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Results for “Protein Aggregation, Pathological”

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PubMed2006

Peroxynitrite-mediated tau modifications stabilize preformed filaments and destabilize microtubules through distinct mechanisms.

Alzheimer's disease (AD) is a progressive amnestic dementia typified by abnormal modifications of the microtubule (MT)-associated tau protein that promote its pathological self-assembly and displacement from the MT lattice. Previously, we showed that peroxynitrite (ONOO-) induces the oxidative 3,3'-dityrosine (3,3'-DT) cross-linking and site-selective nitration of tau monomers [Reynolds et al. (2005) Biochemistry 44, 1690-1700]. In the present study, we examined the effects of ONOO(-)-mediated modifications on two key elements of tau pathobiology: (1) the stability of preformed tau filaments and (2) the ability of monomeric tau to promote tubulin assembly. Here, we report that treatment of synthetic tau filaments with ONOO- generates heat-stable, SDS-insoluble aggregates with a significantly reduced mobility by SDS-PAGE compared to that of nontreated filaments. Ultrastructurally, these aggregates appear to be cross-linked via interfilament bridges. Using LC-MS/MS and HPLC with fluorescent detection, we demonstrate that covalent 3,3'-DT linkages are present within these higher-order aggregates. Similar to monomeric tau, filamentous tau exhibits a hierarchical pattern of nitration following ONOO- treatment with site selectivity toward the amino-terminal residues Tyr18 and Tyr29. Further, select nitration of residues Tyr18, Tyr29, Tyr197, and Tyr394, events known to stabilize the pathological Alz-50 conformation [Reynolds et al. (2005) Biochemistry 44, 13997-14009], inhibits the ability of monomeric tau to promote tubulin assembly. This effect is specific for the 3-NT modification, as mutant tau proteins pseudophosphorylated at each Tyr residue are fully competent to stabilize MTs. Collectively, our results suggest that ONOO(-)-mediated modifications stabilize tau filaments via 3,3'-DT bonding and destabilize MTs by site-selective nitration of tau monomers. Moreover, assumption of the Alz-50 conformation may be the mechanism through which tau nitration modulates MT stability.

Amino Acid Sequence↗

PubMed2005

The stationary phase model of aging in yeast for the study of oxidative stress and age-related neurodegeneration.

Understanding the biochemical and genetic alterations that occur during the aging of post-mitotic cells is critical for understanding the etiology of abnormalities observed during the aging of the central nervous system (CNS). While many theories for cellular aging exist, the free radical theory of aging has proved useful in explaining multiple aspects of post-mitotic cell aging, including the aging of neuronal cells. It is well established that Saccharomyces cerevisiae are an invaluable model system for exploring the regulation of aging in actively dividing cells, but increasing evidence suggests that the chronological lifespan or stationary phase model of aging in S. cerevisiae may also be useful for understanding the aging process in post-mitotic cells. Interestingly, the stationary phase model of aging in S. cerevisiae recapitulates many pathological alterations observed during neuronal aging, including evidence for increased oxidative stress and proteasome inhibition. Studies using proteins relevant to multiple neurodegenerative conditions (prion, alpha-synuclein, huntingtin) have demonstrated the utility of S. cerevisiae as a model system for understanding the genetic regulation of protein aggregation and cell death. Taken together, these data highlight the potential importance of using S. cerevisiae as a model system with which to explore the molecular basis for neuronal alterations observed in normal brain aging as well as multiple age-related diseases of the CNS.

Aging↗

PubMed2005

Effect of vein graft harvesting on endothelial nitric oxide synthase and nitric oxide production.

