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Results for “Protein Aggregation, Pathological”

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PubMed2006

Prostaglandin J2 alters pro-survival and pro-death gene expression patterns and 26 S proteasome assembly in human neuroblastoma cells.

Many neurodegenerative disorders are characterized by two pathological hallmarks: progressive loss of neurons and occurrence of inclusion bodies containing ubiquitinated proteins. Inflammation may be critical to neurodegeneration associated with ubiquitin-protein aggregates. We previously showed that prostaglandin J2 (PGJ2), one of the endogenous products of inflammation, induces neuronal death and the accumulation of ubiquitinated proteins into distinct aggregates. We now report that temporal microarray analysis of human neuroblastoma SK-N-SH revealed that PGJ2 triggered a "repair" response including increased expression of heat shock, protein folding, stress response, detoxification and cysteine metabolism genes. PGJ2 also decreased expression of cell growth/maintenance genes and increased expression of apoptotic genes. Over time pro-death responses prevailed over pro-survival responses, leading to cellular demise. Furthermore, PGJ2 increased the expression of proteasome and other ubiquitin-proteasome pathway genes. This increase failed to overcome PGJ2 inhibition of 26 S proteasome activity. Ubiquitinated proteins are degraded by the 26 S proteasome, shown here to be the most active proteasomal form in SK-N-SH cells. We demonstrate that PGJ2 impairs 26 S proteasome assembly, which is an ATP-dependent process. PGJ2 perturbs mitochondrial function, which could be critical to the observed 26 S proteasome disassembly, suggesting a cross-talk between mitochondrial and proteasomal impairment. In conclusion neurotoxic products of inflammation, such as PGJ2, may play a role in neurodegenerative disorders associated with the aggregation of ubiquitinated proteins by impairing 26 S proteasome activity and inducing a chain of events that culminates in neuronal cell death. Temporal characterization of these events is relevant to understanding the underlying mechanisms and to identifying potential early biomarkers.

Biomarkers↗

PubMed1989

The molecular pathology of scrapie and the biological basis of lesion targeting.

Strains of scrapie virus can only be distinguished from the incubation period and neuropathology of the disease they induce in mice of different Sinc genotypes. There are two main aspects of the neuropathology: a spongiosis or degenerative vacuolation associated with the replication of virus, and cerebral amyloid plaques. The relationship between these two is unclear - they can occupy topographically quite different centres in the CNS and the timing of their occurrence is different. Surprisingly this is also the case using the very precise targeting provided by the intraocular infection route. Here the earliest spongiosis is very localised into the primary and secondary projections from the retina, but plaques, when they occur, occupy quite different sites. The relationship between plaques and spongy degeneration is probably a neuroanatomical one, similar to that suggested between plaques and tangle-bearing neurons in Alzheimer's disease. A third aspect of the pathology is the occurrence of SAF in negative-stained E.M. preparations, formed from the aggregation of a sialoglycoprotein called PrP or SAF-protein. Immunolabelling of plaques with antisera to SAF protein shows as yet that there is no antigenic variation in the protein prepared from different scrapie models with different strains of the virus based on available reagents. Strain variation is only recognised on biological criteria; as yet, there are no independent molecular markers or in vitro tests which can account for the biological variation.

Amyloidosis↗

PubMed1997

Aluminum induces lipid peroxidation and aggregation of human blood platelets.

Aluminum (Al3+) intoxication is thought to play a major role in the development of Alzheimer's disease and in certain pathologic manifestations arising from long-term hemodialysis. Although the metal does not present redox capacity, it can stimulate tissue lipid peroxidation in animal models. Furthermore, in vitro studies have revealed that the fluoroaluminate complex induces diacylglycerol formation, 43-kDa protein phosphorylation and aggregation. Based on these observations, we postulated that Al(3+) -induced blood platelet aggregation was mediated by lipid peroxidation. Using chemiluminescence (CL) of luminol as an index of total lipid peroxidation capacity, we established a correlation between lipid peroxidation capacity and platelet aggregation. Al3+ (20-100 microM) stimulated CL production by human blood platelets as well as their aggregation. Incubation of the platelets with the antioxidants nor-dihydroguaiaretic acid (NDGA) (100 microM) and n-propyl gallate (NPG) (100 microM), inhibitors of the lipoxygenase pathway, completely prevented CL and platelet aggregation. Acetyl salicylic acid (ASA) (100 microM), an inhibitor of the cyclooxygenase pathway, was a weaker inhibitor of both events. These findings suggest that Al3+ stimulates lipid peroxidation and the lipoxygenase pathway in human blood platelets thereby causing their aggregation.

