area were extracted from national data sets held by the Small Area Health Statistics Unit and compared with expected numbers computed by applying national rates stratified for age, sex, and deprivation to the local population (1981 and 1991 censuses). Observed/ expected (O/E) ratios were examined from 0-1 km and 0-7.5 km of the plant, and tests applied for a decline in relative risk with distance up to 7.5 km. RESULTS: There were 12,859 incidence cancers (1977-89) from 0-7.5 km (O/E ratio 1.04; 95% confidence interval (95% CI) 1.02 to 1.06) and 385 from 0-1 km (O/E 1.10; 1.00 to 1.22). There was an excess of skin melanoma from 0-1 km based on 11 cases (O/E 2.13; 1.06 to 3.80), and an excess from 0-7.5 km of cancer of the lung, stomach and pancreas combined, and prostate (O/Es ranged from 1.09 to 1.13). Only the findings from lung cancer were suggestive of a decline in risk with distance, especially in the later period (1982-9). There were 9196 cancer deaths (1981-92) from 0-7.5 km (O/E 1.04; 95% CI 1.02 to 1.06) and 308 from 0-1 km (O/E 1.24; 1.11 to 1.39); and 25507 non-cancer deaths (O/E 1.02; 1.01 to 1.04) from 0-7.5 km and 745 (O/E 1.14; 1.06 to 1.22) from 0-1 km. There was evidence of a decline in mortality with distance for all cancers combined, lung cancer (P = 0.001 for each), and colorectal cancer (P < 0.05), and also for non-cancers (P = 0.001). Proportional mortality analyses suggested a decline in risk with distance for lung cancer (P = 0.003) but not for all cancers or the site specific cancers examined. There was no evidence of an excess in the incidence or mortality from brain cancer. For cancer mortality in the inner-most wards, the findings were, for the most part, well within the range of variation across the region as a whole. CONCLUSIONS: The study provides limited and inconsistent evidence for a localised excess of cancer in the vicinity of the PBI plant. At present, further investigation does not seem warranted other than continued surveillance of mortality and cancer incidence in the locality.
Adolescent↗
Pathological lesions of Alzheimer's disease and dementia with Lewy bodies brains exhibit immunoreactivity to an ATPase that is a regulatory subunit of the 26S proteasome.
MS73 is one of a family of ATPases that act as regulatory subunits of the 26S proteasome. Localisation of this ATPase in histological sections of hippocampus from Alzheimer's disease (AD) and in cingulate gyrus sections of dementia with Lewy bodies (DLB) brains was examined immunohistochemically. In all cases of AD (n = 10) neurofibrillary tangles (NFT), plaque neurites and neuropil threads were immunoreactive for MS73. In seven out of the nine cases of DLB, distinctive MS73-positive structures were detected within cortical Lewy bodies. The association of MS73 with these neuronal abnormalities provides further evidence that proteolytic processing involving the 26S proteasome occurs in lesions of AD and DLB.
Adenosine Triphosphatases↗
Neurofibrillary tangles of Alzheimer's disease brains contain 14-3-3 proteins.
The localisation of 14-3-3 proteins compared to that of tau and ubiquitin-protein conjugates in sections of hippocampus from Alzheimer's disease (AD) brains was examined by immunohistochemistry. In all cases (n = 10), anti-14-3-3 stained a proportion of neurofibrillary tangles (NFT). In general, NFT stained by anti-14-3-3 were smaller than those stained by anti-tau or anti-ubiquitin-protein conjugates and were more confined to the neuronal cell body. Occasionally, cortical Lewy bodies in cases of Lewy body dementia were also found to be 14-3-3-positive. Since 14-3-3 proteins are central to MAP kinase signalling, the results support the proposal that this pathway is in part responsible for the hyperphosphorylation of tau, which leads to the formation of the paired helical filaments seen in AD brains.
14-3-3 Proteins↗
Extraction and protein sequencing of immunoglobulin light chain from formalin-fixed cerebrovascular amyloid deposits.