BACKGROUND: Although the saphenous vein is the most commonly used conduit for coronary artery bypass surgery occlusion rates are high, with more than 50% grafts failing within 10 years. Nitric oxide, a potent vasodilator, also inhibits platelet aggregation, thrombus formation and vascular smooth muscle cell proliferation, is implicated in various vascular pathologies, including graft failure. METHODS: Saphenous veins were obtained from patients undergoing bypass surgery harvested by conventional methods and with minimal handling, using a "no-touch" technique. Tissue distribution and protein expression of endothelial nitric oxide synthase was compared using immunohistochemistry and Western blot analysis. Nitric oxide generation was assessed using the citrulline assay. RESULTS: There was injury to conventional compared with no-touch vein segments, in particular to the lumenal endothelium and tunica adventitia. This injury was accompanied by an absence of endothelial nitric oxide synthase immunostaining at regions of endothelial denudation and damaged adventitial layer of conventional veins and a significant reduction (p < 0.05) in endothelial nitric oxide synthase protein expression. Furthermore, nitric oxide release from conventional tissue extracts was significantly (p < 0.05) lower than no-touch vein segments. CONCLUSIONS: Our results show that there is a reduction in endothelial nitric oxide synthase and nitric oxide release in saphenous veins harvested by conventional surgical methods compared with those prepared atraumatically. These observations may influence graft performance.

Aged↗

PubMed2000

Induction of neuronal death by alpha-synuclein.

The molecular and cellular mechanisms underlying neuronal loss in neurodegenerative diseases are unclear. It is generally thought that aggregation of mutated, abnormally modified or abnormally folded proteins leads to the accumulation of extracellular, intracellular or intranuclear deposits that severely compromise cell physiology, leading to the death of the affected neurons. However, there is growing evidence that neuronal apoptosis in the absence of obvious pathological deposits could have a serious impact on the pathogenesis of neurodegenerative diseases. alpha-Synuclein has been implicated in aetiology and pathogenesis of certain neurodegenerative diseases, although the precise role of this protein in neurodegeneration is uncertain. The normal functions of alpha-synuclein and other members of the synuclein family in the development and function of the nervous system also remain elusive. Here we show that overexpression of wild-type and mutant forms of alpha-synuclein in cultured neurons, but not the closely related persyn (gamma-synuclein), causes apoptosis. These findings suggest that abnormalities of alpha-synuclein metabolism could lead to the neuronal loss occurring in certain forms of neurodegeneration before the formation of characteristic pathological lesions.

Amino Acid Sequence↗

PubMed2005

Biological significance of isoaspartate and its repair system.

Isomerization of L-aspartate and deamidation of L-asparagine in proteins or peptides dominantly give rise to L-isoaspartate by a non-enzymatic reaction via succinimide as a intermediate under physiological conditions. Isoaspartates have been identified in a variety of cellular proteins in vivo as well as pathologically deposited proteins in neurodegenerative brain tissue. We described here that the formation of isoaspartate is enhanced in amyloid-beta (Abeta) peptides in Alzheimer's disease (AD). Specific antibodies recognizing isoaspartate of Abeta revealed that isomerized Abeta peptides were deposited in senile plaques as well as amyloid-bearing vessels. Moreover, it was revealed that Abeta peptides, isomerized at position 7 or 23, were differentially deposited in senile plaques and vascular amyloids in AD brains. In vitro experiments showed that the modification at position 23 greatly enhanced the aggregation of Abeta. Furthermore, systematic proline substitution analyses revealed that the beta-turn structure at positions 22 and 23 of Abeta42 plays a crucial role in the aggregation and neurotoxicity of Abeta peptides. It is suggested that spontaneous isomerization at position 23 induces the conformational change to form a beta-turn at position 23, which plays a pathogenic role in the deposition of Abeta peptides in sporadic AD. Protein L-isoaspartyl methyltransferase (PIMT) is a putative protein repair enzyme, which converts L-isoaspartyl residues in damaged proteins to normal L-aspartyl residues. PIMT-deficient mice manifested neurodegenerative changes concomitant with the accumulation of L-isoaspartate in the brain. We discuss here the pathological implications of the formation of isoaspartate in damaged proteins during neurodegeneration in model mice and AD.

Alzheimer Disease↗

PubMed2006

Parkinson's disease dementia: what's in a Lewy body?