Adult↗

PubMed1992

Amphotericin B treatment dissociates in vivo replication of the scrapie agent from PrP accumulation.

Scrapie and related animal and human disorders are neurodegenerative diseases characterized by the formation of a modified, partly proteinase-resistant protein (PrP) of the host, which tends to aggregate as amyloid fibrils and accumulate in the brain of infected individuals. There is a general consensus that the pathological form of PrP (PrPSc) is essential for the clinical appearance of the disease, but whether it is part of the scrapie agent or a by-product of viral infection is still controversial. Here we report that treatment of scrapie-infected hamsters with amphotericin B delays the accumulation in the brain of the proteinase-resistant portion of PrPSc by about 30 days without affecting scrapie replication. The consequence is that hamsters treated with amphotericin B developed clinical signs of disease later than infected controls. We argue that the proteinase-resistant portion of PrPSc is necessary for the development of the disease but that it is unlikely to be essential for scrapie replication.

Amphotericin B↗

PubMed1999

Evidence for both the nucleus and cytoplasm as subcellular sites of pathogenesis in Huntington's disease in cell culture and in transgenic mice expressing mutant huntingtin.

A unifying feature of the CAG expansion diseases is the formation of intracellular aggregates composed of the mutant polyglutamine-expanded protein. Despite the presence of aggregates in affected patients, the precise relationship between aggregates and disease pathogenesis is unresolved. Results from in vivo and in vitro studies of mutant huntingtin have led to the hypothesis that nuclear localization of aggregates is critical for the pathology of Huntington's disease (HD). We tested this hypothesis using a 293T cell culture model system by comparing the frequency and toxicity of cytoplasmic and nuclear huntingtin aggregates. Insertion of nuclear import or export sequences into huntingtin fragments containing 548 or 151 amino acids was used to reverse the normal localization of these proteins. Changing the subcellular localization of the fragments did not influence their total aggregate frequency. There were also no significant differences in toxicity associated with the presence of nuclear compared with cytoplasmic aggregates. These studies, together with findings in transgenic mice, suggest two phases for the pathogenesis of HD, with the initial toxicity in the cytoplasm followed by proteolytic processing of huntingtin, nuclear translocation with increased nuclear concentration of N-terminal fragments, seeding of aggregates and resultant apoptotic death. These findings support the nucleus and cytosol as subcellular sites for pathogenesis in HD.

Animals↗

PubMed2002

Advances in analysis of glycosaminoglycans: its application for the assessment of physiological and pathological states of connective tissues.

Glycosaminoglycans are a class of biological macromolecules found mainly in connective tissues as constituents of proteoglycans, covalently linked to their core protein. Hyaluronan is the only glycosaminoglycan present under its single form and possesses the ability to aggregate with the class of proteoglycans termed hyalectans. Proteoglycans are localised both at the extracellular and cellular (cell-surface and intracellular) levels and, via either their glycosaminoglycan chains or their core proteins participate in and regulate several cellular events and (patho)physiological processes. Advances in analytical separational techniques, including high-performance liquid chromatography, capillary electrophoresis and fluorophore assisted carbohydrate electrophoresis, make possible to examine alterations of glycosaminoglycans with respect to their amounts and fine structural features in various pathological conditions, thus becoming applicable for diagnosis. In this review we present the chromatographic and electromigration procedures developed to analyse and characterise glycosaminoglycans. Moreover, a critical evaluation of the biological relevance of the results obtained by the developed methodology is discussed.

Chromatography, Liquid↗

PubMed2001

Glycosylation and the immune system.