Substantial amounts of a single protein have been extracted into electrophoresis sample buffer from archived formalin-fixed brain blood vessels, taken from a case of cerebral amyloidosis. Cyanogen bromide cleavage and tryptic digestion of the protein on Western blots allowed amino acid sequences from three resultant peptides to be determined. Comparison of these peptides with database sequences identified the extracted protein as being derived from an immunoglobulin light chain. This is the first demonstration of amino acid sequencing of a polypeptide extracted from formalin-fixed tissue. This case also appears to be unique, since primary cerebrovascular amyloidosis involving immunoglobulin light chains has not been previously described. The amyloid protein had clearly resisted formalin fixation; it is possible that this resistance occurred because the protein was deposited in large amounts as insoluble densely packed aggregates, which may exclude infiltration of the formalin. This technique may therefore have applications in the post-mortem diagnosis of amyloidoses and in the purification of other amyloids.
Adult↗
Endosome-lysosomes, ubiquitin and neurodegeneration.
Before the advent of ubiquitin immunochemistry and immunogold electron microscopy, there was no known intracellular molecular commonality between neurodegenerative diseases. The application of antibodies which primarily detect ubiquitin protein conjugates has shown that all of the human and animal idiopathic and transmissible chronic neurodegenerative diseases, (including Alzheimer's disease (AD), Lewy body disease (LBD), amyotrophic lateral sclerosis (ALS), Creutzfeldt-Jakob disease (CJD) and scrapie) are related by some form of intraneuronal inclusion which contains ubiquitin protein conjugates. In addition, disorders such as Alzheimer's disease, CJD and sheep scrapie, are characterised by deposits of amyloid, arising through incomplete breakdown of membrane proteins which may be associated with cytoskeletal reorganisation. Although our knowledge about these diseases is increasing, they remain largely untreatable. Recently, attention has focused on the mechanisms of production of different types of amyloid and the likely involvement within cells of the endosome-lysosome system, organelles which are immuno-positive for ubiquitin protein conjugates. These organelles may be 'bioreactor' sites for the unfolding and partial degradation of membrane proteins to generate the amyloid materials or their precursors which subsequently become expelled from the cell, or are released from dead cells, and accumulate as pathological entities. Such common features of the disease processes give new direction to therapeutic intervention.
Animals↗
Heat shock proteins, molecular chaperones and the prion encephalopathies.
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Alzheimer Disease↗
The abnormal isoform of the prion protein accumulates in late-endosome-like organelles in scrapie-infected mouse brain.
The prion encephalopathies are characterized by accumulation in the brain of the abnormal form PrPsc of a normal host gene product PrPc. The mechanism and site of formation of PrPsc from PrPc are currently unknown. In this study, ME7 scrapie-infected mouse brain was used to show, both biochemically and by double-labelled immunogold electron microscopy, that proteinase K-resistant PrPsc is enriched in subcellular structures which contain the cation-independent mannose 6-phosphate receptor, ubiquitin-protein conjugates, beta-glucuronidase, and cathepsin B, termed late endosome-like organelles. The glycosylinositol phospholipid membrane-anchored PrPc will enter such compartment for normal degradation and the organelles may therefore act as chambers for the conversion of PrPc into infectious PrPsc in this murine model of scrapie.
Animals↗
Immunohistochemical localization of ubiquitin cross-reactive protein in human tissues.
Ubiquitin cross-reactive protein (UCRP) is an interferon-inducible ubiquitin homologue which is constitutively present in cells and can be conjugated to other proteins. Using a characterized polyclonal antiserum to UCRP, immunohistochemical localization of UCRP was performed on paraffin-processed normal human tissues and in human tissues known to contain ubiquitinated intracellular inclusions. The antibody to UCRP immunostained lymphoid cells, striated and smooth muscle, several epithelia, and neurons. The level of staining varied greatly between tissues but was in a consistent punctate pattern. Localization to neuromuscular junctions and striations is similar to that described for antisera to ubiquitin-protein conjugates. Inclusion bodies characterized by immunoreactivity to anti-ubiquitin were not detected by the antibody to UCRP. Importantly, because UCRP may also be detected by antisera to conjugated ubiquitin, future studies on the distribution of ubiquitin in tissue sections must now take account of possible cross-reactivity with UCRP.