This brief review deals with pathological aspects of dementia associated with Parkinson's disease (PDD). PDD has been variably linked with cortical Lewy body topography and density. alpha-Synuclein and Alzheimer-type pathology frequently co-exist, suggesting that a combination of pathology related to protein dysmetabolism, possibly with synergistic protein-protein interaction, underpins the cognitive impairment in PDD. Dementia may therefore ensue when a "toxic threshold" is reached, irrespective of the combination of pathologies involved in reaching that threshold. The nature of this putative protein-protein interaction needs to be further elucidated, and also whether there are specific clinical correlates of the pathological substrate. Serum and cerebrospinal fluid proteins or imaging techniques may be useful in future as biomarkers to identify the relative contribution of Lewy-related and Alzheimer-type pathology in a given case of PDD and to inform the rational use of drugs that can reduce alpha-synuclein aggregation and beta-amyloid production.

Cerebral Cortex↗

PubMed1984

A morphologic overview of the porcine bioprosthetic valve--before and after its degeneration.

Patients with valvular heart disease have had their diseased, natural heart valves replaced with the porcine aortic bioprosthetic valve (Hancock type) at Henry Ford Hospital since 1971. This commercially available valvular bioprosthesis is characterized by 1) loss of endothelium from both surfaces of the leaflets, 2) modification in the organization of the fibrous connective tissue of the leaflet, and 3) a reduction in the macromolecular complexes (proteoglycan) of the extracellular matrix. Subendothelial components are therefore exposed to the circulating blood. This bioprosthetic heart valve undergoes degeneration after it is inserted into patients for various lengths of time. Prominent features of the degenerated porcine bioprosthetic valve include 1) penetration of plasma proteins into the interior of the leaflet, 2) adhesion of various types of leukocytes (mononuclear cells and granulocytes) to the surfaces of the leaflets, 3) deposition of single platelets, platelet aggregates and microthrombi onto the leaflet's surface, 4) destruction of collagen fibers, and 5) pathologic deposition of calcium salts. The mechanisms responsible for the degeneration of the porcine aortic bioprosthetic valve are incompletely understood at this time.

Animals↗

PubMedDate not supplied

Buerger's disease and protein S deficiency: successful treatment with prostacyclin.

In this report we describe a female patient who presented with peripheral circulatory impairment. She had previously been thought to have a systemic vasculitis, but had failed to respond to corticosteroid and immunosuppressive therapy. Serologic investigations were negative. The patient was a very heavy smoker, and it became clear that her condition had many of the clinical features of Buerger's disease (thrombangiitis obliterans). The diagnosis was confirmed by angiography, and a coagulation screen showed her to have associated protein S deficiency. Her symptoms responded rapidly to cessation of smoking, warfarin therapy, and a series of intravenous prostacyclin infusions. We discuss in detail the possible pathologic mechanisms which might explain the association of protein S deficiency and thrombangiitis obliterans.

Adult↗

PubMed1999

Subfield- and layer-specific changes in parvalbumin, calretinin and calbindin-D28K immunoreactivity in the entorhinal cortex in Alzheimer's disease.

The entorhinal cortex, which is involved in neural systems related to memory, is selectively degenerated in early Alzheimer's disease. Here, we examined neuropathological changes in the eight entorhinal subfields in post mortem Alzheimer's disease subjects using Thionin and Bielschowsky stains and parvalbumin, calretinin and calbindin-D28k immunohistochemistry. Both histological stains revealed the most dramatic cell loss and neurofibrillary tangle formation to be in layers II and V of the lateral, intermediate and caudal subfields. In accordance, immunohistochemical staining showed that neurons and fibres that contain calcium-binding proteins were also more frequently altered in these subfields than in the rostromedial subfields. Detailed analysis further revealed that non-principal cells containing parvalbumin or calbindin-D28k showed morphological alterations early in the entorhinal pathology of Alzheimer's disease, whereas non-principal neurons containing calretinin were better preserved even in Alzheimer's disease patients with severe entorhinal pathology. The degeneration of parvalbumin-immunoreactive neurons and basket-like networks and calbindin-positive non-principal neurons was observed mainly in layer II, where the calretinin-positive non-principal neurons formed aggregates especially at late stages of the disease. The pyramidal-shaped neurons containing either calretinin or calbindin-D28k were often preserved, although morphological alterations were observed. Our findings indicate that specific subfields of the entorhinal cortex involving neurons that contain distinct calcium-binding proteins are differentially vulnerable in Alzheimer's disease. This could have an impact on the topographically organized inputs and outputs of the entorhinal cortex in Alzheimer's patients.