Almost all of the key molecules involved in the innate and adaptive immune response are glycoproteins. In the cellular immune system, specific glycoforms are involved in the folding, quality control, and assembly of peptide-loaded major histocompatibility complex (MHC) antigens and the T cell receptor complex. Although some glycopeptide antigens are presented by the MHC, the generation of peptide antigens from glycoproteins may require enzymatic removal of sugars before the protein can be cleaved. Oligosaccharides attached to glycoproteins in the junction between T cells and antigen-presenting cells help to orient binding faces, provide protease protection, and restrict nonspecific lateral protein-protein interactions. In the humoral immune system, all of the immunoglobulins and most of the complement components are glycosylated. Although a major function for sugars is to contribute to the stability of the proteins to which they are attached, specific glycoforms are involved in recognition events. For example, in rheumatoid arthritis, an autoimmune disease, agalactosylated glycoforms of aggregated immunoglobulin G may induce association with the mannose-binding lectin and contribute to the pathology.

Animals↗

PubMed2000

Metal-catalyzed oxidation of alpha-synuclein in the presence of Copper(II) and hydrogen peroxide.

alpha-Synuclein is a component of abnormal protein depositions of Lewy bodies and senile plaques found in Parkinson's and Alzheimer's diseases, respectively. By using chemical coupling reagents such as dicyclohexylcarbodiimide or N-(ethoxycarbonyl)-2-ethoxy-1, 2-dihydroquinoline, the protein was shown to experience self-oligomerization in the presence of either copper(II) or Abeta25-35. The oligomers which appeared as a ladder on a 10-20% Tricine/SDS-PAGE have been suggested to participate in the formation of protein aggregations by possibly providing a nucleation center. Since oxidatively modified protein could increase its own tendency toward protein aggregation, metal-catalyzed oxidation of alpha-synuclein has been examined with copper(II) and hydrogen peroxide in the absence of the coupling reagent. Intriguingly, the protein was also self-oligomerized into an SDS-resistant ladder on the gel. This biochemically specific copper-mediated oxidative oligomerization was shown to be dependent upon the acidic C-terminus of alpha-synuclein because the C-terminally truncated proteins such as alpha-syn114 and alpha-syn97 were not affected by the metal and hydrogen peroxide. More importantly, the oxidative oligomerization was synergistically enhanced by the presence of Abeta25-35, indicating that the peptide interaction with alpha-synuclein facilitated the copper(II) binding to the acidic C-terminus and subsequent oxidative crosslinking. It has been, therefore, suggested that abnormalities in copper and H(2)O(2) homeostasis and certain pathological factors functionally similar to the Abeta25-35 could play critical roles in the metal-catalyzed oxidative oligomerization of alpha-synuclein, which may lead to possible protein aggregation and neurodegenerations.

Alkylating Agents↗

PubMed2006

Transgenic mice expressing the myotilin T57I mutation unite the pathology associated with LGMD1A and MFM.

Myotilin is a muscle-specific Z-disc protein with putative roles in myofibril assembly and structural upkeep of the sarcomere. Several myotilin point mutations have been described in patients with limb-girdle muscular dystrophy type 1A (LGMD1A), myofibrillar myopathy (MFM), spheroid body myopathy (SBM), three similar adult-onset, progressive and autosomal dominant muscular dystrophies. To further investigate myotilin's role in the pathogenesis of these muscle diseases, we have characterized three independent lines of transgenic mice expressing mutant (T57I) myotilin under the control of the human skeletal actin promoter. Similar to LGMD1A and MFM patients, these mice develop progressive myofibrillar pathology that includes Z-disc streaming, excess myofibrillar vacuolization and plaque-like myofibrillar aggregation. These aggregates become progressively larger and more numerous with age. We show that the mutant myotilin protein properly localizes to the Z-disc and also heavily populates the aggregates, along with several other Z-disc associated proteins. Whole muscle physiological analysis reveals that the extensor digitorum longus muscle of transgenic mice exhibits significantly reduced maximum specific isometric force compared with littermate controls. Intriguingly, the soleus and diaphragm muscles are spared of any abnormal myopathology and show no reductions in maximum specific force. These data provide evidence that myotilin mutations promote aggregate-dependent contractile dysfunction. In sum, we have established a promising patho-physiological mouse model that unifies the phenotypes of LGMD1A, MFM and SBM.