Cross Reactions↗
The expression of alpha B-crystallin in epithelial tumours: a useful tumour marker?
Alpha B-crystallin is a lens protein showing homology with small heat-shock proteins. We have previously demonstrated its expression in non-lenticular normal and diseased human tissues by immunostaining with a polyclonal antibody. In view of the expression seen in normal renal tubular epithelium, we have assessed the immunoreactivity of a variety of epithelial tumours, to determine the usefulness of alpha B-crystallin as a specific renal tumour marker. Carcinomas arising from tissues which normally express alpha B-crystallin, such as colo-rectal and thyroid carcinomas, showed a varying pattern and degree of immunoreactivity. The most consistently positive tumours, however, with typically strong cytoplasmic and cell membrane staining, were renal cell carcinomas, 90 per cent of which showed positive immunoreactivity. This pattern of staining, while not absolutely specific, is a useful aid to the diagnosis of renal carcinoma. When a metastic deposit or a small biopsy is being assessed, anti-alpha B-crystallin may be included in a panel of antibodies, the pattern of staining of which may direct the search for the primary site of the tumour.
Biomarkers, Tumor↗
Endosome-lysosomes and neurodegeneration.
A number of the major human and animal neurodegenerative diseases, such as Alzheimer's disease and sheep scrapie, are characterised by deposits of amyloid, arising through incomplete breakdown of membrane proteins. Although our knowledge concerning these diseases is increasing, they remain largely untreatable. Recently, attention has focussed on the mechanisms of production of different types of amyloid and the likely involvement within cells of acid compartments called endosome-lysosomes. These organelles may be 'bioreactor' sites for the unfolding and partial degradation of membrane proteins to generate the amyloid materials. These subsequently become expelled from the cell, or are released from dead cells, and accumulate as pathological entities. Common features of the disease processes give new direction to therapeutic intervention.
Alzheimer Disease↗
Is there a common intracellular bioreactor in which amyloid formation is initiated in neurodegenerative diseases?
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Alzheimer Disease↗
Expression of polyubiquitin and heat-shock protein 70 genes increases in the later stages of disease progression in scrapie-infected mouse brain.
We have shown by northern analyses that the expression of the mouse polyubiquitin C gene is increased severalfold in the brains of mice infected with both the ME7 and 87V strains of scrapie. Expression of the polyubiquitin gene does not change significantly, compared with controls, until the later stages of disease progression when there is a 2.5-fold increase in ME7-infected brains and a 1.8-fold increase in 87V-infected brains. The patterns of changes of expression of the polyubiquitin genes in brains infected with the two strains of scrapie resemble those of accumulation of ubiquitin-conjugate-positive structures in the brain that are detected immunohistochemically. A similar increase in the expression of a heat-shock protein 70 gene also occurs.
Animals↗
Carbon Source Requirements for Exopolysaccharide Production by Lactobacillus casei CG11 and Partial Structure Analysis of the Polymer.
Exopolysaccharide production by Lactobacillus casei CG11 was studied in basal minimum medium containing various carbon sources (galactose, glucose, lactose, sucrose, maltose, melibiose) at concentrations of 2, 5, 10, and 20 g/liter. L. casei CG11 produced exopolysaccharides in basal minimum medium containing each of the sugars tested; lactose and galactose were the poorest carbon sources, and glucose was by far the most efficient carbon source. Sugar concentrations had a marked effect on polymer yield. Plasmid-cured Muc derivatives grew better in the presence of glucose and attained slightly higher populations than the wild-type strain. The values obtained with lactose were considerably lower for both growth and exopolysaccharide yield. The level of specific polymer production per cell obtained with glucose was distinctively lower for Muc derivatives than for the Muc strain. The polymer produced by L. casei CG11 in the presence of glucose was different from that formed in the presence of lactose. The polysaccharide produced by L. casei CG11 in basal minimum medium containing 20 g of glucose per liter had an intrinsic viscosity of 1.13 dl/g. It was rich in glucose (76%), which was present mostly as 2- or 3-linked residues along with some 2,3 doubly substituted glucose units, and in rhamnose (21%), which was present as 2-linked or terminal rhamnose; traces of mannose and galactose were also present.
Journal Article↗