Aged↗

PubMed1995

Alcoholic liver disease: molecular-pathologic aspects.

Mallory bodies (MBs) are characteristic morphologic features of alcoholic hepatitis but are also associated with non-alcoholic liver diseases including long lasting cholestasis, metabolic and neoplastic disorders. MBs contain in addition to keratins non-keratin components, including microtubule-associated (tau protein) and other not yet characterized proteins in an aggregated form. Aggregation of these components in the cell is promoted by posttranslational modifications, such as partial proteolysis, phosphorylation and cross-linking, and may result in functional and structural disturbances of the cell depending on the physiologic function of the components involved. Several enzymes responsible for these modifications are Ca(++)-dependent. Thus, disturbance of Ca(++)-homeostasis may play an essential role in the pathogenesis of MBs. In some structural aspects MBs closely resemble inclusions associated with degenerative disorders of the central nervous system, including Alzheimer's and Parkinson's disease. Studies on the pathogenesis of MBs, therefore, not only shed light on a peculiar type of liver cell injury but may also assist in the understanding of other chronic degenerative diseases, particularly those of the central nervous system.

Alzheimer Disease↗

PubMed2006

Tau-tubulin kinase 1 (TTBK1), a neuron-specific tau kinase candidate, is involved in tau phosphorylation and aggregation.

Neurofibrillary tangles, which are major pathological hallmarks of Alzheimer's disease (AD), are composed of paired helical filaments (PHFs) containing hyperphosphorylated tau. Specific kinases regulate tau phosphorylation and are closely linked to the pathogenesis of AD. We have characterized a human tau-tubulin kinase 1 (TTBK1) gene located on chromosome 6p21.1. TTBK1 is a serine/threonine/tyrosine kinase that is conserved among species and belongs to the casein kinase 1 superfamily. It is specifically expressed in the brain, especially in the cytoplasm of cortical and hippocampal neurons. TTBK1 phosphorylates tau proteins in both a Mg2+- and a Mn2+-dependent manner. Phosphopeptide mapping and immunoblotting analysis confirmed a direct tau phosphorylation by TTBK1 at Ser198, Ser199, Ser202 and Ser422, which are also phosphorylated in PHFs. TTBK1 also induces tau aggregation in human neuronal cells in a dose-dependent manner. We conclude that TTBK1 is a neuron-specific dual kinase involved in tau phosphorylation at AD-related sites and is also associated with tau aggregation.

Animals↗

PubMed2002

Aberrant tau phosphorylation by glycogen synthase kinase-3beta and JNK3 induces oligomeric tau fibrils in COS-7 cells.

Neurofibrillary tangles (NFTs) are found in a wide range of neurodegenerative disorders, including Alzheimer's disease. The major component of NFTs is aberrantly hyperphosphorylated microtubule-associated protein tau. Because appropriate in vivo models have been lacking, the role of tau phosphorylation in NFTs formation has remained elusive. Here, we describe a new model in which adenovirus-mediated gene expression of tau, DeltaMEKK, JNK3, and GSK-3beta in COS-7 cells produces most of the pathological phosphorylation epitopes of tau including AT100. Furthermore, this co-expression resulted in the formation of tau aggregates having short fibrils that were detergent-insoluble and Thioflavin-S-reactive. These results suggest that aberrant tau phosphorylation by the combination of these kinases may be involved in "pretangle," oligomeric tau fibril formation in vivo.