Animals↗

PubMed1997

Sequence and biological activity of catrocollastatin-C: a disintegrin-like/cysteine-rich two-domain protein from Crotalus atrox venom.

In this study we report on the isolation and biological characterization of a 23.6-kDa protein from the venom of the western diamondback rattlesnake, Crotalus atrox. Primary structural analysis shows the protein to be composed of a spacer/disintegrin-like domain and a cysteine-rich domain. The sequence is identical to the same carboxy-terminal domains found in the C. atrox metalloproteinase, catrocollastatin, and hence we termed the protein catrocollastatin-C. We estimate that catrocollastatin-C represents at least 0.5% of the total protein in C. atrox venom. The protein is an inhibitor of collagen-stimulated but not ADP-stimulated platelet aggregation. Reduction and alkylation of catrocollastatin-C causes a loss of platelet aggregation inhibitor activity. A synthetic, cyclic peptide designed from the catrocollastatin-C disintegrin-like domain has potent platelet aggregation inhibitory activity. This suggests that the corresponding region in the disintegrin-like domain of the protein is at least partially responsible for the inhibition of platelet aggregation previously reported for the protein. These studies underscore the biochemical and functional complexity of crotalid snake venoms due to differential proteolytic processing of precursor proteins and how the processed precursor fragments may contribute to the observed pathological effects of the venom.

Adenosine Diphosphate↗

PubMed2003

Optimized overproduction, purification, characterization and high-pressure sensitivity of the prion protein in the native (PrP(C)-like) or amyloid (PrP(Sc)-like) conformation.

Overproduction and purification of the prion protein is a major concern for biological or biophysical analysis as are the structural specificities of this protein in relation to infectivity. We have developed a method for the effective cloning, overexpression in Escherichia coli and purification to homogeneity of Syrian golden hamster prion protein (SHaPrP(90-231)). A high level of overexpression, resulting in the formation of inclusion bodies, was obtained under the control of the T7-inducible promoter of the pET15b plasmid. The protein required denaturation, reduction and refolding steps to become soluble and attain its native conformation. Purification was carried out by differential centrifugation, gel filtration and reverse phase chromatography. An improved cysteine oxidation protocol using oxidized glutathione under denaturing conditions, resulted in the recovery of a higher yield of chromatographically pure protein. About 10 mg of PrP protein per liter of bacterial culture was obtained. The recombinant protein was identified by monoclonal antibodies and its integrity was confirmed by electrospray mass spectrometry (ES/MS), whereas correct folding was assessed by circular dichroism (CD) spectroscopy. This protein had the structural characteristics of PrP(C) and could be converted to an amyloid structure sharing biophysical and biochemical properties of the pathologic form (PrP(Sc)). The sensitivity of these two forms to high pressure was investigated. We demonstrate the potential of using pressure as a thermodynamic parameter to rescue trapped aggregated prion conformations into a soluble state, and to explore new conformational coordinates of the prion protein conformational landscape.

Animals↗

PubMed2005

Covalent blocking of fibril formation and aggregation of intracellular amyloidgenic proteins by transglutaminase-catalyzed intramolecular cross-linking.

Two different types of physical bonding have been proposed to involve in the formation of neuronal inclusions of patients with neurodegenerative diseases such as Alzheimer's, Parkinson's, and polyglutamine diseases. One is the noncovalent bonding that stabilizes the amyloid-type fibrous aggregates, and the other is the covalent cross-linking catalyzed by tissue transglutaminase. The cross-linking is subdivided into the inter- and intramolecular cross-linking. Little attention has been paid to the pathological roles of the intramolecular cross-linking. To elucidate the possible interplay between the intramolecular cross-linking and the amyloid-type fibril formation, we performed an in vitro aggregation analysis of three intracellular amyloidgenic proteins (a domain of tau protein, alpha-synuclein, and truncated yeast prion Sup35) in the presence of tissue transglutaminase. The analysis was performed in low concentrations of the proteins using techniques including thioflavin T binding and mass spectrometry. The results demonstrated that the amyloid-type fibril formation was strongly inhibited by the transglutaminase-catalyzed intramolecular cross-linking, which blocked both the nucleation and the fiber extension steps of the amyloid formation. Far-UV CD spectroscopy indicated that the cross-linking slightly altered the backbone conformation of the proteins. It is likely that conformational restriction imposed by the intramolecular cross-links has impaired the ordered assembly of the amyloidgenic proteins. Nonamyloid type aggregation was also suppressed by the intramolecular cross-links. On the basis of the results, we proposed that tissue transglutaminase is a modulator for the protein aggregation and can act defensively against the fibril deposition in neurons.