Alzheimer Disease↗

PubMed2005

Cholesterol and amyloid beta fibrillogenesis.

Evidence is accumulating to suggest that cholesterol is a potent risk factor for the development of Alzheimer's disease. An increase in cholesterol level in neuronal membranes may facilitate the generation and aggregation of the amyloid beta-protein (Abeta). Our results and those of other groups suggest that cholesterol has both direct and indirect effects of acceleration of Abeta fibrillogenesis. A novel concept of cholesterol neurobiology is necessary to elucidate the mechanism underlying cholesterol-dependent Abeta pathology.

Alzheimer Disease↗

PubMed2001

Biochemical and structural studies of the prion protein polymorphism.

A hallmark event in transmissible spongiform encephalopathies is the conversion of the physiological prion protein into the disease-associated isoform. A natural polymorphism at codon 129 of the human prion gene, resulting in either methionine or valine, has profound influence on susceptibility and phenotypic expression of the disease in humans. In this study, we investigated the local propensity of synthetic peptides, corresponding to the region of the polymorphism and containing either methionine or valine, to adopt a beta-sheet-rich structure similar to the pathological protein. Circular dichroism studies showed that the methionine-containing peptide has a greater propensity to adopt a beta-sheet conformation in a variety of experimental conditions. The higher beta-sheet tendency of this peptide was also associated with an increased ability to aggregate into amyloid-like fibrils. These results suggest that methionine at position 129 of the prion protein increases its susceptibility to switch to the abnormal conformation, in comparison with the presence of valine at the same position.

Amyloid↗

PubMed1999

Heparin injection into the adult rat hippocampus induces seizures in the absence of macroscopic abnormalities.

The pathological hallmarks of Alzheimer's disease include neurofibrillary tangles, neuropil threads and neuritic plaques. Neurofibrillary tangles and neuropil threads are comprised of paired helical filaments which are themselves composed of a hyperphosphorylated form of the microtubule-associated protein tau. Neuritic plaques are extracellular deposits of aggregated beta amyloid associated with neurites containing hyperphosphorylated tau. The mechanisms by which the neurofibrillary tangles and neuritic plaques develop in Alzhemier's disease are not clear but it is hypothesized that sulphated glycosaminoglycans are important in their formation. This impression is based on the finding that the glycosaminoglycan, heparan sulphate, is found associated with neurofibrillary tangles, neuritic plaques and neuropil threads while dermatan sulphate, chondroitin sulphate and keratan sulphate immunoreactivity is found around neuritic plaques in brains of Alzheimer's disease patients. Furthermore, in vitro studies demonstrate that sulphated glycosaminoglycans such as heparan sulphate and the closely related molecule heparin interact with tau and potentiate its phosphorylation by a number of serine/threonine kinases, reduce its ability to bind to microtubules and induce paired helical filament formation, all properties associated with tau isolated from Alzheimer's disease brain. Thus, we were interested to learn whether intracerebral injection of the sulphated glycosaminoglycan heparin would give rise to alterations in the cytoskeletal protein tau in the rat brain. Although no cytoskeletal changes were observed, to our considerable surprise we found that the intrahippocampal injection of heparin gave rise to seizures. We have investigated this unexpected effect further in vivo and by using in vitro electrophysiological techniques.

Animals↗

PubMed1999

Thermodynamic instability of human lambda 6 light chains: correlation with fibrillogenicity.