Adult↗

PubMedDate not supplied

Development of a new treatment for Alzheimer's disease and Parkinson's disease using anti-aggregatory beta-synuclein-derived peptides.

The synaptic protein alpha-synuclein is a major constituent of Lewy bodies (LB), pathological neuronal inclusion bodies found in Parkinson's disease (PD), Alzheimer's disease (AD), and other neurodegenerative disorders. Owing to data from patient brains, it was speculated that an imbalance between alpha-synuclein and beta-synuclein might be one of the reasons for formation of LBs and the consequent functional deficits. This was supported by the fact that beta-synuclein is able to prevent abnormal alpha-synuclein aggregation. Transgenic mice overexpressing alpha-synuclein display LB-like inclusions in different brain regions and motor deficits. To verify if re-establishing a normal relation between alpha-synuclein and beta-synuclein is able to prevent the pathology, bigenic mice have been created that overexpress both synucleins. Beta-synuclein decreased formation of LBs by 40% and prevented functional deficits. This is considered as preliminary in vivo proof of antiaggregatory function of beta-synuclein and its potential as therapeutic substance for treatment of neurodegenerative disorders linked with abnormal protein aggregation. Peptide libraries have been synthesized to explore the active structures of beta-synuclein. The first 15 N-terminal amino-acids turned out to be important for the antiaggregatory effect. Further smaller beta-synuclein-derived peptides have screened for antiaggregatory and neuroprotective potency in different tissue-culture systems. Preliminary data suggest some of them can be used as leads for further drug development.

Alzheimer Disease↗

PubMed2001

Partially folded intermediates as critical precursors of light chain amyloid fibrils and amorphous aggregates.

Light chain, or AL, amyloidosis is a pathological condition arising from systemic extracellular deposition of monoclonal immunoglobulin light chain variable domains in the form of insoluble amyloid fibrils, especially in the kidneys. Substantial evidence suggests that amyloid fibril formation from native proteins occurs via a conformational change leading to a partially folded intermediate conformation, whose subsequent association is a key step in fibrillation. In the present investigation, we have examined the properties of a recombinant amyloidogenic light chain variable domain, SMA, to determine whether partially folded intermediates can be detected and correlated with aggregation. The results from spectroscopic and hydrodynamic measurements, including far- and near-UV circular dichroism, FTIR, NMR, and intrinsic tryptophan fluorescence and small-angle X-ray scattering, reveal the build-up of two partially folded intermediate conformational states as the pH is decreased (low pH destabilized the protein and accelerated the kinetics of aggregation). A relatively nativelike intermediate, I(N), was observed between pH 4 and 6, with little loss of secondary structure, but with significant tertiary structure changes and enhanced ANS binding, indicating exposed hydrophobic surfaces. At pH below 3, we observed a relatively unfolded, but compact, intermediate, I(U), which was characterized by decreased tertiary and secondary structure. The I(U) intermediate readily forms amyloid fibrils, whereas I(N) preferentially leads to amorphous aggregates. Except at pH 2, where negligible amorphous aggregate is formed, the amorphous aggregates formed significantly more rapidly than the fibrils. This is the first indication that different partially folded intermediates may be responsible for different aggregation pathways (amorphous and fibrillar). The data support the hypothesis that amyloid fibril formation involves the ordered self-assembly of partially folded species that are critical soluble precursors of fibrils.

Amyloid↗

PubMed2002

Early accumulation of p62 in neurofibrillary tangles in Alzheimer's disease: possible role in tangle formation.

Neurofibrillary tangles (NFTs) and neuritic plaques (NPs) are two major histopathological lesions in Alzheimer's disease (AD). Although their aetiological relationship is unclear, both NFTs and dystrophic neurites of NPs display immunoreactivity for ubiquitin. This suggests that dysfunction in ubiquitin-mediated proteolysis and the resulting accumulation of ubiquitin-conjugated proteins may contribute to the origination of dystrophic neurites and NFTs. We recently discovered a novel constituent of neuropathological protein aggregates, ubiquitin-binding protein p62, with evidence that the accumulation of ubiquitin-conjugated proteins and p62 into cytoplasmic inclusions might be interconnected. In the present work we examined in detail the role of p62 in AD-type pathology, i.e. NFTs, NPs and neuropil threads. Using immunohistochemistry for p62, ubiquitin and hyperphosphorylated tau, we analysed parietal cortical samples of 15 clinicopathologically verified AD cases and nine nondemented aged subjects with abundant NPs. We found that p62 immunoreactivity appears early during neurofibrillary pathogenesis and is invariably and stably present in NFTs. In contrast, p62 was absent or barely detectable in neuropil threads. Furthermore, NP-associated dystrophic neurites were generally devoid of p62, regardless of their content of hyperphosphorylated tau and/or ubiquitin. The results suggest that early involvement of p62 might be critical in the aggregation of hyperphosphorylated tau into perikaryal aggregates, i.e. NFTs.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Autophagosomes: biogenesis from scratch?

To survive extreme environmental conditions, and in response to certain developmental and pathological situations, eukaryotic organisms employ the catabolic process of autophagy. This degradative pathway allows cells to eliminate large portions of the cytoplasm, from aberrant protein aggregates to superfluous or damaged organelles and even entire organisms such as invading bacteria. Structures targeted for destruction are sequestered into large double-membrane vesicles called autophagosomes and then delivered into the interior of the lysosome or vacuole, where they are consumed by resident hydrolases. Autophagosome formation during selective autophagy is dependent upon the cargoes, and in all cases seems to involve expansion of the sequestering membrane.

Animals↗

PubMed2006

The roles of intracellular protein-degradation pathways in neurodegeneration.

Many late-onset neurodegenerative diseases, including Parkinson's disease and Huntington's disease, are associated with the formation of intracellular aggregates by toxic proteins. It is therefore crucial to understand the factors that regulate the steady-state levels of these 'toxins', at both the synthetic and degradation stages. The degradation pathways acting on such aggregate-prone cytosolic proteins include the ubiquitin-proteasome system and macroautophagy. Dysfunction of the ubiquitin-proteasome or macroautophagy pathways might contribute to the pathology of various neurodegenerative conditions. However, enhancing macroautophagy with drugs such as rapamycin could offer a tractable therapeutic strategy for a number of these diseases.

Animals↗

PubMed2005

A crucial role of mitochondrial Hsp40 in preventing dilated cardiomyopathy.

Many heat-shock proteins (Hsp) are members of evolutionarily conserved families of chaperone proteins that inhibit the aggregation of unfolded polypeptides and refold denatured proteins, thereby remedying phenotypic effects that may result from protein aggregation or protein instability. Here we report that the mitochondrial chaperone Hsp40, also known as Dnaja3 or Tid1, is differentially expressed during cardiac development and pathological hypertrophy. Mice deficient in Dnaja3 developed dilated cardiomyopathy (DCM) and died before 10 weeks of age. Progressive respiratory chain deficiency and decreased copy number of mitochondrial DNA were evident in cardiomyocytes lacking Dnaja3. Profiling of Dnaja3-interacting proteins identified the alpha-subunit of DNA polymerase gamma (Polga) as a client protein. These findings suggest that Dnaja3 is crucial for mitochondrial biogenesis, at least in part, through its chaperone activity on Polga and provide genetic evidence of the necessity for mitochondrial Hsp40 in preventing DCM.

Animals↗