Certain types of human light chains have the propensity to deposit pathologically as amyloid fibrils as evidenced by the preferential association of monoclonal lambda 6 proteins with AL amyloidosis. However, the molecular features that render such proteins amyloidogenic have not been elucidated. Based upon the demonstrated relationship between the thermodynamic stability of light chains and their propensity to aggregate in vitro, we have initiated studies where the thermodynamic properties and fibrillogenic potential of two recombinant (r) V lambda 6 molecules were compared. The first protein was generated from cDNA cloned from marrow-derived plasma cells from a patient (Wil) who had AL amyloidosis and renal amyloid deposits; the second was from a patient (Jto) with multiple myeloma in whom the lambda 6 protein was deposited not as amyloid but in the form of renal tubular casts. The thermodynamic stabilities of rV lambda 6Wil and -Jto were determined from chaotropic and thermal denaturation studies. Based upon the Delta GH2O, Delta H, Delta G25 degrees C, Tm, and Cm values, the rV lambda 6Wil was less stable than its nonamyloidogenic counterpart, rV lambda 6Jto. Measurement of fibril formation using a novel in vitro fibril forming assay demonstrated that although both rV lambda 6 proteins formed fibrils in vitro, Wil had a shorter lag time and exhibited faster kinetics under physiologic conditions. Comparative amino acid sequence analyses of these two components and other lambda 6 amyloid-associated light chains revealed that the Jto protein had certain primary structural features that we posit contributed to its increased stability and thus rendered this protein nonamyloidogenic. Our studies provide the first evidence that stabilizing interactions within the V L domain can influence the kinetics of light chain fibrillogenicity.

Amino Acid Sequence↗

PubMed2005

Function of the cysteine-rich domain of the haemorrhagic metalloproteinase atrolysin A: targeting adhesion proteins collagen I and von Willebrand factor.

The cysteine-rich domain of the haemorrhagic metalloproteinase atrolysin A was shown to inhibit collagen-stimulated platelet aggregation and to interact with MG-63 osteosarcoma cells via integrin alpha2beta1 to inhibit adhesion to collagen I. In addition, we demonstrate by solid-phase binding assays that atrolysin A binds to collagen I and to vWF (von Willebrand factor) via exosites in the cysteine-rich domain. Interestingly, the binding site of the cysteine-rich domain on collagen I is distinct from the cell adhesion site, since the incubation of collagen-I-coated plates with the cysteine-rich domain did not prevent the adhesion of MG-63 cells to collagen. Finally, we show by surface plasmon resonance (BIAcore) analyses that the cysteine-rich domain can block vWF binding to collagen I as well as the binding of collagen I to vWF. Taken together, these results indicate that this domain may function as a cell-surface-receptor-binding site and/or a substrate recognition exosite and may thus play a role in the pathologies associated with atrolysin A.

Amino Acid Sequence↗

PubMed2001

Conformational change, aggregation and fibril formation induced by detergent treatments of cellular prion protein.

The conversion of protease-sensitive prion protein (PrP-sen) to a high beta-sheet, protease-resistant and often fibrillar form (PrP-res) is a central event in transmissible spongiform encephalopathies (TSE) or prion diseases. This conversion can be induced by PrP-res itself in cell-free conversion reactions. The detergent sodium N-lauroyl sarkosinate (sarkosyl) is a detergent that is widely used in PrP-res purifications and is known to stimulate the PrP-res-induced conversion reaction. Here we report effects of sarkosyl and other detergents on recombinant hamster PrP-sen purified from mammalian cells under oxidizing conditions that maintain the single native disulfide bond. Low concentrations of sarkosyl (0.001-0.1%) induced aggregation of PrP-sen molecules, increased light scattering, altered fluorescence excitation and emission spectra, and enhanced the proportion of beta-sheet secondary structure according to circular dichroism and infrared spectroscopies. An enhancement of beta-sheet content was also seen with 0.001% sodium dodecyl sulfate (SDS) but not several other types of detergents. Electron microscopy revealed that sarkosyl induced the formation of both amorphous and fibrillar aggregates. The fibrils appeared to be constructed from spherical bead-like protofibrils. Neither TSE infectivity nor the characteristic partial proteinase K resistance of PrP-res was detected in the sarkosyl-induced PrP aggregates. We conclude that certain anionic detergents can disrupt the conformation of PrP-sen and induce high beta-sheet aggregates that are distinct from scrapie-associated PrP-res in terms of protease-resistance, infrared spectrum and infectivity. These results reinforce the idea that not all high-beta aggregates of PrP are equivalent to the pathologic form, PrP-res.

Animals↗